Propranolol, chlorpromazine and diclofenac restore susceptibility of extensively drug-resistant (XDR)-Acinetobacter baumannii to fluoroquinolones

Mostafa A Mohammed; Mohammed T Ahmed; Bahaa E Anwer; Khaled M Aboshanab; Mohammad M Aboulwafa

doi:10.1371/journal.pone.0238195

. 2020 Aug 26;15(8):e0238195. doi: 10.1371/journal.pone.0238195

Propranolol, chlorpromazine and diclofenac restore susceptibility of extensively drug-resistant (XDR)-Acinetobacter baumannii to fluoroquinolones

Mostafa A Mohammed ¹, Mohammed T Ahmed ¹, Bahaa E Anwer ¹, Khaled M Aboshanab ^2,^*, Mohammad M Aboulwafa ²

Editor: Monica Cartelle Gestal³

PMCID: PMC7449414 PMID: 32845920

Abstract

Nosocomial infections caused by extensively drug-resistant (XDR) or Pan-Drug resistant (PDR) Acinetobacter (A.) baumannii have recently increased dramatically creating a medical challenge as therapeutic options became very limited. The aim of our study was to investigate the antibiotic-resistance profiles and evaluate the various combinations of ciprofloxacin (CIP) or levofloxacin (LEV) with antimicrobial agents and non-antimicrobial agents to combat antimicrobial resistance of XDR A. baumannii. A total of 100 (6.25%) A. baumannii clinical isolates were recovered from 1600 clinical specimens collected from hospitalized patients of two major university hospitals in Upper Egypt. Antimicrobial susceptibility tests were carried out according to CLSI guidelines. Antimicrobial susceptibility testing of the respective isolates showed a high percentage of bacterial resistance to 19 antimicrobial agents ranging from 76 to99%. However, a lower percentage of resistance was observed for only colistin (5%) and doxycycline (57%). The isolates were categorized as PDR (2; 2%), XDR (68; 68%), and multi-drug resistant (MDR) (30; 30%). Genotypic analysis using ERIC-PCR on 2 PDR and 32 selected XDR isolates showed that they were not clonal. Combinations of CIP or LEV with antibiotics (including, ampicillin, ceftriaxone, amikacin, or doxycycline) were tested on these A. baumannii non-clonal isolates using standard protocols where fractional inhibitory concentrations (−FICs) were calculated. Results of the respective combinations showed synergism in 23.5%, 17.65%, 32.35%, 17.65% and 26.47%, 8.28%, 14.71%, 26.47%, of the tested isolates, respectively. CIP or LEV combinations with either chlorpromazine (CPZ) 200 μg/ml, propranolol (PR) in two concentrations, 0.5 mg/ml and 1.0 mg/ml or diclofenac (DIC) 4 mg/ml were carried out and the MIC decrease factor (MDF) of each isolate was calculated and results showed synergism in 44%, 50%, 100%, 100% and 94%, 85%, 100%, 100%, of the tested isolates, respectively. In conclusion, combinations of CIP or LEV with CPZ, PR, or DIC showed synergism in most of the selected PDR and XDR A. baumannii clinical isolates. However, these combinations have to be re-evaluated in vivo using appropriate animal models infected by XDR- or PDR- A. baumannii.

Introduction

A. baumannii is a Gram-negative, strictly aerobic bacterial pathogen that can survive for prolonged periods under a wide range of environmental conditions and on surfaces making it a frequent cause of nosocomial infections and outbreaks [1]. A. baumannii is one of the most common bacteria causing hospital acquired and difficult to treat infections, due to its endless capacity to acquire antimicrobial resistance owing to the plasticity of its genome [1]. Many acquired resistance mechanisms have been reported for this pathogen, rendering it able to express MDR or XDR phenotypes which are associated with significant morbidities and mortalities [2].

In the last four decades, fluoroquinolones (FQs) have shown good activity against A. baumannii isolates, even better than penicillin derivatives, cephalosporins, and aminoglycosides. However, resistance to these drugs has rapidly emerged in recent years [3]. The developing resistance of A. baumannii to polymyxins has been described at least during the last two decades and this was attributed, besides their misuse, to the abundance of these antibiotics in multiple pharmaceutical markets [4] Consequently, researchers have been focusing their work on finding new therapeutic options to overcome this health problem [5–7]. However, little information is available on the treatment regimens of A. baumannii by FQs combination with antimicrobial or non-antimicrobial agents. For example, the combination of LEV with amikacin in north Egypt to control A. baumannii resistance was barely successful [8], while several other attempts failed [9]. However, a fully successful combination of CIP with amikacin against Gram-negative bacteria in Upper Egypt was reported [10]. On the other hand, another study reported the successful use of polymyxin B and doxycycline combination in patients with MDR A. baumannii infections [11]. Recently, various reports confirmed that PR, a non-selective beta-blocker, had potent negative effects on the cell growth viability and progression, and was suggested with evidence to reduce cancer types [12, 13]. Some studies were conducted on the use of a combination of a non-selective beta-blocker, including PR [7] or carvedilol [14], with other drugs for the treatment of rosacea. Most patients with bacterial infections suffer pain and fever that require complex treatment with antibiotics, antipyretics, and analgesics. Antipyretics and non-steroidal anti-inflammatory drugs (NSAIDs) commonly co-administered with antimicrobial therapy often modify the susceptibility of microbes to antimicrobial therapy [15–17] by changing the hydrophobicity of microbes [18], influencing biofilm production [19], and interacting with the transport and release of antibiotics [20]. The antibacterial activity of antipsychotic agents, such as CPZ has been recently evaluated on certain Gram-negative pathogens [5, 6], Gram-positive pathogens and Mycobacterium tuberculosis [21]. However, the effect of FQs in combination with non-antimicrobial agents such as PR, DIC, or CPZ on A. baumannii clinical isolates particularly, XDR or PDR phenotypes is still not yet explored.

Therefore, this study aimed to determine the antibiotic-resistant phenotypes of A. baumannii recovered from hospitalized patients of two major University Hospitals in Upper Egypt, followed by evaluating various CIP-LEV combinations with several antibiotics and non-antibiotics particularly, PR, DIC, and CPZ as an attempt to control the resistance of this pathogen.

Methods

Bacterial isolates

One thousand and six hundred clinical specimens including blood, ventral venous catheters, endotracheal tube, urinary tract catheter, sputum, urine, skin, wound and throat swabs were collected from hospitalized febrile patients (oral temperature >38°C for at least 1 hour) between January 2014 and March 2019 from Al Azhar and Assiut University Hospitals, Upper Egypt. The study was approved by the Research Ethics Committee Faculty of Pharmacy, Ain Shams University ENREC-ASU-63 where both informed and written consents were obtained from parents of patients after explaining the study purpose. All specimens were streak-plated on MacConky agar (Oxoid Limited, England), then the non-lactose fermenter colonies sub-cultured on blood agar and finally on Herellea agar (Himedia, India), and incubated at 37°C for 24 hours [22].

Phenotypic and genotypic identification of A. baumannii

For phenotypic identification, separate colonies were processed for qualitative conventional diagnostic tests for A. baumannii; including Gram staining and biochemical tests such as catalase, citrate utilization, oxidase, and indole tests [22]. For genotypic identification, the amplification of the intrinsic bla_OXA-51-like gene was used [23]. The primers used for detection of bla_OXA-51 gene were F-(5-TAATGCTTTGATCGGCCTTG-3) and R-(5-TGGATTGCACTTCATCTTGG-3) [24]. DNA was extracted and purified using the GeneJet PCR purification kit (Thermo, USA, catalog No. K0721), following instructions of the manufacturer. The extracted DNA was stored at -20°C for further use. PCR amplification was performed in the thermal cycler (Nyx Technik ATC 401, USA) using 25 pmol of each primer, 100 ng of genomic DNA, the PCR mixture (25 μl) formulated according to the protocol supplied with the Dream Taq master mix kit (Thermo Fisher, UK) and 1 μl template DNA. PCR conditions were 94°C for 3 min, 35 cycles at 94°C for 45 s at 57°C for 45 s, and at 72°C for 1 min, followed by a final extension step at 72°C for 5 min. PCR products were analyzed using agarose gel electrophoresis [23]. The characteristic band at 353 bp for bla_OXA-51-like genes was considered positive only for A. baumannii. A. baumannii ATCC 19606 was used for quality control.

Antimicrobial susceptibility testing

Disc diffusion

This is was carried out using Kirby-Bauer disk diffusion method as recommended by the Clinical and Laboratory Standards Institute (CLSI) [25]. The antibiotic discs used for susceptibility testing were imipenem (10 μg), meropenem (10 μg), piperacillin (100 μg), piperacillin/tazobactam (100/10 μg), ampicillin/sulbactam (10/10 μg), ceftazidime (30 μg), cefotaxime (30 μg), ceftriaxone (30 μg), cefepime (30μg), amikacin (10μg), tobramycin (10 μg), gentamicin (10 mg), CIP (5 μg), LEV (5 μg), gatifloxacin (5 μg), trimethoprim/sulfamethoxazole (12.5/23.75 μg), tigecycline (15 μg), doxycycline (30 mcg) and colistin (10 units). All antimicrobial discs were purchased from Oxoid (UK) except gatifloxacin discs which were purchased from Himedia (India). MDR, XDR, and PDR phenotypes were identified as previously determined [26].

Minimum inhibitory concentrations (MICs)

MICs of CIP, LEV, ampicillin (AMP), ceftriaxone (CRO), amikacin (AK), doxycycline (DO), and vancomycin (VC) against some selected XDR- and PDR- A. baumannii isolates were determined by broth micro-dilution method according to CLSI guidelines [27]. A. baumannii ATCC 19606 was used for quality control. The MIC for antimicrobial agents ranged from 0.125–256 μg/ml.

Molecular typing of recovered isolates

To investigate the clonal relationship, clonal expansion, and diversity of the recovered A. baumannii isolates, molecular typing using ERIC-PCR was carried out on some of these isolates that showed PDR and XDR phenotypes [28]. Genomic DNA was extracted using the Genomic DNA Purification Kit (Thermo Fisher Scientific, UK) according to the manufacturer’s instructions. ERIC-PCR was carried out using the ERIC-1 (5’-ATGTAAGCTCCTGGGGATTCAC-3’) and ERIC-2 (5’-AAGTAAGTG ACTGGGGTGAGCG-3’) primers as previously described [28]. The PCR products were analyzed using agarose gel electrophoresis using 1.5% (w/v) agarose containing ethidium bromide (0.5 mg/ml) and visualized on a UV transilluminator. Analysis of ERIC-PCR dendrogram was constructed by the use of the UPGMA clustering method, Bionumeric program version 7.6 (Applied Maths). The Percentage of similarity among 34 strains of A. baumannii was calculated by the use of Jaccard's Coefficient [29].

Evaluation of drug combinations

Evaluation of FQs-antibiotic combinations

Firstly, the MIC of each antimicrobial agent CIP, LEV, AMP, CRO, VC, AK, and DO was determined using the broth microdilution technique according to the CLSI guidelines 2011 [27]. In vitro, combinations of FQs members including either CIP or LEV with either AMP, CRO, VC, AK, or DO were evaluated through the checkerboard method and the protocol described by Hsieh et al [30]. The resulting checkerboard included each combination of two antibiotics in opposite directions where one antibiotic was serially diluted horizontally and the other was diluted vertically. The sum of the fractional inhibitory concentration (−FICs) was calculated according to the following equation:

\sum {FICs = FIC}_{A} {+FIC}_{B} = \frac{A}{M I C A} + \frac{B}{M I C B}

FIC_A is the MIC of drug A in the combination /MIC of drug A alone, and FIC_B is the MIC of drug B in the combination /MIC of drug B alone. The combination is considered synergistic when −FIC is ≤ 0.5, additive when −FIC is > 0.5 and ≤ 1, indifferent when −FIC is >1 and ≤ 4, and antagonistic when −FIC is > 4 [9].

Evaluation of FQs- non-antibiotic combinations

FQs combinations with different non-antibiotic such as the non-selective β blocker PR, 25 μg-1 mg/ml (Sigma, Aldrich, UK), selective β blocker labetalol, 25 μg-1 mg/ml (Sigma, Aldrich, UK), the NSAIDs DIC, 25 μg-4 mg/ml (Novartis, Egypt), acetylsalicylic acid, 100–400 μg/ml (Adwia, Egypt), the proton pump inhibitor omeprazole and esomeprazole 25–200 μg/ml (Sigma, Aldrich, UK), the diuretic furosemide, 25 μg-1 mg/ml (Sanofi Aventis, Egypt) and the antipsychotic CPZ, 25–200 μg/ml (Sigma, Aldrich, UK) against some selected A. baumannii were evaluated by calculating the MDF [31]. The MDF of each isolate was calculated according to the following formula MDF = MIC_{without non-antibiotic} / MIC_{with non-antibiotic}. An MDF value greater than 4 was defined as a significant inhibition according to the protocol of Huguet [31]. The MIC of tested FQs ranged from 0.03–64 μg/ml.

