ESBL Genes Research Articles

Antibiotic resistance and virulence characteristics of four carbapenem-resistant Klebsiella pneumoniae strains coharbouring blaKPC and blaNDM based on whole genome sequences from a tertiary general teaching hospital in central China between 2019 and 2021.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) infection is a worldwide health issue that poses a serious threat to public health. This study summarizes the clinical features of four patients with CRKP coproducing NDM and KPC infections and further analyses the molecular typing, resistance and virulence factors of the four CRKP strains. Of the twenty-two CRKP isolates, four strains coharbouring blaKPC and blaNDM isolated from four patients were screened by Sanger sequencing between October 2019 and April 2021. Demographics, clinical and pathological data of the four patients were collected through electronic medical records. Antimicrobial susceptibility testing, biofilm formation assays and serum bactericidal assays were performed on the four isolates. The antibiotic resistance and virulence genes were investigated by whole-genome sequencing. Sequence types (STs) were determined by multilocus sequence typing, and serotypes were identified by wzi gene sequencing. Three patients recovered, and one patient stopped treatment. Four strains were multiple carbapenemase producers: KPC-2, NDM-4, SME-5 and IMI-4 coproducer; KPC-2, NDM-1 and SME-3 coproducer; KPC-2, NDM-1 and IMI-3 coproducer; KPC-2 and NDM-5 coproducer. They also harboured ESBL genes and mutations in the efflux pump regulator genes. They were multidrug resistant but sensitive to tigecycline and colistin. Four isolates had moderate biofilm-forming abilities and carried various virulence genes, including siderophores, type 1 fimbriae and E. coli common pilus. Only the NO. 3 strain was resistant to the serum. The STs and serotypes of the four strains were ST11 and KL64, ST337 and none, ST307 and KL102KL149KL155, and ST29 and K54, respectively. Four CRKP strains coharbouring blaKPC and blaNDM also carried other carbapenemase genes. Notably, the NO. 1 isolate carrying four carbapenemase genes has not been reported globally until now. Four strains exhibited a high level of resistance to multiple antibiotics. Additionally, three of the four patients were exposed to invasive medical devices that provided an environment for biofilm formation. Meanwhile, three strains with adhesion genes as moderate biofilm formers might form biofilms resulting in long hospital stays, increasing therapeutic difficulty, and even treatment failure. This study reminds clinicians that CRKP strains with multiple carbapenemase genes emerged in our hospital, and stronger measures should be taken to the control of nosocomial infections.

Surveillance of antibiotic resistance in the northern zone of Senegal: Molecular characterization of beta-lactam resistance genes in Enterobacteriaceae (extended spectrum beta-lactamase, plasmid cephalosporinases, carbapenemases).

Introduction: With the increase in bacterial resistance to antibiotics, policies are being implemented on a national scale. These resistances are multiple, especially in enterobacteria, and represent a real public health problem due to the difficulty of their treatment. The objective of this work was to identify ESBL, cephalosporinase and carbapenemase producing enterobacteria in laboratories in the northern part of Senegal and to compare the mechanisms involved. Materials and methods: This is a retrospective study conducted at the Saint-Louis Regional Hospital and the Microbiology Unit of the Pasteur Institute of Dakar. Bacterial strains were collected between January and June 2021 in eight bacteriology laboratories in the regions of Saint-Louis, Louga and Matam. Bacterial identification was done by studying morphological, cultural and biochemical characteristics using an API® 20 E gallery and the MALDI-TOFF technique. The antibiogram was performed by the disc diffusion method according to the CA-SFM version 2021 procedure including the search for specific resistances (ESBL). The ESBL genes (CTX, TEM, OXA, and SHV) were identified by polymerase chain reaction (PCR) with specific primers and then analyzed by gel electrophoresis. Results: Forty-five strains of Enterobacteriaceae were ESBL producers and each had at least one beta-lactam resistance gene. The CTX-M gene was found in all strains, followed by the OXA (64.44%), TEM (53.33%) and SHV (20%) genes. A simultaneous presence of these 4 genes was noted in 6 isolates (13.33%). Apart from resistance to ertapenem, all strains were sensitive to imipenem. For quinolones, almost all strains (93%) were resistant. Conclusion: These results show the need to reinforce the surveillance of ESBL-producing Enterobacteriaceae in the Senegal area. These important data should help to orientate strategies for the reduction of MRBs and their dissemination in the community.

Resistance to critically important antibiotics in hospital wastewater from the largest Croatian city

