Prevalence and characterisation of carbapenemase encoding genes in multidrug-resistant Gram-negative bacilli

Sayran Hamad Haji; Safaa Toma Hanna Aka; Fattma A Ali

doi:10.1371/journal.pone.0259005

. 2021 Nov 1;16(11):e0259005. doi: 10.1371/journal.pone.0259005

Prevalence and characterisation of carbapenemase encoding genes in multidrug-resistant Gram-negative bacilli

Sayran Hamad Haji ^1,^*, Safaa Toma Hanna Aka ¹, Fattma A Ali ²

Editor: Abdelazeem Mohamed Algammal³

PMCID: PMC8559951 PMID: 34723978

Abstract

Background

Emerging worldwide in the past decade, there has been a significant increase in multidrug-resistant bacteria from serious nosocomial infections, especially carbapenemase-producing Gram-negative bacilli that have emerged worldwide. The objective of this study is to investigate carbapenem resistance in Gram-negative bacilli bacteria using phenotypic detection, antimicrobial resistance profiles and genotypic characterisation methods.

Methods

200 Gram-negative bacilli isolates were collected from different clinical specimens. All clinical samples were exposed to isolation and identification of significant pathogens applying bacteriological examination and an automated Vitek-2 system. The isolates were subjected to susceptibility tests by the Vitek-2 automated system and those isolates that were resistant to beta-lactam drugs, including carbapenems, third-generation cephalosporines or cefoxitin, were selected for phenotyping using Carba plus disc system assay for detection of carbapenemase-producing isolates. These isolates were further confirmed by molecular detection. PCR was used for the detection carbapenem-resistant genes (OXA-48, IMP, NDM, VIM, and KPC).

Results

110 (55%) of 200 Gram-negative bacilli were identified as beta-lactam-resistant isolates. The frequency of carbapenem-resistant isolates was calculated to be 30.9% (n = 34/110). A collection totalling 65/110 (59%) isolates were identified as carbapenemase producers by phenotypic method. Moreover, among the 65 carbapenemase-producing Gram-negative isolates with a positive phenotype-based result, 30 (46%), 20 (30%) and 18 (27%) isolates were positive for OXA-48, KPC and MBL enzymes, respectively, as well as the production of 27% of AmpC with porin loss. Tigecycline was the most effective antibiotic that affected 70% of MDR isolates, but high rates of resistance were detected to other tested antimicrobials. Of interest, a high incidence of MDR, XDR and PDR profiles were observed among all carbapenemase-producing isolates. 36% (24/65) of the tested isolates were MDR to 3 to 5 antimicrobial classes. 29% (17/65) of the recovered isolates were XDR to 6 to 7 antimicrobial classes. Alarmingly, 24% (16/65) of isolates displayed PDR to all the tested 8 antimicrobial classes. Genotype assay, including 53 phenotypically confirmed carbapenemase-producing isolates of Gram-negative bacilli, found 51(96%) isolates were harbouring one or more genes. The most common carbapenemase gene was bla _NDM 83% (44/53) followed by bla _OXA-48 75% (40/53), bla _VIM 49% (26/53) and bla _IMP 43% (23/53), while the gene bla _KPC was least frequent 7% (4/53). 92% (46/51) of isolates were involved in the production of more than one carbapenemase gene.

Conclusion

This study demonstrated the emergence of carbapenemase-producing Gram-negative pathogens implicated in healthcare-related infections. Accurate identification of carbapenem-resistant bacterial pathogens is essential for patient treatment, as well as the development of appropriate contamination control measures to limit the rapid spread of pathogens. Tigecycline exhibited potent antimicrobial activity against MDR, XDR and PDR-producing strains that establish a threatening alert which indicates the complex therapy of infections caused by these pathogens.

Introduction

Multidrug resistance has increased globally and is considered a public health threat. Several recent studies have reported the existence of multidrug-resistant bacterial pathogens from different origins including humans, poultry, cattle and fish. This has increased the need for routine application of antimicrobial susceptibility testing to detect the antibiotic of choice as well as screening of the emerging MDR strains [1, 2].

In general, the phenomenon of MDR is primarily attributed to the recurrent and indiscriminate use of antibiotics as well as coding for some antimicrobial resistance genes [3].

In the last decade, an alarming increase in the prevalence of multidrug-resistant bacteria of serious nosocomial infections, especially carbapenemase-producing Gram-negative bacilli has been shown worldwide [4, 5]. Carbapenems (i.e. meropenem, imipenem, ertapenem and doripenem) are a safe and highly effective class of antibiotics and considered last line drugs for controlling multidrug-resistant Gram-negative pathogens including extended-spectrum beta-lactamases-producing Enterobacteriaceae [6]. Owing to their broad-spectrum activity, carbapenems are frequently used in the treatment of life-threatening infections. Over-prescribing of these drugs has increased carbapenem resistance. This problem is one of the main reasons for the expression of carbapenemase genes among members of this family [7]. Carbapenemase enzymes are the most common mechanism by which resistance to carbapenems arises [8, 9]. Carbapenem resistance has been labelled in Enterobacteriaceae, mostly in Klebsiella pneumoniae compared to Escherichia coli or other Enterobacterial species, and in non-fermentative Gram-negative bacilli such as Pseudomonas aeruginosa and Acinetobacter baumannii [4]. These Gram-negative bacteria are the main nosocomial pathogens and the most prevalent bacteria responsible for a range of hospital-acquired and community-acquired difficult to treat infections such as meningitis, pneumonia, peritonitis, septicaemia and urinary tract infections. Infection acquired by carbapenemase-producing Gram-negative bacilli is associated with high in-hospital mortality (up to 70%) due to the inefficiency of initial treatment regimens [10].

Clinically relevant Gram-negative bacilli infections include a variety of species. Urinary tract infections (Escherichia coli, Enterobacter species, Proteus species, Serratia marcescens), gastrointestinal infections (Salmonella typhi, Shigella sp., Salmonella enteritidis, Helicobacter pylori) and respiratory tract infections (Pseudomonas aeruginosa, Klebsiella species, Haemophilus, Legionella pneumophila) are Gram-negative primary infections in most hospitals [11].

Recently, a non-fermenting Gram-negative bacterium, Acinetobacter baumannii, has been linked with nosocomial infections. Likewise, other Gram-negative organisms such as Aeromonas sp. and Stenotrophomonas maltophilia have been increasingly reported in many incidents of bacteraemia, pneumonia, meningitis, and surgical or wound site infections in hospital intensive care settings. Most of these Gram-negative microorganisms are resistant to nearly all antimicrobial drugs available worldwide [12].

Various mechanisms are found, for instance acquisition of genes encoding beta-lactamases that destroy the antibiotic, efflux pumps to eliminate agents from the bacterial cell before the target site is reached, and modification of binding sites that may occur before adequate concentrations are reached to remove the whole polymicrobial community. Gram-negative organisms are no exception to these interactions [13].

Carbapenems have become the last resort antibiotics in the treatment of many serious bacterial infections, particularly in hospital settings. However, most drugs, whether beta-lactam or other non-beta-lactam antimicrobials have been ineffective against multi-resistant Gram-negative bacteria [12]. Therefore, acquired carbapenemase-producing Gram-negative bacilli are an actual clinical concern for antimicrobial administration. Specifically those resistant to carbapenems may be associated with resistance to other classes of antibiotics, such as fluoroquinolones and aminoglycosides [4].

Initially, carbapenemases were chromosomally mediated in a few specific species, but now they mediate a plasmid, or both a chromosomal and a plasmid. This results in a more violent spread of resistance due to horizontal transfer between different bacterial species and genera [9]. Since 1993, different types of carbapenemases have been recognised belonging to three molecular classes, the Ambler classes A, B and D beta-lactamases [14]: class A carbapenemases, Klebsiella pneumoniae carbapenemase (KPC) enzymes; class B metallo-beta-lactamases (MBLs) such as New Delhi metallo-beta-lactamases (NDM), Verona integron-encoded metallo-beta-lactamase (VIM) and imipenemase (IMP) type enzymes; and class D carbapenem-hydrolysing oxacillinase (OXA) such as OXA-48 enzymes [15].

Class A beta-lactamase has the ability to degrade penicillins, carbapenems and cephalosporins. Class B metallo-beta-lactamases exhibit different hydrolysis activities against all beta-lactams except for monobactam (aztreonam). Their activity is inhibited by EDTA but not by clavulanic acid [14, 16]. The OXA-48 enzyme shows low activity against carbapenems, high activity against penicillin but only low activity against 3rd and 4th generation cephalosporins. However, this is rarely a treatment option because other beta-lactamases, such as ESBL, are often associated [9]. Globally, most common carbapenemases consist of MBLs (IMP, VIM and NDM), OXA and KPC. The production of carbapenemases including OXA-48, NDM and KPC are the predominant resistance mechanisms among carbapenem-resistant Enterobacteriaceae clinical isolates [17]. In addition, the production of carbapenemases may be related to their hydrolytic action and levels of resistance to broad-spectrum beta-lactams, along with the potential migration of these genes [10].

Different types of mechanisms can mediate carbapenem resistance among the non-carbapenemase-producing or carbapenemase-producing Gram-negative microorganisms. Carbapenemases are beta-lactamases enzymes that are capable of actively cleaving the amide bond in beta-lactam rings of nearly all beta-lactam antibiotics, including the carbapenems, rendering them inactive [16, 18]. The non-carbapenemase-producing carbapenem-resistant microorganisms produce other mechanisms of resistance that can be related to porin mutation in the outer membrane or the presence of an efflux pump, or both, with the production of ESBL or AmpC beta-lactamase dependent on the specific Gram-negative microorganism. All of the carbapenem-resistant microorganisms are of concern, as they are likely to be multidrug-resistant [4, 18].

Nevertheless, carbapenemase-producing microorganisms are of most concern and therefore targeted by antibiotic stewardship and infection control programs, as it is believed to be the primary mechanism responsible for the spread of carbapenem resistance among Gram-negative microorganisms globally [18]. Carbapenem-resistant Enterobacteriaceae are highly lethal and treatment choices are limited [19]. Since in one study the mortality rate was 22%, it was necessary to assess the propensity for carbapenem resistance in hospitals in Erbil city, Iraqi Kurdistan Region, and the genetic characteristics responsible for resistance [20].

