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. 2022 Nov 17;10(11):2283. doi: 10.3390/microorganisms10112283

Characterization of Genomic Diversity among Carbapenem-Resistant Escherichia coli Clinical Isolates and Antibacterial Efficacy of Silver Nanoparticles from Pakistan

Aamir Jamal Gondal 1, Nakhshab Choudhry 2, Hina Bukhari 3, Zainab Rizvi 4, Nighat Yasmin 1,*
Editor: Chang-Ro Lee
PMCID: PMC9699514  PMID: 36422353

Abstract

The emergence of carbapenem-resistant Escherichia coli (E. coli) is considered an important threat to public health resulting in resistance accumulation due to antibiotics misuse and selection pressure. This warrants periodic efforts to investigate and develop strategies for infection control. A total of 184 carbapenem-resistant clinical strains of E. coli were characterized for resistance pattern, resistance genes, plasmids, sequence types and in vitro efficacy of silver nanoparticles (AgNPs). Carbapenem resistance was prevalent in E. coli isolated from female patients (64.7%), urine samples (40.8%) and surgical wards (32.1%). Polymyxin-B showed higher susceptibility. ESBLs and carbapenemases were produced in 179 and 119 isolates, respectively. Carbapenemase-encoding genes were observed among 104 strains with blaNDM-1 (45.1%), blaOXA-48 (27%), blaNDM-7 (3.8%), blaNDM-1/blaOXA-48 (15.4%), blaNDM-7/blaOXA-48 (2.9%), blaOXA-48/blaVIM (3.8%) and blaNDM-1/blaVIM (2%). ESBL resistance genes were detected in 147 isolates, namely blaSHV (24.9%), blaCTX-M (17.7%), blaTEM (4.8%), blaSHV/blaCTX-M (29.2%), blaSHV/blaTEM (15%) and blaCTX-M/blaTEM (8.8%). ST405 (44.4%) and ST131 (29.2%) were more frequent sequence types with ST101 (9.7%), ST10 (9.7%) and ST648 (7%). The replicon types IncFII, IncFIIK, IncA/C, IncN and IncL/M were detected. The combination of MEM/AgNPs remained effective against carbapenemase-positive E. coli. We reported genetically diverse E. coli strains coharboring carbapenemases/ESBLs from Pakistan. Moreover, this study highlights the enhanced antibacterial activity of MEM/AgNPs and may be used to manage bacterial infections.

Keywords: Escherichia coli (E. coli), ST405, ST131, carbapenemases, silver nanoparticles

1. Introduction

The repeated exposure of bacterial species to antibiotics results in selection pressure ultimately modifying their antimicrobial physiology. Carbapenem resistance has been accumulated among the Enterobacteriaceae (CRE) due to the significant spread of core carbapenemase-encoding genes present on plasmids or mobile genetic elements such as integrons and transposons. Therefore, carbapenemases presented a stable and transferable form of resistance due to carbapenem hydrolysis and geographic spread such as blaNDM, blaOXA-48, blaVIM, blaIMP and blaKPC [1,2]. Since the identification of blaNDM, its spread has been commonly observed among Enterobacteriaceae, especially from Asian territories such as Pakistan, India and China. blaIMP-carrying CRE were predominantly detected in Taiwan and Japan with worldwide sporadic reports. Greece was identified as a center of blaVIM-positive CRE leading to the outbreaks in Europe and China. On the other hand, the eruption of blaKPC-producing CRE was recorded in the USA, Europe and China, while Turkey, Europe and the Mediterranean region are the core places for OXA-48-producing CRE [3,4]. This geographical distribution of carbapenemases showed that CRE members have mastered the art of hiding through interchangeable resistance features responsible for the dispersion of carbapenem resistance. Common CRE pathogens responsible for amplified resistance spread through the distribution of carbapenemases, including E. coli, K. pneumoniae and Enterobacter spp. [1,2]. Therefore, the detection and pursuing of such pathogens has been recommended as a critical priority by the CDC and WHO [4].

E. coli is a part of normal human gut microbiota; however, several supremely adapted E. coli clones with multiple resistant genes were shown to accustom the new niches, thus causing far-ranging diseases such as urinary/respiratory tract infections, intestinal infections and sepsis [5,6]. Most studies reported antimicrobial resistance among E. coli in the non-clinical settings from Pakistan such as blaOXA-1, blaCTX-M15 and blaTEM in chicken meat [7], ST10 with mcr-1 and blaTEM in cattle farm environment [8] and blaNDM, blaOXA-48, blaTEM and blaSHV from sewage water [9]. However, there are few reports regarding high-risk clones of E. coli with multiple resistance genes in hospital-acquired infections from Pakistan, such as ST131, which has been linked to blaKPC-2, while ST1196 has been linked to blaNDM-1 [10].

The efficacy of the currently available antibiotics has reduced due to the emergence of resistant bacterial clones, forcing the search for other methods to combat such dangerous clones. In this regard, nanoparticles are attractive candidates due to their stability and biocompatibility, as evidenced by widespread antimicrobial, industrial and biomedical applications [11]. It was shown that nanoparticles, in combination with antibiotics, enhanced the antimicrobial efficiency against resistant microorganisms [12]. A powerful antibacterial response has been observed when myco-synthesized AgNPs in combination with imipenem were used against imipenem-resistant K. pneumoniae isolates. The MIC values of imipenem were reduced for imipenem-resistant K. pneumoniae strains with an FIC index of 0.07 [13]. Biologically synthesized AgNPs from plant extracts have been used with tetracycline against S. aureus and K. pneumoniae, showing significantly increased activity [14]. Another study reported the combined elevated effect of AgNPs with kanamycin [15] and colistin against MDR pathogens [16]. Therefore, the use of AgNPs may be considered as one of the useful therapeutic strategies for treating microbial infections and the reversal of bacterial resistance.

Globally widespread carbapenem-resistant E. coli strains necessitate novel approaches to stop the spread of these dangerous infections. Therefore, continuous surveillance studies and improved antimicrobial usage plans are required so that the proper measures can be adopted to overcome the spread of high-risk clones. The distribution of carbapenemases among Pakistan E. coli is, however, only partially and sparsely studied [17]. Therefore, constant surveillance investigations are required in terms of genotyping, plasmid and sequence typing to address the resistance situation in this country. Keeping this in view, this study aims to investigate the burden of carbapenem resistance, genomic diversity and efficacy of antimicrobials with silver nanoparticles among E. coli clinical isolates from Pakistan, so that applicable strategies can be devised for appropriate infection control.

2. Materials and Methods

A total of 184 carbapenem-resistant clinical strains of E. coli were collected during May-2019 till August-2020 from routine diagnostic facility of Mayo hospital, Lahore, Pakistan. Clinical samples were collected from different hospital sections such as surgery (59/184, 32.1%), nephrology (36/184, 19.6%), medicine (27/184, 14.7%), urology (23/184, 12.5%), ICU (9/184, 4.9%), pediatric medicine (9/184, 4.9%), chest medicine (8/184, 4.3%), cardiology (7/184, 3.8%) and oncology (6/184, 3.3%). Sample types included urine (75/184, 40.8%), wounds (28/184, 15.2%), pus (27/184, 14.7%), pleural fluids (17/184, 9.2%), tip cells (17/192, 9.2%), blood (13/184, 7.1%), sputum (4/184, 2.2%) and tissue (3/184, 1.6%). The clinical samples were cultured on MacConkey agar (Oxoid Ltd., Basingstoke, UK) while cysteine lactose electrolyte-deficient (CLED) media (Oxoid Ltd., Basingstoke, UK) were used for urine samples. CHROMagarTM ESBL media (CHROMagar, Paris, France) were used to identify ESBL producer strains. The bacterial cultures were characterized by Gram’s staining and API-20E (BioMerieux, Marcy-l’Étoile, France).