The emergence of extended-spectrum β-lactamase (ESBL)- and especially carbapenemases in Enterobacterales has led to limited therapeutic options. Therefore, it is critical to fully understand all potential routes of transmission, especially in high-risk sources such as hospital wastewater. This study aimed to quantify four enteric opportunistic pathogens (EOPs), total, ESBL- and carbapenem-resistant coliforms and their corresponding resistance genes (two ESBL and five carbapenemase genes) and to characterize enterobacterial isolates from hospital wastewater from two large hospitals in Zagreb over two seasons. Culturing revealed similar average levels of total and carbapenem-resistant coliforms (3.4 × 104 CFU/mL), and 10-fold lower levels of presumptive ESBL coliforms (3 × 103 CFU/mL). Real-time PCR revealed the highest E. coli levels among EOPs (105 cell equivalents/mL) and the highest levels of the blaKPC gene (up to 10−1 gene copies/16S copies) among all resistance genes examined. Of the 69 ESBL- and 90 carbapenemase-producing Enterobacterales (CPE) isolates from hospital wastewater, all were multidrug-resistant and most were identified as Escherichia coli, Citrobacter, Enterobacter, and Klebsiella. Among ESBL isolates, blaCTX-M-15 was the most prevalent ESBL gene, whereas in CPE isolates, blaKPC-2 and blaNDM-1 were the most frequently detected CP genes, followed by blaOXA-48. Molecular epidemiology using PFGE, MLST and whole-genome sequencing (WGS) revealed that clinically relevant variants such as E. coli ST131 (blaCTX-M-15/blaTEM-116) and ST541 (blaKPC-2), K. pneumoniae ST101 (blaOXA-48/blaNDM-1), and Enterobacter cloacae complex ST277 (blaKPC-2/blaNDM-1) were among the most frequently detected clone types. WGS also revealed a diverse range of resistance genes and plasmids in these and other isolates, as well as transposons and insertion sequences in the flanking regions of the blaCTX-M, blaOXA-48, and blaKPC-2 genes, suggesting the potential for mobilization. We conclude that hospital wastewater is a potential secondary reservoir of clinically important pathogens and resistance genes and therefore requires effective pretreatment before discharge to the municipal sewer system.

Molecular characterization of antimicrobial resistance related genes in E. coli, Salmonella and Klebsiella isolates from broilers in the West Region of Cameroon.

Antibiotic resistance has become an enduring threat to human health. This has prompted extensive research to identify the determinants responsible in a bid to fight the spread of resistance and also develop new antibiotics. However, routine procedures focus on identifying genetic determinants of resistance only on phenotypically resistant isolates. We aimed to characterise plasmid mediated resistance determinants in key Enterobacteriaceae isolates with differential phenotypic susceptibility profiles and evaluated the contribution of resistance genes on phenotypic expression of susceptibility. The study was carried out on 200 Enterobacteriaceae isolates belonging to the genera E. coli, Salmonella, and Klebsiella; 100 resistant and 100 susceptible to quinolones, aminoglycosides, and ESBL-producing as determined by disk diffusion. Reduced susceptibility in susceptible isolates was determined as an increased MIC by broth microdilution. Plasmid-borne resistance genes were sought in all isolates by endpoint PCR. We performed correlations tests to determine the relationship between the occurrence of resistance genes and increased MIC in susceptible isolates. We then used the notion of penetrance to show adequacy between resistance gene carriage and phenotypic resistance as well as diagnostic odds ratio to evaluate how predictable phenotypic susceptibility profile could determine the presence of resistant genes in the isolates. Reduced susceptibility was detected in 30% (9/30) ESBL negative, 50% (20/40) quinolone-susceptible and 53.33% (16/30) aminoglycoside-susceptible isolates. Plasmid-borne resistance genes were detected in 50% (15/30) of ESBL negative, 65% (26/40) quinolone susceptible and 66.67% (20/30) aminoglycoside susceptible isolates. Reduced susceptibility increased the risk of susceptible isolates carrying resistance genes (ORs 4.125, 8.36, and 8.89 respectively for ESBL, quinolone, and aminoglycoside resistance genes). Resistance gene carriage correlated significantly to reduced susceptibility for quinolone and aminoglycoside resistance genes (0.002 and 0.015 at CI95). Gene carriage correlated with phenotypic resistance at an estimated 64.28% for ESBL, 56.90% for quinolone, and 58.33% for aminoglycoside resistance genes. A high carriage of plasmid-mediated genes for ESBL, quinolone, and aminoglycoside resistance was found among the Enterobacteriaceae tested. However, gene carriage was not always correlated with phenotypic expression. This allows us to suggest that assessing genetic determinants of resistance should not be based on AST profile only. Further studies, including assessing the role of chromosomal determinants will shed light on other factors that undermine antimicrobial susceptibility locally.

A Novel Single-Tube Eicosaplex/Octaplex PCR System for the Detection of Extended-Spectrum β-Lactamases, Plasmid-Mediated AmpC β-Lactamases, and Integrons in Gram-Negative Bacteria.

We developed two multiplex polymerase chain reactions (PCRs) for the detection of extended-spectrum β-lactamases (ESBLs), plasmid-mediated AmpC β-lactamases, aac(6')-Ib gene, and integrase genes (intI1, intI2, and intI3) in class 1, 2, and 3 integrons in Gram-negative bacteria. We evaluated the PCRs using 109 Gram-negative isolates from non-organic (ANO) and organic (AO) vegetables and fruits. Screening of ANO substances identified five SHV, one TEM-1, one CTX-M, 20 AmpC-CS, and two intI1 positives. DNA sequencing revealed CTX-M in Pantoea spp. was blaRANH-2, a plasmid-mediated CTX-M related ESBL gene only found in Rahnella spp. Of the 20 AmpC-CS positives, 10 were CMY/MIR/ACT/EC (3 new variants), eight were ACT, one was AZECL, and one was new Pseudomonas-related AmpC family. Screening of AO substances identified 11 SHV, two TEM-1, three CTX-M (one OXY-2, two CTX-M-14/-15), two OXA-9, 13 AmpC-CS and one intI1 positives. The 13 AmpC-CS positives were five CMY/MIR/ACT/EC, three ACT, one MOX-12 variant, and four ADC (one ADC-25 and three new variants). We developed a rapid, easy-to-perform, low-cost, and reliable multiplex PCR system for screening clinically relevant β-lactamases and integrons in Gram-negative bacteria. We showed the prevalence of ESBLs and AmpC β-lactamases among our panel of ampicillin-resistant Gram-negative strains and detection of NDM and OXA carbapenemases.