Practical and rapid detection of carbapenemases is essential for targeted antimicrobial therapy and to ensure early control of infection in hospitals. The detection of carbapenemases and the accurate and rapid identification of their genes in Enterobacteriaceae remains a major public health challenge for clinical laboratories [10]. The MASTDISCS combi Carba plus disc system (MAST-Carba plus, Mast Group Ltd., UK), is an initial phenotypic assay and is a practical and fast method for distinguishing carbapenemase activity regardless of carbapenemase genes from Enterobacteriaceae in the clinical laboratory. This method is based on the use of a combination of faropenem (disc A) and MBLs (disc B), KPC inhibitor (disc C), AmpC inhibitor (disc D), and temocillin in combination with MBLs inhibitor (disc E). These have recently been described as promising methods for the detection of organisms with MBLs, KPC and OXA-48 enzymes [21]. Molecular techniques remain the gold standard for identifying and characterising states of carbapenemases. The most common of them is polymerase chain reactions [9].

The polymerase chain reaction is a specific and highly dependable diagnostic instrument that simplifies the detection of pathogenic microorganisms, virulence genes as well as antimicrobial resistance genes [22].

The overall objective of the study is to investigate the incidence of carbapenem resistance in Gram-negative bacilli pathogens isolated in hospitals in Erbil city, Iraqi Kurdistan Region. This process was conducted using phenotype-based detection, along with their antimicrobial resistance profiles to different classes of antibiotics, to characterise the prevalence of carbapenemase resistance genes (KPC, IMP, NDM, VIM, OXA-48) using polymerase chain reaction (PCR).

Materials and methods

A total of 200 cultures with positive growth of Gram-negative bacilli isolates (i.e. non-fermenter and Enterobacteriaceae) were collected from different clinical specimens. All of the clinical samples were exposed to isolation and identification of significant pathogens using bacteriological investigation, including microscopy of the Gram-stained preparations and biochemical analysis using Vitek-2 automated system with the AST-GN card (bioMerieux, USA) according to the manufacturer’s instructions from the microbiology laboratory at hospitals. Bacteria were stored from single colonies in trypticase soy broth with 40% glycerol at -70°C until used [23].

These isolates were subjected to susceptibility tests by the Vitek-2 automated system with the AST-GN card (bioMerieux, USA) and those that were resistant to broad-spectrum beta-lactam drugs including carbapenems, third-generation cephalosporines or cefoxitin, were selected for further characterisation. 110/200 (55%) beta-lactam-resistant isolates of Gram-negative bacilli were recovered from different clinical specimens: urine 69 (62%), sputum 18 (16%), swab 16 (14%) and blood 7 (6%). The frequency of carbapenem-resistant isolates was calculated to be 30.9% (n = 34). A collection totalling 65/110 (59%) were identified as carbapenemase-producing isolates by the phenotype-based method using the Carba plus disc system assay, and further confirmed by molecular detection. The median age of the patients was 40 years, with a range of 1–80 years. Among 65 patients, 39 (60%) were female. The bacterial strains were isolated from a variety of infection sites including urine (38), sputum (15), wound swab (8) and blood (4). The isolates were collected from community and hospitalised patients at hospitals in Erbil city, Iraqi Kurdistan Region, during 2019–2020.

Antibacterial susceptibility test of carbapenemase-producing Gram-negative bacilli

In the current study, the following antimicrobials were tested: piperacillin and piperacillin-tazobactam (β-lactams and β-lactamase-inhibitor combination); ceftazidime, ceftriaxone, cefazoline, cefepime and cefoxitin (cephalosporins); gentamicin and amikacin (aminoglycosides); imipenem, ertapenem and meropenem (carbapenems); ciprofloxacin (quinolone); trimethoprim/sulphamethoxazole (sulphonamide); tigecycline (tetracycline); and nitrofurantoin (in urinary infection). They were determined for all these isolates using the Vitek-2 automated (GN-20 and 22 AST card) system (bioMerieux, USA) according to the manufacturer’s instructions [10]. The resistance profiles were categorised into MDR, XDR and PDR according to Algammal [1].

Phenotype detection for carbapenemase production using commercial combination disc assay

Carbapenemase production was examined phenotypically for all strains that showed resistance or intermediate resistance to carbapenem drugs, or demonstrated resistance to either third-generation cephalosporins or cefoxitin, using the MASTDISCS® Combi Carba plus (Carba plus) commercial combination disc assay, according to the manufacturer’s instructions [10, 21].

MASTDISCS® Combi Carba plus (MASTDISCS combi-D73C, Bootle, UK) is a combined-disc test of a five-disc system: penem (disc A), penem+MBLs inhibitor (disc B), penem+KPC inhibitor (disc C), penem+AmpC inhibitor (disc D) and temocillin+MBLs inhibitor (disc E). Evaluation was to detect KPC, MBL and OXA-48-like carbapenemases formed by Enterobacteriaceae, including the reliable differentiation of KPC from AmpC-producing isolates. Adding the temocillin disc in combination with MBL inhibitor (E disc), instead of just the temocillin disc, improves the discrimination of OXA-48 by removing the confusion of MBLs incorrectly identified as OXA-48. MASTDISCS® Combi Carba plus D73C was performed according to the manufacturer’s instructions. First, a 0.5 McFarland inoculum of the test organism was prepared and uniformly spread across the surface of Mueller–Hinton agar plates. Five prepared discs were placed onto the inoculated medium by sterile forceps, ensuring sufficient space between discs to allow obviously clear inhibition zones to be formed. After 18 to 24 hours of incubation at 37°C, the diameter of any zones of inhibition observed were measured and recorded in millimetres (mm) disregarding any small colonies within the region. The results were interpreted according to the manufacturer’s instructions [21]: discs showing no zone of inhibition were recorded as 6 mm, ≥5 mm difference in the disc zone diameters observed only between disc B and disc A (less than 5 mm difference between disc C or disc D and disc A) indicated the production of MBLs. Likewise, ≥5 mm difference only between disc C and disc A (less than 5 mm difference between disc B or disc D and disc A) indicated KPC production. AmpC production with porin loss is indicated when both discs C and D show a zone difference of ≥5 mm compared to disc A. In the absence of such synergistic effects among discs A–D, the diameter of the inhibition zone of ≤10 mm around disc E indicated OXA-48 carbapenemase production [10]. K. pneumoniae NCTC 13438 (KPC positive), K. pneumoniae NCTC 13440 (MBL positive) and K. pneumoniae NCTC 13442 (OXA-48 positive) were used as positive controls, and E. coli ATCC25922 as negative controls.

PCR-based detection of carbapenemase genes

53 isolates screened as carbapenemase-positive by the phenotype-based method were further investigated for the detection of carbapenemase genes using PCR-based methods. Singleplex and multiplex PCR were carried out to examine for the presence of five predominant carbapenemase genes namely KPC (bla _KPC gene), MBLs (bla _IMP, bla _VIM, and bla _NDM genes) and _OXA-48 (bla _OXA-48 gene) using a panel of primers as previously described [7, 24, 25]. Multiplex PCR for bla _IMP and bla _VIM detection was planned. Screening of isolates containing the bla _NDM, bla _OXA-48 and bla _KPC genes was tested by singleplex PCR. Extraction of the total DNA from a bacterial culture in the logarithmic phase was conducted with the commercial genomic DNA extraction kit (DNALandScientfic Cat No. GG2001) according to the manufacturer’s instructions. All the strains were identified for the mentioned genes using the PCR technique through amplification of the gene-specific targeted sequence in a thermocycler machine (Techne/UK).

In singleplex PCR (bla _OXA-48, bla _NDM and bla _KPC genes) the premix was subjected in the total volume of 25μl, 12.5 μl Gotaq Green Master Mix (Promega/USA), 3μl of genomic DNA, 1.5 μl for each primer and the volume completed with 6.5μl nuclease-free water. The PCR for bla _OXA-48 and bla _NDM genes were conducted according to the following conditions, initial denaturation 95°C for 5min, 35 cycles amplification for denaturation at 95°C for 30 sec, annealing at 56°C for 1 min, extension step at 72°C for 1 min then the final extension at 72°C for 5 min. The multiplex master mix was prepared in total volume of 25μl, 12.5 μl Gotaq Green Master Mix (Promega/USA), 3μl of genomic DNA, 1 μl for each primer and the volume completed with 5.5μl DNase, RNase free water [25].

The Multiplex PCR program reaction for bla _IMP and bla _VIM were conducted as the following: initial denaturation at 94°C for 8 min; denaturation at 94°C for 30 sec, annealing at 54°C for 55 sec and extension at 72°C for 1 min; and the final extension at 72°C for 10 min. The PCR product amplicons were then analysed by agarose gel electrophoresis (2%) and the DNA stained with safe dye (Bioland/USA). The presence and absence of the carbapenemase genes were determined using a UV transilluminator (Syngene/UK) and the PCR product band for all genes identified on the gel as compared to the standard DNA ladder/1kb (Norgenbiotek/Canada).

Statistical analysis

GraphPad Prism (version 5; GraphPad Software, San Diego, CA). Chi-square test was applied to analyse the collected data. A P-value of < 0.05 was considered statistically significant.

Ethical issues

This study was approved by the ethics committee for the College of Pharmacy, Hawler Medical University. During data collection, all patients who attended treatment were asked to obtain verbal consent. The purpose of the study was explained to the participants and details about anonymity and the right to withdraw from the study at any stage were provided. Finally, verbal consent was obtained from the participants in this research.

Results

Demographic factors and characteristics of bacterial isolates

A collection of 200 Gram-negative bacilli were collected from different clinical specimens. 110 (55%) broad-spectrum beta-lactam resistant isolates of Gram-negative bacilli were multidrug-resistant (MDR) to different classes of antimicrobials tested, including carbapenems. The isolates comprised Escherichia coli (E. coli) (n = 49, 44%), Klebsiella sp. (K. pneumoniae and K. oxytoca) (n = 23, 20%), Acinetobacter baumanii (A. baumanii) (n = 13, 11%), Pseudomonas aeruginosa (P. aeruginosa) (n = 10, 9%), Proteus sp. (P. vulgaris and P. mirablis) (n = 5, 4%) and Serratia marcescens (n = 3, 2%). Less prevalent microorganisms were, Enterobacter cloacae, Stenotrophomonas maltophilia, Morganella morganii, Achromobacter dentrificans, Ewingella americana, Sphingomonas sp. and Pantoea agglomerans and included one isolate each (n = 1, 0.9%). The frequency of carbapenem-resistant isolates was calculated to be 30.9% (n = 34). The uppermost resistance isolates to carbapenems were detected for Acinetobacter baumanii at 9, while Klebsiella sp., E. coli and Pseudomonas aeruginosa ranked second in terms of gaining resistance to imipenem with a frequency of 6 for each. Urine samples were found to be the dominant type (62%), followed by sputum (16%). Statistical analysis showed that there was a significant difference in the prevalence of different bacterial pathogens and carbapenemase-resistant isolates recovered from different types of samples (p < 0.05) (Table 1).

Table 1. Prevalence of broad-spectrum beta-lactam resistant Gram-negative bacilli isolates and isolation of carbapenem-resistant isolates from different types of clinical samples.