Results

Specimen collection and identification of the recovered A. baumannii isolates

The endotracheal tube specimen showed the highest percentage (29%) of the recovered A. baumannii isolates while throat swab, skin, and central venous catheter specimens gave the lowest percentages (2.0% each) (Fig 1). Out of 1600 samples cultivated on MacConky agar, only 623 isolates (38.94%) were identified as non-lactose fermenters, of which 151 isolates were phenotypically identified as Acinetobacter isolates. Of these, only 100 isolates (6.25%) were PCR positive for the bla_OXA-51-like gene and therefore were identified as A. baumannii (Fig 2).

Fig 1 — CVC: Central Venus Catheters. ETT: Endotracheal tube, UTC: Urinary Tract Catheter.

Antimicrobial susceptibility testing

Disc diffusion

As shown in Fig 3, all the A. baumannii isolates exhibited high resistance to most of the tested antimicrobial agents (76–99%). However, the lowest resistance recorded was to colistin (5%) followed by doxycycline (57%). Phenotypically, 30 (30%), 68(68%), and 2 (2%) isolates were reported as MDR, XDR, and PDR, respectively (Fig 4). Analysis of the resulting susceptibility to 19 antimicrobial agents showed the resistance diversity of these isolates. They were divided into 12 major profiles, according to the number of antimicrobial agents they exhibited resistance to, ranging from 19 to 6 antimicrobial agents. The first profile represented PDR isolates (two isolates) that were resistant to all 19 antimicrobial agents tested. The second profile represented some of XDR isolates (32 isolates) that were resistant to all tested antimicrobial agents except colistin. Finally, profile number 12 was resistant to only 6 out of the 19 tested antimicrobial agents (Fig 5).

Fig 4 — MDR (Multi-Drug resistant), XDR; extensively–Drug-resistant and PDR (Pan-Drug resistant).

Minimum inhibitory concentration

The MIC values of 34 A. baumannii isolates including 2 PDR (profile 1, Fig 5) and 32 XDR isolates (Profile 2; Fig 5) are outlined in Table 1. The MIC values of LEV, CIP, AMP, CRO, AK, DO and CV against the selected 34 isolates ranged from 4–32, 8–64, 32–128, 64–256, 32–256, 16–256, and 16–256 μg/ml, respectively.

Table 1. MIC of selected antimicrobial agents against PDR and some selected XDR isolates (n = 34).

No.	Isolates code	MICs (μg/ml)
No.	Isolates code	CIP	AMP	LEV	CRO	AK	DO	VC
1	AS-07	16	64	8	64	256	64	128
2	AS-09	32	64	8	128	256	256	128
3	AS-15	16	64	8	64	256	256	128
4	AS-18	32	64	16	64	256	256	128
5	AS-19	32	64	16	256	256	256	128
6	AS-24	32	64	16	64	32	16	16
7	AS-25	16	64	16	64	64	16	16
8	AS-26	32	64	8	64	256	256	16
9	AS-30	32	64	16	64	256	256	16
10	AS-31	8	64	4	128	64	256	16
11	AS-32	64	128	32	256	256	256	64
12	AS-34	16	64	4	64	64	256	16
13	AS-35	16	64	4	64	256	256	16
14	AS-36	16	64	4	64	256	256	16
15	AS-37	16	64	4	64	256	64	16
16	AS-38	16	32	4	64	128	64	64
17	AS-39	16	32	4	64	128	64	128
18	AS-42	32	128	8	64	256	64	256
19	AS-47	32	128	8	64	256	64	256
20	AS-50	16	128	8	64	256	64	256
21	AS-51	16	128	8	64	128	16	256
22	AS-52	32	128	16	64	128	16	256
23	AS-54	16	128	8	64	128	16	256
24	AZ-02	16	32	8	256	64	16	32
25	AZ-06	32	32	8	256	64	16	256
26	AZ-10	16	32	8	64	256	16	128
27	AZ-25	32	64	4	64	128	32	256
28	AZ-26	16	32	8	64	256	32	256
29	AZ-36	16	32	8	64	256	16	128
30	AZ-41	32	32	4	64	256	16	64
31	AZ-42	32	32	8	64	256	16	128
32	AZ-43	32	32	4	64	256	64	16
33	AZ-44	32	64	4	64	128	64	16
34	AZ-46	16	32	16	64	128	64	16

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MICs, minimum inhibitory concentrations; CIP, ciprofloxacin; LEV, levofloxacin; AMP, ampicillin; CRO, ceftriaxone; AK, amikacin; DO, doxycycline; and VC, vancomycin.

Genotyping of isolates

A genotypic analysis of the 34 selected isolates using ERIC-PCR is shown in Fig 6. The obtained isolates were divided into three major clusters (Cluster I, II, and III) with similarity ranging from 0% to 97.3%. Cluster I was a major cluster representing 58.82% (20/34) of the isolates. Cluster II represented 5.88% (2/34) while cluster III represented 35.29% of the total isolates. Clusters I and II were completely recovered from Assiut University hospitals. Cluster III was recovered from Al Azhar University hospital except for one isolate (AS-47) which was recovered from Assiut University hospital.

Evaluation of drug combinations

Evaluation of FQs-antibiotic combinations

The results of the antimicrobial combination with FQs (LEV, CIP) using the checkerboard method were presented in the Tables 2 and 3.

Table 2. Effects of CIP combinations with different antimicrobial agents by the checkerboard method.

No.	Isolates code	CIP-AMP μg/ml	∑FIC	Int	CIP-CRO μg/ml	∑FIC	Int	CIP-AK μg/ml	∑FIC	Int	CIP-DO μg/ml	∑FIC	Int	CIP-VC μg/ml	∑FIC	Int
1	AS-07	8(64)	1.50	I	8(64)	1.50	I	4(64)	0.50	S	4(32)	0.75	D	16(128)	2.00	I
2	AS-09	16(64)	1.50	I	16(64)	1.00	D	8(64)	0.50	S	16(64)	0.75	D	16(128)	1.50	I
3	AS-15	8(64)	1.50	I	16(64)	2.00	I	8(128)	1.00	D	8(32)	0.63	D	32(128)	3.00	I
4	AS-18	8(32)	0.75	D	16(64)	1.50	I	32(64)	1.25	I	32(64)	1.25	I	16(128)	1.50	I
5	AS-19	16(32)	1.00	D	16(128)	1.00	D	8(64)	0.50	S	16(64)	0.75	D	16(128)	1.50	I
6	AS-24	8(16)	0.50	S	16(64)	1.50	I	16(16)	1.00	D	4(8)	0.63	D	16(16)	1.50	I
7	AS-25	4(16)	0.50	S	4(64)	1.25	I	32(32)	2.50	I	2(4)	0.38	S	16(16)	2.00	I
8	AS-26	8(16)	0.50	S	4(16)	0.38	S	8(64)	0.50	S	16(64)	0.75	D	8(8)	0.75	D
9	AS-30	4(16)	0.38	S	4(16)	0.38	S	8(256)	1.25	I	16(64)	0.75	D	8(8)	0.75	D
10	AS-31	2(16)	0.50	S	4(16)	0.63	D	2(16)	0.50	S	16(64)	2.25	I	2(16)	1.25	I
11	AS-32	32(64)	1.00	D	16(128)	0.75	D	16(128)	0.75	D	32(64)	0.75	D	32(32)	1.00	D
12	AS-34	8(32)	1.00	D	4(64)	1.25	I	2(32)	0.63	D	4(128)	0.75	D	4(16)	1.25	I
13	AS-35	8(32)	1.00	D	4(32)	0.75	D	16(64)	1.25	I	16(64)	1.25	I	4(16)	1.25	I
14	AS-36	8(64)	1.50	I	4(8)	0.38	S	4(64)	0.50	S	16(64)	1.25	I	4(16)	1.25	I
15	AS-37	4(64)	1.25	I	4(16)	0.50	S	4(128)	0.75	D	8(16)	0.75	D	4(16)	1.25	I
16	AS-38	1(8)	0.31	S	4(32)	0.75	D	4(64)	0.75	D	8(32)	1.00	D	4(64)	1.25	I
17	AS-39	1(8)	0.31	S	4(64)	1.25	I	4(64)	0.75	D	4(64)	1.25	I	4(128)	1.25	I
18	AS-42	16(64)	1.00	D	8(64)	1.25	I	8(128)	0.75	D	8(32)	0.75	D	32(256)	2.00	I
19	AS-47	16(64)	1.00	D	4(64)	1.13	I	8(64)	0.50	S	8(64)	1.25	I	16(256)	1.50	I
20	AS-50	8(64)	1.00	D	8(64)	1.50	I	2(32)	0.25	S	2(32)	0.63	D	8(128)	1.00	D
21	AS-51	4(32)	0.50	S	8(32)	1.00	D	2(64)	0.63	D	2(4)	0.38	S	32(128)	2.50	I
22	AS-52	8(64)	0.75	D	8(64)	1.25	I	8(64)	0.75	D	4(8)	0.63	D	16(128)	1.00	D
23	AS-54	8(64)	1.00	D	4(16)	0.50	S	8(64)	1.00	D	8(4)	0.75	D	8(256)	1.50	I
24	AZ-02	8(32)	1.50	I	8(64)	0.75	D	2(16)	0.38	S	2(4)	0.38	S	8(16)	1.00	D
25	AZ-06	16(16)	1.00	D	8(64)	0.50	S	8(32)	0.75	D	4(4)	0.38	S	8(256)	1.25	I
26	AZ-10	4(32)	1.25	I	8(32)	1.00	D	16(64)	1.25	I	4(8)	0.75	D	8(128)	1.50	I
27	AZ-25	8(32)	0.75	D	8(64)	1.25	I	4(64)	0.63	D	4(16)	0.63	D	4(256)	1.13	I
28	AZ-26	8(16)	1.00	D	8(64)	1.50	I	8(64)	0.75	D	8(16)	1.00	D	8(256)	1.50	I
29	AZ-36	1(32)	1.06	I	8(64)	1.50	I	8(64)	0.75	D	8(8)	1.00	D	16(128)	2.00	I
30	AZ-41	16(32)	1.50	I	8(64)	1.25	I	8(64)	0.50	S	4(4)	0.38	S	16(64)	1.50	I
31	AZ-42	16(32)	1.50	I	8(64)	1.25	I	8(64)	0.50	S	8(8)	0.75	D	8(128)	1.25	I
32	AZ-43	8(32)	1.25	I	8(64)	1.25	I	8(128)	0.75	D	8(64)	1.25	I	16(16)	1.50	I
33	AZ-44	16(32)	1.00	D	8(32)	0.75	D	4(64)	0.63	D	8(64)	1.25	I	4(16)	1.13	I
34	AZ-46	1(32)	1.06	I	8(16)	0.75	D	4(64)	0.75	D	4(16)	0.50	S	16(16)	2.00	I

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AS. Isolates recovered from Assuit University; AZ, Isolates recovered from Al-Azhar University; CIP, ciprofloxacin; FIC, fractional inhibitory concentration; Int. Interpretation; CIP-AMP μg/ml (MIC of ciprofloxacin-ampicillin after combination; CIP-CRO μg/ml (MIC of ciprofloxacin-ceftriaxone after combination; CIP-AK μg/ml (MIC of ciprofloxacin-amikacin after combination; CIP-DO μg/ml (MIC of ciprofloxacin-doxycycline- after combination; CIP-VC μg/ml (MIC of ciprofloxacin-vancomycin after combination; S, Synergism ≤0.5; D, Additive >0.5 ≥1; I, Indifference >1 and ≤4.0.

Table 3. Effects of LEV combinations with different antimicrobial agents by the checkerboard method.