An abnormal multidrug-resistant and hypervirulent <i>Klebsiella pneumoniae</i> clinical isolate without <i>rmpA</i> or <i>rmpA2</i>

<i>Klebsiella pneumoniae</i> is a notorious opportunistic pathogen, especially hypervirulent <i>K</i>. <i>pneumoniae</i> (hvKp). Fortunately, most classical hvKp strains are antibiotic-susceptible. However, in recent years, reports of multidrug-resistant hvKp (MDR-hvKp) have increased dramatically, threatening the health and safety of people worldwide. Here, we report the discovery of MDR-hvKp without <i>rmpA</i> and <i>rmpA2</i> in a 92-year-old patient with chronic obstructive pulmonary disease. The patient died on the seventh day of hospitalization. Phenotyping experiments and whole-genome sequencing of <i>K</i>. <i>pneumoniae</i> isolate 21072329 isolated from the patient’s sputum were performed. Moreover, 21072329 belongs to ST11-KL47 MDR-hvKp, which was highly lethal to <i>Galleria mellonella</i>. Meanwhile, 21072329 had a strong viscosity, and it was difficult to completely centrifuge it; 21072329 carried ESBL genes (<i>bla</i><sub>CTX-M-65</sub>, <i>bla</i><sub>SHV-158</sub>, and <i>bla</i><sub>TEM-1</sub>) and a carbapenemase gene (<i>bla</i><sub>KPC-2</sub>), and it was resistant to carbapenem antibiotics and third- and fourth-generation cephalosporins. Although 21072329 had the characteristics of hvKp, <i>rmpA</i> and <i>rmpA2</i> could not be found in its genome; it also only carried a siderophore of yersiniabactin. This may indicate that other hypervirulence factors promote the formation of hvKp. MDR-hvKp has already brought an enormous burden to global medical care, and those carrying unknown hypervirulence factors are new threats, so urgent prevention and control with research are urgently needed.

High burden of extended spectrum β–lactamase (ESBL)–encoding genes in third–generation cephalosporin–resistant Escherichia coli recovered from frequently contacted surfaces and wastewater of selected healthcare institutions in Nigeria

Objective: This study aimed to determine the carriage of extended spectrum β-lactamase (ESBL) genes in third-generation cephalosporin-resistant (3GCR) Escherichia (E.) coli from frequently contacted surfaces, wastewater and disinfectant-cleaning solutions of selected healthcare institutions in South-western Nigeria. Methods: Samples were collected over three months for the isolation of 3GCR E. coli on MacConkey agar containing 6 μg/mL of cefotaxime. 3GCR E. coli isolates were identified by detection of uidA gene and susceptibility to selected antibiotics was performed using disc-diffusion method. Detection of ESBL genes was done using primer-specific PCR. Results: A total of 22 ESBL-producing E. coli (11 each from the frequently contacted surfaces and wastewater) were obtained from the pool of 3GCR isolates in this study. No isolate was recovered from the disinfectant-cleaning solution. All the ESBL-producing E. coli obtained from the frequently contacted surfaces and wastewater were multidrug resistant, with complete resistance observed to ampicillin, cefotaxime, cefpodoxime, tetracycline and ertapenem. The ESBL genotyping showed that 54.5% carried bla CTX-M, 63.6% carried bla TEM and 9.1% carried bla SHV in isolates from the frequently contacted surfaces, while 63.6%, 9.1% and 18.2% carried bla CTX-M, bla TEM and bla SHV, respectively, in the isolates obtained from the wastewater. Conclusions: This study showed a high burden of multidrug resistance E. coli on frequently contacted surfaces and wastewater of the studied healthcare institutions, indicating the need for good hygiene and proper mitigation measures to prevent potential public health and environmental challenges.

A one-year genomic investigation of Escherichia coli epidemiology and nosocomial spread at a large US healthcare network