Bacteria	No. of isolates (%)	Carbapenem resistant	Specimen no. and types
Bacteria	No. of isolates (%)	Carbapenem resistant	Urine	Sputum	Swab	Blood
E. coli	49 (44)	6	42	_	3	4
Klebsiella sp.	23 (20)	6	13	5	6	_
*Acinetobacter baumanii*	13 (11)	9	1	9	2	1
*Pseudomonas aeruginosa*	10 (9)	6	7	1	2	_
Proteus sp.	5 (4)	3	2	2	1	_
*Serratia marcescens*	3 (2)	1	2	1		_
*Enterobacter cloacae*	1 (0.9)	_	_	_	1	_
*Stenotrophomonas maltophilia*	1 (0.9)	1	_	_		1
*Morganella morganii*	1 (0.9)	_	1	_		_
*Achromobacter dentrificans*	1 (0.9)	_	_	_	1	_
*Ewingella americana*	1 (0.9)	_	1	_	_	_
Sphingomonas sp.	1 (0.9)	1	_	_	_	1
*Pantoea agglomerans*	1 (0.9)	1	1	_	_	_
Total no. (%)	110 (55)	34 (30.9)	69 (62)	18 (16)	16 (14)	7 (6)

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P value < 0.0086.

All cases of resistance to carbapenems and second or third-generation cephalosporins were first tested by a phenotype-based method using Carba plus test, 65/110 were identified as carbapenemase-producing isolates. These isolates belonged to different species: E. coli (n = 20, 30.7%); Klebsiella sp. (12 K. pneumoniae and 3 K. oxytoca), (n = 15, 23%) and (n = 3, 4.6%); Acinetobacter baumanii (n = 11, 16.9%); Pseudomonas aeruginosa (n = 8, 12.3%); and Proteus sp. (P. vulgaris and P. mirablis) (n = 4, 6.1%). The less predominant microorganisms were, Serratia marcescens, Enterobacter cloacae, Stenotrophomonas maltophilia, Morganella morganii, Achromobacter dentrificans, Sphingomonas sp. and Pantoea agglomerans, and included one isolate each (n = 1, 1.5%). The predominant bacteria were E. coli, K. pneumoniae and A. baumannii (20, 30%, 15, 23%, 11, 16% strains, respectively).

It was found that females were more likely to acquire imipenem-resistant infections (60%) than males (40%). Infection rates were diverse among different age groups, with the maximum average being observed among patients of the 60–70 years age group. The isolates were predominantly isolated from urine (38/65), sputum (15/65), swabs (8/65) and blood (4/65) (Table 2).

Table 2. Characteristic and antibiogram of 65 carbapenemase-producing Gram negative bacilli.

Bacteria	Total no. of isolates	Specimen no. and types	Antimicrobial classes and number of resistance isolates [%]
			β-lactam and β- lactamase inhibitor combination		Cephalosporins					Carbapenems			Aminoglycosides		Quinolone	Nitrofurantoin	Sulphonamide	Tetracycline
			piperacillin	piperacillin- tazobactam	cefazoline	ceftriaxone	cefepime	ceftazidime	cefoxitin	imipenem	meropenem	ertapenem	amikacin	gentamicin	ciprofloxacin	nitrofurantoin	trimethoprim/ Sulphamethoxazole	tigecycline
E. coli	20 [30.7]	17 urines	20 [100]	10 [50]	20 [100]	20 [100]	19 [95]	20 [100]	6 [30]	1 [5]	1 [5]	1 [5]	5 [25]	8 [40]	10 [50]	3 [15]	14 [70]	3 [15]
		2 bloods
		1 swab
Klebsiella sp.	15 [23]	12 urines	15 [100]	6 [40]	15 [100]	15 [100]	15 [100]	15 [100]	5 [33]	4 [26]	2 [13]	2 [13]	5 [33]	10 [66]	11 [73]	8 [53]	12 [80]	3 [20]
		2 sputum
		1 swab
*Acinetobacter baumanii*	11 [16.9]	1 urine	11 [100]	11 [100]	11 [100]	11 [100]	11 [100]	11 [100]	10 [90]	11 [100]	11 [100]	11 [100]	11 [100]	11 [100]	11 [100]	11 [100]	11 [100]	9 [81]
		1 swab
		9 sputum
*Pseudomonas aeruginosa*	8 [12.3]	5 urines	8 [100]	4 [50]	8 [100]	7 [87]	8 [100]	4 [50]	0	4 [50]	4 [50]	4 [50]	5 [62]	6 [75]	5 [62]	7 [87]	4 [50]	2 [25]
		2 swabs
		1 sputum
Proteus sp.	4 [6.1]	1urine	4 [100]	2 [50]	4 [100]	4 [100]	3 [75]	3 [75]	1 [25]	3 [75]	3 [75]	2 [50]	2 [50]	1 [25]	3 [75]	3 [75]	3 [75]	1 [25]
		1 swab
		2sputum
*Serratia marcescens*	1 [1.5]	sputum	1 [100]	0	1 [100]	1 [100]	1 [100]	1 [100]	0	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	0	1 [100]	1 [100]	0
*Enterobacter cloacae*	1 [1.5]	swab	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]
*Stenotrophomonas maltophilia*	1 [1.5]	blood	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	0	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]
*Morganella morganii*	1 [1.5]	urine	0	0	1 [100]	0	0	0	0	0	0	0	0	1 [100]	1 [100]	1 [100]	1 [100]	0
*Achromobacter dentrificans*	1 [1.5]	swab	0	0	0	0	0	0	0	0	0	0	0	1 [100]	0	0	0	0
Sphingomonas sp.	1 [1.5]	blood	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	0	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]	1 [100]
*Pantoea agglomerans*	1 [1.5]	urine	1 [100]	0	0	1 [100]	-	-	1 [100]	0	1 [100]	0	0	1 [100]	0	0	0	0
Total no. [%]	65	38 urines	63 [96]	36 [55]	63 [96]	62 [95]	61 [93]	58 [89]	24 [36]	27 [41]	26 [40]	24 [36]	32 [49]	43 [66]	44 [67]	37 [56]	49 [75]	20 [32]
		15 sputum
		8 swabs
		4 bloods

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P value < 0.0001.

Antimicrobial susceptibility profile results

According to the Vitek-2 automated system test results, all 65 carbapenemase-producing isolates were multidrug-resistant. The susceptibility of the isolates to different antimicrobial agents revealed that almost all strains were highly resistant to beta-lactam antibiotics (piperacillin and cefazolin 96%, ceftriaxone 95%, cefepime 93% and ceftazidime 89%). Among the isolates, the percentages of resistance to imipenem, meropenem and ertapenem were 27 (41%), 26 (40%) and 24 (36%), respectively. Carbapenems resistance has been associated with resistance to other antibiotic classes. The rates of resistance to other antibiotics were: trimethoprim/sulphamethoxazole 75%; ciprofloxacin 67%; gentamicin 66%; nitrofurantoin 56%; piperacillin/tazobactam 55%; amikacin 49%; cefoxitin 36%; and tigecycline 32%. The isolated carbapenemase-producing Gram-negative bacilli exhibited a high significance difference in resistance to several tested antimicrobial agents (p < 0.05) (Table 2).

Patterns of phenotypic resistance of carbapenemase-producing isolates

In general, almost all carbapenemase-producing strains were resistant to at least three classes of antimicrobials and were considered multidrug-resistant (MDR). In comparison, extensive drug-resistant (XDR) is classified as resistance to all but two or less classes of antimicrobials, and resistant to all classes of available antimicrobial agents is known as pan drug-resistant (PDR) [1, 26].

Our findings revealed that of 65 carbapenemase-producing Gram-negative bacilli isolates, 24 (36%) were defined as multidrug-resistant to 3 from 5 classes of antimicrobials. The most common phenotypic resistance patterns (β-lactams and β-lactamase inhibitor combinations, cephalosporins, quinolone and sulphonamides) were represented among the E. coli isolates, consisting of 4 classes of antimicrobials.

Furthermore, 17 (29%) of the recovered carbapenemase-producing isolates were extensively drug-resistant from 6 to 7 antimicrobial classes. The most common phenotypic resistance patterns were six (β-lactams and β-lactamase inhibitor combinations, cephalosporins, aminoglycosides, quinolone, sulphonamides and tetracyclines) among Klebsiella sp.

Unfortunately, 16 (24%) of isolates were PDR to all the tested antimicrobial classes (β-lactams and β-lactamase inhibitor combinations, cephalosporins, aminoglycosides, quinolone, sulphonamides, tetracyclines, nitrofurantoin and carbapenems) which were prevalent among Acinetobacter baumannii. Statistically, there was a significant difference in the distribution of MDR, XDR and PDR among carbapenemase-producing Gram-negative bacilli strains (p < 0.05) (Table 3).

Table 3. Distribution of MDR, XDR, and PDR patterns among 65 carbapenemase-producing Gram-negative bacilli strains.