No.	Isolates code	LEV-AMP μg/ml	∑FIC	Int	LEV-CRO μg/ml	∑FIC	Int	LEV-AK μg/ml	∑FIC	Int	LEV-DO μg/ml	∑FIC	Int	LEV-VC μg/ml	∑FIC	Int
1	AS-07	8(64)	2.00	I	8(64)	2.00	I	4(128)	1.00	D	8(64)	2.00	I	8(128)	2.00	I
2	AS-09	1(64)	1.13	I	8(128)	2.00	I	4(128)	1.00	D	8(64)	1.25	I	8(128)	2.00	I
3	AS-15	8(64)	2.00	I	8(64	2.00	I	4(256)	1.50	I	2(64)	0.50	S	8(128)	2.00	I
4	AS-18	8(32)	1.00	D	16(64)	2.00	I	32(256)	3.00	I	32(64)	2.25	I	16(128)	2.00	I
5	AS-19	16(32)	1.50	I	16(128)	1.50	I	8(256)	1.50	I	16(64)	1.25	I	16(128)	2.00	I
6	AS-24	8(32)	1.00	D	16(64)	2.00	I	4(8)	0.50	S	4(8)	0.75	D	16(32)	3.00	I
7	AS-25	8(32)	1.00	D	4(64)	1.25	I	32(64)	3.00	I	2(4)	0.38	S	16(16)	2.00	I
8	AS-26	8(64)	2.00	I	4(64)	1.50	I	4(256)	1.50	I	8(64)	1.25	I	8(16)	2.00	I
9	AS-30	8(64)	2.00	I	4(64)	1.25	I	8(256)	1.50	I	8(64)	0.75	D	8(8)	1.00	D
10	AS-31	1(16)	0.50	S	4(128)	2.00	I	1(16)	0.50	S	4(64)	1.25	I	2(8)	1.00	D
11	AS-32	16(128)	1.50	I	16(256)	1.50	I	16(128)	1.00	D	16(64)	0.75	D	32(32)	1.50	I
12	AS-34	1(16)	0.50	S	2(16)	0.75	D	1(16)	0.50	S	4(128)	1.50	I	2(16)	1.50	I
13	AS-35	1(16)	0.50	S	4(64)	2.00	I	8(256)	3.00	I	4(64)	1.25	I	2(16)	1.50	I
14	AS-36	1(16)	0.50	S	4(64)	2.00	I	2(64)	0.75	D	4(64)	1.25	I	4(16)	2.00	I
15	AS-37	2(32)	1.00	D	4(32)	1.50	I	2(64)	0.75	D	1(8)	0.38	S	4(16)	2.00	I
16	AS-38	1(8)	0.50	S	1(16)	0.50	S	2(32)	0.75	D	1(4)	0.31	S	4(64)	2.00	I
17	AS-39	1(4)	0.38	S	2(64)	1.50	I	1(128)	1.25	I	2(64)	1.50	I	4(64)	1.50	I
18	AS-42	8(64)	1.50	I	8(64)	2.00	I	8(256)	2.00	I	4(32)	1.00	D	8(128)	1.50	I
19	AS-47	4(128)	1.50	I	4(64)	1.50	I	8(256)	2.00	I	8(64)	2.00	I	8(128)	1.50	I
20	AS-50	8(128)	2.00	I	8(64)	2.00	I	8(256)	2.00	I	2(32)	0.75	D	8(256)	2.00	I
21	AS-51	8(128)	2.00	I	8(64)	2.00	I	4(128)	1.50	I	1(4)	0.38	S	32(256)	5.00	A
22	AS-52	16(128)	2.00	I	8(64)	1.50	I	8(128)	1.50	I	2(8)	0.63	D	16(256)	2.00	I
23	AS-54	8(128)	2.00	I	4(64)	1.50	I	8(128)	2.00	I	2(4)	0.50	S	8(256)	2.00	I
24	AZ-02	4(32)	1.50	I	8(128)	1.50	I	2(16)	0.50	S	2(2)	0.38	S	4(16)	1.00	D
25	AZ-06	4(16)	1.00	D	8(128)	1.50	I	2(16)	0.50	S	2(8)	0.75	D	8(256)	2.00	I
26	AZ-10	8(32)	2.00	I	8(64)	2.00	I	16(256)	3.00	I	4(8)	1.00	D	8(128)	2.00	I
27	AZ-25	1(8)	0.38	S	8(128)	4.00	I	4(128)	2.00	I	4(16)	1.50	I	4(256)	2.00	I
28	AZ-26	8(16)	1.50	I	8(128)	3.00	I	8(256)	2.00	I	8(16)	1.50	I	8(256)	2.00	I
29	AZ-36	16(32)	3.00	I	8(64)	2.00	I	8(256)	2.00	I	4(8)	1.00	D	16(128)	3.00	I
30	AZ-41	1(8)	0.50	S	1(16)	0.50	S	2(128)	1.00	D	1(4)	0.50	S	4(64)	2.00	I
31	AZ-42	8(32)	2.00	I	8(64)	2.00	I	8(256)	2.00	I	2(4)	0.50	S	8(128)	2.00	I
32	AZ-43	1(8)	0.50	S	1(16)	0.50	S	8(256)	3.00	I	8(64)	3.00	I	2(16)	1.50	I
33	AZ-44	2(32)	1.00	D	4(64)	2.00	I	4(128)	2.00	I	8(64)	3.00	I	2(8)	1.00	D
34	AZ-46	16(32)	2.00	I	8(32)	1.00	D	8(128)	1.50	I	4(32)	0.75	D	16(16)	2.00	I

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AS. Isolates recovered from Assuit University; AZ, Isolates recovered from Al-Azhar University; LEV, levofloxacin; FIC, fractional inhibitory concentration; Int. Interpretation; LEV-AMP μg/ml (MIC of levofloxacin—ampicillin after combination); LEV-CRO μg/ml (MIC of levofloxacin -ceftriaxone after combination); LEV-AK μg/ml (MIC of levofloxacin—amikacin after combination; LEV-DO μg/ml (MIC of levofloxacin—doxycycline after combination); LEV-VC μg/ml (MIC of levofloxacin—vancomycin after combination); S, Synergism ≤0.5; D, Additive >0.5 ≥1; I, Indifference >1 and ≤4.0; A, Antagonism >4.

Analysis of Tables 1–3 were carried out and the MICs of FQs after the addition of antimicrobial agents were shown in Table 4.

Table 4. Summary of antibiotic combinations with FQs by checkerboard method on antibiotic resistant isolates.

Antibiotics combination	MICs range of FQs after addition other antibiotics	∑FIC Range	Activity
Antibiotics combination	MICs range of FQs after addition other antibiotics	∑FIC Range	Synergy No.(%)	Additive No.(%)	Indifference No.(%)	Antagonist No. (%)
CIP-AMP	1–32	0.31–1.5	8(23.53)	14(41.18)	12(35.29)	0
LEV-AMP	1–16	0.38–3	9(26.47)	6(17.65)	19(55.88)	0
CIP-CRO	4–16	0.38–2	6(17.65)	11(32.35)	17(50)	0
LEV-CRO	1–16	0.5–4	3(8.28)	2(5.88)	29(85.29)	0
CIP-AK	2–32	0.25–2.5	11(32.35)	18(52.94)	5(14.71)	0
LEV-AK	1–32	0.5–3	5(14.71)	7(20.59)	22(64.71)	0
CIP-DO	2–32	0.38–2.25	6(17.65)	20(58.82)	8(23.53)	0
LEV-DO	1–32	0.31–3	9(26.47)	10(29.41)	15(44.12)	0
CIP-VC	2–32	0.75–3	0	(17.65)6	(82.35)28	0
LEV-VC	2–32	1–5	0	29(85.29)	4(11.76)	1(2.94)

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FQs: fluoroquinolones; MICs: minimum inhibitory concentrations; FIC, fractional inhibitory concentration; CIP: ciprofloxacin, LEV: levofloxacin; AMP: ampicillin; CRO: ceftriaxone; AK: Amikacin; DO: doxycycline; VC: vancomycin.

Evaluation of FQs- non-antibiotic combinations

The results of FQs in combination with non-antibiotics are presented in Tables 5 and 6. Results revealed that, CPZ 200 μg/ml, when combined with CIP or LEV, increased the susceptibilities of the isolates to the antimicrobials by 44.12% and 94.12%, respectively. No effect was obtained by using concentrations ranging from 25–150 μg/ml. PR, in a concentration of 0.5 mg /ml, diminished the resistance by 50% and 85.29% when combined with CIP and LEV, respectively (Table 7). Interestingly, PR (1 mg/ml) and sodium DIC (4 mg/ml) completely diminished FQ resistance when each was used in combination with either CIP or LEV (Tables 5 and 6). No significant effects were observed when each of omeprazole, esomeprazole, acetylsalicylic acid, furosemide, and labetalol were used in the combination with CIP or LEV.

Table 5. Effects of non-antibiotics on the MIC of CIP.

No.	Isolates code	MICs (μg/ml) CIP alone	CPZ 200μg/ml		PR 0.5 mg /ml		PR 1mg /ml		DIC 4mg/ ml
No.	Isolates code	MICs (μg/ml) CIP alone	MICs (μg/ml) CIP+CPZ	MDF	MICs (μg/ml) CIP+PR	MDF	MICs (μg/ml) CIP+PR	MDF	MICs (μg/ml) CIP+ DIC	MDF
1	AS-07	16	8	2	0.5	32	0.03	533.3	0.25	64
2	AS-09	32	2	16	0.5	64	0.03	1066.7	0.03	1066.7
3	AS-15	16	8	2	4	4	0.06	266.7	0.125	128
4	AS-18	32	8	4	4	8	0.25	128	0.125	256
5	AS-19	32	8	4	4	8	0.25	128	0.125	256
6	AS-24	32	8	4	0.5	64	0.03	1066.7	0.125	256
7	AS-25	16	2	8	1	16	0.06	266.7	0.03	533.3
8	AS-26	32	2	16	1	32	0.06	533.3	0.125	256
9	AS-30	32	2	16	1	32	0.06	533.3	0.06	533.3
10	AS-31	8	4	2	0.125	64	0.03	266.7	0.03	266.7
11	AS-32	64	16	4	8	8	0.25	256	0.125	512
12	AS-34	16	8	2	2	8	0.03	533.3	0.03	533.3
13	AS-35	16	8	2	8	2	0.06	266.7	0.06	266.7
14	AS-36	16	8	2	8	2	0.06	266.7	0.03	533.3
15	AS-37	16	8	2	8	2	0.06	266.7	0.06	266.7
16	AS-38	16	8	2	0.5	32	0.03	533.3	0.06	266.7
17	AS-39	16	8	2	2	8	0.06	266.7	0.06	266.7
18	AS-42	32	4	8	1	32	0.25	128	0.06	533.3
19	AS-47	32	0.5	64	0.5	64	0.03	1066.7	0.125	256
20	AS-50	16	8	2	8	2	0.06	266.7	0.03	533.33
21	AS-51	16	1	16	1	16	0.25	64	0.125	128
22	AS-52	32	1	32	1	32	0.25	128	0.03	1066.7
23	AS-54	16	1	16	0.5	32	0.03	533.3	0.125	128
24	AZ-02	16	1	16	0.5	32	0.03	533.3	0.125	128
25	AZ-06	32	0.5	64	0.5	64	0.03	1066.7	0.125	256
26	AZ-10	16	0.5	32	0.5	32	0.03	533.3	0.06	266.67
27	AZ-25	32	0.125	256	4	8	0.06	533.3	0.125	256
28	AZ-26	16	0.125	128	4	4	0.03	533.3	0.03	533.3
29	AZ-36	16	0.125	128	0.125	128	0.06	266.7	0.03	533.3
30	AZ-41	32	0.125	256	4	8	0.125	256	0.125	256
31	AZ-42	32	0.125	256	4	8	0.125	256	0.125	256
32	AZ-43	32	0.125	256	4	8	0.03	1066.7	0.125	256
33	AZ-44	32	0.125	256	2	16	0.03	1066.7	0.125	256
34	AZ-46	16	0.125	128	4	4	0.03	533.3	0.03	533.3

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AS. Isolates recovered from Assuit University; AZ, Isolates recovered from Al-Azhar University; MICs, minimum inhibitory concentrations; MDF, MIC decrease factor; CIP, ciprofloxacin; CPZ, chlorpromazine; PR. Propranolol; DIC, diclofenac.

Table 6. Effects of non -antibiotic combinations on the MIC of LEV.

No.	Isolates code	MICs (μg/ml) LEV	CPZ 200μg/ml		PR 0.5 mg /ml		PR 1mg /ml		DIC 4mg/ ml
No.	Isolates code	MICs (μg/ml) LEV	MICs (μg/ml) LEV+CPZ	MDF	MICs (μg/ml) LEV+PR	MDF	MICs (μg/ml) LEV+PR	MDF	MICs (μg/ml) LEV+DIC	MDF
1	AS-07	8	2	4	0.5	16	0.03	266.7	0.125	64
2	AS-09	8	0.5	16	0.5	16	0.03	266.7	0.03	266.7
3	AS-15	8	0.25	32	4	2	0.06	133.3	0.06	133.3
4	AS-18	16	0.5	32	4	4	0.125	128	0.06	266.7
5	AS-19	16	0.5	32	4	4	0.125	128	0.06	266.7
6	AS-24	16	0.5	32	0.5	32	0.03	533.3	0.03	533.3
7	AS-25	16	0.06	266.7	1	16	0.06	266.7	0.03	533.3
8	AS-26	8	0.5	16	1	8	0.06	133.3	0.25	32
9	AS-30	16	0.25	64	1	16	0.06	266.7	0.06	266.7
10	AS-31	4	0.125	32	0.125	32	0.03	133.3	0.03	133.3
11	AS-32	32	8	4	4	8	0.125	256	0.125	256
12	AS-34	4	0.5	8	2	2	0.03	133.3	0.03	133.3
13	AS-35	4	0.5	8	2	2	0.06	66.7	0.06	66.7
14	AS-36	4	1	4	2	2	0.06	66.7	0.03	133.3
15	AS-37	4	1	4	1	4	0.06	66.7	0.06	66.7
16	AS-38	4	1	4	0.5	8	0.03	133.3	0.06	66.7
17	AS-39	4	1	4	2	2	0.06	66.7	0.06	66.7
18	AS-42	8	2	4	1	8	0.03	266.7	0.06	133.3
19	AS-47	8	0.5	16	0.5	16	0.03	266.7	0.125	64
20	AS-50	8	8	1	1	8	0.06	133.3	0.03	266.7
21	AS-51	8	0.25	32	1	8	0.125	64	0.125	64
22	AS-52	16	0.25	64	4	4	0.125	128	0.03	533.3
23	AS-54	8	0.25	32	0.5	16	0.03	266.7	0.06	133.3
24	AZ-02	8	0.25	32	0.5	16	0.03	266.7	0.03	266.7
25	AZ-06	8	0.125	64	0.5	16	0.03	266.7	0.06	133.3
26	AZ-10	8	0.25	32	0.5	16	0.03	266.7	0.06	133.3
27	AZ-25	4	0.125	32	1	4	0.06	66.7	0.06	66.7
28	AZ-26	8	0.125	64	2	4	0.03	266.7	0.03	266.7
29	AZ-36	8	0.125	64	0.06	133.3	0.03	266.7	0.03	266.7
30	AZ-41	4	0.125	32	0.03	133.3	0.03	133.3	0.125	32
31	AZ-42	8	0.125	64	2	4	0.125	64	0.06	133.3
32	AZ-43	4	0.125	32	0.5	8	0.03	133.3	0.125	32
33	AZ-44	4	0.125	32	0.5	8	0.03	133.3	0.06	66.7
34	AZ-46	16	0.125	128	1	16	0.03	533.3	0.03	533.3

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AS. Isolates recovered from Assuit University; AZ, Isolates recovered from Al-Azhar University; MICs, minimum inhibitory concentrations; MDF, MIC decrease factor; LEV, levofloxacin; CPZ, chlorpromazine; PR. Propranolol; DIC, diclofenac.