BackgroundExtra-intestinal pathogenic Escherichia coli (ExPEC) are a leading cause of bloodstream and urinary tract infections worldwide. Over the last two decades, increased rates of antibiotic resistance in E. coli have been reported, further complicating treatment. Worryingly, specific lineages expressing extended-spectrum β-lactamases (ESBLs) and fluoroquinolone resistance have proliferated and are now considered a serious threat. Obtaining contemporary information on the epidemiology and prevalence of these circulating lineages is critical for containing their spread globally and within the clinic.MethodsWhole-genome sequencing (WGS), phylogenetic analysis, and antibiotic susceptibility testing were performed for a complete set of 2075 E. coli clinical isolates collected from 1776 patients at a large tertiary healthcare network in the USA between October 2019 and September 2020.ResultsThe isolates represented two main phylogenetic groups, B2 and D, with six lineages accounting for 53% of strains: ST-69, ST-73, ST-95, ST-131, ST-127, and ST-1193. Twenty-seven percent of the primary isolates were multidrug resistant (MDR) and 5% carried an ESBL gene. Importantly, 74% of the ESBL-E.coli were co-resistant to fluoroquinolones and mostly belonged to pandemic ST-131 and emerging ST-1193. SNP-based detection of possible outbreaks identified 95 potential transmission clusters totaling 258 isolates (12% of the whole population) from ≥ 2 patients. While the proportion of MDR isolates was enriched in the set of putative transmission isolates compared to sporadic infections (35 vs 27%, p = 0.007), a large fraction (61%) of the predicted outbreaks (including the largest cluster grouping isolates from 12 patients) were caused by the transmission of non-MDR clones.ConclusionBy coupling in-depth genomic characterization with a complete sampling of clinical isolates for a full year, this study provides a rare and contemporary survey on the epidemiology and spread of E. coli in a large US healthcare network. While surveillance and infection control efforts often focus on ESBL and MDR lineages, our findings reveal that non-MDR isolates represent a large burden of infections, including those of predicted nosocomial origins. This increased awareness is key for implementing effective WGS-based surveillance as a routine technology for infection control.

Antimicrobial Resistance Profiles and Molecular Characteristics of Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolated from Healthy Cattle and Pigs in Korea.

Antimicrobial-resistant bacteria isolated from food animals pose a major health threat to the public on this planet. This study aimed to determine the susceptibility profiles of Escherichia coli isolated from cattle and pig fecal samples and investigate the molecular characteristics of extended-spectrum β-lactamase (ESBL)-producing E. coli using gene identification, conjugation, and Southern blot approach. Overall 293 E. coli were recovered from cattle (120 isolates) and pigs (173 isolates) in 7 provinces of Korea during 2017-2018. Ampicillin, chloramphenicol, streptomycin, and sulfisoxazole resistance rates were the highest in pigs' isolates (>60%, p ≤ 0.001) compared to that in cattle (3-39%). Multidrug resistance (MDR) was higher in pig isolates (73%) than in cattle (31%), and the MDR profile usually includes streptomycin, sulfisoxazole, and tetracycline. Resistance to critically important antimicrobials such as ceftiofur, colistin, and ciprofloxacin was higher in weaners than those from finishers in pigs. The qnrS gene was detected in 13% of the pig isolates. Eight isolates from pigs and one isolate from cattle were identified as ESBL-producers and ESBL genes belonged to blaCTX-M-55 (n = 4), blaCTX-M-14 (n = 3), and blaCTX-M-65 (n = 2). Notably, the blaCTX-M-65 and qnrS1 genes were found to be carried together in an identical plasmid (IncHI2) in two isolates from finisher pigs. The blaCTX-M-carrying isolates belonged to phylogenetic groups B1 (n = 4), B2 (n = 2), A (n = 2), and D (n = 1). The blaCTX-M genes and non-β-lactam resistance traits were transferred to the E. coli J53 recipient from seven blaCTX-M-positive strains isolated from pigs. The blaCTX-M genes belonged to the IncI1α, IncFII, and IncHI2 plasmids and are also associated with the ISEcp1, IS26, IS903, and orf477 elements. These findings suggested the possibility of blaCTX-M-carrying E. coli transmission to humans through direct contact with cattle and pigs or contamination of food products.

Antimicrobial Susceptibility and Genetic Prevalence of Extended-Spectrum β-Lactamases in Gram-Negative Rods Isolated from Clinical Specimens in Pakistan.

The prevalence of extended-spectrum β-lactamase (ESBL) genes has increased remarkably, resulting in multidrug-resistant gram-negative rods (GNRs) in clinical specimens. This cross-sectional study aimed to determine the antimicrobial susceptibility of ESBL-producing GNRs and its correlation with corresponding genes. Two hundred and seventy-two (n = 272) samples were evaluated for the molecular identification of ESBL genes by polymerase chain reaction after confirmation with the modified double-disc synergy test. E. coli 64.0% (n = 174) was the most prevalent ESBL producer, followed by Klebsiella species 27.2% (n = seventy-four), Acinetobacter species 6.6% (n = eighteen) and others 2.2% (n = six). These ESBL-producing isolates showed resistance to β-lactam antibiotics, i.e., sulbactam/cefoperazone (41.5%), piperacillin/tazobactam (39.3%), meropenem (36.0%), imipenem (34.2%) and non- β-lactam antibiotics, i.e., nalidixic acid (89.0%), co-trimoxazole (84.9%), ciprofloxacin (82.4%), gentamicin (46.3%), nitrofurantoin (24.6%), amikacin (19.9%) and fosfomycin (19.9%). The incidences of the ESBLs-producing genes blaCTX-M, blaTEM, blaOXA and blaSHV were 91.2%, 61.8%, 39.3% and 17.6%, respectively. Among nine multiple-gene combinations, blaCTX-M + blaTEM (30.5%) was the most prevalent combination, followed by blaCTX-M + blaOXA + blaTEM (14.0%), blaCTX-M + blaOXA (13.6%), blaCTX-M + blaTEM + blaSHV (7.0%), blaCTX-M + blaSHV (2.2%), blaCTX-M + blaOXA + blaSHV (2.2%) and blaOXA + blaTEM (1.8%). ESBLs producing GNRs carrying blaCTX-M, blaTEM, blaOXA and blaSHV showed resistances to β-lactam antibiotics, i.e., ampicillin, amoxillin-clavulanic acid, cefotaxime and ceftazidime but were susceptible to carbapenems (meropenem and imipenem), β-lactam-β-lactamase inhibitor combination (piperacillin/tazobactam) and non-β-lactam antibiotics i.e., aminoglycoside (amikacin and gentamicin), nitrofurantoin and fosfomycin. These antibiotics that demonstrated activity may be used to treat infections in clinical settings.