Bacteria	Resistance profile	The most common phenotypic resistance patterns	Number of antimicrobial resistance classes
*E. coli* n = 20**	13MDR	β-lactams and β-lactamase inhibitor combinations: Piperacillin and Piperacillin /Tazobactam	3–4
		Cephalosporins: ceftazidime, ceftriaxone, cefazoline, cefepime and cefoxitin
		Sulphonamides: trimethoprim-sulphamethoxazole
		Quinolone: ciprofloxacin
	1XDR	β-lactams and β-lactamase inhibitor combinations: Piperacillin and Piperacillin/Tazobactam	6
		Cephalosporins: ceftazidime, ceftriaxone, cefazoline, and cefepime
		Aminoglycosides: gentamicin and amikacin
		Quinolone: ciprofloxacin
		Sulphonamides: trimethoprim-sulphamethoxazole
		Tetracyclines: tigecycline
	1PDR	^*β-lactams & β-lactamase inhibitor combinations: Piperacillin and Piperacillin/Tazobactam	8
		Cephalosporins: ceftazidime, ceftriaxone, cefazoline, cefepime and cefoxitin
		Carbapenems: imipenem, ertapenem and meropenem
		Sulphonamides: trimethoprim-sulphamethoxazole
		Tetracyclines: tigecycline
		Aminoglycosides: gentamicin and amikacin
		Quinolone: ciprofloxacin
		Nitrofurantoin: (in urinary infection)
Klebsiella sp. n = 15	5MDR	β-lactams: Piperacillin	3–5
		Cephalosporins: ceftazidime, ceftriaxone, cefazoline, and cefepime
		Sulphonamides: trimethoprim-sulphamethoxazole
		Aminoglycosides: gentamicin `
		Quinolone: ciprofloxacin
	6XDR	β-lactams and β-lactamase inhibitor combinations: Piperacillin and Piperacillin/Tazobactam	6
		Cephalosporins: ceftazidime, ceftriaxone, cefazoline, cefepime and cefoxitin
		Sulphonamides: trimethoprim-sulphamethoxazole
		Aminoglycosides: gentamicin and amikacin
		Quinolone: ciprofloxacin
		Nitrofurantoin: (in urinary infection)
	2 PDR	^*	8
Acinetobacter baumanii n = 11	2XDR	β-lactams and β-lactamase inhibitor combinations: Piperacillin and Piperacillin/Tazobactam	7
		Cephalosporins: ceftazidime, ceftriaxone, cefazoline, cefepime and cefoxitin
		Carbapenems: imipenem, ertapenem and meropenem
		Sulphonamides: trimethoprim-sulphamethoxazole
		Aminoglycosides: gentamicin and amikacin
		Quinolone: ciprofloxacin
		Nitrofurantoin: (in urinary infection)
	9PDR	^*	8
Pseudomonas aeruginosa n = 8	3MDR	β-lactams and β-lactamase inhibitor combinations: Piperacillin and Piperacillin/Tazobactam	3–5
		Cephalosporins: ceftazidime, ceftriaxone, cefazoline, and cefepime
		Nitrofurantoin: (in urinary infection)
		Sulphonamides: trimethoprim-sulphamethoxazole
	4XDR	β-lactams and β-lactamase inhibitor combinations: Piperacillin and Piperacillin/Tazobactam	6–7
		Cephalosporins: ceftazidime, ceftriaxone, cefazoline, cefepime and cefoxitin
		Carbapenems: imipenem, ertapenem and meropenem
		Nitrofurantoin: (in urinary infection)
		Aminoglycosides: gentamicin and amikacin
		Quinolone: ciprofloxacin
	1 PDR	^*	8
Proteus sp. n = 4	1MDR	β-lactams and β-lactamase inhibitor combinations: Piperacillin and Piperacillin/Tazobactam	3–4
		Cephalosporins: ceftriaxone, and cefazoline
		Carbapenems: imipenem, ertapenem and meropenem
		Quinolone: ciprofloxacin
	3XDR	β-lactams and β-lactamase inhibitor combinations: Piperacillin and Piperacillin/Tazobactam	6–7
		Cephalosporins: ceftazidime, ceftriaxone, cefazoline, and cefepime
		Carbapenems: imipenem, ertapenem and meropenem
		Sulphonamides: trimethoprim-Sulphamethoxazole
		Aminoglycosides: gentamicin and amikacin
		Quinolone: ciprofloxacin
		Nitrofurantoin: (in urinary infection)
Serratia marcescens n = 1	1XDR	β-lactams: Piperacillin	6
		Cephalosporins: ceftazidime, ceftriaxone, cefazoline, and cefepime
		Carbapenems: imipenem, ertapenem and meropenem
		Sulphonamides: trimethoprim-Sulphamethoxazole
		Aminoglycosides: gentamicin and amikacin
		Nitrofurantoin: (in urinary infection)
Enterobacter n = 1	1PDR	^*	8
Stenotrophomonas n = 1	1PDR	^*	8
Morganella morganii n = 1	1MDR	Cephalosporins: cefazoline	5
		Sulphonamides: trimethoprim-sulphamethoxazole
		Tetracyclines: tigecycline
		Aminoglycosides: gentamicin and amikacin
		Quinolone: ciprofloxacin
Achromobacter n = 1	_	_	_
Sphingomonas sp. n = 1	1PDR	^*	8
Pantoea agglomerans n = 1	1MDR	β-lactams: Piperacillin	4
		Carbapenems: imipenem, ertapenem and meropenem
		Cephalosporins: ceftazidime, ceftriaxone, cefazoline, and cefepime
		Aminoglycosides: gentamicin
Total no. (%)	24MDR
	17XDR
	16PDR

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* Most common phenotypic resistance patterns in relation to PDR are the same for all bacteria.

P value < 0.0182.

Phenotype detection of carbapenemase activity

The phenotype-based detection results of the Carba plus assay showed that 65 of 110 beta-lactams-resistant isolates of Gram-negative bacilli produced one or more carbapenemases, including 20 (30%) with Class A carbapenemase KPC (E. coli, n = 8; Klebsiella sp., n = 5; Pseudomonas aeruginosa, n = 3; Proteus sp., n = 2; Acinetobacter baumanii., n = 1; and Achromobacter dentrificans, n = 1); 18 (27%) with Class B carbapenemase MBL (Klebsiella sp., n = 5; Acinetobacter baumanii., n = 9; Pseudomonas aeruginosa n = 1; and Stenotrophomonas maltophilia; Serratia marcescens; and Proteus sp., n = 1, respectively); and 30 (46%) with Class D carbapenemase OXA-48 (E. coli, n = 8; Klebsiella sp., n = 4; Acinetobacter baumanii, n = 9; Pseudomonas aeruginosa, n = 4; Proteus sp., n = 2; and n = 1 for each of Serratia marcescens; Sphingomonas sp.; and Pantoea agglomerans). In addition, AmpC beta-lactamase production with porin loss was detected in 18 (27%) isolates (Table 4).

Table 4. Phenotype detection of carbapenemase-producing isolates of Gram-negative bacilli.

Bacteria	Tested isolates (%)	Carbapenemase Classes			AmpC + Porin loss (%)	Total no. of carbapenemase producing isolates (%)
		Class A KPC (%)	Class B MBL (%)	Class D OXA-48(%)
		KPC	MBLs	OXA-48
E. coli	49(40)	8(40)	_	8(26)	8(44)	20(30.7)
Klebsiella sp.	23(20)	5(25)	5(27)	4(13)	4(22)	15(23)
*Acinetobacter baumanii*	13(11)	1(5)	9(50)	9(30)	1(5)	11(16.9)
*Pseudomonas aeruginosa*	10(9)	3(15)	1(5)	4 (13)	2(11)	8(12.3)
Proteus sp.	5(4)	2(10)	1(5)	2 (6)	_	4(8.1)
*Serratia marcescens*	3 (2)	_	1(5)	1(3)	_	1(1.5)
*Pantoea agglomerans*	1(0.9	_	_	1(3)	1(5)	1(1.5)
*Enterobacter cloacae*	1(0.9)	_	_	_	1(5)	1(1.5)
Sphingomonas sp.	1(0.9)	_	_	1(3)	_	1(1.5)
*Stenotrophomonas maltophilia*	1(0.9)	_	1(5)	_	_	1(1.5)
*Achromobacter dentrificans*	1(0.9)	1(5)	_	_	_	1(1.5)
*Ewingella americana*	1(0.9)	_	_	_	_	_
*Morganella morganii*	1(0.9)	_	_	_	1 (5)	1(1.5)
Total no. (%)	110	20(30)	18(27)	30(46)	18(27)	65(59%)

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P value < 0.0182.

The highest rates of KPC and AmpC were found among E. coli isolates, while the maximum rates for MBL and OXA-48 were recorded among A. baumanii isolates. Detailed results of carbapenemase phenotype detection by Carba plus are shown in (Table 4). Statistically, there is a significant difference in the frequency of carbapenemase classes A, B and D with AmpC production among beta-lactam-resistant isolates of Gram-negative bacilli (p < 0.0001).

The results of the Carba plus test are revealed in (Fig 1). The positive result of KPC-producing isolates indicates that there is ≥5 mm difference only between disc C and disc A (<5 mm difference between disc B or disc D and disc A) (Fig 1A). The positive result of MBL-producing isolates shows that there is ≥5 mm difference in the disc zone diameters detected only between disc B and disc A (<5 mm difference between disc C or disc D and disc A) (Fig 1B). The positive result of the OXA-48-producing isolate displays that there is the absence of synergistic effects among discs A–D, inhibition zone diameter of ≤10 mm around disc E (Fig 1C). The positive result of AmpC with porin loss, shows that there is ≥5 mm difference in both discs D and C zone diameter compared with disc A (<5 mm difference between disc B and disc A) (Fig 1D). In contrast, the negative results of isolates of non-carbapenemase-producing Gram-negative bacilli isolate display that disc A, B, C and D inhibition zone diameters vary by ≤2 mm, and the disc E >10 mm (Fig 1E). Thus, when looking at the PCR results, the MAST-Carba plus can correctly identify the 50/53 carbapenemase producers regardless of carbapenemase types.

Prevalence of carbapenemase genes

After gene extraction, 53 phenotypically confirmed carbapenemase-producing isolates were examined genotypically based on the primers described previously for five carbapenemase genes KPC (bla KPC gene), MBLs (bla IMP, bla VIM and bla NDM genes) and OXA-48 (bla OXA-48 gene), by singleplex and multiplex PCR methods (Figs 2 and 3). Overall detection of genes, 51/53 (96%) of the isolates were positive for one or more carbapenemase genes. The result was including 50 (94%) isolates producing MBL (44 NDM producers, 26 VIM producers, and 23 IMP producers), 40 (75%) isolates producing OXA-48 and 4 (7%) with KPC. MBL producers belong to twelve diverse Gram-negative bacilli isolates, the OXA-48 producers belong to ten different Gram-negative bacilli isolates, while the remaining four KPCs belong to three different Gram-negative bacilli isolates. In general, Klebsiella sp. 12 (22%) and A. baumanii 11 (20%), were the two species with the largest number of these genes, followed by E. coli 10 (18%), Pseudomonas aeruginosa 8 (15%), Proteus sp. 3 (5%) and one isolate (1%) for each of Serratia marcescens, Stenotrophomonas malophilia, Sphingomonas sp., Achromobacter dentrificans, Enterobacter cloacae, Morganella morganii and Pantoea agglomerans. Moreover, most of the carbapenemase-producing isolates produced more than one carbapenemase gene 46 (92%). General features of carbapenemase-producing isolates are shown in (Table 5). Statistical study verified that there is a highly significant difference in the prevalence of carbapenemase genes among the tested isolates (p < 0.0001).

Fig 2 — Lanes 3–5 represent the positive IMP carbapenemase gene, Lanes 8–10 represent the positive VIM carbapenemase gene, Lanes 6 and 7 represent co-expression of IMP and VIM genes. The molecular size of the IMP and VIM genes are 232 and 390 bp respectively. Lane 1 and 11 are 1-kb DNA ladders. Lane 2 corresponds to the negative control.

Fig 3 — Lanes 3–5 represent the positive OXA-48 gene, Lanes 6–9 represent the positive NDM gene, and Lanes 10–13 represent the positive KPC gene. Lane 1 is a 1-kb DNA ladder. Lane 2 corresponds to the negative control. The molecular size of the tested *bla* _OXA-48, *bla* _NDM, and *bla* _KPC carbapenemase genes are 438, 621 and 798 bp respectively.

Table 5. Genotype detection of carbapenemase-producing Gram-negative bacilli.