Table 7. Summary of CIP and LEV combinations with non-antibiotics.

Non-antibiotic	FQs(MIC before, μg/ml)		No. (% Susceptible) by
Non-antibiotic	CIP (8–64)	LEV (4–32)	CIP	LEV
MIC range (μg/ml) after CPZ 200 μg/ml	0.125–16	0.06–8	15(44.12)	32(94.12)
MIC range (μg /ml) after PR 0.5 mg/ml	0.125–8	0.03–4	17(50)	29(85.29)
MIC range (μg/ml) after PR 1 mg/ml	0.03–0.25	0.03–0.125	34(100)	34(100)
MIC range (μg/ml) after sodium dilofenac 4 mg/ml	0.03–0.125	0.03–0.25	34(100)	34(100)

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FQs: fluoroquinolones, MIC: minimum inhibitory concentration, CIP: ciprofloxacin, LEV: levofloxacin, CPZ: Chlorpromazine.

Discussion

Respiratory tract, urinary tract, and blood infections are the most frequent clinical complications of A. baumannii. In our study, a total of 100 A. baumannii MDR clinical isolates were recovered from respiratory tract specimens (including endotracheal tubes, nasal, sputum and throat), urinary tract specimens (including urine and urinary tract catheter), blood, wound, skin, and central venous catheter specimens. Previous studies reported that, the respiratory tract, blood, and urine specimens were the main sources of A. baumannii pathogens [32, 33]. Moreover, wounds or soft-tissue infections, skin and catheter-associated infections (including, a central venous catheter, and urinary tract catheter) were recently reported to be caused by A. baumannii and therefore, highlighted the importance of environmental contamination in disseminating such infections. Another study revealed that, the epidemiology of A. baumannii infection differs according to the anatomical site as well as the clinical conditions of the patients worldwide [34]. The A. baumannii pathogen was recorded as an opportunistic pathogen often susceptible to colistin and having a low susceptibility to other antimicrobial agents [35, 36]. Therefore, A. baumannii’s growing resistance is a worldwide problem [37]. The incurable strains of A. baumannii endanger the lives of millions of hospitalized patients every year [38]. Continuous study of their resistance pattern is a must to control or at least decrease their devastating effect on the quality of medical treatment. In the current study, the antimicrobial susceptibility patterns of 100 A. baumannii isolates were determined revealing a high resistance to most of the tested antimicrobial agents (76–99%). On the other hand, colistin was the most effective anti-microbial agent against A. baumannii 95% followed by doxycycline 43%. Our study agrees with the previous study which recorded that, A. baumannii pathogen as an opportunistic pathogen often susceptible to colistin and having a low susceptibility to other antimicrobial agents causing radical morbidity and mortality [35]. The non-rational use of antimicrobial agents may be considered as the main cause for resistance development of this harmful pathogen.

The XDR and PDR isolates of A. baumannii are a leading cause of hospital-acquired infections [39]. PDR and XDR are increasingly being reported worldwide in clinical [40] or environmental isolates [41]. In the present study, 2, 68 and 30% of the recovered isolates exhibited PDR, XDR and MDR phenotypes, respectively. Phenotypic analysis of their susceptibility to the 19 antimicrobial agents showed the diversity of the resistance of these isolates. They were divided into 12 major profiles according to the number of antimicrobial agents to which they were resistant. It was previously reported that, the high prevalence of MDR, XDR and PDR A. baumannii was due to the misuse of the antimicrobial agents [42, 43], or due to the differences in rates of infections by the respective pathogen (which could be an indications of how strictly the hygiene protocols and good manufacturing practice are applied in different hospitals) [44], in addition to the plasticity and endless capacity of A. baumannii genomes [45].

The use of ERIC-PCR as a genotyping technique to inspect the epidemics of hospital-acquired infection depends on its ability to epidemiologically relate the collected isolates during a nosocomial outbreak and investigate if the involved isolates are genetically related or originated from different strains [46, 47]. In our study, the phylogenetic dendrogram of ERIC-PCR showed that the isolates can be divided into three major clusters. The respective diversity of the collected isolates into three major clusters may be due to the multiple contamination sources of A. baumannii. This result could be an indication of clonal expansion and microbial colonization from different sources as previously reported [48]. Therefore, the obtained results emphasized the urgent need for a new scenario of drug combinations, new therapeutics options, as well as accelerating the development of the new infection control strategies to combat MDR resistance particularly of clinically relevant pathogens such as A. baumannii.

Little information is available on successful treatment regimens of A. baumannii by FQs combinations with antimicrobial or non-antimicrobial agents. Few attempts have been made so far to control the resistance of this strain [10, 49]. Our results revealed that both doxycycline and colistin still retain good activities against A. baumannii. However, both antibiotics have not been recommended in the treatment of severe infections caused by A. baumannii, particularly in the intensive care unit and cardiac care unit [50]. Several studies have been conducted to evaluate various antibiotic combinations to be used as options for the treatment of MDR A. baumannii clinical isolates. However, the results obtained were controversial and were attributed to many other factors [51–53]. In this study, a new scenario has been attempted for finding possible therapeutic options to control infections caused by XDR- and PDR- A. baumannii clinical isolates. This was carried out through an evaluation of the use of CIP or LEV, the two most widely used antimicrobial agents for the treatment of A. baumannii-associated infections, in combination with either antibiotics or non- antibiotics including, PR, DIC, and CPZ.

In our study, 34 A. baumannii clinical isolates were selected based on their different antibiotic resistance profiles as well as results of ERIC-PCR that revealed that these isolates were not clonal. Drug combinations of either CIP or LEV with ampicillin, ceftriaxone, amikacin, doxycycline or vancomycin tested on the 34 isolates (including 2 PDR and 32 XDR) showed synergism in 23.53, 17.65, 32.35, 17.65 and 26.47, 8.28, 14.71, 26.47%, respectively. Several previous in vitro studies reported synergic effects between diverse drugs for the treatment of A. baumannii [51–53]. The results of the previous studies of drug combinations were classified as synergistic, additive, indifferent, or even antagonistic. FQs are major antimicrobial agents associated with induction of resistance, but when used in combination, may prevent resistance [54, 55]. Our study revealed that, AMP reduced MICs of CIP (8–64 μg/ml) and LEV (4–32 μg/ml) to 1–32 μg/ml and 1–16 μg/ml, enhancing their efficacy by 23.53% and 26.47%, respectively. However, these results do not agree with a previous study that was conducted in north Egypt [9]. This contradiction could be due to intrinsic resistance, genetic differentiation of the isolates as well as the geographical factor. CRO also reduced MICs of CIP and LEV and showed a synergistic effect by 17.65 and 8.28%, respectively. These results agree with a previous study that reported successful combinations of CIP with cephalosporins against Pseudomonas spp. [56]. In the present study, successful combination of AMK with CIP and LEV were obtained, which reduced MICs to 2–32 and 1–32 μg/ml with synergies of 32.35 and 14.71%, respectively. Our results are in accordance with a previous study conducted in Upper Egypt, which reported a successful CIP-AMK combination against Gram-negative pathogens [10]. It was previously stated that, amikacin combination with FQs is used to expand the antimicrobial spectrum, reduce toxicity, and prevent or diminish the emergence of resistant mutants of FQs [57].

Different mechanisms in A. baumannii isolates were reported to be responsible to resistance to beta-lactams and other important classes of antibiotics, leading to the emergence of PDR A. baumannii causing nosocomial infections [58]. Doxycycline is a bacteriostatic antibiotic and showed high activity against A. baumannii either in monotherapy or combinations [11, 59]. The DO-FQs combination reduced the MICs of CIP and LEV from 8–64 and 4–32 μg/ml to 2–32 and 1–32 μg/ml, respectively. Our results agreed with previous studies which showed the ability of doxycycline to potentiate the efficacy of other antimicrobial agents when used in combination with FQs [60, 61]. Although vancomycin acts by hindering cell wall synthesis in the Gram-positive pathogens, it showed a synergistic effect when used in combination with colistin against A. baumannii [11]. Moreover, the membrane-permeabilizing properties of colistin was reported to enhance the activity of vancomycin against A. baumannii [11]. In this study, in vitro combinations of vancomycin with two members of FQs, the CIP and LEV did not show any synergistic effect against PDR A. baumannii clinical isolates.

Several successful combinations of FQs with natural products against MDR A. baumannii were previously reported [62, 63]. However, few studies were conducted on the use of some non-selective beta-blockers such as PR in combination with antimicrobial agents [7] or carvedilol alone [14]. In the present study, combination of CIP or LEV with PR successfully overcame bacterial resistance of XDR and PDR A. baumannii clinical isolates. CIP or LEV combination with PR at concentration 0.5 mg/ml significantly reduced the MICs of CIP and LEV and at 1 mg/ml completely inhibited the resistance of A. buamannii. The effect of PR as an efflux pump inhibitor [64], or as an antibacterial [65] has been investigated. Different chemotherapeutics are nowadays known to inhibit or diminish the microbial resistance of XDR and PDR of different microorganisms. Various drugs have got consideration recently in different research fields, including cancer therapy as a transporter and drug delivery as PR [13, 66], calcium channel blockers [67] natural products such as reserpine [68]. In this study, a combination of CIP or LEV with DIC at 4 mg/ml restored the susceptibility of tested XDR, and PDR A. baumannii to FQs. Our results fully agreed with Dutta et al which showed successful synergism of DIC with antimycobacterial drugs [69]. Furthermore, antipyretics and NSAIDs are commonly co-administered with antimicrobial therapy and primarily act by inhibiting prostaglandin synthesis. Although their exact function is uncertain, several suggestions have been explained [70, 71]. They include changing the surface hydrophobicity of microbes [18], altering the expression of virulence factor, influencing biofilm production [19], affecting motility and metabolism, inhibiting quorum sensing among microbes [72], interacting with the transport and release of antibiotics by polymorphonuclear leukocytes (PMNL) [20], and modifying the susceptibility of microbes to antimicrobial therapy [15–17]. The effect of NSAIDs on the antibiotic susceptibility of pathogens was investigated which mostly resulted from a change in direct antimicrobial penetration through cell membranes of bacteria or from an increase or decrease in efflux through the membranes [15, 16, 73]. By understanding these mechanisms, these synergistic effects can be exploited in the treatment of infectious diseases and potential compromising effects on antimicrobial efficacy can be avoided [15]. However, decreased susceptibility can also result from induced β-lactamase activity [74]. DIC has analgesic, antipyretic, as well as anti-inflammatory characters. This non-steroidal anti-inflammatory drug has demonstrated strong antimicrobial property when tested against a large number of bacteria and has bactericidal activity in nature due to inhibition of DNA synthesis [5, 75].

Antipsychotics of different groups such as phenothiazines (CPZ hydrochloride) have significant antibacterial activity [6, 76] and act as efflux pump inhibitors [77]. In this study, CPZ combinations with CIP or LEV have markedly reduced the MIC of resistance isolates. Our result agreed with studies previously carried out against Mycobacterium spp [78, 79]. It has been found that CPZ had a significant bactericidal effect [6, 75] in addition to the efflux pump inhibitor action when tested for their effect on antibiotic resistance [79, 80].

Conclusion

Several combination regimens have been successfully evaluated in vitro for combating antimicrobial resistance of PDR and XDR isolates. In this research, high prevalence of PDR and XDR A. baumannii isolates associated with nosocomial infections in Upper Egypt was observed and therefore, exerted a negative impact on patient health and disease prognosis. Propranolol, chlorpromazine and diclofenac restore susceptibility of some selected XDR A. baumannii to CIP and LEV. More pharmacokinetic/pharmacodynamics studies are needed to guide the use of these combinations against these life-threatening pathogens.