Evaluation of Antimicrobial Resistance of Different Phylogroups of Escherichia coli Isolates from Feces of Breeding and Laying Hens.

Animal and food sources are seen as a potential transmission pathway of multi-drug resistance (MDR) micro-organisms to humans. Escherichia. coli is frequently used as an indicator of fecal contamination in the food industry and known as a reservoir of antimicrobial resistance genes (ARGs). Microbial contamination as a major outcome for the poultry and egg industry and is a serious public health problem. In the present study we performed the quantification of β-glucoronidase positive E. coli in 60 fecal samples of breeding and laying hens collected in Portugal in 2019. Phylogenetic and pathotypic characterization, antimicrobial susceptibility, and detection of resistant extended-spectrum β-lactamase (ESBL) genes were assessed. The phylogenetic and pathogenic characterization and detection of ESBL genes were assessed by real-time PCR and antimicrobial susceptibility was evaluated using the disk diffusion method. Overall, E. coli quantification was 6.03 log CFU/g in breeding hens and 6.02 log CFU/g in laying hens. The most frequent phylogroups were B1. None of the isolates was classified as diarrheagenic E. coli (DEC). In total, 57% of the isolates showed MDR and 3.8% were positive for ESBL. Our study highlights that consumers may be exposed to MDR E. coli, presenting a major hazard to food safety and a risk to public health.

Whole Genome Sequencing of Antibiotic Resistant Genes in Isolates from Surfaces in a Science Laboratory.

Isolates obtained from laboratory surfaces were identified and characterized. Ten consecutive isolates were obtained from 30 sample surfaces of a University Science Laboratory in Edo State Nigeria in May, 2021. Swabs of surfaces from the laboratory were obtained aseptically. The sample swabs were streaked on MacConkey, eosin methylene blue, mannitol salt, and nutrient agar plates, respectively, and incubated appropriately. Distinct colonies were randomly obtained from culture plates and characterized phenotypically. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was used to analyze four isolates (40%) obtained by selection criteria. Susceptibility testing using antibiotics was performed for the identified isolates by Kirby-Bauer method for 15 antibiotics. Isolate characterization and identification of resistance determinants were determined using whole genome sequencing (WGS). Microorganisms identified included Leclercia adecarboxylata, Enterobacter hormaechei, Atlantibacter hermanii, and Stenotrophomonas maltophilia. Three identified isolates were antibiotics-resistant and were investigated by WGS. Resistance genes were found in all (100%) of the resistant laboratory isolates. The resistance determinants included β-lactamase genes, aminoglycoside modifying enzymes, qnr genes, sulfonamide, tetracycline, and trimethoprim resistance genes, respectively. Two isolates carried ESBL genes and blaCTX-M-15 was detected. Our study displays the dissemination of antibiotic resistance among isolates obtained from surface of a University Science Laboratory. To the best of our knowledge, we have reported the first genomic characterization of resistance to antibiotics in isolates obtained from surfaces of a University Science Laboratory in Nigeria.

Phenotypic ESBL and non-phenotypic ESBL isolates of Klebsiella pneumoniae exhibit differing responses to induced antimicrobials resistance and subsequent antibiotic cross-resistance.

To investigate the effect of adapting Klebsiella pneumoniae clinical isolates harboring ESBL genes to cetrimide (CT) in terms of subsequent cross-resistance to other biocides and antibiotics, and to investigate changes to virulence markers, such as biofilm formation and efflux activity. The changes between phenotypic extended spectrum β-lactamases (pESBL) expressing isolates and non-phenotypic ESBL (npESBL) isolates was compared. Kl. pneumoniae isolates (14 pESBL and 17 npESBL) were adapted to increasing concentrations of CT, until 4 × MIC was reached. The MIC of the adapted isolates was tested against chloroxylenol (CX) and chlorhexidine. Disk diffusion techniques were used to determine the susceptibility of the isolates to different antibiotics. Biofilm formation was assessed for the isolates using the crystal violet method and efflux pump activity was studied using the ethidium bromide assay. After CT adaptation, 100% of npESBL isolates and 85.7% of pESBL isolates showed increase in CT MIC after CT adaptation. While 41.2% of npESBL and 57.1% of the pESBL isolates showed a cross-resistance with chlorhexidine. CT adaptation resulted in a significant decrease in the susceptibility of npESBL isolates to aztreonam and cefotaxime compared to pESBL isolates, which could be linked to the increase in efflux activity of npESBL compared to pESBL. Biofilm formation was significantly increased after CT adaptation regardless of the type of isolate. The extensive use of biocides in the environment can induce cross-resistance to other biocides and antibiotics, and can increase the ability of bacteria to form biofilms. The response of bacteria to biocide adaptation differs between pESBL and npESBL isolates, although the effect is strain specific.