Bacteria	Tested isolates	Carbapenemase genes					Total genes no. (%)	Two or more genes
		KPC	MBL n = 50			OXA-48
		KPC	NDM	IMP	VIM	OXA-48
E. coli	10(33)	1 (10)	8(80)	5(50)	4 (40)	7 (70)	10(18)	8
Klebsiella sp.	14(26)	-	10(71)	1(7)	11(78)	8 (57)	12(22)	10
*Acinetobacter baumanii*	11(20)	2 (18)	9 (81)	8(72)	5 (45)	10 (90)	11(20)	11
*Pseudomonas aeruginosa*	8(15)	-	7 (87)	3 (37)	2 (25)	7 (87)	8(15)	7
Proteus sp.	3(5)	-	3(100)	2 (66)	2 (66)	3 (100)	3(5)	3
*Serratia marcescens*	1(1.8)	-	1(100)	1 (100)	-	1(100)	1(1)	1
*Enterobacter cloacae*	1(1.8)	-	1(100)	-	-	1 (100)	1(1)	1
*Stenotrophomonas maltophilia*	1(1.8)	-	1(100)	1 (100)	-	1 (100)	1(1)	1
*Morganella morganii*	1(1.8)	-	1(100)	-	1 (100)	1 (100)	1(1)	1
*Achromobacter dentrificans*	1(1.8)	1 (100)	1(100)	-	1 (100)	1 (100)	1(1)	1
Sphingomonas sp.	1(1.8)	-	1(100)	1 (100)	-	1 (100)	1(1)	1
*Pantoea agglomerans*	1(1.8)	-	1(100)	1 (100)	-	1 (100)	1(1)	1
Total no. (%)	53	4(7)	44 (83)	23(43)	26 (49)	40(75)	51(96)	46(92)

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P value < 0.0001.

Discussion

Increased drug resistance is creating a real problem for treatment options, especially with high carbapenem resistance among Gram-negative bacilli isolates, which has been demonstrated and reported in many countries. As a result, the clinical effect of carbapenem resistance has become a serious public health crisis on a global scale [27]. Currently, the proliferation of carbapenemases is the most pressing resistance problem in Gram-negative bacteria.

Clinically important carbapenemase-producing pathogens are a triple risk as a result of their MDR profile, increased occurrence, and rapid distribution from species to species and even between different genera of Gram-negative bacteria through transmissible genetic elements (e.g., insertion sequences, transposons and plasmids). Furthermore, transmissible genetic elements harbour many additional antimicrobial resistance genes, subsequent in MDR or XDR traits, which greatly restricts treatment selection [18].

The microbiological examination in the present study revealed that 55% (110/200) broad-spectrum beta-lactam resistant isolates of Gram-negative bacilli were recovered from different clinical specimens. The most predominant bacterial pathogen retrieved was E. coli (44%), followed by K. pneumoniae (20%) and A. baumannii (11%). Statistical analysis showed that there was a significant difference in the prevalence of different bacterial pathogens and carbapenemase-resistant isolates recovered from different types of specimens (p < 0.05). The uppermost number of clinical isolates was collected from urine and sputum from the urinary tract infections and respiratory tract infections, respectively (Table 1). These results are in agreement with those obtained by Jalalvand et al. and Rimrang et al. [7, 28] who demonstrated that the most predominant Gram-negative bacilli were E. coli and K. pneumoniae.

The detection rate of beta-lactam drug-resistant isolates of Gram-negative bacilli in clinical settings has gradually increased. This is possibly due to repeated exposure to drugs such as penicillin and 3rd generation cephalosporins. Similar resistance mechanisms have been detected in other studies where the loss of porin expression or ESBL enzymes in combination with AmpC beta-lactamases and expression of common efflux pumps, are a factor in the acquisition of carbapenemase resistance in Gram-negative pathogens [12].

Carbapenemases have been widely reported in Enterobacteriaceae worldwide [29]. However, limited studies of carbapenemase-producing Gram-negative bacilli have been conducted in the Kurdistan region of northern Iraq. Our study revolved around the dissemination of carbapenem-resistant Gram-negative bacilli bacteria, which provides information on the extent of the problem in the field from a microbiological and epidemiological point of view.

The frequency of carbapenem resistance in our community throughout the study period was 34/110 (30.9%) (Table 1). The isolation rates of carbapenem-resistant Gram-negative bacilli from various countries was found to be: 36% in Egypt [30], 13.6% and 37.9% in Iran [7, 26], respectively, 56% in Pakistan [31], 24.6% in China [27], 19% in Algeria [4], 2.82% in Turkey [19], 86.3% in Tunis [23], 5.99% in Morocco [29] and 2.9% in Ghana [12].

The most prevalent carbapenem-resistant isolated Gram-negative bacilli were A. baumannii, followed by K. pneumoniae, P. aeruginosa and E. coli. Conforming with other studies, the most common isolated species in this study was A. baumanii [4, 5].

The current emergence of carbapenemase-producing bacteria, especially Enterobacteriaceae, is of concern because it is often associated with the occurrence of multidrug-resistant isolates, where there are very few, if any, drug options available for them. Therefore, detection and initial identification of carbapenemase-producing bacteria are important [12].

Overall, 59% (65/110) of strains, including carbapenem-resistant strains of Gram-negative bacilli, were expressing phenotypic production of carbapenemases. The results showed that E. coli ranked first among Gram-negative bacteria, while K. pneumoniae ranked second in terms of the number of detected isolates (Table 2). Similar to data from Iran and Lebanon, the most common isolated species is E. coli and the second is Klebsiella pneumoniae [7, 24]. Urinary tract infections were found to be the dominant type of infection, 38 out of 65 isolates (58%) were from urine cultures, followed by respiratory tract infections (23%), wound infections (12%) and blood infections associated with systemic disease (6%). Urinary tract infections were particularly found to be associated with E. coli and Klebsiella sp. This finding is consistent with studies on invasive urinary tract infections documenting E. coli as a major pathogen among Gram-negative isolates [7, 14].

Concerning the antimicrobial susceptibility profiles (Table 2), the retrieved carbapenemase-producing isolates demonstrated the increased resistance level for penicillin as well as 1st, 3rd and 4th generation cephalosporins, sulphonamide, quinolones and aminoglycosides. The resistance rates for imipenem, meropenem and ertapenem were 41%, 40% and 36%, respectively. The progress of such resistant isolates is reflected in public health warnings.

Significant antimicrobial resistance was detected among carbapenem-resistant Gram-negative isolates compared to other MDR isolates. The resistance pattern of A. baumanii to imipenem in the current study (100%) is consistent with the reports of Boral et al. [32] but lower rates (85%, 77.5% and 61.89%) of imipenem resistance were reported by Shokri et al. [26], Shamim et al. [33] and Ain et al. [31], respectively. Acinetobacter spp. has been found to be a pathogen notorious for nosocomial infections. This is most often associated with wound infections and was the causative agent of 92% of infections associated with imipenem resistance [31]. Furthermore, the recovered carbapenemase-producing isolates were susceptible to tigecycline which affected approximately 70% of MDR isolates. These results were in agreement with the observations of Shokri et al. [26].

Of interest, a high incidence of MDR, XDR and PDR profiles was observed among all carbapenemase-producing isolates. 65 isolates of the Gram-negative bacteria exhibited different patterns of resistance including 24 (36%) MDR, 17 (26%) XDR and 16 (24%) PDR patterns (Table 3).

The remarkable prevailing resistance regarding MDR to 4 classes, XDR to 6 classes and PDR to 8 classes of tested antimicrobials was observed among E. coli, Klebsiella sp. and A. baumannii isolates, respectively. This is supported by observations in the literature [26].

The higher rate of MDR pattern among the strains of Gram-negative bacteria carrying the carbapenemase gene in the current study, may be attributed to higher selection pressure due to self-medication, empirical use and overuse of carbapenems and 3rd generation cephalosporins, and a lack of careful monitoring of resident MDR isolates in hospital settings, particularly in the surgery ward, ICU and burns ward. For these reasons, more monitoring programs are needed in this area [34].

MDR and XDR isolates of these bacteria are increasingly being reported globally, but according to available results, higher rates than those previously reported were observed. A study conducted in Tehran Hospital in 2020 found that two types of bacteria, one A. baumannii and one of the P. aeruginosa strains were PDR [26].

The development of these PDR isolates is worrying as there is practically no antibiotic to treat them, and therefore morbidity and mortality rates due to infection with these strains are expected to be high. Some studies display high death rates (between 40 and 65%) among infected patients with P. aeruginosa and Acinetobacter spp. [35].

Among the 65 carbapenemase-producing Gram-negative isolates with a positive phenotype-based result, 30 (46%), 20 (30%), and 18 (27%) isolates were positive for OXA-48, KPC and MBL enzymes, respectively, or containing two of these enzymes, as well as the production of 27% of AmpC with porin loss (Table 4).

Despite the difference in detecting carbapenemase classes using the phenotypic method when compared with the genotyping method, in general, when looking at PCR results, Carba plus can correctly identify 51/53 (96%) carbapenemase-producers. It is interesting that through the application of the phenotyping method in the current study, it was demonstrated that almost all isolates of carbapenemase-producing Gram-negative bacilli selected for the genotyping method were able to produce different types of carbapenemases by the genotype-based method. It is important to note that this study points to Carba plus as potentially offering an effective in situ method for discriminating KPC, MBLs and OXA-48 carbapenemase genes in clinical Enterobacteriaceae isolates. This is ideal for application in the basic microbiology laboratory, providing real laboratory resolutions to help in the stewardship of antibiotics [10].

We have shown that Carba plus is appropriate for the detection of carbapenemase classes, which is comparable to previous results by Ohsaki et al. [21]. Carba plus displayed a brilliant discriminatory capability of carbapenemase types among multiple and single carbapenemase producers. Identification accuracy was found to be mostly high for IMP MBL producers, and for OXA-48 producers which may be difficult to detect due to reduced MICs to carbapenems [36].

To confirm the accuracy and dependability of identification results, we used molecular approaches. Reliable identification of carbapenemase genes is essential for disease outbreak detection. Out of 53 phenotypically confirmed carbapenemase-producing Gram-negative bacilli isolates, 51/53 (96%) isolates were harbouring one or more than one gene, while in two isolates none of the genes were detected. The most common carbapenemase gene was bla _NDM 83% (44/53) followed by bla _OXA-48 75% (40/53), bla _VIM 49% (26/53) and bla _IMP 43% (23/53), while the gene bla _KPC was least frequent (4/53) (Table 5). Interestingly, bla _NDM was the most commonly detected carbapenemase and bla _OXA-48 was second, which is consistent with other reports [12, 16, 19, 37]. In addition, similar to previous analyses, the two genes bla _OXA-48 and bla _NDM were commonly detected with a high predominance of bla _OXA-48 [23, 26]. The New Delhi metallo-β-lactamase (bla _NDM-1) gene was identified in New Delhi in an XDR K. pneumoniae in 2009 [5]. The gene is currently prevalent in other Enterobacteriaceae, A. baumannii, K. oxytoca, P. aeruginosa, M. morganii, Citrobacter freundii and E. cloacae, as well as in other countries including the United States and United Kingdom. This may be because NDM enzymes are encoded on highly mobile, conjugative plasmids which ease horizontal inter and intra-species transfer between bacteria rather than clonal spread [19].