2.1. Determination of Antibiotic Susceptibility Pattern

Antibiotic susceptibility testing was conducted by using the standard disc diffusion method as per CLSI instructions [18]. The antimicrobial discs meropenem (MEM; 10 µg), ertapenem (ETP; 10 µg), imipenem (IMP; 10 µg), cefzolin (CFZ; 30 µg), ceftaroline (CPT; 30 µg), cefotaxime (CTX; 30 µg), ceftazidime (CAZ; 30 µg), amikacin (AK; 10 µg), ciprofloxacin (CIP; 5 µg), ampicillin (AMP; 10 µg), doxycycline (DO; 30 µg), aztreonam (ATM; 30 µg), amoxicillin–clavulanic acid (AMC; 20/10 µg), piperacillin–tazobactam (TZP; 100/10 µg), tigecycline (TGC; 15 µg), polymyxin-B (PB; 300U) and trimethoprim–sulfamethoxazole (SXT; 1.25/23.75 µg) (Oxoid, Basingstoke, UK) were used. The standard broth microdilution method was used to ascertain antimicrobials MIC. The double-disc synergy test and carbapenem inactivation method were used to identify extended spectrum β-lactamases (ESBLs) and carbapenemase-producing strains. Quality control strains were E. coli ATCC 25922 and P. aeruginosa ATCC 27853.

2.2. Detection of Antimicrobial Resistance-Encoding Genes

Genomic DNA was isolated by the heat lysis method [19]. Briefly, 2 to 3 colonies of bacterial culture were mixed with 500 µL sterile distilled water and heated at 98 °C for 10 min at 300 rpm (ThermoMixer, Fischer Scientific, Waltham, MA, USA). Tubes were centrifuged at 1000 rpm for 10 min and supernatant collected in newly labeled tube. DNA was stored at −80 °C. The carbapenemase-encoding genes blaNDM-1, blaOXA-48, blaIMP, blaVIM and blaKPC-2 and the ESBLs blaCTX-M, blaSHV and blaTEM were detected by using a 50 µL of PCR reaction mixture containing 25 µL of 2 × PCR Master Mix (Cat # K0171, Thermoscientific, Waltham, MA, USA), 1 µL of each primer (10 µM), 2 µL of DNA and dH2O up to 50 µL in PCR (Thermal Cycler, Proflex, ABI). PCR cycling conditions were 95 °C for 40 sec, melting temperature (Tm) 30 sec and 72 °C for 30 sec (30 cycles). Agarose gel electrophoresis (1–1.5%) was used to resolve PCR products. Allelic discrimination of blaNDM was performed by Sanger’s sequencing method. The cycle sequencing was performed by using BigDye terminator v3.1 kit with 10 µL PCR reaction mixture containing BigDye terminator 3.1 Ready Reaction Mix 4 µL, forward primer (3.2 pmol) 0.5 µL, purified DNA template (5–20 ng) 2 µL and dH2O 3.5 µL. PCR cycling conditions were 96 °C for 1 min, 96 °C for 10 sec, 50 °C for 5 sec and 60 °C for 2 min (35 cycles). PCR product was purified by using BigDye XTerminator purification kit as per kit instructions and capillary electrophoresis was conducted by Genetic Analyzer (ABI-3500, Thermo Fischer, Waltham, MA, USA). Sequencing analysis software v6.1 and basic local alignment tool (BLAST, NCBI) was used for data analysis and interpretation. The primer sequences with Tm are given in Table S1.

2.3. Multilocus Sequence Typing (MLST) and Plasmid Analysis

MLST was used for sequence typing analysis of E. coli strains harboring blaNDM based on the allelic profile resemblance of eight housekeeping genes (E. coli Pasteur Scheme): DNA polymerase (dinB), isocitrate dehydrogenase (icdA), p-aminobenzoate synthase (pabB), polymerase PolII (polB), proline permease (putP), tryptophan synthase subunit A (trpA), tryptophan synthase subunit B (trpB) and beta-glucuronidase (uidA) [20]. The primer sequences are given in Table S1. The same primers were used for sequencing analysis as described above. Allele number and STs were assigned by online MLST database https://pubmlst.org/bigsdb?db=pubmlst_mlst_seqdef (accessed on 16 December 2021). Plasmid analysis was carried out by using PCR-based replicon typing kit based on their incompatibility groups [21].

2.4. Determination of Efficacy of Silver Nanoparticles (AgNPs)

The activity of AgNPs and MEM was evaluated by the broth microdilution checkerboard method. Commercially available AgNPs (particle size: 10 nm, solution concentration 20 μg/mL in aqueous buffer containing sodium citrate as stabilizer) were used (Cat # 730785, Sigma-Aldrich, St. Louis, MO, USA). The dilutions of MEM and AgNPs were prepared in Mueller Hinton broth with final concentration ranges of 1024, 512, 256, 128, 64, 32, 16 and 8 μg/mL for MEM and 10, 5, 2.5, 1.25, 0.625 and 0.312 μg/mL for AgNPs (Table S2). Bacterial cultures were prepared at a concentration of 0.5 McFarland (equivalent to 108 CFU/mL) and diluted to 1:100 (106 CFU/mL). In sterile 96-well microtiter plate, each well was inoculated with 100 μL of diluted bacterial suspension and mixed with equal volumes of antibiotic solution. All tests were conducted in duplicate with a growth control without addition of antibiotics and with sodium citrate addition. The inoculated microtiter plate was incubated at 37 °C for 18 h. After incubation, the fractional inhibitory concentration index (ΣFIC) was calculated by dividing individual MIC of treatments with MIC of the combination drugs. ΣFIC value ≤1 was considered to have a synergistic, 1.1 to 2.0 indifferent and ≥2 antagonistic effect [22].

3. Results

3.1. Characteristics of Sample

In the current study, the isolates were screened and included on the basis of their carbapenem susceptibility profile. All the isolates were resistant to at least one of the carbapenems (MEM, IMP and ETP). On the basis of this selection criterion, 184 carbapenem-resistant E. coli strains were retrieved out of total 650 collected samples. Carbapenem-resistant samples were mainly obtained from females (119/184, 64.7%), the major sample type was urine (75/184, 40.8%) and the main hospital section was surgery (59/184, 32.1%), followed by nephrology (36/184, 19.6%). Carbapenemase production was recorded in 64.7% (119/184) of the carbapenem-resistant E. coli isolates, while 35.3% (65/184) did not produce carbapenemase, indicating that other mechanisms are involved for resistance development against carbapenems in these isolates. On other hand, ESBL production was observed in 97.3% (179/184) of the isolates.