Antibiogram And Prevalence Of ESBL Genes In Escherichia Coli From Clinical Specimen

The members of the Enterobacteriaceae family are among the most common causative agents of various clinical infections. Some pathogens produce enzymes to escape the effect of antibiotics and the ESBL enzyme is the new strategy of the bacterial pathogen which is responsible for emerging antimicrobial resistance. The prevalence of Gram Negative bacteria that are usually associated with the production of ESBLs enzymes is on the rise around the globe, both in residing area and hospital setups, as a result, multiple therapeutic problems including antibiotic resistance are being faced. The current study was aimed to look at the characteristics, the pattern of antibiotic resistance, and to estimate the prevalence of ESBL-producing Escherichia coli in clinical isolates. The samples after collection were streaked and cultured on MacConkey, and chocolate, and blood agar. The identification of the isolates was done based on various biochemical characteristics, and the API 20-E test. The modified Disc Diffusion method was used to confirm ESBL enzyme synthesis, and the Kirby Bauer disc diffusion method was used to assess antibiotic susceptibility. Out of 121 isolates of E. coli, 51.2% (n=62) were recovered from female patients and 48.8% (n=59) were obtained from male patients. The genotypic detection of ESBL genes was done by PCR followed by gel electrophoresis. Of total 47 E. coli confirmed for the production of ESBLs, 91.4% (n=43) were found harbouring blaCTX-M followed by blaTEM and blaSHV with 21.2% (n=10) and 8.5% (n=4) prevalence. The table gives an overview of ESBL gene distribution in clinically isolated E. coli. The current study indicates the magnitude of antimicrobial resistance and the underlying genes especially the blaCTX-M gene among the E. coli isolates.

642. In Vitro Activity of Cefiderocol and Comparator Agents Against Molecularly Characterized Enterobacterales Clinical Isolates Causing Infection in United States Hospitals (2020-2021)

Abstract Background Cefiderocol (CFDC) is a siderophore cephalosporin that hijacks the Gram-negative bacteria iron transport system to facilitate cell entry and reach its target. CFDC remains stable to hydrolysis in the presence of serine β-lactamases (ESBLs, KPC, and OXA-type carbapenemases) and metallo-β-lactamases (MBL). CFDC and comparator activities were analyzed against Enterobacterales (ENT), including molecularly characterized isolates, as part of the SENTRY Antimicrobial Surveillance Program in the USA. Methods 8,328 ENT were collected from 32 sites in 2020-2021. Susceptibility testing was performed by broth microdilution. CFDC testing used iron-depleted media. CLSI breakpoints were used. E. coli, K. pneumoniae, and P. mirabilis with ceftriaxone, ceftazidime, or aztreonam MIC ≥2 μg/mL, and any ENT displaying MIC ≥2 μg/mL for imipenem (excluded for P. mirabilis, P. penneri, and indole-positive Proteeae) or meropenem (MER), were subjected to genome sequencing and screening of β-lactamase genes. Results In general, CFDC (≥ 99.9% susceptible [S]), imipenem-relebactam (IMR; 99.4-100%S), meropenem-vaborbactam (MEV; 100%S), and ceftazidime-avibactam (CZA; 100%S) were active against carbapenem-susceptible ENT that carried ESBL and/or AmpC genes (Table). CFDC (MIC50/90, 0.5/4 μg/mL; 98.4%S) and CZA (MIC50/90, 1/8 μg/mL; 91.2%S) were the most active agents against carbapenem-nonS isolates, whereas IMR (MIC50/90, 0.25/4 μg/mL; 81.6%S) and MEV (MIC50/90, 0.12/8 μg/mL; 86.4%S) had suboptimal activity. CFDC (MIC50/90, 0.5/2 μg/mL), IMR (MIC50/90, 0.12/0.5 μg/mL), MEV (MIC50/90, 0.03/0.5 μg/mL), and CZA (MIC50/90, 1/2 μg/mL) were active (100%S) against the KPC subset. CFDC (MIC, 0.5-4 μg/mL; 100%S) was also active against ENT carrying MBL genes, whereas CFDC (MIC, 0.5-2 μg/mL; 100%S) and CZA (1-4 μg/mL; 100%S) were active against isolates carrying blaOXA-48-like. Conclusion CFDC activity was consistent, regardless of phenotypes or genotypes, including against isolates carrying carbapenemase genes other than blaKPC, where approved β-lactam/β-lactamase inhibitor combinations showed limited activity. These data reinforce CFDC as an important option for the treatment of infections caused by ENT and resistant subsets. Disclosures Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Office for Assistant Secretary of Defense for Health Affairs: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support|Spero Therapeutics: Grant/Research Support John H. Kimbrough, PhD, AbbVie: Grant/Research Support|GSK: Grant/Research Support Valerie Kantro, BA, AbbVie: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Shionogi: Grant/Research Support Dee Shortridge, PhD, AbbVie: Grant/Research Support|JMI Laboratory: Employee|Melinta: Grant/Research Support|Menarini: Grant/Research Support|Shionogi: Grant/Research Support Helio S. Sader, MD, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Pfizer: Grant/Research Support Jennifer M. Streit, BS, MT(ASCP), Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Shionogi: Grant/Research Support Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support.