We detected carbapenemases genes in different MDR Gram-negative bacilli bacteria. The most prevalent bacteria were Klebsiella sp., A. baumanii, followed by E. coli and P. aeruginosa, which is similar to data from recent studies conducted in other countries [4, 23, 26, 29, 38]. Comparable to previous reports [14, 38], the KPC gene was the least detected among carbapenemase-producing bacteria, even in some studies none of bla _KPC was detected [28, 37]. A probable explanation for the low detection rate of the KPC gene among carbapenemase-producing isolates might be that these isolates contain other class A carbapenemases (other than KPC), for example GES, Sme, NMC-A and IMI. This is backed up by reports elsewhere [39].

This result was in agreement with the observations of Mahrach et al. that the bla _OXA-48 gene was detected in 75% of isolates [29]. According to the study by El-Badawi et al. the bla _OXA-48 gene was detected in 73.68% of carbapenemase-producing isolates [39].

Finding OXA-48 producers is a major and worrisome issue. We observed a high frequency of infection with OXA-48 carbapenemase-producing isolates, the majority of OXA-48-producing isolates were A. baumannii, which is comparable to what was reported in a study by Bourafa et al. [4]. However, in contrast to our study, K. pneumoniae were the main OXA-48-producing isolates in other studies [12, 19].

Understanding the contribution of other factors, such as the production of ESBLs or defects in cell wall permeability, is necessary to increase the level of resistance to carbapenems and cephalosporins. In other studies, a close correlation was demonstrated between the expression of ESBLs (SHV and CTX-M-15) and OXA-48 enzyme production [31, 34]. Co-expression of AmpC enzymes and OXA-48 in carbapenem-resistant Enterobacteriaceae has previously been shown [19]. These results confirm the existence of other resistance mechanisms in our isolates.

OXA-48 is widely spread all over the world and has been a source of hospital infections globally. Of late, OXA-48 has been documented in a number of the Mediterranean nation-states as an epidemic, including in Turkey, Iran, Morocco, Lebanon and Tunisia [29].

This study revealed a high prevalence of MBL genes (bla _VIM, bla _IMP, and bla _NDM), 50/53 (94%) of carbapenemase-producing isolates (Table 5) and all the three genes assayed were detected in the study samples. Based on the data from this study, there is a rapid emergence of MBL-producing isolates in some Erbil city hospitals. It appears that a similar pattern of distribution of the bla _NDM gene may exist in other countries of the world [26, 31, 37, 38].

It is interesting that not all isolates producing carbapenemase are resistant to carbapenem. This is because carbapenemase production always raises the MICs of carbapenems, but may not be sufficiently high to be classified as resistant or intermediate resistant [20].

We observed a large number of isolates 92% (46/51) that were involved in the production of more than one carbapenemase gene (Table 5). The co-harbouring of carbapenemase isolates has been detected in several studies [19, 34]. It is worth noting that the coexistence of these carbapenemase genes is a therapeutic challenge to clinicians, due to restricted treatment options and the potential for global spread by horizontal transfer [40]. Interestingly, some isolates were found to contain two unrelated carbapenemase genes (bla _OXA-48 and bla _NDM) as previously observed in other countries [4, 5, 12, 38].

In the present work, isolates carrying 2 to 4 carbapenemase genes were commonly detected, especially among Klebsiella sp. and A. baumannii. This indicates that these microorganisms were able to accumulate numerous pan drug or multidrug-resistant determinants [34]. These isolates have already been described in other reports [5, 31, 34, 37].

Single isolates harbouring multiple genes have been described and identified as a key threat to antibacterial chemotherapy [34]. In accordance with the observations of Aruhomukama et al., Okoche et al., and Sadeghi et al. [8, 34, 38], the most important combinations were observed among K. pneumoniae isolates frequently carrying multiple and broad-spectrum beta-lactamases.

Conclusions

The results of our study illustrate the emergence of carbapenemase producing Gram-negative bacilli isolates from patients at the referral hospitals in the Kurdistan Region of Iraq. The most common carbapenem-resistant bacteria that have been identified as responsible in healthcare-related infections are A. baumannii, K. pneumoniae and E. coli. The recovered clinical isolates demonstrated noteworthy multidrug resistance to penicillins, cephalosporins, sulphonamides, quinolones and aminoglycosides, which are considered a threat to public health. Retrieved isolates of carbapenemase-producing Gram-negative bacilli were markedly MDR, XDR or PDR to many classes of antimicrobials, a warning signal indicating complex therapy for diseases caused by these resistant microorganisms. Tigecycline showed promising antimicrobial activity against MDR, XDR and PDR Gram-negative bacilli. Carbapenem resistance by phenotypic detection is usually associated with production of classes D, A and B carbapenemases as well as AmpC production with porin loss.

Moreover, all carbapenem-resistant genes (bla _OXA, bla _IMP, bla _NDM, bla _VIM and bla _KPC) were identified in the study specimen. The most prevalent gene was bla _NDM and the lowest was bla _KPC.

The combination of phenotypic and genotypic detection of carbapenem-resistant isolates is an effective and reliable epidemiological process. Therefore, this study recommends continuous monitoring of antimicrobial susceptibility testing to detect multidrug-resistant isolates along with limited and appropriate use of antibiotics in hygienic practices. Accurate identification of carbapenem-resistant bacterial pathogens is essential in treating patients, as well as advancing appropriate contamination control measures to curb the rapid spread of these strains.

Supporting information

S1 Fig

(PDF)

Click here for additional data file.^{(243.9KB, pdf)}

S1 Table. Characteristic and antibiogram profile of 65 carbapenemase-producing Gram-negative bacilli.

(XLSX)

Click here for additional data file.^{(17.1KB, xlsx)}

S2 Table. The number of bacteria positive for carbapenemase genes and number of genes per organism n = 51.

(XLSX)

Click here for additional data file.^{(11.6KB, xlsx)}

Acknowledgments

The authors would like to thank the management and staff at the Research Centre for their assistance.

Data Availability

The minimal dataset underlying this study is available on Mendeley (https://data.mendeley.com/datasets/dp7g8rntyb/1).