Supporting information

S1 Fig

(PDF)

Click here for additional data file.^{(362KB, pdf)}

Acknowledgments

The authors are grateful to the Medical Research Center, Faculty of Medicine Assiut University, and Al Azhar University hospitals for providing A. buamannii isolates. More grateful is extended to Prof. Enas A Deaf, Prof. Nahla M Elsherbiny, and Assist. Prof. Mohamed A. EL-Mokhtar; Department of microbiology and immunology, Faculty of Medicine, Assiut University, and Dr. Mohamed A. Abdel-Lateef; Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Al-Azhar University, Assiut branch for their helping during the study.

Data Availability

All relevant data are within the manuscript

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0238195.r001

Decision Letter 0

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2 Jun 2020

PONE-D-20-04012

Propranolol and chlorpromazine restore susceptibility of extensively drug-resistant (XDR)-Acinetobacter baumannii to fluoroquinolones

PLOS ONE

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Dear Aboshanab,

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: N/A

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: No

Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: MANUSCRIPT PONE-D-20-04012

Manuscript PONE-D-20-04012 describes the effect of different drug combinations with ciprofloxacin and levofloxacin on the susceptibility of PDR and XDR A. baumannii to the fluoroquinolones CIP and LEV. Propranolol and diclofenac sodium particularly obliterated the resistance of the PDR and XDR strains. No doubt, any study reporting alternative therapies in the treatment of infections caused by A. baumannii, a species of bacteria notorious for causing difficult to treat infections in immune-compromised patients is important judging from the current global scourge antimicrobial resistance. Unfortunately, not minding the importance of the area of focus of this manuscript, it is difficult for me to recommend this paper for publication in PLOS ONE in its present form.

1. The authors did not make judicious use of existing literature to provide adequate background for their study and relate the findings to previous studies in this area.

2. They also did not follow the PLOS ONE guidelines for authors in the preparation of their manuscript. For example, PLOS ONE do not use structured abstract as they did in this manuscript. Similarly, the figure titles were not inserted in the manuscript as spelt out in the authors guidelines

3. The manuscript was not line-numbered, making reference to specific sections difficult in the course of revising the manuscript.

4. The writing style and language use need considerable improvement as the present presentation is unclear making it difficult to follow the thoughts and ideas that the authors are trying to present in some parts of the manuscript. There are important word omissions, use of the noun resistant in place of the adjectival form of the word, sentences that do not read well etc.

5. Authors should please define all abbreviations at first use

6. Most important, I am concerned about the number and narrow geographical origin of the isolates of A. baumannii (34) included in the evaluation of drug combination part of the experiment. In my own opinion, this number is not enough to make the broad conclusion reached by the authors in this study about the effects of those combined drug in ameliorating the resistance of the isolates to antimicrobial drugs.

Specific comments for the authors

Abstract

Methods section

1. Method section not detailed enough leaving out important details about the drug combination trial which is supposed to be the main focus of the manuscript. This critical omission made some of the results presented in the results section of the structured abstract orphans and unlinked to the methods section.

Results section

1. This section contained information that could have been included in the methods section of the abstract.

2. What informed the choice of the 34 isolates included in the drug combination trial? It will be important to know how the authors selected the 34 isolates, especially the 32 XDR isolates from among the 68 isolates that were XDR since the authors found out that the isolates are not clonal through ERIC-PCR. Why did they not include any of the MDR isolates in this test? Including as many isolates as possible will certainly have strengthened and validated the findings of this study (see comment no.6 above).

3. ........including 2 PDR and 32 XDR showed synergism in 23.5%, 17.65%............ : I could not understand what the authors are trying to pass across to readers here. Do they mean that the drug combination showed synergism in 23.5% etc. of the isolates? Same goes for the combination with CPZ and propranolol.

4. Does the concentration range after propranolol (0.5-1mg/ml) refer to both CPZ and propranolol?

5. The authors concluded that their results will greatly help physicians for (sic) the proper choice of antibiotic combinations for combating A. baumannii but some of the drugs combined such as CPZ and Propranolol are not antibiotics.

Keywords

1. extensively drug resistant should be qualified

Introduction

1. The information presented in this section to provide a background upon which this study is built is scanty. Authors should please add more recent published information to this section to provide a more robust background for their study and to set the study within the context of previous studies in the area.

2. Line 3-4: A. baumannii is not a hospital acquired infection but a bacterium that cause HAI

Methods

Bacterial isolates

1. What did the authors do with the incubated plates? This question becomes important in the light of the next section where they reported carrying out phenotypic and genotypic tests to identify the colonies. How many colonies did the authors subject to this identification step? From which of the three different media used did they selected the colonies? How did they prevent the selection of clonal replicates from the three agar media?

2. Can the authors supply the name of the positive control used in addition to the ATCC accession number?

Antimicrobial susceptibility testing

1. The authors mentioned colistin among the antibiotics used for this test by disc diffusion. This method is not recommended for testing of colistin susceptibility because of the large molecular weight of colistin which hinders its diffusion

2. It should not be MIC of XDR and PDR isolates against CIP and LEV, rather it is the MIC of CIP and LEV against the bacterial isolates.

3. The authors should supply the range of concentrations used for MIC determination

4. The authors mentioned vancomycin among the antibiotics used for MIC determination. Vancomycin is not recommended for use against gram negative bacteria.

Molecular typing of recovered isolates

1. In the abstract section, the authors mentioned that all isolates were not clonal while in this section, it appears that only 34 isolates were subjected to ERIC-PCR. The authors need to clarify this to let us know if they subjected all or 34 isolates to ERIC-PCR

Evaluation of drug combinations

1. The authors should please provide a brief summary of how the evaluation of drug combinations was carried out. For example how were the drugs combined? Was the combination carried out in-vitro or in-vivo? These are critical details needed to interpret the results

2. CPZ was conspicuously missing in the list of drugs evaluated in this section. Interestingly, the main conclusion of the manuscript centered on CPZ and propranolol .

Results

Specimen collection and identification of recovered A. baumannii isolates

1. The authors mentioned isolating 623 non-lactose fermenters and 977 lactose fermenters of which a total of 151 isolates were phenotypically identified Acinetobacter isolates (sic). It is important to clarify if the 151 isolates identified as Acinetobacter were a subset of the non-lactose fermenters or a subset of the lactose fermenters?

Antimicrobial susceptibility testing

1. The level of resistance to doxycycline was reported as 47% in the abstract but was reported as 57% here. Authors should please clarify which is the correct figure.

2. I am confused that the authors are referring to multiple contamination sources of A. baumannii in Upper Egypt. I am not sure of how this relate to the isolation of the bacteria of the present study from clinical specimen.

MIC

1. In the methods section, the authors mentioned that they determined the MIC using 7 antimicrobial drugs namely CIP, LEV, ampicillin, ceftriaxone, amikacin, doxycycline, and vancomycin. Curiously in the results section, results for only 2 (CIP and LEV) were given. The authors should please supply results for the remaining 5 antibiotics used in MIC determination.

Genotyping

1. Again, it becomes very important for the authors to clarify how many isolates were genotyped in the methods section. The authors made references to “obtained isolates” and “% of total isolates”. To my own understanding 100 isolates were obtained in the study, but the information here showed only 34 isolates were genotyped which does not represent all the obtained isolates or the total isolates as alluded to by the authors in this section.

Evaluation of drug combinations

1. Two antimicrobial profiles selected including...... Selected for what?

2. Table 1- I think before we can conclude on whether there is synergy or antagonism and the extent of the synergy as reported in this table, there is need for baseline references (i.e. the baseline MIC for CIP and LEV before addition of the combination antibiotics) for each isolate. That I did not see in the table. The authors should kindly correct me if I am wrong.

3. The authors should provide legends to define all abbreviations in the tables and figures.

4. Table 2- Please refer to comment no2 above. Table 2 is more like what is expected. One wonders why Table 1 is completely different from Table 2 even though they are used to report similar results.

5. The results presented by the authors in Table 2 and the manuscript showed that propranolol and diclofenac sodium completely reduced the MIC of CIP and LEV below the susceptibility cut-off value. One is therefore curious as to the reason(s) why the authors decided to lay emphasis on CPZ in the title without any mention of diclofenac sodium.

6. Labels on the axes of some of the figures are not expressing any meaning. Both axes of figure 5 carry exactly the same label.

Discussion

1. Again, the discussion of results is not clear and the authors did not use existing literature to place their result in proper perspective.

2. If the authors are reporting a total of 100 A. baumannii clinical isolates and 61 were from RTI, UTI and blood, where did the remaining 39 isolates came from?

References

1. The guidelines for authors as regards reference listing was not followed by the authors

2. The doi provided for reference no 18 is not correct.

Reviewer #2: 1. The manuscript has several typographical errors and English should be reviewed.

a) Page 4, line 2: the coma should be after "progression" not before.

b) Page 4, line 16: delete the point after "Catheters".

c) Page 4, line 24: missing space between "hours" and the reference "[10]".

d) Page 6, line 14: missing space between "guidelines" and the reference "[15]".

e) Page 7, line 19: delete the point before "(figure 1)".

f) Page 10, line 3: delete the semicolon after "CPZ".

g) Page 10, line 3: the second "CIP" should be changed by "LEV".

h) Page 10, line 4: omit semicolon after "44.12%".

i) Page 11, line 11: delete double space in "and mortality".

j) Page 11, line 12: delete double space in "due to misuse".

k) Page 11, line 19: misspelling of "originating" it should be "originated"

l) Page 12, line 1: include a reference to figure 5 when the three major clusters are mentioned.

m) Page 12, line 23: avoid the second "and".

n) Page 14, line 6: add the meaning of NSAIDs.

o) Page 14, line 21: missing space between "[50]." and "However".

p) Page 15, line 5: missing space between "inhibitors" and the reference "[57]".

2. Acinetobacter species are non-fermenting bacteria. The authors on page 7 mention that they selected lactose fermenting bacteria, which by definition are not Acinetobacter species.

3. The authors divided their strains into 12 major profiles, but they do not explain the criteria followed.

4. The authors must explain why they only choose 34 isolates.

5. If "Evaluation of FQs-antibiotic combinations" and "Evaluation of FQs- non-antibiotic combinations" are subsections of "Evaluation of drug combinations", they should be marked as such.

6. Table 1: the title of the second column is misspelled the words "and" and "before" must change their position; the "D" from "FQDs" should be eliminated.

7. There are missing data in table 2.

8. Figure 5 should be redone, the X-axis has the same name as the Y-axis, and the legend is not clear.

9. The quality of the figures should be improved.

10. The conclusion should not include results.

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Aug 26;15(8):e0238195. doi: 10.1371/journal.pone.0238195.r002

Author response to Decision Letter 0

17 Jun 2020

Author's Response to Reviewer's comments

On the behalf of all authors, we would like to thank the reviewers for their valuable comments and suggestions that will actually improve and add to this manuscript. Corrections are highlighted in yellow color and all have been included in the revised manuscript

Reviewer #1: MANUSCRIPT PONE-D-20-04012

Author response:

Thank you for the reviewer comments and recommendations. As known, the extensive drug-resistant (XDR) pathogens particularly A. baumannii is a major health problem and currently still remains a great obstacle for the physicians for their management and cure since the therapeutics options remain very limited. Therefore, the main objective of this research was to find new alternatives for the control of the respective superbug through testing various drug combinations. All comments elicited by the reviewer have been taken into full consideration and the required corrections have been made and included in the revised manuscript (corrections are highlighted in yellow color in the revised manuscript).

1. The authors did not make judicious use of existing literature to provide adequate background for their study and relate the findings to previous studies in this area.

Author response:

Thank you for the reviewer comment. We updated both introduction and discussion sections to include all relevant literature available and relate the obtained findings to previous literature (introduction; page 4 lines 77-84 and lines 86-98), Discussion; page 24, lines 313-344; p28; lines 414-418, P29, lines 426-429; P30; lines 469-473)

Author response:

We have noticed that both types of abstracts are accepted for PLOS ONE. We have previously published an article in PLOS ONE with graphical abstract. (https://pubmed.ncbi.nlm.nih.gov/30157188/?from_term=aboshanab&from_sort=date&from_pos=9) . however, we changed the abstract style based on the reviewer suggestion. All figure legends have been inserted at the end of the manuscript as recommended (page 40)

3. The manuscript was not line-numbered, making reference to specific sections difficult in the course of revising the manuscript.

Author response:

We have included line numbering in the revised manuscript as recommended.

Author response:

Thank you for your valuable comment. The whole manuscript has been revised thoroughly for any grammar or any spelling mistake

5. Authors should please define all abbreviations at first use

Author response:

All abbreviations were firstly mentioned in full sentences and abbreviations were inserted between brackets, and were used consequently in the whole manuscript thereafter.

Author response:

We thank the reviewer for his valuable comment. Actually, we have done these combinations on the 34 A. baumannii isolates, the clinical relevant isolates which included, 2 PDR and 32 XDR isolates of the highest resistant pattern (all were resistance to the 19 antibiotics tested except colistin) and genetically not identical as determined by ERIC-PCR. Besides, these antibiotic combinations take a lot of time, effort, and resources and because of limited resources, we carried out these experiments on the respective isolates that isolated from different patients of two largest clinical hospitals in Upper Egypt were not clonal as confirmed by ERIC-PCR. However, we are willing to conduct the obtained promising combinations on a further large number of isolates in the future. This part has been clarified inserted in the manuscript (page27, lines 377-384)

Specific comments for the authors

Abstract

Do not use structured abstract as they did in this manuscript

Author response:

The abstract has been modified into non-graphical one.