641. Activity of Gepotidacin Tested Against Molecularly Characterized Escherichia coli Isolates Resistant to Commonly Used Oral Therapies for UTI in the US (2019-2020)

Abstract Background Gepotidacin is a novel first in class triazaacenaphthylene antibiotic in Phase 3 clinical trials for the treatment of gonorrhea and uncomplicated urinary tract infection (UTI). This study evaluates the epidemiology of E. coli (EC) causing UTI in US patients and the activity of gepotidacin and comparators against various subsets, including those with characterized resistance mechanisms. Methods 1,993 EC collected from 45 US sites were included as part of the Gepotidacin UTI Global Surveillance Study as part of the SENTRY Antimicrobial Surveillance Program (2019-2020). Isolates were tested for susceptibility (S) by CLSI methods and CLSI interpretations were applied. Isolates that met MIC criteria for screening of extended-spectrum β-lactamase (ESBL) genes were subjected to genome sequencing followed by ESBL gene screening and epidemiology typing (MLST, O:H, and fimH). Results A total of 84.4% (1,682/1,993) EC were ESBL negative (Table). Among these isolates, 16.5% (278/1,682) were not S to the fluoroquinolones (FQ) ciprofloxacin and/or levofloxacin, whereas 26.2% (440/1,682) were not S to trimethoprim-sulfamethoxazole (SXT). An ESBL phenotype was noted in 15.6% (311/1,993) of EC, which tended to be mostly not S to the FQs (79.7%), SXT (61.7%), amoxicillin-clavulanate (52.9%), and oral cephalosporins (99.7%). Most ESBL isolates carried CTX-M alleles alone (81.4%; 253/311), whereas 9.6% (30/311) had plasmid AmpC genes. Approximately half (56.3%; 175/311) of ESBL isolates belonged to clonal complex (CC) 131, of which 70.9% (124/175) were O25b:H4 and carried fimH30. Overall, gepotidacin had MIC90 of 2-4 mg/L against various phenotypic/genotypic subsets. Conclusion High rates of EC not S to commonly used oral agents (FQs and SXT) were observed. ESBL phenotype further compromised the activity of oral agents, including oral cephalosporins. Gepotidacin had potent and stable in vitro activity against various subsets, including the resistant CC131 O25b:H4 clone. These data support the further clinical development of gepotidacin as a treatment option for UTI caused by EC, including resistant isolates against which other oral treatment options are limited. Disclosures Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Office for Assistant Secretary of Defense for Health Affairs: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support|Spero Therapeutics: Grant/Research Support SJ Ryan Arends, PhD, AbbVie: Grant/Research Support|GSK: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Shionogi: Grant/Research Support John H. Kimbrough, PhD, AbbVie: Grant/Research Support|GSK: Grant/Research Support Valerie Kantro, BA, AbbVie: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Shionogi: Grant/Research Support Nicole E. Scangarella-Oman, MS, GlaxoSmithKline plc.: Employee and shareholder Jennifer M. Streit, BS, MT(ASCP), Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Shionogi: Grant/Research Support Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support.

Antimicrobial Resistance Profile of Extended-spectrum Beta-lactamase Genes in Escherichia coli Isolates Using Multiplex PCR Technique

Background: Broad-spectrum antibiotic resistance genes are one of the most common developing resistance genes worldwide. Accordingly, it is of paramount importance to study the extended-spectrum beta-lactamase genes to report them to physicians to select the most appropriate treatment. Objectives: This study aimed to detect three genes of ESBL such as TEM, AmpC, and KPC simultaneously. Methods: Primers were designed for ESBL genes such as TEM, AmpC, and KPC with Genscript software. In this study, control-positive genes were used for the PCR set-up. Fifty isolates of Escherichia coli isolated in the Baqiyatallah Hospital were confirmed and checked by Multiplex PCR. Results: This study revealed that TEM, AmpC, and KPC primers could detect positive control genes. The sensitivity and specificity of the multiplex PCR technique for these genes were 0.001 ng and 100%, respectively. Conclusions: This study revealed that a Multiplex PCR with a sensitivity of 0.001 ng and 100% specificity can detect ESBL genes precisely. Accordingly, the rapid and precise detection of the antibiotic resistance genes and the recommendation of an appropriate treatment pattern can decrease the distribution of antibiotic resistance occurrence and economic cost.