Funding Statement

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

References

1.Algammal A.M., et al., atp D gene sequencing, multidrug resistance traits, virulence-determinants, and antimicrobial resistance genes of emerging XDR and MDR-Proteus mirabilis. Scientific reports, 2021. 11(1): p. 1–15. 10.1038/s41598-021-88861-w [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Algammal A.M., et al., Emerging MDR-Pseudomonas aeruginosa in fish commonly harbor opr L and tox A virulence genes and bla TEM, bla CTX-M, and tet A antibiotic-resistance genes. Scientific Reports, 2020. 10(1): p. 1–12. 10.1038/s41598-020-72264-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Algammal A.M., et al., Methicillin-Resistant Staphylococcus aureus (MRSA): one health perspective approach to the bacterium epidemiology, virulence factors, antibiotic-resistance, and zoonotic impact. Infection and Drug Resistance, 2020. 13: p. 3255. doi: 10.2147/IDR.S272733 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Bourafa N., et al., Molecular characterization of carbapenem-resistant Gram-negative bacilli clinical isolates in Algeria. Infection and drug resistance, 2018. 11: p. 735. doi: 10.2147/IDR.S150005 [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Karuniawati A., Saharman Y.R., and Lestari D.C., Detection of carbapenemase encoding genes in Enterobacteriace, Pseudomonas aeruginosa, and Acinetobacter baumanii isolated from patients at Intensive Care Unit Cipto Mangunkusumo Hospital in 2011. Acta Med Indones, 2013. 45(2): p. 101–6. . [PubMed] [Google Scholar]
6.McKibben L., et al., Guidance on public reporting of healthcare-associated infections: recommendations of the Healthcare Infection Control Practices Advisory Committee. American journal of infection control, 2005. 33(4): p. 217–226. 10.1086/502585 [DOI] [PubMed] [Google Scholar]
7.Jalalvand K., et al., Evaluation of phenotypic and genotypic characteristics of carbapnemases-producing enterobacteriaceae and its prevalence in a referral hospital in Tehran city. Iranian journal of pathology, 2020. 15(2): p. 86. doi: 10.30699/ijp.2020.111181.2188 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Aruhomukama D., et al., Bla VIM-and bla OXA-mediated carbapenem resistance among Acinetobacter baumannii and Pseudomonas aeruginosa isolates from the Mulago hospital intensive care unit in Kampala, Uganda. BMC infectious diseases, 2019. 19(1): p. 1–8. 10.1186/s12879-019-4510-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Shaker O., et al., Evaluation of Combined Use of Temocillin Disk and Mastdisks Inhibitor Combination Set Against Polymerase Chain Reaction for Detection of Carbapenem-Resistant Enterobacteriaceae. Open Access Maced J Med Sci. 2018. Feb 15; 6 (2): 242–247. 2018. doi: 10.3889/oamjms.2018.090 [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Hu W., et al., Evaluation of MASTDISCS combi Carba plus for the identification of metallo-β-lactamases, KPC and OXA-48 carbapenemase genes in Enterobacteriaceae clinical isolates. Letters in applied microbiology, 2020. 70(1): p. 42–47. 10.1111/lam.13240 [DOI] [PubMed] [Google Scholar]
11.Peleg A.Y. and Hooper D.C., Hospital-acquired infections due to gram-negative bacteria. New England Journal of Medicine, 2010. 362(19): p. 1804–1813. doi: 10.1056/NEJMra0904124 [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Codjoe, F.S., Detection and characterisation of carbapenem-resistant gram-negative bacilli infections in Ghana. 2016, Sheffield Hallam University. http://shura.shu.ac.uk/id/eprint/15577
13.Walsh F., The multiple roles of antibiotics and antibiotic resistance in nature. Frontiers in microbiology, 2013. 4: p. 255. 10.3389/fmicb.2013.00255 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Deldar Abad Paskeh M., Mehdipour Moghaddam M.J., and Salehi Z., Prevalence of plasmid-encoded carbapenemases in multi-drug resistant Escherichia coli from patients with urinary tract infection in northern Iran. Iranian Journal of Basic Medical Sciences, 2020. 23(5): p. 586–593. http://ijbms.mums.ac.ir/article_14823.html doi: 10.22038/ijbms.2020.34563.8199 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Sheikh A.F., et al., Emerge of NDM-1-producing multidrug-resistant pseudomonas aeruginosa and co-harboring of carbapenemase genes in South of Iran. Iranian Journal of Public Health, 2020. 49(5): p. 959. [PMC free article] [PubMed] [Google Scholar]
16.Braun S.D., et al., Prevalence of carbapenemase-producing organisms at the Kidney Center of Rawalpindi (Pakistan) and evaluation of an advanced molecular microarray-based carbapenemase assay. Future microbiology, 2018. 13(11): p. 1225–1246. 10.2217/fmb-2018-0082 [DOI] [PubMed] [Google Scholar]
17.Han R., et al., Dissemination of Carbapenemases (KPC, NDM, OXA-48, IMP, and VIM) among Carbapenem-Resistant Enterobacteriaceae isolated from adult and children patients in China. Frontiers in Cellular and Infection Microbiology, 2020. 10: p. 314. 10.3389/fcimb.2020.00314 [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Workneh M., Yee R., and Simner P.J., Phenotypic methods for detection of carbapenemase production in carbapenem-resistant organisms: what method should your laboratory choose? Clinical microbiology newsletter, 2019. 41(2): p. 11–22. 10.1016/j.clinmicnews.2019.01.001 [Google Scholar]
19.Karabay O., et al., The carbapenem-resistant Enterobacteriaceae threat is growing: NDM-1 epidemic at a training hospital in Turkey. Annals of clinical microbiology and antimicrobials, 2016. 15(1): p. 1–6. 10.1186/s12941-016-0118-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Gong X., et al., Molecular characterization and epidemiology of carbapenem non-susceptible Enterobacteriaceae isolated from the Eastern region of Heilongjiang Province, China. BMC infectious diseases, 2018. 18(1): p. 1–10. 10.1186/s12879-018-3294-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Ohsaki Y., et al., MASTDISCS combi Carba plus, a simple method for discriminating carbapenemase-producing Enterobacteriaceae, including OXA-48-type producers. Microbiology and immunology, 2018. 62(1): p. 60–65. 10.1111/1348-0421.12553 [DOI] [PubMed] [Google Scholar]
22.Algammal A.M., et al., Prevalence, the antibiogram and the frequency of virulence genes of the most predominant bacterial pathogens incriminated in calf pneumonia. AMB Express, 2020. 10(1): p. 1–8. 10.1186/s13568-020-01037-z [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Kollenda H., et al., Screening for carbapenemases in ertapenem-resistant Enterobacteriaceae collected at a Tunisian hospital between 2014 and 2018. European Journal of Microbiology and Immunology, 2019. 9(1): p. 9–13. 10.1556/1886.2018.00033 [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Dandachi I., et al., Carriage of beta-lactamase-producing Enterobacteriaceae among nursing home residents in north Lebanon. International Journal of Infectious Diseases, 2016. 45: p. 24–31. 10.1016/j.ijid.2016.02.007 [DOI] [PubMed] [Google Scholar]
25.Poirel L., et al., Multiplex PCR for detection of acquired carbapenemase genes. Diagnostic microbiology and infectious disease, 2011. 70(1): p. 119–123. 10.1016/j.diagmicrobio.2010.12.002 [DOI] [PubMed] [Google Scholar]
26.Shokri D., et al., Resistotyping, phenotyping and genotyping of New Delhi metallo-β-lactamase (NDM) among Gram-negative bacilli from Iranian patients. Journal of medical microbiology, 2017. 66(4): p. 402–411. 10.1099/jmm.0.000444 [DOI] [PubMed] [Google Scholar]
27.Jin C., et al., Molecular Characteristics of Carbapenem-Resistant Enterobacter cloacae in a Tertiary Hospital in China. Infection and drug resistance, 2020. 13: p. 1575. doi: 10.2147/IDR.S254056 [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Rimrang B., et al., Emergence of NDM-1-and IMP-14a-producing Enterobacteriaceae in Thailand. Journal of antimicrobial chemotherapy, 2012. 67(11): p. 2626–2630. 10.1093/jac/dks267 [DOI] [PubMed] [Google Scholar]
29.Mahrach Y., et al., Phenotypic and molecular study of carbapenemase-producing Enterobacteriaceae in a regional hospital in northern Morocco. J Clin Med Sci, 2019. 3: p. 113. [Google Scholar]
30.Makharita R.R., et al., Antibiogram and genetic characterization of carbapenem-resistant gram-negative pathogens incriminated in healthcare-associated infections. Infection and drug resistance, 2020. 13: p. 3991. doi: 10.2147/IDR.S276975 [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Ain N.U., et al., High frequency and molecular epidemiology of metallo-β-lactamase-producing gram-negative bacilli in a tertiary care hospital in Lahore, Pakistan. Antimicrobial Resistance & Infection Control, 2018. 7(1): p. 1–9. 10.1186/s13756-018-0417-y [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Boral B., et al., A prospective multicenter study on the evaluation of antimicrobial resistance and molecular epidemiology of multidrug-resistant Acinetobacter baumannii infections in intensive care units with clinical and environmental features. Annals of clinical microbiology and antimicrobials, 2019. 18(1): p. 1–9. 10.1186/s12941-019-0319-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Shamim S., Abbas M., and Qazi M.H., Prevalence of multidrug resistant Acinetobacter baumannii in hospitalized patients in Lahore, Pakistan. Pakistan J Mol Med, 2015. 2(1): p. 23–8. www.pjmm.uol.edu.pk [Google Scholar]
34.Sadeghi M.R., et al., Molecular characterization of extended-spectrum β-lactamase, plasmid-mediated AmpC cephalosporinase and carbapenemase genes among Enterobacteriaceae isolates in five medical centres of East and West Azerbaijan, Iran. Journal of medical microbiology, 2016. 65(11): p. 1322–1331. 10.1099/jmm.0.000356 [DOI] [PubMed] [Google Scholar]
35.Leão A.C., et al., Acinetobacter spp. are associated with a higher mortality in intensive care patients with bacteremia: a survival analysis. BMC infectious diseases, 2016. 16(1): p. 1–8. 10.1186/s12879-016-1695-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Nagano N., et al., First report of OXA-48 carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in Japan from a patient returned from Southeast Asia. Japanese journal of infectious diseases, 2013. 66(1): p. 79–81. 10.7883/yoken.66.79 [DOI] [PubMed] [Google Scholar]
37.Masseron A., et al., Molecular characterization of multidrug-resistance in Gram-negative bacteria from the Peshawar teaching hospital, Pakistan. New microbes and new infections, 2019. 32: p. 100605. 10.1016/j.nmni.2019.100605 [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Okoche D., et al., Prevalence and characterization of carbapenem-resistant Enterobacteriaceae isolated from Mulago National Referral Hospital, Uganda. PLoS One, 2015. 10(8): p. e0135745. 10.1371/journal.pone.0135745 [DOI] [PMC free article] [PubMed] [Google Scholar]
39.El-Badawy M.F., et al., The First Egyptian Report Showing the Co-Existence of blaNDM-25, blaOXA-23, blaOXA-181, and blaGES-1 Among Carbapenem-Resistant K. pneumoniae Clinical Isolates Genotyped by BOX-PCR. Infection and Drug Resistance, 2020. 13: p. 1237. doi: 10.2147/IDR.S244064 [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Solgi H., et al., Molecular characterization of intestinal carriage of carbapenem-resistant Enterobacteriaceae among inpatients at two Iranian university hospitals: first report of co-production of bla NDM-7 and bla OXA-48. European Journal of Clinical Microbiology & Infectious Diseases, 2017. 36(11): p. 2127–2135. 10.1007/s10096-017-3035-3 [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0259005.r001

Decision Letter 0

Abdelazeem Mohamed Algammal

23 Jul 2021

PONE-D-21-22590

Evaluation of carbapenemase-producing clinical isolates of Gram-negative bacilli using phenotypic and genotypic methods at hospitals in Erbil city, Kurdistan region of Iraq

PLOS ONE

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Reviewer #1: Comments to authors:

- The current study is very interesting; however, the authors should address the following comments to improve the quality of the manuscript:

- The manuscript should be revised for language editing and grammar mistakes by a native English speaker.

- Please write the genes name in correct form all over the manuscript (the 1st 3 letters must be small and italic and the other 3 letter are capital and subscripted)

Title:

I think the work would benefit from the title that contains main conclusion of the study (should be derived from the conclusion), please modify the title.

Abstract:

- The abstract must illustrates the used methods and the most prevalent results (give more hints about methods and results). Besides, rephrase the main conclusion of your findings.

Introduction:

-Give a hint about different infections caused by common pathogenic members of Enterobacteriaceae (such as E. coli and K. pneumonia) and Pseudomonas aeruginosa, their virulence factors, and the mechanism of disease occurrence.

- The introduction needs to be more informative:

-The authors should illustrate the public heath importance concerning the emergence of multidrug-resistant (MDR) bacterial pathogens that reflecting the necessary of new potent and safe antimicrobial agents. Several studies proved the widespread MDR- bacterial pathogens;

Authors could add the following paragraph:

Multidrug resistance has been increased globally that is considered public health threat. Several recent studies reported the existence of multidrug-resistant bacterial pathogens from different origins including humans, poultry, cattle, and fish that increase the need for routine application of the antimicrobial susceptibility testing to detect the antibiotic of choice as well as screening of the emerging MDR strains. You should cite the following valuable studies:

1-PMID: 33177849

2-PMID: 32497922

3-PMID:33061472

4-PMID: 33947875

5-PMID: 32472209

6-PMID: 32994450

-Rephrase the aim of work to be clear and better sound.

Material and methods

-Table 1 must be placed in the results section.

-It is not acceptable to illustrate the prevalence of the bacterial pathogens in the methods; it should be placed to the results section.

-Where are the methods of the isolation and identification of 200 Gram-negative bacteria?? You must illustrate in details and support your methods with specific references.

- Phenotype-based method for carbapenemases: should be modified to be: Phenotypedetection carbapenemases using Disc diffusion method. Rephrase this section; add more details, discuss in details and specific references.

- Confirmation of carbapenemase production using commercial

combination disk assay; add specific references to this section.

-Molecular detection of genes should be modified:

PCR-based detection of carbapenemase genes. Besides, add specific references.

-Where are the statistical analyses?

-Results:

- Where are the results of phenotypic characters and the prevalence of the isolated bacterial pathogens from the examined samples? You must illustrate their prevalence in a new table.

-Table 1 must be placed in the results section.

- Illustrate in a new table the test results of the phenotypic MDR (illustrate the antimicrobial classes of the used antimicrobial agents).

-Illustrate the distribution of the carbapenemase genes among the recovered isolates in a new table.