3.2. Resistance Gene Profile

The carbapenemase-producing strains were further evaluated for the presence of resistance genes. It was observed that 87.4% (104/119) of isolates carried resistance genes with the higher prevalence of blaNDM-1 (45.1%, 47/104), while blaNDM-7 was detected in 3.8% (4/104) of strains. blaOXA-48 represented the second highest carbapenemase-resistance gene (27%, 28/104). On the other hand, the co-production of blaNDM-1 and blaOXA-48 was observed in 15.4% (16/104) of the strains. We also detected the presence of blaNDM-7/blaOXA-48 (2.9%, 3/104), blaOXA-48/blaVIM (3.8%, 4/104) and blaNDM-1/blaVIM (2%, 2/104). blaKPC-2 and blaIMP were not detected.

Furthermore, the frequency of the selected β-lactamase-encoding genes was assessed. ESBL-resistance genes were detected in 82.1% (147/184) of the isolates. The dominance was observed for blaSHV (24.9%, 36/147), followed by blaCTX-M (17.7%, 26/147) and blaTEM (4.8%, 7/147), while in case of coharbored ESBL genes, blaSHV/blaCTX-M (29.2%, 43/147) were in abundance together with blaSHV/blaTEM (15%, 22/147) and blaCTX-M/blaTEM (8.8%, 13/147). The distribution of resistance genes with respect to the samples and wards is given in Table 1.

Table 1.

Molecular profile of carbapenem-resistant E. coli isolates.

Sample Ward Strain Antimicrobial Resistance Gene
Urine Urology/Nephrology EC-75, EC-73, EC-95, EC-101 blaNDM-1/blaOXA-48/blaSHV/blaCTX-M
EC-89 blaNDM-7/blaOXA-48/blaSHV/blaCTX-M
EC-02 blaNDM-1/blaOXA-48/blaSHV
EC-71 blaNDM-1/blaVIM/blaSHV
EC-11, EC-67, EC-68, EC-94, EC-102 blaNDM-1/blaSHV/blaCTX-M
EC-88 blaNDM-1/blaCTX-M/blaTEM
EC-50 blaNDM-7/blaSHV/blaCTX-M
EC-49, EC-63 blaNDM-1/blaCTX-M
EC-86 blaNDM-7/blaCTX-M
EC-48 blaOXA-48/blaVIM/blaSHV
EC-01, EC-92 blaOXA-48/blaSHV/blaCTX-M
EC-51, EC-79, EC-91 blaOXA-48/blaSHV
EC-69 blaOXA-48/blaTEM
Surgery EC-97 blaNDM-1/blaOXA-48/blaCTX-M/blaTEM
EC-10, EC-29 blaNDM-1/blaSHV/blaCTX-M/blaTEM
EC-80 blaNDM-1/blaCTX-M
EC-78 blaNDM-1/blaSHV
EC-60 blaOXA-48/blaSHV/blaCTX-M
EC-08, EC-82 blaOXA-48/blaCTX-M
EC-99 blaOXA-48/blaSHV
Medicine EC-56 blaNDM-1/blaOXA-48/blaCTX-M/blaTEM
EC-57 blaNDM-1/blaOXA-48/blaSHV/blaCTX-M
EC-98 blaNDM-7/blaOXA-48/blaSHV/blaCTX-M
EC-44, EC-77, EC-104 blaNDM-1/blaSHV/blaCTX-M
EC-22 blaNDM-1/blaCTX-M
EC-45 blaNDM-1/blaSHV
EC-81 blaOXA-48/blaSHV/blaCTX-M
EC-58 blaOXA-48/blaSHV
Chest medicine EC-103 blaNDM-1/blaVIM/blaSHV
Pus Surgery EC-17 blaNDM-7/blaOXA-48/blaSHV/blaCTX-M/blaTEM
EC-39 blaNDM-1/blaOXA-48/blaSHV/blaCTX-M
EC-23 blaNDM-1/blaOXA-48/blaCTX-M/blaTEM
EC-09, EC-13, EC-31, EC-34, EC-40 blaNDM-1/blaSHV/blaCTX-M
EC-14, EC-16 blaNDM-1/blaCTX-M
EC-43 blaNDM-1/blaSHV
Cardiology EC-38 blaNDM-1/blaSHV/blaCTX-M
EC-37 blaOXA-48/blaSHV/blaCTX-M
EC-26 blaOXA-48/blaSHV
Medicine EC-27 blaNDM-1/blaSHV
Wound Surgery EC-33 blaNDM-1/blaOXA-48/blaSHV
EC-12, EC-83 blaNDM-1/blaCTX-M/blaTEM
EC-06 blaOXA-48/blaVIM/blaCTX-M
EC-04 blaOXA-48/blaSHV/blaCTX-M
EC-21 blaNDM-1/blaCTX-M
EC-30 blaNDM-1/blaSHV
Medicine EC-15 blaNDM-1/blaSHV/blaCTX-M
EC-07 blaNDM-1/blaTEM
ICU EC-84 blaNDM-1/blaOXA-48/blaSHV/blaCTX-M
EC-32 blaNDM-1/blaOXA-48/blaSHV
EC-35 blaNDM-1/blaSHV/blaCTX-M/blaTEM
EC-24 blaOXA-48/blaSHV/blaCTX-M/blaTEM
Pediatrics medicine EC-42 blaNDM-1/blaSHV/blaCTX-M/blaTEM
EC-20 blaOXA-48/blaVIM/blaSHV
Blood Surgery EC-76 blaNDM-1/blaOXA-48/blaTEM
EC-93 blaNDM-1/blaCTX-M/blaTEM
EC-19 blaOXA-48/blaVIM/blaSHV
EC-28 blaOXA-48/blaSHV/blaCTX-M
Nephrology EC-25 blaOXA-48/blaSHV/blaCTX-M/blaTEM
EC-36 blaNDM-1/blaSHV
Pediatrics medicine EC-18 blaOXA-48/blaCTX-M
Oncology EC-90 blaNDM-1/blaOXA-48/blaSHV
Tip-cells Surgery EC-05 blaNDM-1/blaSHV/blaCTX-M
EC-03 blaOXA-48/blaSHV/blaCTX-M
EC-96 blaNDM-1/blaSHV
Medicine EC-47 blaNDM-1/blaSHV
EC-53 blaOXA-48/blaSHV
Nephrology EC-64 blaOXA-48/blaSHV/blaCTX-M
EC-72 blaNDM-7/blaSHV
ICU EC-65 blaNDM-1/blaSHV/blaCTX-M
EC-61 blaOXA-48/blaSHV
Pleural-fluid Surgery EC-54 blaNDM-1/blaOXA-48/blaSHV/blaCTX-M
EC-41, EC-55 blaNDM-1/blaSHV
Medicine EC-85 blaNDM-1/blaOXA-48/blaCTX-M/blaTEM
EC-59 blaOXA-48/blaCTX-M
Chest medicine EC-70 blaNDM-1/blaOXA-48/blaSHV/blaCTX-M
EC-100 blaNDM-1/blaSHV
Urology EC-66 blaOXA-48/blaSHV
Tissue Oncology EC-62 blaNDM-1/blaSHV/blaCTX-M
EC-52 blaOXA-48/blaVIM/blaSHV
EC-87 blaNDM-1/blaSHV
Sputum Chest medicine EC-46, EC-74 blaOXA-48/blaSHV/blaCTX-M
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3.3. Genetic Profiling and Antimicrobial Susceptibility Analysis

In order to identify the genetic variability based on clonal lineage among E. coli isolates, NDM-positive strains (n = 72) were subjected to a sequence type analysis. ST405 (44.4%, 32/72) and ST131 (29.2%, 21/72) were found to be the dominant sequence types. Other sequence types were also detected, including ST101 (9.7%, 7/72), ST10 (9.7%, 7/72) and ST648 (7%, 5/72). The detected replicon types included IncFII, IncFIIK, IncA/C, IncN and IncL/M.