Prevalence of Extended-spectrum b-lactamase Producing E. coli and Klebsiella spp. Isolated from Buffaloes in Eastern Plain Zone of Uttar Pradesh

Background: Antimicrobial resistance (AMR) due to emergence and spread of Extended-spectrum β-lactamase (ESBL) producing bacteria are point of discussion in both human as well as in animals across the world as it is one of the latest challenges faced by scientific community. The present study highlighted the prevalence of ESBL producing E. coli and Klebsiella spp. isolated from buffaloes in Uttar Pradesh. Methods: Total 240 samples were collected from two district of eastern plain zone of Uttar Pradesh during January 2020 to March 2021. E. coli and Klebsiella spp. isolates were confirmed by cultural characteristics in selective media and biochemical tests. ESBL producing isolates were confirmed by DDST, ESBL E-strip and PCR analysis using specific primer for (bla-CTX-M-1, bla-CTX-M-2, bla-CTX-M-9, bla-TEM and bla-SHV). Antibiogram of all confirmed ESBL producing isolates was performed against 20 antimicrobials under 12 different classes. Result: In the present study, a total of 147 (61.25%) E. coli and 19 (7.91%) Klebsiella spp. were isolated and identified based on cultural and biochemical characteristics. A total of 104 (62.65%) and 99 (59.63%) isolates were confirmed as ESBL producers using DDST and ESBL E-strip tests respectively. PCR analysis, revealed the presence of selected ESBL genes in 92 (55.42%) isolates, among them bla-CTX-M-1 was found most dominant gene (69.41%) in E. coli and (71.42%) in Klebsiella spp. All the ESBL positive E. coli and Klebsiella spp. isolates were found to be 99-100% and 86-100% sensitive to chloramphenicol, polypeptides and aminoglycosides classes respectively. Both isolates of E. coli and Klebsiella spp. showed 100% resistance to cefotaxime, cefpodoxime ceftriazone and ampicillin, 76.47% and 71.42% to ceftazidime respectively and 81.52% isolates were found to be MDR.

Prevalence of Antibiotic-Resistant E. coli Strains in a Local Farm and Packing Facilities of Honeydew Melon in Hermosillo, Sonora, Mexico.

Pathogenic strains of Escherichia coli threaten public health due to their virulence factors and antibiotic resistance. Additionally, the virulence of this bacterium varies by region depending on environmental conditions, agricultural practices, and the use of antibiotics and disinfectants. However, there is limited research on the prevalence of antibiotic-resistant E. coli in agriculture. Therefore, this research aimed to determine the antibiotic resistance of E. coli isolated from the Honeydew melon production system in Hermosillo, Sonora, Mexico. Thirty-two E. coli strains were isolated from 445 samples obtained from irrigation water, harvested melons, the hands of packaging workers, boxes, and discarded melons. The resistance profile of the E. coli strains was carried out to 12 antibiotics used in antimicrobial therapeutics against this bacterium; a high level of resistance to ertapenem (100%) was detected, followed by meropenem (97%), and ampicillin (94%); 47% of the strains were classified as multidrug-resistant. It was possible to identify the prevalence of the extended-spectrum β-lactamase (ESBLs) gene blaTEM (15.6%), as well as the non-ESBL genes qepA (3.1%) and aac(6')lb-cr (3.1%). The E. coli strains isolated from irrigation water were significantly associated with resistance to aztreonam, cefuroxime, amikacin, and sulfamethoxazole/trimethoprim. Irrigation water, packing workers' hands, and discarded melons showed a higher prevalence of antibiotic-resistant, ESBL, and non-ESBL genes of E. coli strains in a farm and packing facility of Honeydew melon in Hermosillo, Sonora.

Molecular detection and characterization of Shigella spp. harboring extended-spectrum β-lactamase genes in children with diarrhea in northwest Iran.

Shigellosis is one of the acute bowel infections and remains a serious public health problem in resource-poor countries. The present study aimed to survey the distribution of extended-spectrum β-lactamase (ESBL)-producing Shigella strains isolated from patients with diarrhea in northwest Iran. In the present cross-sectional study, from January 2019 to December 2020, 1280 fecal samples were collected from children with diarrhea in Ardabil, Iran. Multiplex PCR assay was applied for the presence of ipaH, invC, wbgZ, rfpB, and rfc genes to detect Shigella spp., Shigella sonnei, Shigella dysenteriae, Shigella flexneri, and Shigella boydii, respectively. Phenotypic detection of ESBL-producing isolates was carried out using the Double Disc Test (DDT). The frequency of main ESBL encoding genes including blaCTX-M, blaSHV, and blaTEM was detected using multiplex PCR. The genetic similarity of S. sonnei isolates was determined using ERIC PCR. A total of 49 Shigella isolates (3.8%; 49/1280) including 42 (85.7%) S. sonnei, 5 (10.2%) S. flexneri, and 2 (4%) S. dysenteriae were identified. S. boydii was not detected in any fecal samples. ESBLs were produced by 10.2% of Shigella spp. including 3 S. sonnei, 1 S. flexneri, and 1 S. dysenteriae. The ESBL encoding genes include blaCTX-M and blaTEM found in 65.3% and 61.2% of isolates, respectively. blaSHV gene was not detected in any isolates. The ERIC-PCR profiles allowed the differentiation of 42 S. sonnei strains into 6 clusters. Our study revealed a high frequency of ESBL-encoding genes among Shigella spp. in northwest Iran. The high prevalence of S. sonnei harboring ESBL genes, in the present work, is the main challenge for dysentery treatment, and this concern justifies the need for effective and regular monitoring of antibiotic usage among patients.