Methods section. Method section not detailed enough leaving out important details about the drug combination trial which is supposed to be the main focus of the manuscript. This critical omission made some of the results presented in the results section of the structured abstract orphans and unlinked to the methods section.

Author response:

We thank the reviewer for his valuable comment. This part has been modified according to reviewer recommendation where more details about the method of evaluation of both antibiotic (FIC) and non-antibiotic (MIC decrease factor, MDF) were inserted (page 2, line 41-50). However, due to the word number limitation of the abstract, more details were inserted in the method section of the manuscript (page 8, lines 178-207)

Results section

1. This section contained information that could have been included in the methods section of the abstract.

Author response:

This part has been modified in the non-graphical abstract where both methods and findings were included (page 2, lines, 39-50)

Author response:

We thank the reviewer for his valuable comment. Actually, we have done these combinations on the 34 A. baumannii isolates including, 2 PDR and 32 XDR isolates representing the 12 different resistance profiles obtained. In addition, we have selected the 32 XDR isolates based on their antibiogram profile where we selected those of different patterns of antibiotic sensitivity as previously determined by disc and MIC measurement. We do agree if this experiment was done on the 100 isolates, results will be more reliable and confident. However, each experiment took a lot of time, effort, and resources and was done in triplicate and because of limited resources, we carried out these experiments on respective isolates of different antibiotic sensitivity profiles and not clonal as confirmed by ERIC PCR. However, we are willing to conduct the obtained promising combinations on a further large number of isolates in the future. This part has been clarified inserted in the manuscript (page27, lines 377-384)

Author response:

This part has been clarified in the abstract (highlighted in yellow color) page 23 lines 44-47.

4. Does the concentration range after propranolol (0.5-1mg/ml) refer to both CPZ and propranolol?

Author response:

No, we have tested two concentrations of propranolol (0.5 mg/ml and 1 mg/ml). This part has been corrected and clarified in the whole manuscript (page 2, line 48; table 6)

Author response:

Actually, we thank the reviewer for this valuable comment. This part has been modified and corrected in the conclusion into "Combinations of CIP or LEV with CPZ, PR or DIC showed synergism in most of the selected PDR and XDR A. baumannii clinical isolates. The obtained results are still promising in vitro study however, these combinations have to be re-evaluated in vivo using appropriate animal models infected by XDR- or PDR- A. baumannii, a life-threatening pathogen. (page 3, line 55-57)

This a general conclusion.

Keywords

extensively drug-resistant should be qualified

Author response:

Extensively drug-resistant has been changed into extensive drug-resistant in the whole manuscript.

Introduction

Author response:

Thank you for the reviewer valuable comment. The introduction section has been modified and updated by adding more recent published information and citations to provide a more robust background of other previous studies. (Page 4, lines; 77-84; 86-94; 96-98)

2. Line 3-4: A. baumannii is not a hospital-acquired infection but a bacterium that cause HAI

Author response:

This sentence has been corrected (page 3; line 64-65)

Methods

Bacterial isolates

Author response:

We thank the reviewer for his valuable comment. Actually, each of the collected clinical specimen was streak-plated on MacConky agar, (Oxoid Limited, England), then only one the suspected colonies of non-lactose fermenter was again steak-plate on blood agar (to ensure no contamination and presence of homologous colonies) and then one colony out of the blood agar (enriched media) was streak-plated on Herellea agar (selective media) for final growth characteristics and colony morphology of A. baumannii. Himedia, India), and incubated at 37°C for 24 hours (reference 20; Gerner-Smidt P, Tjernberg I, Using J. Reliability of phenotypic tests for identification of Acinetobacter species. J Clin microbiol. 1991;29(2):277-82. PMID: 2007635).

Then one colony out of Herellea was subjected to biochemical tests and finally for genotypic identification via the selection of blaOXA-51 gene using PCR for final confirmation (so one one colony was picked out and run the whole process for final identification) (page 5; lines 115-118).

2. Can the authors supply the name of the positive control used in addition to the ATCC accession number?

Author response:

A. baumannii ATCC 19606 standard strain was used as a positive control (inserted in the manuscript, page 6; lines 138-139)

3. Antimicrobial susceptibility testing

Author response:

We thank the reviewer for his valuable comment. We agree with the reviewer that colistin has large molecular weight and this hinders its diffusion to some extend from the disc however, colistin by disc sensitivity was recommended by CLSI 2016 at page 63 under the title “Zone Diameter and Minimal Inhibitory Concentration Interpretive Standards for Pseudomonas aeruginosa”.

In addition, other studies have used colistin disc diffusion in order to discriminate between XDR and PDR pathogens and these include:

• Falagas ME, Karageorgopoulos DE. Pandrug resistance (PDR), extensive drug resistance (XDR), and multidrug resistance (MDR) among Gram-negative bacilli: the need for international harmonization in terminology. Clin Infect Dis. 2008;46(7):1121-2.

• Piewngam, P., & Kiratisin, P. (2014). Comparative assessment of antimicrobial susceptibility testing for tigecycline and colistin against Acinetobacter baumannii clinical isolates, including multidrug-resistant isolates. International journal of antimicrobial agents, 44(5), 396-401.‏

• Galani, I., Kontopidou, F., Souli, M., Rekatsina, P. D., Koratzanis, E., Deliolanis, J., & Giamarellou, H. (2008). Colistin susceptibility testing by Etest and disk diffusion methods. International journal of antimicrobial agents, 31(5), 434-439.

2. It should not be MIC of XDR and PDR isolates against CIP and LEV, rather it is the MIC of CIP and LEV against the bacterial isolates.

Author response:

Corrected (page 7; lines 155-157)

3. The authors should supply the range of concentrations used for MIC determination

Author response:

The MIC of test concentrations for antimicrobial agents was ranged from 0.125-256 μg/ml. (Inserted in the revised manuscript (page 7; lines, 159-160)

4. The authors mentioned vancomycin among the antibiotics used for MIC determination. Vancomycin is not recommended for use against gram-negative bacteria.

Author response:

We thank the reviewer for his valuable comment. Although vancomycin acts by hindering cell wall synthesis in the Gram-positive pathogens only, however, in previous study vancomycin showed a synergistic effect with colistin against A. baumannii (11) and it was reported that the membrane-permeabilizing properties of colistin could enhance the activity of vancomycin against A. baumannii [11]. Therefore, we included vancomycin here to test its effect in combination with CIP or LEV, and to achieve this had to measure its MIC before measuring the FIC when combined with CIP or LEV. (This part was clarified in the discussion section; page 29 (lines 418-424)

In addition there are some study reports for the effect of vancomycin combination with colistin against MDR pathogens

1. Chen, F., Tang, Y., Zheng, H., Xu, Y., Wang, J., & Wang, C. (2019). Roles of the Conserved Amino Acid Residues in Reduced Human Defensin 5: Cysteine and Arginine Are Indispensable for Its Antibacterial Action and LPS Neutralization. ChemMedChem, 14(15), 1457-1465.‏

2. Gordon, N. C., Png, K., & Wareham, D. W. (2010). Potent synergy and sustained bactericidal activity of a vancomycin-colistin combination versus multidrug-resistant strains of Acinetobacter baumannii. Antimicrobial agents and chemotherapy, 54(12), 5316-5322.‏

Molecular typing of recovered isolates

Author response:

We thank the reviewer for his valuable comment. ERIC PCR has been carried out on the selected 34 isolates that were selected ( 2 PDR and 32 XDR with different antibiotic susceptibility patterns). This part was clarified in both the abstract (p2; lines 39-40) and in the Method sections (p7; line 166-167).

2. . Evaluation of drug combinations

Author response:

We thank the reviewer for his valuable comment. A combination of FQs with antibiotics was evaluated by using by Checkerboard method while FQs combination with non-antibiotics was evaluated by MDF method. And both combinations were carried out in-vitro. However, more details have been inserted in the methods and results in order to clarify how the respective drug combinations were evaluated (p8, lines 179-1946 for antibiotic combinations) and (p9; lines 195-207 for non-antibiotic combinations)

2. CPZ was conspicuously missing in the list of drugs evaluated in this section. Interestingly, the main conclusion of the manuscript centered on CPZ and propranolol.

CPZ was inserted and included (p9; line 201)

3. Results

Specimen collection and identification of recovered A. baumannii isolates

Author response:

They were a subset of the non-lactose fermenters. Was clarified this issue in the revised manuscript (p9; lines 216-217).

2. Antimicrobial susceptibility testing

1. The level of resistance to doxycycline was reported as 47% in the abstract but was reported as 57% here. Authors should please clarify which is the correct figure.

Author response:

We are sorry for this typing error. It was corrected in the abstract to 57% (p2; line 38)

2. I am confused that the authors are referring to multiple contamination sources of A. baumannii in Upper Egypt. I am not sure of how this relates to the isolation of the bacteria of the present study from clinical specimens.

Author response:

We thank the reviewer for his valuable comment. Analysis of the resulting susceptibility to 19 antimicrobial agents showed the diversity of the resistance of the isolates. They were divided into 12 major profiles according to the resistance number of antimicrobial agents the resistance extended from 19 to 6 antimicrobial agents. The first profile represents PDR (two isolates) was resistant to 19 antimicrobial agents. The second profile represents some of XDR (32 isolates) was resistant to all tests antimicrobial agents except colistin. Finally, profile number 12 was resistant only to 6 antimicrobial agents. (Fig. 5). (page 10; lines 226-232)

MIC

Author response:

The MIC of the remaining 5 antibiotics has done in order to measure the FIC for evaluating the outcomes of antibiotics combinations. As requested, the MIC values of the 7 antibiotics have been included in table 1 of the revised manuscript.

1. Genotyping

Again, it becomes very important for the authors to clarify how many isolates were genotyped in the methods section. The authors made references to “obtained isolates” and “% of total isolates”. To my own understanding 100 isolates were obtained in the study, but the information here showed only 34 isolates were genotyped which does not represent all the obtained isolates or the total isolates as alluded to by the authors in this section.

Author response:

We thank the reviewer for his valuable comment. Genotyping was done on the selected 34 isolates (corrected, page 12 line 253) and also in Fig. 6

2. Evaluation of drug combinations

1. Two antimicrobial profiles selected including...... Selected for what?

Author response:

We thank the reviewer for his valuable comment. This paragraph was rephrased into (Two antimicrobial profiles including, profile 1 ( PDR profile; 2 isolates were resistant to 19 antimicrobial agents) and profile 2 (XDR profile; 32 isolates were resistant to 18 antimicrobial agents) were selected for further study and evaluating the effect of CIP or LEV combination with some selected antibiotics and non-antibiotics (Fig. 5); page 12 (lines 262-265)

3. Table 1- I think before we can conclude on whether there is synergy or antagonism and the extent of the synergy as reported in this table, there is need for baseline references (i.e. the baseline MIC for CIP and LEV before addition of the combination antibiotics) for each isolate. That I did not see in the table. The authors should kindly correct me if I am wrong.

Author response:

Thank you for your comments. Actually, we have included the final outcomes of antibiotic combinations in only one table. However, we included the required data in three tables instead of one table (the baseline MIC for CIP and LEV before the addition of the combination antibiotics) for each isolate are now included in the revised manuscript in tables 1, 2, & 3)

4. The authors should provide legends to define all abbreviations in the tables and figures.

Author response:

All abbreviations have been included in the footnotes of each table and figure.

Table 2- Please refer to comment no2 above. Table 2 is more like what is expected. One wonders why Table 1 is completely different from Table 2 even though they are used to report similar results.

Author response:

Corrected and updated.

Table 2 became table 7 besides of tables 5 and 6 as baseline data to calculate MIC decrease factor (MDF) after adding MIC of CIP and LEVO before and after combination with non-antibiotics. Table 1 is completely different from Table 2 due to Table 1 calculates ƩFIC a result combination of antimicrobial agents combination according to Hsieh protocol. On the other hand table, 2 calculates MDF as a result combination of antimicrobial agents with non-antimicrobial agents according to Huguet protocol.

Author response:

We thank the reviewer for his valuable comment. Our rationale for this is that both CPZ and PR were effective at a low concentration of 200 µg/m for CPZ, 0.5 mg/ml, and 1 mg/ml for PR, respectively) while DIC was only effective only at high concentration (4mg/ ml). However, we have updated this information and included DIC in the title.

6. Labels on the axes of some of the figures are not expressing any meaning. Both axes of figure 5 carry exactly the same label.

Author response:

We thank the reviewer for his valuable comment. Labels are corrected and updated in the revised manuscript.

Discussion:

1. Again, the discussion of results is not clear and the authors did not use existing literature to place their result in proper perspective.

Author response:

We thank the reviewer for his valuable comment. The discussion section have been thoroughly revised and updated to include all relevant date from previous existing literatures (P24, lines 313-344; page 27, lines 377-384; page 28 (lines 414-429), P29 (lines 426-429), P30 (lines 469-473)

2. If the authors are reporting a total of 100 A. baumannii clinical isolates and 61 were from RTI, UTI and blood, where did the remaining 39 isolates came from?