Carbapenem-resistant clinical strains displayed a varied antimicrobial resistance profile with 100% resistance to third-generation cephalosporins, including CPT, CFZ, CAZ, CTX, FOX, ATM, AMP and AMC. The discrete susceptibility pattern for other antimicrobials exhibited a multifarious resistance profile such as TZP (76.3%), SXT (62.5%), TGC (54.1%) and CIP (51.4%), whereas the highest susceptibility was recorded for PB (16.6%) accompanied by DO (47.2%) and AK (45.8%). In order to investigate the antimicrobial resistance pattern in relation to sequence types, we classified the strains into diverse sets of isolates arbitrarily based on their resistance pattern. All isolates represented an MDR phenotype. The detailed results are given in Table 2.

Table 2.

Antimicrobial resistance and genetic profile of NDM-positive E. coli isolates.

Clinical Strains Resistance Profile MLST Replicon Type
EC-02, EC-14, EC-17, EC-23, EC-30, EC-33, EC-34, EC-35, EC-42, EC-45, EC-47, EC-56, EC-62, EC-75, EC-83 CPT, CFZ, CAZ, CTX, FOX, ATM, AMP, AMC, AK, TZP, TGC ST405 IncFII, IncA/C, IncN, IncL/M
EC-05, EC-21, EC-22, EC-36, EC-38, EC-41, EC-44, EC-78, EC-86, EC-88, EC-98, EC-103 CPT, CFZ, CAZ, CTX, FOX, ATM, AMP, AMC, CIP, DO, SXT, TZP ST405 IncFII, IncA/C, IncN, IncL/M
EC-49, EC-55, EC-71, EC-76, EC-90 CPT, CFZ, CAZ, CTX, FOX, ATM, AMP, AMC, AK, PB, CIP, TGC ST405 IncFII, IncA/C, IncN, IncL/M
EC-07, EC-13, EC-15, EC-40, EC-68, EC-72, EC-77, EC-84, EC-87, EC-89, EC-95, EC-101, CPT, CFZ, CAZ, CTX, FOX, ATM, AMP, AMC, DO, CIP, SXT, TZP ST131 IncFII, IncA/C, IncL/M
EC-16, EC-27, EC-50, EC-67, EC-73, EC-85, EC-96, EC-97, EC-102 CPT, CFZ, CAZ, CTX, FOX, ATM, AMP, AMC, AK, TZP, SXT, TGC ST131 IncFII, IncA/C, IncL/M
EC-09, EC-10, EC-12, EC-29, EC-31, EC-80, EC-104 CPT, CFZ, CAZ, CTX, FOX, ATM, AMP, AMC, PB, TGC, SXT, DO ST10 IncFII, IncN
EC-32, EC-39, EC-65, EC-100 CPT, CFZ, CAZ, CTX, FOX, ATM, AMP, AMC, AK, TZP ST101 IncFII, IncN
EC-43, EC-54, EC-70 CPT, CFZ, CAZ, CTX, FOX, ATM, AMP, AMC, TZP, CIP, DO, TGC ST101 IncFII, IncN
EC-11, EC-57, EC-63, EC-93, EC-94 CPT, CFZ, CAZ, CTX, FOX, ATM, AMP, AMC, SXT, CIP ST648 IncFII, IncN, IncFIIK
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Furthermore, the correlation of carbapenemase-resistance genes with sequence types was analyzed, by which we identified different combinations of resistance genes within different detected sequence types. Most importantly, we found that ST405 and ST131 were more prevalent comprising isolates with blaNDM-7/blaOXA-48 and blaNDM-1/blaOXA-48 which coharbored different combinations of ESBLs, while ST10, ST101 and ST648 were prevalent among the blaNDM-1 harboring isolates. The distribution of sequence types in relation to resistance genes is given in Table 3.

Table 3.

Distribution of sequence types with resistance gene pattern.

MLST Resistance Gene Pattern No. of Isolates
ST405 blaNDM-7/blaOXA-48/blaSHV/blaCTX-M/blaTEM EC-17
blaNDM-7/blaOXA-48/blaSHV/blaCTX-M EC-98
blaNDM-7/blaCTX-M EC-86
blaNDM-1/blaOXA-48/blaCTX-M/blaTEM EC-23, EC-56
blaNDM-1/blaOXA-48/blaSHV/blaCTX-M EC-75, EC-98
blaNDM-1/blaOXA-48/blaTEM EC-76
blaNDM-1/blaOXA-48/blaSHV EC-02, EC-33, EC-90
blaNDM-1/blaVIM/blaSHV EC-71, EC-103
blaNDM-1/blaSHV/blaCTX-M/blaTEM EC-35, EC-42
blaNDM-1/blaSHV/blaCTX-M EC-05, EC-34, EC-38, EC-44, EC-62
blaNDM-1/blaCTX-M/blaTEM EC-83, EC-88
blaNDM-1/blaCTX-M EC-14, EC-21, EC-22, EC-49
blaNDM-1/blaSHV EC-30, EC-36, EC-41, EC-45, EC-47, EC-55, EC-78
ST131 blaNDM-7/blaOXA-48/blaSHV/blaCTX-M EC-89
blaNDM-7/blaSHV/blaCTX-M EC-50, EC-102
blaNDM-7/blaSHV EC-72
blaNDM-1/blaOXA-48/blaSHV/blaCTX-M EC-73, EC-84, EC-95
blaNDM-1/blaOXA-48/blaCTX-M/blaTEM EC-85, EC-97, EC-101
blaNDM-1/blaSHV/blaCTX-M EC-13, EC-15, EC-40, EC-67, EC-68, EC-77
blaNDM-1/blaSHV EC-27, EC-87, EC-96
blaNDM-1/blaCTX-M EC-16
blaNDM-1/blaTEM EC-07
ST10 blaNDM-1/blaSHV/blaCTX-M/blaTEM EC-10, EC-29
blaNDM-1/blaSHV/blaCTX-M EC-09, EC-31, EC-104
blaNDM-1/blaCTX-M/blaTEM EC-12
blaNDM-1/blaCTX-M EC-80
ST101 blaNDM-1/blaOXA-48/blaSHV/blaCTX-M EC-39, EC-54, EC-70
blaNDM-1/blaOXA-48/blaSHV EC-32
blaNDM-1/blaSHV/blaCTX-M EC-65
blaNDM-1/blaSHV EC-43, EC-100
ST648 blaNDM-1/blaOXA-48/blaSHV/blaCTX-M EC-57
blaNDM-1/blaSHV/blaCTX-M EC-11, EC-94
blaNDM-1/blaCTX-M/blaTEM EC-93
blaNDM-1/blaCTX-M EC-63
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Carbapenem-resistant strains of E. coli containing carbapenemases (n = 16) were selected randomly for the determination of the AgNPs’ efficacy. Carbapenem-sensitive strains of E. coli (n = 6) were used as controls. The MIC values of bacterial cultures grown in the presence of MEM, AgNPs and MEM/AgNPs were determined. The MIC values of the cultures were the highest in the presence of MEM and AgNPs alone. However, when a combination of MEM/AgNPs was used, there was a reduction in the MIC values. The detailed results are described in Table 4.