- Please increase the resolution of all figures (1200 dpi).

-Where are the statistical analyses?

-Discussion:

-The discussion is too long and poor; the authors are advised to illustrate the real impact of their findings without repetition of results.

-Conclusion

- Should be rephrased to be sounded. A real conclusion should focus on the question or claim you articulated in your study, whose resolution has been the main objective of your paper?

Reviewer #2: - The current study has a significant impact, but it needs a major revision:

- The manuscript should be revised for grammar mistakes.

-The title is broad, please modify the title.

-In the introduction: discuss the public health importance of the members of Enterobacteriaceae and other Gram negative bacterial pathogens.

-Improve the aim of work.

Methods:

-Transfer Table 1 to the results.

-Transfer the prevalence of bacterial species to the results section.

-Discuss the isolation and identification of the bacterial pathogens.

-Specific references should be added to all the used methods and techniques.

-Where is the statistical analysis?

-Results:

- Discuss the prevalence of the retrieved isolates in a new table.

- Transfer Table 1 to the results

- Please increase the resolution of all figures (1200 dpi).

-Where is the statistical analysis?

-Discussion:

-Very long; please improve

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2021 Nov 1;16(11):e0259005. doi: 10.1371/journal.pone.0259005.r002

Author response to Decision Letter 0

4 Sep 2021

Dear Dr. Abdelazeem

Thank you for reviewing my manuscript. I appreciate your efforts and the efforts of the reviewers.

I have worked hard to edit the revised manuscript and I hope I have done well

I look forward to having my research accepted in PLOS ONE journal.

Sayran H. Haji

Author

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When submitting your revision, we need you to address these additional requirements.

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I tried to be in accordance with the requirements of PLOS ONE style

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2. In your Methods section, please provide additional information on the verbal consent provided by the patients, including as to how this verbal consent was documented or witnessed.

I added regarding oral consent in the ethical issues section

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My cover letter contains the DOI of the original, uncropped, unedited images of the blot /gel data published in a public data repository.

https://data.mendeley.com/datasets/fhnkrf3jdr/1

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Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

The minimal data set is available in public repository and include the relevant DOIs, within my revised cover letter.

Dataset: https://data.mendeley.com/datasets/dp7g8rntyb/1

I don’t have any ethical or legal restrictions

We will update your Data Availability statement to reflect the information you provide in your cover letter.

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5. Please ensure that you refer to Figure 1 in your text as, if accepted, production will need this reference to link the reader to the figure.

I referenced all the Figures in my text to link the reader to the figure.

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I referenced all the tables in my script to connect the reader to the figure.

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Reviewer #1: Comments to authors:

- The current study is very interesting; however, the authors should address the following comments to improve the quality of the manuscript:

- The manuscript should be revised for language editing and grammar mistakes by a native English speaker.

I have checked for language editing and grammatical errors by native British speakers using online proofreading;

Professional editing and proofreading

services at your fingertips

https://app.proofreadmyessay.co.uk/my-documents/

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- Please write the genes name in correct form all over the manuscript (the 1st 3 letters must be small and italic and the other 3 letter are capital and subscripted)

I corrected the name of the genes throughout the manuscript

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Title:

I think the work would benefit from the title that contains main conclusion of the study (should be derived from the conclusion), please modify the title.

I modified the title that contains main conclusion

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Abstract:

- The abstract must illustrates the used methods and the most prevalent results (give more hints about methods and results). Besides, rephrase the main conclusion of your findings.

More hints were given about the methods and results. Besides, I reformulated the main conclusion of the results.

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Introduction:

Hints were given about the common pathogenic members of Enterobacteriaceae in the introduction part

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- The introduction needs to be more informative:

Authors could add the following paragraph:

1-PMID: 33177849

2-PMID: 32497922

3-PMID:33061472

4-PMID: 33947875

5-PMID: 32472209

6-PMID: 32994450

I have illustrated the public health importance of the emergence of multidrug-resistant (MDR) bacterial pathogens by adding your paragraph, and I really appreciate your help in providing these valuable studies, which I used as references.

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-Rephrase the aim of work to be clear and better sound.

I paraphrased the aim of the work

Material and methods

-Table 1 must be placed in the results section.

I have moved the table to the resulting part

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-It is not acceptable to illustrate the prevalence of the bacterial pathogens in the methods; it should be placed to the results section. I have moved the prevalence of bacterial pathogens to the results section.

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-Where are the methods of the isolation and identification of 200 Gram-negative bacteria?? You must illustrate in details and support your methods with specific references.

The methods for isolating and identifying 200 Gram-negative bacteria have been explained with adding specific references. Furthermore, I added a new table to identify 200 Gram-negative bacteria.

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I have modified the phenotypic-based method for carbapenemes to the phenotype detection of carbapenemes using the disc diffusion method, and more details have been added to this section with references.

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- Confirmation of carbapenemase production using commercial

combination disk assay; add specific references to this section.

The phenotypic-based method has been combined with confirmation of carbapenemase production using commercial combination disk checked, because I think it was duplicated, and specific references were added

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-Molecular detection of genes should be modified:

PCR-based detection of carbapenemase genes. Besides, add specific references.

Molecular gene detection was changed to PCR-based detection of carbapenemase genes. Besides, specific references have been added.

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-Where are the statistical analyses?

Statistical analyzes for this study were designed using GraphPad Prism (version 5; GraphPad Software, San Diego, CA). The Chi-square test was applied to analyze the obtained data.

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-Results:

- Where are the results of phenotypic characters and the prevalence of the isolated bacterial pathogens from the examined samples? You must illustrate their prevalence in a new table.

The results of the phenotypic characteristics and prevalence of bacterial pathogens isolated from the examined samples were added to a new table with a clear explanation of the correlation in the text. ------------------------------------------------------------------------------------

-Table 1 must be placed in the results section.

Table 1 has been converted to Table 2 and moved to the result section.

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- Illustrate in a new table the test results of the phenotypic MDR (illustrate the antimicrobial classes of the used antimicrobial agents).

I have developed a new table of MDR phenotypic test results identifying the antimicrobial classes of the antimicrobial agents used.

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-Illustrate the distribution of the carbapenemase genes among the recovered isolates in a new table.

The distribution of carbapenemase genes among the recovered isolates is illustrated in a new table

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- Please increase the resolution of all figures (1200 dpi).

I have increased the resolution. Hope it is clear now

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-Where are the statistical analyses?

Statistical analyzes for this study were designed using GraphPad Prism (version 5; GraphPad Software, San Diego, CA). The Chi-square test was applied to analyze the obtained data.

------------------------------------------------------------------------------------

-Discussion:

-The discussion is too long and poor; the authors are advised to illustrate the real impact of their findings without repetition of results.

I've shortened the discussion and deleted all the duplicates as much as I can

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-Conclusion

- Should be rephrased to be sounded. A real conclusion should focus on the question or claim you articulated in your study, whose resolution has been the main objective of your paper?

The conclusion has been reformulated to be presented and includes the main resolution of my study.

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Reviewer #2: - The current study has a significant impact, but it needs a major revision:

- The manuscript should be revised for grammar mistakes.

I have checked for language editing and grammatical errors by native British speakers using online proofreading;

Professional editing and proofreading

services at your fingertips

https://app.proofreadmyessay.co.uk/my-documents/

------------------------------------------------------------------------------------

-The title is broad, please modify the title.

I have modified and shortened the title

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-In the introduction: discuss the public health importance of the members of Enterobacteriaceae and other Gram-negative bacterial pathogens.

The public health importance of the members of Enterobacteriaceae and other Gram-negative bacterial pathogens was discussed in the introduction part

------------------------------------------------------------------------------------

-Improve the aim of work.

The purpose of the work has improved

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Methods:

-Transfer Table 1 to the results.

I have moved the table to the resulting part

------------------------------------------------------------------------------------

-Transfer the prevalence of bacterial species to the results section.

I have moved the prevalence of bacterial pathogens to the results section.

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-Discuss the isolation and identification of the bacterial pathogens.

The methods for isolating and identifying Gram-negative bacteria have been explained with adding specific references. Furthermore, I added a new table to identify 200 Gram-negative bacteria.

------------------------------------------------------------------------------------

-Specific references should be added to all the used methods and techniques.

I added specific references to all the methods and techniques used

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-Where is the statistical analysis?

Statistical analyzes for this study were designed using GraphPad Prism (version 5; GraphPad Software, San Diego, CA). The Chi-square test was applied to analyze the obtained data.

------------------------------------------------------------------------------------

-Results:

- Discuss the prevalence of the retrieved isolates in a new table.

The results of the prevalence of bacterial pathogens isolated from the examined samples were added to a new table with a clear explanation in the text.

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- Transfer Table 1 to the results

Table 1 has been converted to Table 2 and moved to the result section.

------------------------------------------------------------------------------------

- Please increase the resolution of all figures (1200 dpi).

I have increased the resolution. Hope it is clear now

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-Discussion:

-Very long; please improve

I've shortened and improved the discussion as much as I can________________________________________

Attachment

Submitted filename: Response to Reviewers30.08.docx

Click here for additional data file.^{(26KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0259005.r003

Decision Letter 1

Abdelazeem Mohamed Algammal

7 Sep 2021

PONE-D-21-22590R1Prevalence and characterisation of carbapenemase encoding genes in multidrug-resistant Gram-negative bacilliPLOS ONE

Dear Dr. haji,

Please submit your revised manuscript by Oct 22 2021 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'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Abdelazeem Mohamed Algammal, Prof, Ph.D

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)

**********

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

Reviewer #1: Yes

**********

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

Reviewer #1: Yes

**********

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

Reviewer #1: Yes

**********

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

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors have addressed some of my comments; however the have

ignored a major comment concerning the introduction.

Please address the following comment:

The introduction needs to be more informative:

-The authors should illustrate the public heath importance concerning the emergence

of multidrug-resistant (MDR) bacterial pathogens that reflecting the necessary of new

potent and safe antimicrobial agents. Several studies proved the widespread MDRbacterial pathogens;

Authors could add the following paragraph:

Multidrug resistance has been increased globally that is considered public health

threat. Several recent studies reported the existence of multidrug-resistant bacterial

pathogens from different origins including humans, poultry, cattle, and fish that

increase the need for routine application of the antimicrobial susceptibility testing to

detect the antibiotic of choice as well as screening of the emerging MDR strains. You

should cite the following valuable studies:

1-PMID: 33177849

2-PMID: 32497922

3-PMID:33061472

4-PMID: 33947875

5-PMID: 32472209

6-PMID: 32994450

**********

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

PLoS One. 2021 Nov 1;16(11):e0259005. doi: 10.1371/journal.pone.0259005.r004

Author response to Decision Letter 1

11 Sep 2021