Author response:

We thank the reviewer for his valuable comment. In our study, a total of 100 A. baumannii MDR clinical isolates of which, 61, 17, 12, 6,2 and 2 isolates were recovered from respiratory tract infection (including Enotreacheal tubes, nasal, sputum and throat, urinary tract infections (including urine and urinary tract catheter), blood, wound, skin, and central venous catheter, respectively. (Inserted in Discussion section P24; lines 299-303)

References

1. The guidelines for authors as regards reference listing was not followed by the authors

Author response:

We thank the reviewer for his valuable comment. All references have been revised and adjusted according to the journal guidelines

2. The doi provided for reference no 18 is not correct.

Author response:

We thank the reviewer for his valuable comment. It was corrected: Hsieh MH, Chen MY, Victor LY, Chow JW. Synergy assessed by checkerboard a critical analysis. Diagn Microbiol Infect Dis. 1993;16(4):343-9.

https://doi.org/10.1016/0732-8893(93)90087-N

Reviewer #2:

1. The manuscript has several typographical errors and English should be reviewed.

Author response:

Thank you for your valuable comment. The whole manuscript has been revised thoroughly for any grammar or any spelling mistake

a) Page 4, line 2: the coma should be after "progression" not before.

Author response:

Corrected (P4, line 86)

b) Page 4, line 16: delete the point after "Catheters".

Author response:

deleted (P5, line 109)

c) Page 4, line 24: missing space between "hours" and the reference "[10]".

Author response:

Corrected (P5, line 118)

d) Page 6, line 14: missing space between "guidelines" and the reference "[15]".

Author response:

Corrected page 7 (line 158)

e) Page 7, line 19: delete the point before "(figure 1)".

Author response:

Done (page 9, line 214)

f) Page 10, line 3: delete the semicolon after "CPZ".

Author response:

deleted (page 17, line 273)

g) Page 10, line 3: the second "CIP" should be changed by "LEV".

Corrected (page 17, line 273)

h) Page 10, line 4: omit semicolon after "44.12%".

Author response:

done (page 17 line 274)

i) Page 11, line 11: delete double space in "and mortality".

Author response:

done (page 25 line 324)

j) Page 11, line 12: delete double space in "due to misuse".

Author response:

done (page 25 line 340)

k) Page 11, line 19: misspelling of "originating" it should be "originated"

corrected (page 26 line 348)

l) Page 12, line 1: include a reference to figure 5 when the three major clusters are mentioned.

Author response:

done (page 26, line 356)

m) Page 12, line 23: avoid the second "and".

Author response:

removed (page 27 line 390)

n) Page 14, line 6: add the meaning of NSAIDs.

Author response:

added (page 29 line 443)

o) Page 14, line 21: missing space between "[50]." and "However".

Author response:

done (page 30, line 463)

p) Page 15, line 5: missing space between "inhibitors" and the reference "[57]".

Author response:

done (page 30 line 471)

2. Acinetobacter species are non-fermenting bacteria. The authors on page 7 mention that they selected lactose fermenting bacteria, which by definition are not Acinetobacter species.

Author response:

They were a subset of the non-lactose fermenters. Was clarified this issue in the revised manuscript (p9; lines 216-217).

3. The authors divided their strains into 12 major profiles, but they do not explain the criteria followed.

Author response:

The collected 100 A. baumanni isolates were categorized into 12 resistance profiles based on their resistance patterns to the 19 antibiotics as illustrated in figure 5 and also clarified in the text (page 10, lines 229-7-232)

4. The authors must explain why they only choose 34 isolates.

Author response:

We thank the reviewer for his valuable comment. The same author reply to reviewer 1 (query 6).

Actually, we have done these combinations on the 34 A. baumannii isolates, the clinical relevant isolates which included, 2 PDR and 32 XDR isolates of the highest resistant pattern (all were resistance to the 19 antibiotics tested except colistin) and genetically not identical as determined by ERIC-PCR. Besides, these antibiotic combinations take a lot of time, effort and resources and because of limited resources, we carried out these experiments on the respective isolates that isolated from different patients of two largest clinical hospitals in the Upper Egypt were not clonal as confirmed by ERIC-PCR. However, we are willing to conduct the obtained promising combinations on further large number of isolates in future. This part has been clarified inserted in the manuscript (page27, lines 377-384)

5. If "Evaluation of FQs-antibiotic combinations" and "Evaluation of FQs- non-antibiotic combinations" are subsections of "Evaluation of drug combinations", they should be marked as such.

Author response:

We thank the reviewer for his valuable comment. This part has been clarified and the two evaluations were separated in both methods (page 8; lines 180-209) and results (page 12, lines 261-281) sections.

6. Table 1: the title of the second column is misspelled the words "and" and "before" must change their position; the "D" from "FQDs" should be eliminated.

Author response:

Table 1 has been changed and updated according to reviewe1 1 request

7. There are missing data in table 2.

Author response:

Table 2 has been changed and updated according to reviewe1 1 request

7. Figure 5 should be redone, the X-axis has the same name as the Y-axis, and the legend is not clear.

Author response:

Thank you for the reviewer comment. Both label axes are modified and corrected in the revised manuscript.

9. The quality of the figures should be improved.

Author response:

We have modified and increased all figure resolutions as possible.

10. The conclusion should not include results.

Author response:

We thank the reviewer for his valuable comment The conclusion section has been modified and updated in the revised manuscript and the result data has been removed however, sometimes we included our findings in order to compare them with the previous research or literature.

Dear Dr. Monica Cartelle Gestal, Ph.D., Academic Editor, PLOS ONE,

On behalf of all authors who participated in writing this manuscript, we would like to express our deepest gratitude to the reviewers for their valuable comments that enriched the revising process. All comments and recommendations by reviewers were done and the whole manuscript was revised thoroughly for any typing or grammatical mistakes. On behalf of all co-authors, we appreciate your kelp and effort and we hope that manuscript become suitable to be accepted for publication and we are looking forwards hearing from you soon.

Kind regards

Corresponding author,

Prof Dr. Khaled Aboshanab (Ph.D.)

Attachment

Submitted filename: Response letter to Reviewers comments.docx

Click here for additional data file.^{(55.3KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0238195.r003

Decision Letter 1

Monica Cartelle Gestal

9 Jul 2020

PONE-D-20-04012R1

Propranolol, chlorpromazine and diclofenac restore susceptibility of extensive drug-resistant (XDR)-Acinetobacter baumannii to fluoroquinolones

PLOS ONE

Dear Dr. Aboshanab,

Please submit your revised manuscript by Aug 23 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Monica Cartelle Gestal, PhD

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

Reviewer #2: No

**********

6. Review Comments to the Author

Reviewer #1: MANUSCRIPT PONE-D-20-04012-R1

I commend the efforts put in by the authors to address the issues raised in the original submission of this manuscript. However, I will say by my own assessment that we are only getting close to our goal of generating a publishable manuscript, we are not yet there. The manuscript still needs thorough language editing to make it readable. While the authors have made commendable efforts in addressing the lapses on the science side of the manuscript, I have not really seen much improvement in the writing style and language use aspect of the paper. Largely, the manuscript is still very difficult to follow and readability has not been significantly improved. I appreciate the fact that the authors, like many other authors are not native English speakers, but it is important that they communicate with their audience, it is my considered opinion that the manuscript is not communicating in its present format. My candid suggestion is for the authors is to seek the assistance of a native English speaker with experience in scientific writing, or if they can afford it, a manuscript editing service. Some specific issues that needed attention are enumerated below

Abstract :

Title: Please change extensive to extensively. The authors probably misinterpret my suggestion that they should qualify “extensively drug resistant” in the keyword, hence they went ahead to change the phrase throughout the manuscript to “extensive”. What I meant is that they should add what is extensively resistant to complete the keyword, in this case A. baumannii.

Line 27: change “of” to “caused by”

Line 29-30: “....and evaluating the various combinations to combat ....” evaluating the various combination of what? In addition, authors should please change “evaluating” to “evaluate”

Line 32: Delete “a total of” before 1600

Line 36: Change to “high percentage of bacterial resistance to 19.....”

Line 42: Please include CPZ, PR and DIC among drugs tested in combination with CIP and LEV

Lines 43-44: Please change “have been tested” to “was tested” and delete “including 2 PDR and 32 XDR

Lines 51-53: Delete the sentence starting with in conclusion, it is not necessary.

Line 55: Delete “ The obtained results are still promising in vitro study” and start the sentence with However.......

Line 57: Please delete “a life threatening pathogen”.

Line 58: Delete “extensive drug-resistant” it is more like a repetition of the first keyword XDR-A. baumannii

Selection of isolates for inclusion in the drug combination experiment

I am still not comfortable with the criteria used by the authors in selecting isolates for the drug combination experiment. According to them, they selected the 34 isolates based on drug susceptibility profile. While this is scientific enough, it remains a curiosity how they selected which among the 68 XDR isolates to include since according to them (Lines 230-231) all the 68 XDR isolates shared exactly the same susceptibility profile i.e. resistance to all antibiotics except colistin. Again, one wonders why representatives of the MDR strains are not included in the study as a 3rd category since the work is about antibiotic resistant A. baumannii?

Introduction

I appreciate that the authors have updates this section of the manuscript with new information. However, I still have concerns about the writing style and presentation which needed considerable improvement.

Line 62: insert the word “bacteria” after aerobic and after common in line 65

Line 157: Delete “against” after isolates

Lines 159-160: Delete “of”

Line 180: Change “a MIC” to “the MIC”

Line 185: did the authors mean to write two instead of tow

Line 193: ....antagonistic when ΣFIC is ˃ than what?

Line 196: The authors should mention the different concentrations of each non-antibiotic drugs tested may be as supplementary information. While they may think this is not very important, it does provide a valuable guide for others who may want to repeat this experiment.

Line 207: Range should be from low to high and not otherwise as presented here.

Lines 214-216: In view of the fact that the target organism in this study is a non-lactose fermeter, I wonder why the authors are mentioning the isolation of lactose fermenting colonies in this section. I will rather they delete all reference to lactose fermenters as it has no relevance in this work.

Lines 223-224: The authors claimed that the lowest resistance was exhibited towards doxycycline at 57% followed by colistin at 5%. 57% could not be lower than 5%

Discussion

The discussion section, much like the whole manuscript still needs considerable improvement in writing style. In addition, much of this section is just a combination of results with previous studies without the authors providing the much needed depth of insight into their results. They also devoted a lot of space to discussion on isolation, antibiotic susceptibility and genotyping of the isolates through ERIC-PCR at the detriment of the real focus of their research which is the evaluation of drug combination in the control of resistant A. baumannii. The resultant effect of this is an overly long discussion section.

Line 321: The authors should please correct the doxycycline susceptibility data to 43% since they earlier mentioned that resistance to doxyxcycline was 57%.

Lines 377-384: This explanation as presently offered does not help make a case for the low number of isolates included in this study. Claiming that the experiment took a lot of time, effort and resources and was carried out in triplicates is at best not tenable. In my own opinion, these are essential ingredients one may not be able to avoid in the business of science. Pointing out that the study provided baseline information upon which a wider study could be based would have been a better alternative.

Line 397: Authors mentioned that their results did not agree previous study in north Egypt. They should please cite that study for reference here. Similarly in Lines 404-405 authors claimed their results agree with previous study in upper Egypt, they should cite that study also for reference.

Lines 414-416: An incomplete sentence

I suppose the values in bracket in Tables 2 and 3 are the baseline MICs of CIP and LEV? If this is so, this should be indicated in the legends to the table.

Figures Legend- I think the authors should check again the journal requirement about figures is that the figure caption and legend be inserted immediately after the paragraph where the figure was first mentioned.

Figure 3. The authors should please correct the value for CT, 95% would not have been that small on the chart.

Reviewer #2: This manuscript has been improved, and has very interesting results, but:

1. The authors should revise again the format of the manuscript. Some spaces are missing, incorrect used punctuation marks, missing words and repetition of others (clearly from copy and paste some sentences), as well as words misspelling.

2. It is not needed to repeat the meaning of the abbreviations along with the text as far they explained the first time.

3. The term “our findings” is not properly used.

4. Line 132. The authors repeat the amount of DNA added to the PCR, this information was provided in line 130.

5. Line 135-136. The sentence implies that they used the UV illuminator before doing the electrophoresis gel.

6. Line 197, 387, 433. “&” is not appropriate for a text, should be changed for “and”, “as well as” or other synonym chosen by the authors.

7. Tables 2 and 3. Authors should add to the table legend the meaning of the numbers between brackets, and use the same names in the table and the legend (CIP-AMP or AMP-CIP).

8. Table 2. There is no A in the table, so there is no point in explaining it in the legend.

9. Table 4. FIC meaning missing.

10. Line 299. I think the authors may want to say bacteremia or blood infection.

11. Lines 397-406. This paragraph was not clearly written, some sentences are incomplete.

12. Lines 426-436. Sentences incomplete. Paragraph unintelligible, clearly a result of copy and paste sentences.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

PLoS One. 2020 Aug 26;15(8):e0238195. doi: 10.1371/journal.pone.0238195.r004

Author response to Decision Letter 1

14 Jul 2020

Author's Response to Reviewer's comments

On the behalf of all authors, we would like to thank the reviewers for their valuable comments and suggestions that will improve and add to this manuscript. Corrections are highlighted in yellow color and all have been included in the revised manuscript.