Table 4.

AgNPs and MEM interaction determined by ΣFIC values.

Sr. # Strain ID ΣFIC Interpretation
1 EC-98 (blaNDM-1/blaOXA-48) 0.75 Synergism
2 EC-17 (blaNDM-7/blaOXA-48) 1 Synergism
3 EC-89 (blaNDM-7/blaOXA-48) 1 Synergism
4 EC-98 (blaNDM-7/blaOXA-48) 0.5 Synergism
5 EC-23 (blaNDM-1/blaOXA-48) 1 Synergism
6 EC-56 (blaNDM-1/blaOXA-48) 1.5 Indifferent
7 EC-73 (blaNDM-1/blaOXA-48) 1 Synergism
8 EC-75 (blaNDM-1/blaOXA-48) 1 Synergism
9 EC-50 (blaNDM-7) 0.75 Synergism
10 EC-86 (blaNDM-7) 0.5 Synergism
11 EC-12 (blaNDM-1) 0.75 Synergism
12 EC-32 (blaNDM-1) 1 Synergism
13 EC-42 (blaNDM-1) 1.2 Indifferent
14 EC-44 (blaNDM-1) 1 Synergism
15 EC-62 (blaNDM-1) 0.5 Synergism
16 EC-63 (blaNDM-1) 0.75 Synergism
17 EC-261 0.5 Synergism
18 EC-423 0.5 Synergism
19 EC-438 0.5 Synergism
20 EC-503 1 Synergism
21 EC-510 3 Antagonism
22 EC-587 2 Indifferent
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synergism ≤ 1, indifferent 1.1 to 2.0, antagonism effect ≥ 2.

4. Discussion

The eventual outcome of carbapenem resistance among Enterobacteriaceae was evidenced globally by the rapid circulation of plasmid-encoded carbapenemases, making them critical for nosocomial outbreaks. Although carbapenemase detection is essential for infection control purposes, their precise characterization among different species is helpful in clinical practice as it impacts therapeutic decisions. The presence of different clinical strains in the genetic context among E. coli has not been revealed in the study area. In the present study, carbapenemase production was observed in 64.7% of the E. coli isolates. Previously, a variable range of carbapenemase production among E. coli strains has been documented from Pakistan, such as 37.1% in 2015, 93% in 2018, 81% in 2019 and 22.02% in 2021 [5,10,23,24] with the observation of similar trends globally, such as 89% from China, 9.82% from Morocco and 20.2% from Germany [20,21,22]. Similarly, reports from Pakistan showed higher rates of carbapenemase production among other Enterobacteriales such as K. pneumoniae 27.2% (34/125) [10], 61.8% (68/110) [25], 77.7% (91/117) [26] and 82.8% [27]. On the other hand, we found 28.3% of carbapenem-resistant E. coli clinical isolates. Previous studies from Pakistan demonstrated an increasing trend of carbapenem resistance in E. coli with 6% to 37.9% resistance from 2014 until 2018, and thus identified E. coli as a major contributor to the carbapenem resistance in Pakistan [23,28,29]. The leading cause for the growing trend of increased carbapenem resistance is the excessive use of carbapenems that result in the survival of complex clones with highly endured resistant strains [30].

Our results indicate a high resistance to third-generation cephalosporins, while PB displayed a higher susceptibility. In agreement with our results, previous studies from Pakistan also reported higher susceptible rates for colistin and fosfomycin [10,31,32]. On the other hand, avian-derived E. coli isolates from Pakistan showed resistance to SXT, TE, CTX and CAZ, while chicken-originated isolates were more susceptible to chloramphenicol and lower levels of resistance against third-generation cephalosporins [33,34]. Moreover, we found that surgery (32.1%) and nephrology (19.6%) were the main hospital sections responsible for the spread of E. coli infections. However, the ICU has been linked to the dissemination of carbapenem-resistant strains in China [6,35].

We observed that the most widely circulating carbapenem-resistance genes were blaNDM-1 (45.1%), blaNDM-7 (3.8%) and blaOXA-48 (27%), while the co-existence of blaNDM-1/blaOXA-48 (15.4%), blaNDM-7/blaOXA-48 (2.9%) and blaOXA-48/blaVIM (3.8%) was recorded. Data from Pakistan depicted that the main drivers of carbapenem resistance in E. coli are NDM and OXA-48, as evidenced by a number of reports such as blaNDM-1, blaNDM-4, blaNDM-5, blaNDM-7, blaOXA-48 and blaNDM-1/blaOXA-48 [10,36,37,38]. Few reports described the presence of blaVIM and blaIMP, while only one study reported the presence of blaKPC-2 with the coexistence of ESBLE genes in E. coli from Pakistan [5,10,24,39]. However, we reported the co-existence of blaNDM-7/blaOXA-48 and blaOXA-48/blaVIM for the first time in E. coli isolates from Pakistan.

In the current analysis of E. coli strains coharboring carbapenemase genes, the co-existence of ESBLs was detected such as blaNDM-1/blaOXA-48/blaSHV, blaNDM-1/blaOXA-48/blaCTX-M/blaTEM, blaNDM-1/blaOXA-48/blaCTX-M/blaSHV and blaNDM-7/blaOXA-48/blaCTX-M/blaSHV/blaTEM. Hitherto, the presence of carbapenemases and beta lactamases has been described from Pakistan including blaNDM-1/blaCTXM-15/blaOXA-1 and blaNDM-1/blaCTXM-15 [10]. In our strains, the coexistence of blaNDM-7/blaOXA-48/blaCTX-M/blaSHV/blaTEM is described for the first time.

Antimicrobial pressure has the ability to single-out clonal lineages and plasmids with resistance determinants, resulting in an enhanced transmission capacity. In our study, the clonal lineage analysis showed the sequence types ST405 and ST131 predominantly coharboring blaNDM-7/blaOXA-48 and blaNDM-1/blaOXA-48, while ST101, ST10 and ST648 were prevalent among blaNDM-1 harboring isolates. Previously, ST131 and ST405 in blaNDM-1 and blaKPC-2 positive E. coli strains with IncH12 and IncN replicon types have been reported, while ST648 has been described in blaNDM-7 containing E. coli isolates. [10,36,40]. ST101 and ST648 were reported in NDM-positive E. coli isolates [41]. On the other hand, ST10 was reported in avian-derived E. coli isolates [33] and ST131 among poultry birds from Pakistan [42], while we observed ST10 among clinical isolates for the first time. Furthermore, we detected IncFII, IncFIIK, IncA/C, IncN and IncL/M replicon types among our study isolates. Other replicon types reported from Pakistan include IncL/M, IncA/c, Inc and IncF-II [26,33,36,43,44].

Regardless of the significant efforts for the improvement and control of infections, carbapenem-resistant bacteria remain an alarming threat. Thus, few treatment options are left due to limited resources. Mostly, β-lactam antimicrobials showed inconsequential treatment effects against carbapenem-resistant microbes [45]. Emerging clinical evidence suggests that treatment with combination therapy may be beneficial against carbapenem-resistant pathogens [46]. Since ancient times, silver is known for its antimicrobial effects; therefore, in order to overcome the resistance development by the extensive use of antibiotics, silver nanoparticles can be used as an alternative approach to antibiotic combination therapy against MDR organisms [15,47,48]. Our data indicated that the combination of MEM/AgNPs resulted in the reduction of MIC values as compared to the presence of MEM and AgNPs alone against NDM-positive E. coli isolates. It has been shown that AgNPs and ciprofloxacin have better antimicrobial efficiency against E. coli [49]. Moreover, it has been suggested that biosynthesized AgNPs may work as antimicrobials to control E. coli infections [50,51,52,53]. However, we reported for the first time the effect of AgNPs in combination with MEM against E. coli clinical isolates.

5. Conclusions

We reported the co-existence of blaNDM-7/blaOXA-48 and blaOXA-48/blaVIM in E. coli isolates from Pakistan with a novel ST405 E. coli strain coharboring blaNDM-7/blaOXA-48/blaCTX-M/blaSHV/blaTEM. Moreover, ST10 was identified in clinical isolates coharboring blaNDM-1/blaCTX-M/blaSHV/blaTEM for the first time. The resistance pattern observed in our study suggested that surprisingly powerful strains evolved in Pakistan with time, which may indicate a complicated survival mechanism, particularly in the scenario wherein antibiotics misuse is rising. However, due to funding issues, we could not explore other resistance mechanisms and invasive genes. Moreover, a large number of strains may be tested further for AgNPs synergism by time kill assay. Our results also show that the antimicrobial efficacy can be improved when used in combination with silver nanoparticles.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms10112283/s1, Table S1: Nucleotide sequences for PCR and sequencing [12,13,14,15]; Table S2: Concentration of MEM and AgNPs

Click here for additional data file. (149.1KB, zip)

Author Contributions

Conceptualization, A.J.G., N.C. and N.Y.; Data curation, A.J.G. and N.Y.; Formal analysis, A.J.G., N.C., H.B., Z.R. and N.Y.; Funding acquisition, N.C.; Investigation, A.J.G., Z.R. and N.Y.; Methodology, A.J.G. and N.Y.; Project administration, N.C.; Resources, A.J.G., N.C., H.B. and N.Y.; Software, A.J.G.; Supervision, A.J.G. and N.Y.; Validation, A.J.G.; Writing—original draft, N.Y.; Writing—review and editing, A.J.G. and N.Y. All authors have read and agreed to the published version of the manuscript.

Informed Consent Statement

This study was approved by the institutional review board of the King Edward Medical University (41/RC/KEMU), Lahore, Pakistan. Informed consent was obtained from the participants for inclusion in this study.

Data Availability Statement

The data used to support the findings of this study are available from the corresponding authors upon request.

Conflicts of Interest

The authors declare no conflict of interest.

Funding Statement

The research work was funded by King Edward Medical University, Lahore, Pakistan via grant no. 41/RC/KEMU, dated 20 July 2017.