Abstract :

Author response:

Thank you for the reviewer's comment. We updated the title and updated the key words

Line 27: change “of” to “caused by”

Author response:

Thank you for the reviewer's comment. done (page2, line 27)

Line 29-30: “....and evaluating the various combinations to combat ....” evaluating the various combination of what? In addition, authors should please change “evaluating” to “evaluate”

Author response:

Thank you for your valuable comment. We updated to “ evaluate the various combinations of ciprofloxacin or levofloxacin with antimicrobial agents and non-antimicrobial agents to combat antimicrobial resistance of XDR A. baumannii”.(page 2; lines 30-31)

Line 32: Delete “a total of” before 1600

Author response:

Thank you for the reviewer's comment. We deleted “a total of”. (page 2, line 33)

Line 36: Change to “high percentage of bacterial resistance to 19.....”

Author response:

Thank you for your valuable comment. We updated to “a high percentage of bacterial resistance to 19”(page 2, line 37)

Line 42: Please include CPZ, PR and DIC among drugs tested in combination with CIP and LEV

Author response:

Thank you for the reviewer's comment. this part was updated since CPZ, PR, and DIC are among drugs that were tested according to MIC decrease factor (MDF) protocol however the antibiotic combinations were evaluated according to standard protocol of fractional inhibitory concentrations (ƩFICs) (line 43). So we put them in aseprate senctence for more clarifications (page2, lines 47-49)

Lines 43-44: Please change “have been tested” to “was tested” and delete “including 2 PDR and 32 XDR

Author response:

Thank you for your valuable comment. We updated the sentence (page2, line 43).

Lines 51-53: Delete the sentence starting with in conclusion, it is not necessary.

Author response:

Thank you for the reviewer's comment. We deleted the sentence.

Line 55: Delete “ The obtained results are still promising in vitro study” and start the sentence with However.......

Author response:

Thank you for your valuable comment. We updated the sentence (page 3, lines 51-53).

Line 57: Please delete “a life-threatening pathogen”.

Author response:

Thank you for the reviewer's comment. We deleted the sentence (page 3, line 55).

Line 58: Delete “extensive drug-resistant” it is more like a repetition of the first keyword XDR-A. baumannii

Author response:

Thank you for the reviewer's comment. We deleted the sentence.

Selection of isolates for inclusion in the drug combination experiment

Author response:

Thank you for your valuable comment. Yes, we selected the 34 isolates based on the drug susceptibility profile. The clinical isolates which were included, (2 PDR and 32 XDR) The selected 32 showed the highest resistant patterns (all were resistant to 19 teste antibiotics except colistin) while other resistant profiles were those which were susceptible to 2 drugs of the 13 drugs tested. So, we selected the isolates of the highest resistance patterns (life-threatening isolates from medical pont of view). In addition , the the selected isolates non-clonal. Future studies will be conducted for testing these combinations on large number of isolates and to evaluated in vivo using appropriate animal model.

Introduction

Author response:

Thank you for your valuable comment. The whole manuscript has been revised thoroughly for any grammar or any spelling or amy possible typing errors.

Line 62: insert the word “bacteria” after aerobic and after common in line 65

Author response:

Thank you for the reviewer's comment. added . (page 3, line 59)

Line 157: Delete “against” after isolates

Author response:

Thank you for the reviewer's comment. deleted . (page 7, line 151)

Lines 159-160: Delete “of”

Author response:

Thank you for the reviewer's comment. We deleted "of". (page 7, line 156)

Line 180: Change “a MIC” to “the MIC”

Author response:

Thank you for the reviewer's comment. We changed it. (page 8, line 170)

Line 185: did the authors mean to write two instead of tow

Author response:

Thank you for the reviewer's comment. Yes, we changed it (page 8, line 175).

Line 193: ....antagonistic when ΣFIC is ˃ than what?

Author response:

Thank you for the reviewer comment. “when ƩFIC is > 4”, we changed it (page 8, line 183).

Author response:

We added the different concentrations of non-antimicrobial agents (page 8, lines 186-191)..

Line 207: Range should be from low to high and not otherwise as presented here.

Author response: corrected (page 9, line 195)

Author response:

Thank you for the reviewer's comment. we updated the sentence. (page 9, line 203)

Lines 223-224: The authors claimed that the lowest resistance was exhibited towards doxycycline at 57% followed by colistin at 5%. 57% could not be lower than 5%.

Author response:

Thank you for the reviewer comment. we updated the sentence (page 9, line 211)

Discussion

Author response:

Thank you for your valuable comment. We revised the discussion and we discussed the results according to the available literature however, there were only few studies on fluoroquinolone combination with antimicrobial agents are available. We revised the dicsusion and we did our best to make it more concise about the drug combinations.

Line 321: The authors should please correct the doxycycline susceptibility data to 43% since they earlier mentioned that resistance to doxyxcycline was 57%.

Author response:

Thank you for the reviewer's comment. we changed it. (page 24, line 320)

Author response:

Thank you for the reviewer's comment. this part of in the discussion have been updated (page 27, lines, 380-384)

Line 397: Authors mentioned that their results did not agree previous study in north Egypt. They should please cite that study for reference here.

Author response:

Citation has been include.(page 27, line 380)

Similarly in Lines 404-405 authors claimed their results agree with previous study in upper Egypt, they should cite that study also for reference.

Author response:

Citation has been included (page 27, line 389)

Lines 414-416: An incomplete sentence

Author response:

Corrected (page 27, line 383-384)

I suppose the values in bracket in Tables 2 and 3 are the baseline MICs of CIP and LEV? If this is so, this should be indicated in the legends to the table.

Author response:

The baselines od MICs of CIP and LEV are provided in table 1. In tables, 2 and 3 are the MICs of the two tested antibiotics after their combinations (this was clarified in the legends of tables 2 and 3).

Author response:

The legends of all figures are provided at the end of the manuscript according to the journal requirement.

Figure 3. The authors should please correct the value for CT, 95% would not have been that small on the chart.

Author response:

It was provided as % resistance (5% resistance) = 95% sensetive

Reviewer #2:

This manuscript has been improved, and has very interesting results, but:

Author response:

Thank you for the reviewer's comment. Thank you for your valuable comment. The whole manuscript has been revised thoroughly for any grammar or any spelling or typing errors

2. It is not needed to repeat the meaning of the abbreviations along with the text as far they explained the first time.

Author response:

Thank you for the reviewer's comment. all have been revised and corrected

3. The term “our findings” is not properly used.

Author response:

Thank you for the reviewer's comment. We replaced “our findings” by “present study”, and “in this study”. (page 24; line 320, apge 25, line 329)

4. Line 132. The authors repeat the amount of DNA added to the PCR, this information was provided in line 130.

Author response:

Thank you for the reviewer's comment. Information was provided in line 130, represent final concentration of DNA (100 ng of genomic DNA,), while Information was provided in line 132, represent the volume of DNA was added to PCR mixture (and 1 µl template DNA). However, we deleted it (containing 100-200 ng of chromosomal DNA) (page 6, line 128).

5. Line 135-136. The sentence implies that they used the UV illuminator before doing the electrophoresis gel.

Author response:

Corrected to " PCR products were analyzed using agarose gel electrophoresis [23]. " (page 6, line 131)

6. Line 197, 387, 433. “&” is not appropriate for a text, should be changed for “and”, “as well as” or other synonym chosen by the authors.

Author response:

Thank you for your valuable comment. we changed it to “and”in whole manuscript

7. Tables 2 and 3. Authors should add to the table legend the meaning of the numbers between brackets, and use the same names in the table and the legend (CIP-AMP or AMP-CIP).

Author response:

There are the MIC of the two antibiotics after their cobmibations. (was clarified and included in the legends of tables 2 and 3)

8. Table 2. There is no A in the table, so there is no point in explaining it in the legend.

Author response:

Deleted since we did not have antagonism (deleted)

9. Table 4. FIC meaning missing.

Author response:

Thank you for the reviewer's comment. FIC, fractional inhibitory concentration (We added it in the legend of table 4)

10. Line 299. I think the authors may want to say bacteremia or blood infection.

Author response:

Thank you for your valuable comment. We changed it to a blood infection (page 24, line 298).

11. Lines 397-406. This paragraph was not clearly written, some sentences are incomplete.

Author response:

Thank you for your valuable comment. We changed it It has been amended according to reviewer 1 by added references 9 and 10.

12. Lines 426-436. Sentences incomplete. Paragraph unintelligible, clearly a result of copy and paste sentences.

Author response:

Revised and corrected.

Dear Dr. Monica Cartelle Gestal, Ph.D., Academic Editor, PLOS ONE,

Kind regards

Corresponding author,

Prof Dr. Khaled Aboshanab (Ph.D.)

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(36.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0238195.r005

Decision Letter 2

Monica Cartelle Gestal

12 Aug 2020

Propranolol, chlorpromazine and diclofenac restore susceptibility of extensive drug-resistant (XDR)-Acinetobacter baumannii to fluoroquinolones

PONE-D-20-04012R2

Dear Dr. Aboshanab,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Monica Cartelle Gestal, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

6. Review Comments to the Author

Reviewer #1: I commend the efforts of the authors in attending to the comments and suggestions of this reviewer which has greatly improve the quality of this manuscript. Still in the same spirit of improving the quality of the manuscript, authors should just attend to this minor corrections after which the manuscript should be accepted for publication.

Line 40: Authors should please define MDR at first use.

Line 165: please delete “of” after percentage

Line 265: Change resistance to resistant

Line 330: Delete the antimicrobial before susceptibility

Line 350: “comate”? did the authors mean to say Combat?

Figure 5 legend: Change “No of resistance.....” to “No of Resistant....”

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

PLoS One. doi: 10.1371/journal.pone.0238195.r006

Acceptance letter

Monica Cartelle Gestal

14 Aug 2020

PONE-D-20-04012R2

Propranolol, chlorpromazine and diclofenac restore susceptibility of extensively drug-resistant (XDR)-Acinetobacter baumannii to fluoroquinolones

Dear Dr. Aboshanab:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Monica Cartelle Gestal

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PERMALINK

Propranolol, chlorpromazine and diclofenac restore susceptibility of extensively drug-resistant (XDR)-Acinetobacter baumannii to fluoroquinolones

Mostafa A Mohammed

Mohammed T Ahmed

Bahaa E Anwer

Khaled M Aboshanab

Mohammad M Aboulwafa

Roles

Abstract

Introduction

Methods

Bacterial isolates

Phenotypic and genotypic identification of A. baumannii

Antimicrobial susceptibility testing

Disc diffusion

Minimum inhibitory concentrations (MICs)

Molecular typing of recovered isolates

Evaluation of drug combinations

Evaluation of FQs-antibiotic combinations

Evaluation of FQs- non-antibiotic combinations

Results

Specimen collection and identification of the recovered A. baumannii isolates

Fig 1. Frequencies of A. baumannii isolates from patients with different clinical samples.

Fig 2. PCR amplification of blaOXA-51 like gene in some A. baumannii clinical isolates, lane M, a gene ruler 100 bp ladder; lane 1, a positive control; lane 2, a negative control; lanes 3 to 11, positive results with an expected size of 353 bp; lane 12, negative results.

Antimicrobial susceptibility testing

Disc diffusion

Fig 3. Antimicrobial susceptibility testing of A. baumannii isolates (n = 100).

Fig 4. Phenotypic analysis of the recovered A. baumannii isolates (n = 100).

Fig 5. Antimicrobial agents resistance profiles scored for different A. baumannii isolates.

Minimum inhibitory concentration

Table 1. MIC of selected antimicrobial agents against PDR and some selected XDR isolates (n = 34).

Genotyping of isolates

Fig 6. ERIC-PCR dendrogram analysis for 34 A. baumanni was constructed by the UPGMA clustering method.

Evaluation of drug combinations

Evaluation of FQs-antibiotic combinations

Table 2. Effects of CIP combinations with different antimicrobial agents by the checkerboard method.

Table 3. Effects of LEV combinations with different antimicrobial agents by the checkerboard method.

Table 4. Summary of antibiotic combinations with FQs by checkerboard method on antibiotic resistant isolates.

Evaluation of FQs- non-antibiotic combinations

Table 5. Effects of non-antibiotics on the MIC of CIP.

Table 6. Effects of non -antibiotic combinations on the MIC of LEV.

Table 7. Summary of CIP and LEV combinations with non-antibiotics.

Discussion

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Monica Cartelle Gestal

Roles

Author response to Decision Letter 0

Decision Letter 1

Monica Cartelle Gestal

Roles

Author response to Decision Letter 1

Decision Letter 2

Monica Cartelle Gestal

Roles

Acceptance letter

Monica Cartelle Gestal

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Fig 2. PCR amplification of bla_OXA-51 like gene in some A. baumannii clinical isolates, lane M, a gene ruler 100 bp ladder; lane 1, a positive control; lane 2, a negative control; lanes 3 to 11, positive results with an expected size of 353 bp; lane 12, negative results.