Footnotes

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  • 1.Hansen G.T. Continuous Evolution: Perspective on the Epidemiology of Carbapenemase Resistance Among Enterobacterales and Other Gram-Negative Bacteria. Infect. Dis. Ther. 2021;10:75–92. doi: 10.1007/s40121-020-00395-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Lynch J.P., Clark N.M., Zhanel G.G. Escalating antimicrobial resistance among Enterobacteriaceae: Focus on carbapenemases. Expert Opin. Pharmacother. 2021;22:1455–1474. doi: 10.1080/14656566.2021.1904891. [DOI] [PubMed] [Google Scholar]
  • 3.Cui X., Zhang H., Du H. Carbapenemases in Enterobacteriaceae: Detection and Antimicrobial Therapy. Front. Microbiol. 2019;10:1823. doi: 10.3389/fmicb.2019.01823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Le Thanh Dong H.V.E., Espinoza J.L. Emerging superbugs: The threat of carbapenem resistant enterobacteriaceae. AIMS Microbiol. 2020;6:176. doi: 10.3934/microbiol.2020012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Khan S.H., Jahan S., Ahmad I., Rahman S.U., Rehman T.U. Incidence of blaIMP and blaVIM Genes among Carbapenemase Producing Escherichia coli in Lahore, Pakistan. Pak. J. Zool. 2019;51:1959. doi: 10.17582/journal.pjz/2019.51.5.sc1. [DOI] [Google Scholar]
  • 6.Tian X., Zheng X., Sun Y., Fang R., Zhang S., Zhang X., Lin J., Cao J., Zhou T. Molecular Mechanisms and Epidemiology of Carbapenem-Resistant Escherichia coli Isolated from Chinese Patients During 2002–2017. Infect. Drug Resist. 2020;13:501–512. doi: 10.2147/IDR.S232010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Zainab L., Ibrar K., Sadiq A., Hamid A., Ullah M., Noor R. Extended spectrum beta lactamases-producing Escherichia coli in retail chicken meat from Khyber Pakhtunkhwa, Pakistan. Saudi J. Biol. Sci. 2022;29:103280. doi: 10.1016/j.sjbs.2022.103280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Ali A., Fontana H., Sano E., Li R., Humayon M., Rahman S., Lincopan N., Mohsin M. Genomic features of a high-risk mcr-1.1-positive Escherichia coli ST10 isolated from cattle farm environment. Environ. Sci. Pollut. Res. 2021;28:54147–54152. doi: 10.1007/s11356-021-15437-6. [DOI] [PubMed] [Google Scholar]
  • 9.Yasmin S., Karim A. Temporal Variation of Meropenem Resistance in E. coli Isolated from Sewage Water in Islamabad, Pakistan. Antibiotics. 2022;11:635. doi: 10.3390/antibiotics11050635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Bilal H., Rehman T.U., Khan M.A., Hameed F., Jian Z.G., Han J., Yang X. Molecular Epidemiology of mcr-1, blaKPC-2, and blaNDM-1 Harboring Clinically Isolated Escherichia coli from Pakistan. Infect. Drug Resist. 2021;14:1467. doi: 10.2147/IDR.S302687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Lekha C.D., Lekha C. Review on silver nanoparticle synthesis method, antibacterial activity, drug delivery vehicles and toxicity pathways: Recent advances and future aspects. J. Nanomater. 2021;5:4401829. doi: 10.1155/2021/4401829. [DOI] [Google Scholar]
  • 12.Singh P., Mijakovic I. Antibacterial Effect of Silver Nanoparticles Is Stronger If the Production Host and the Targeted Pathogen Are Closely Related. Biomedicines. 2022;10:628. doi: 10.3390/biomedicines10030628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Abdel-Aziz M.M., Yosri M., Amin B.H. Control of imipenem resistant-Klebsiella pneumoniae pulmonary infection by oral treatment using a combination of mycosynthesized Ag-nanoparticles and imipenem. J. Radiat. Res. Appl. Sci. 2017;10:353–360. doi: 10.1016/j.jrras.2017.09.002. [DOI] [Google Scholar]
  • 14.Hussein E.A.M., Mohammad A.A.-H., Harraz F.A., Ahsan M.F. Biologically Synthesized Silver Nanoparticles for Enhancing Tetracycline Activity Against Staphylococcus aureus and Klebsiella pneumoniae. Braz. Arch. Biol. Technol. 2019;62:97. doi: 10.1590/1678-4324-2019180266. [DOI] [Google Scholar]
  • 15.Vazquez-Muñoz R., Meza-Villezcas A., Fournier P., Soria-Castro E., Juarez-Moreno K., Gallego-Hernandez A., Bogdanchikova N., Vazquez-Duhalt R., Huerta-Saquero A. Enhancement of antibiotics antimicrobial activity due to the silver nanoparticles impact on the cell membrane. PLoS ONE. 2019;14:e0224904. doi: 10.1371/journal.pone.0224904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Yassin M.T., Mostafa A.A.-F., Al-Askar A.A., Al-Otibi F.O. Synergistic Antibacterial Activity of Green Synthesized Silver Nanomaterials with Colistin Antibiotic against Multidrug-Resistant Bacterial Pathogens. Crystals. 2022;12:1057. doi: 10.3390/cryst12081057. [DOI] [Google Scholar]
  • 17.Bilal H., Khan M.N., Rehman T., Hameed M.F., Yang X. Antibiotic resistance in Pakistan: A systematic review of past decade. BMC Infect. Dis. 2021;21:1–19. doi: 10.1186/s12879-021-05906-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.CLSI . Performance Standards for Antimicrobial Susceptibility Testing. 28th ed. Clinical and Laboratory Standards Institute; Wayne, PA, USA: 2018. CLSI supplement M100. [Google Scholar]
  • 19.Dashti A.A., Jadaon M.M. Heat treatment of bacteria: A simple method of DNA extraction for molecular techniques. Kuwait Med. J. 2009;41:117–122. [Google Scholar]
  • 20.Brolund A., Rajer F., Giske C.G., Melefors A., Titelman E., Sandegren L. Dynamics of Resistance Plasmids in Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae during Postinfection Colonization. Antimicrob. Agents Chemother. 2019;63:e02201-18. doi: 10.1128/AAC.02201-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Carloni E., Andreoni F., Omiccioli E., Villa L., Magnani M., Carattoli A. Comparative analysis of the standard PCR-Based Replicon Typing (PBRT) with the commercial PBRT-KIT. Plasmid. 2017;90:10–14. doi: 10.1016/j.plasmid.2017.01.005. [DOI] [PubMed] [Google Scholar]
  • 22.Jamaran S., Zarif R.B. Synergistic effect of silver nanoparticles with neomycin or gentamicin antibiotics on mastitis-causing Staphylococcus aureus. Open J. Ecol. 2016;6:452–459. doi: 10.4236/oje.2016.67043. [DOI] [Google Scholar]
  • 23.Ain N.U., Iftikhar A., Bukhari S.S., Abrar S., Hussain S., Haider M.H., Rasheed F., Riaz S. High frequency and molecular epidemiology of metallo-β-lactamase-producing gram-negative bacilli in a tertiary care hospital in Lahore, Pakistan. Antimicrob. Resist. Infect. Control. 2018;7:1–9. doi: 10.1186/s13756-018-0417-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Akhtar J., Saleem S., Shahzad N., Waheed A., Jameel I., Rasheed F., Jahan S. Prevalence of Metallo-β-Lactamase IMP and VIM Producing Gram Negative Bacteria in Different Hospitals of Lahore, Pakistan. Pak. J. Zool. 2018;50:6. doi: 10.17582/journal.pjz/2018.50.6.2343.2349. [DOI] [Google Scholar]
  • 25.Naeem S., Bilal H., Muhammad H., Khan M.A., Hameed F., Bahadur S., Rehman T.U. Detection of blaNDM-1 gene in ESBL producing Escherichia coli and Klebsiella pneumoniae isolated from urine samples. J. Infect. Dev. Ctries. 2021;15:516–522. doi: 10.3855/jidc.12850. [DOI] [PubMed] [Google Scholar]
  • 26.Gondal A.J., Saleem S., Jahan S., Choudhry N., Yasmin N. Novel Carbapenem-Resistant Klebsiella pneumoniae ST147 Coharboring blaNDM-1, blaOXA-48 and Extended-Spectrum β-Lactamases from Pakistan. Infect. Drug Resist. 2020;13:2105–2115. doi: 10.2147/IDR.S251532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Imtiaz W., Syed Z., Rafaque Z., Andrews S.C., Dasti J.I. Analysis of Antibiotic Resistance and Virulence Traits (Genetic and Phenotypic) in Klebsiella pneumoniae Clinical Isolates from Pakistan: Identification of Significant Levels of Carbapenem and Colistin Resistance. Infect. Drug Resist. 2021;14:227–236. doi: 10.2147/IDR.S293290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Uddin F., Imam S.H., Khan S., Khan T.A., Ahmed Z., Sohail M., Elnaggar A.Y., Fallatah A.M., El-Bahy Z.M. NDM Production as a Dominant Feature in Carbapenem-Resistant Enterobacteriaceae Isolates from a Tertiary Care Hospital. Antibiotics. 2021;11:48. doi: 10.3390/antibiotics11010048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Durrani M.A., Ahmed R., Kumar M., Bakar I. Frequency of Class B Carbapenemases (MbL) in enterobacteriacae. J. Pak. Med. Assoc. 2014;64:519–523. [PubMed] [Google Scholar]
  • 30.Kong H.-K., Pan Q., Lo W.-U., Liu X., Law C.O.K., Chan T.-F., Ho P.-L., Lau T.C.-K. Fine-tuning carbapenem resistance by reducing porin permeability of bacteria activated in the selection process of conjugation. Sci. Rep. 2018;8:15248. doi: 10.1038/s41598-018-33568-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Iqbal S., Bhatti S.M., Amir M., Zaman Q.U., Raza A. Scenario of Antibiotic Resistance in Pakistan: A Systematic Review. Pak. J. Med. Health Sci. 2022;16:643–649. doi: 10.53350/pjmhs22165643. [DOI] [Google Scholar]
  • 32.Haq I., Haq M., Farooq M., Javaid A., Zafar S., Ahmad A., Khan A.M.K., Abbas M., Khan B.B., Haq M. Multi-Drug Resistance of Escherichia coli (E. coli) Isolated from Clinical Isolates in District Peshawar Kp Pakistan. Pak. J. Med. Health Sci. 2022;16:830–835. doi: 10.53350/pjmhs22161830. [DOI] [Google Scholar]
  • 33.Mohsin M., Raza S., Schaufler K., Roschanski N., Sarwar F., Semmler T., Schierack P., Guenther S. High Prevalence of CTX-M-15-Type ESBL-Producing E. coli from Migratory Avian Species in Pakistan. Front. Microbiol. 2017;8:2476. doi: 10.3389/fmicb.2017.02476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Ahmad K., Khattak F., Ali A., Rahat S., Noor S., Mahsood N., Somayya R. Carbapenemases and Extended-Spectrum β-Lactamase–Producing Multidrug-Resistant Escherichia coli Isolated from Retail Chicken in Peshawar: First Report from Pakistan. J. Food Prot. 2018;81:1339–1345. doi: 10.4315/0362-028X.JFP-18-045. [DOI] [PubMed] [Google Scholar]
  • 35.Hoang C.Q., Nguyen H.D., Vu H.Q., Nguyen A.T., Pham B.T., Tran T.L., Nguyen H.T.H., Dao Y.M., Nguyen T.S.M., Nguyen D.A., et al. Emergence of New Delhi Metallo-Beta-Lactamase (NDM) and Klebsiella pneumoniae Carbapenemase (KPC) Production by Escherichia coli and Klebsiella pneumoniae in Southern Vietnam and Appropriate Methods of Detection: A Cross-Sectional Study. BioMed. Res. Int. 2019;2019:e9757625. doi: 10.1155/2019/9757625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Qamar M.U., Walsh T., Toleman M., Saleem S., Jahan S. First identification of clinical isolate of a Novel “NDM-4” producing Escherichia coli ST405 from urine sample in Pakistan. Braz. J. Microbiol. 2018;49:949–950. doi: 10.1016/j.bjm.2018.02.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Qamar M.U., Walsh T.R., Toleman M.A., Tyrrell J.M., Saleem S., Aboklaish A., Jahan S. Dissemination of genetically diverse NDM-1, -5, -7 producing-Gram-negative pathogens isolated from pediatric patients in Pakistan. Futur. Microbiol. 2019;14:691–704. doi: 10.2217/fmb-2019-0012. [DOI] [PubMed] [Google Scholar]
  • 38.Masseron A., Poirel L., Ali B.J., Syed M., Nordmann P. Molecular characterization of multidrug-resistance in Gram-negative bacteria from the Peshawar teaching hospital, Pakistan. New Microbes New Infect. 2019;32:100605. doi: 10.1016/j.nmni.2019.100605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Ejaz H., Younas S., Qamar M., Junaid K., Abdalla A., Abosalif K., Alameen A., Elamir M., Ahmad N., Hamam S., et al. Molecular Epidemiology of Extensively Drug-Resistant mcr Encoded Colistin-Resistant Bacterial Strains Co-Expressing Multifarious β-Lactamases. Antibiotics. 2021;10:467. doi: 10.3390/antibiotics10040467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Ali I., Rafaque Z., Dasti J.I., Graham S.E., Salzman E., Foxman B. Uropathogenic E. coli from Pakistan Have High Prevalence of Multidrug Resistance, ESBL, and O25b-ST131. Open Forum Infect. Dis. 2016;3:1. doi: 10.1093/ofid/ofw172.1556. [DOI] [Google Scholar]
  • 41.Mushtaq S., Irfan S., Sarma J.B., Doumith M., Pike R., Pitout J., Livermore D.M., Woodford N. Phylogenetic diversity of Escherichia coli strains producing NDM-type carbapenemases. J. Antimicrob. Chemother. 2011;66:2002–2005. doi: 10.1093/jac/dkr226. [DOI] [PubMed] [Google Scholar]
  • 42.Ilyas S., Rasool M.H. The Escherichia coli sequence type 131 harboring extended-spectrum beta-lactamases and carbapenemases genes from poultry birds. Infect. Drug Resist. 2021;14:805. doi: 10.2147/IDR.S296219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Nahid F., Zahra R., Sandegren L. A bla OXA-181-harbouring multi-resistant ST147 Klebsiella pneumoniae isolate from Pakistan that represent an intermediate stage towards pan-drug resistance. PLoS ONE. 2017;12:e0189438. doi: 10.1371/journal.pone.0189438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Umair M., Mohsin M. Prevalence and genetic relatedness of extended spectrum-β-lactamase-producing Escherichia coli among humans, cattle, and poultry in Pakistan. Microb. Drug Resist. 2019;25:1374–1381. doi: 10.1089/mdr.2018.0450. [DOI] [PubMed] [Google Scholar]
  • 45.Zhang R., Liu L., Zhou H., Chan E.W., Li J., Fang Y., Li Y., Liao K., Chen S. Nationwide Surveillance of Clinical Carbapenem-resistant Enterobacteriaceae (CRE) Strains in China. eBioMedicine. 2017;19:98–106. doi: 10.1016/j.ebiom.2017.04.032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Tzouvelekis L.S., Markogiannakis A., Piperaki E., Souli M., Daikos G.L. Treating infections caused by carbapenemase-producing Enterobacteriaceae. Clin. Microbiol. Infect. 2014;20:862–872. doi: 10.1111/1469-0691.12697. [DOI] [PubMed] [Google Scholar]
  • 47.Matsumura Y., Yoshikata K., Kunisaki S.-I., Tsuchido T. Mode of Bactericidal Action of Silver Zeolite and Its Comparison with That of Silver Nitrate. Appl. Environ. Microbiol. 2003;69:4278–4281. doi: 10.1128/AEM.69.7.4278-4281.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Stensberg M.C., Wei Q., McLamore E.S., Porterfield D.M., Wei A., Sepúlveda M.S. Toxicological studies on silver nanoparticles: Challenges and opportunities in assessment, monitoring and imaging. Nanomedicine. 2011;6:879–898. doi: 10.2217/nnm.11.78. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Adil M., Khan T. Evaluation of the antibacterial potential of silver nanoparticles synthesized through the interaction of antibiotic and aqueous callus extract of Fagonia indica. AMB Express. 2019;9:1–12. doi: 10.1186/s13568-019-0797-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Sharma G., Nam J.-S., Sharma A.R., Lee S.-S. Antimicrobial Potential of Silver Nanoparticles Synthesized Using Medicinal Herb Coptidis rhizome. Molecules. 2018;23:2268. doi: 10.3390/molecules23092268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Vu X.H., Duong T.T.T., Pham T.T.H., Trinh D.K., Nguyen X.H., Dang V.-S. Synthesis and study of silver nanoparticles for antibacterial activity against Escherichia coli and Staphylococcus aureus. Adv. Nat. Sci. Nanosci. Nanotechnol. 2018;9:025019. doi: 10.1088/2043-6254/aac58f. [DOI] [Google Scholar]
  • 52.Elsayed A., Safwat A., Abdelsattar A.S., Essam K., Nofal R., Makky S., El-Shibiny A. The antibacterial and biofilm inhibition activity of encapsulated silver nanoparticles in emulsions and its synergistic effect with E. coli bacteriophage. Inorg. Nano Metal Chem. 2022;5:1–11. doi: 10.1080/24701556.2022.2081191. [DOI] [Google Scholar]
  • 53.Wang X., Lee S.-Y., Akter S., Huq A. Probiotic-Mediated Biosynthesis of Silver Nanoparticles and Their Antibacterial Applications against Pathogenic Strains of Escherichia coli O157:H7. Polymers. 2022;14:1834. doi: 10.3390/polym14091834. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

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Data Availability Statement

The data used to support the findings of this study are available from the corresponding authors upon request.

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