Prevalent ESBL Genes Research Articles

Prevalence and molecular characterization of ESBL-producing Escherichia coli isolated from broiler chicken and their respective farms environment in Malaysia.

Extended spectrum beta-lactamase-producing Escherichia coli (ESBL-EC) is an increasing public health threat. This study aimed to determine the prevalence and characterization of ESBL-producing Escherichia coli (E. coli) isolated from broiler chicken and their farm environment, in Kelantan Malaysia. Escherichia coliwas isolated from 453 collected samples, including 210 cloacal swabs and 243 environmental samples. The antimicrobial susceptibility profile of the E. coli isolates was assessed for sixteen antibiotics using the disc diffusion method. The E. coli isolates were evaluated for phenotypic ESBL production using modified double disc synergy. After extraction of genomic DNA, ESBL resistance genes, phylogenetic group, and virulence genes were detected by PCR using appropriate primers. ESBL genes were further confirmed by sequencing. The molecular typing of E. coli strains was determined by Multilocus Sequence Typing (MLST). A total of 93.8% (425/453) E. coli were isolated from the collected samples. Out of 334 E. coli isolates screened, 14.7% (49/334) were phenotypically ESBL producers. All the ESBL-EC were resistant to tetracycline, ciprofloxacin, and ampicillin. Thus, 100% of the ESBL-EC were multidrug resistant. Of the ESBL-EC 81.6% were positive for at least one ESBL encoding gene. The most prevalent ESBL gene detected was blaTEM (77.6%; 38/49) followed by blaCTX-M (32.7%; 16/49) and blaSHV (18.4%; 9/49). The majority of ESBL-EC belonged to phylogenic groups A followed by B1 accounting for 44.9% and 12.2%, respectively. The most frequently identified sequence types were ST10 (n = 3) and ST206 (n = 3). The most detected virulence genes in the E. coli isolates were astA (33.3%; 22/66) followed by iss (15.2%; 10/66). Our results show both broiler chicken and their respective farms environment were reservoirs of multi-drug resistant ESBL-producing E. coli and ESBL resistance genes.

Extended-spectrum beta-lactamase-producing Enterobacterales in human health: Experience from the tricycle project, Ghana.

Vulnerable groups, such as pregnant women, are at increased risk of potentially life-threatening infections with extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E) for both mother and newborn. However, data regarding ESBL-E carriage and associated risk factors in Ghanaian pregnant women remain scarce. This study aimed to determine the prevalence of ESBL-E carriage and its associated risk factors among pregnant women attending the antenatal clinic at the Korle Bu Teaching Hospital. A systematic sample of 700 pregnant women with gestational age ≥ 34 weeks attending the antenatal clinic at Korle Bu Teaching Hospital was included in the study. After administering a structured questionnaire to assess potential risk factors associated with ESBL-E carriage, patients were given a sterile stool container to submit at least 1 g of stool specimen. Recovered isolates from faecal specimens were identified using MALDI-TOF-MS technology. These isolates were then subjected to susceptibility testing and ESBL identification. A random subset of 24 ESBL-producing Escherichia coli isolates was whole-genome sequenced on the MiSeq Illumina platform. Risk factors associated with ESBL-E carriage were determined using multivariable logistic regression analysis. Among the 700 pregnant women, 42% (294) carried ESBL-E. The predominant ESBL-producing Enterobacterales were Escherichia coli (95%). Fifty percent (50%) of ESBL-E were multidrug resistant isolates (MDRs). Whole-genome sequencing of 24 ESBL-producing E. coli isolates revealed that blaCTX-M-15 (96%) was the most prevalent ESBL gene type. Notably, most isolates belonged to commensal phylogenetic groups (A, B1, and C; 88%). Having a primary level of education (aOR 1.45, 95% CI 1.05-1.96) and consuming legumes as the main source of protein (aOR 0.17, 0.40-0.83) were significantly associated with intestinal carriage of ESBL-E. This study identified a high prevalence of ESBL-E and MDR-ESBL-E carriage among pregnant women. Our findings underscore the urgent need for public health interventions to control the spread of AMR.

Prevalence of bacteremia and antimicrobial resistance pattern among patients in South Lebanon

Bacteremia is a leading cause of morbidity and mortality worldwide. Rising prevalence and antimicrobial resistance (AMR) are critical public health issues. This study aims to determine the prevalence of bacteremia and the AMR pattern among patients in South Lebanon. A cross-sectional study analyzed 76 positive blood cultures from Hammoud and Labib Hospitals in South Lebanon between September 2023 and March 2024. The phenotype and antimicrobial susceptibility of gram-positive and gram-negative were determined by using disk diffusion. Genotypically, polymerase chain reaction was used to detect the carbapenemase-resistant Enterobacterales (CRE), extended-spectrum β-lactamases (ESBL), and methicillin-resistant Staphylococcus aureus genes. Out of 76 isolates, 38 (50%) were gram-positive and 38 (50%) were gram-negative. Escherichia coli was the most common among gram-negative (18. 42%), with 10.52% ESBL and 3.94% CRE. Staphylococcus coagulase negative was the most common among gram-positive (40.78%), followed by Staphylococcus aureus (6.57%), with 3.94% methicillin-resistant S. aureus. The prevalent ESBL gene was CTX-M (100%), and for the CRE, NDM (66.66%) was the most common gene. Regarding S. aureus, 66.66% were mecA. The diverse bacteremia isolates and resistance genes in South Lebanon reflect global variability in incidence and resistance profiles. High rates of bacteremia and AMR in South Lebanon underscore the need for effective antibiotic stewardship programs.

Treated municipal wastewater as a source of high-risk and emerging multidrug-resistant clones of E. coli and other Enterobacterales producing extended-spectrum β-lactamases

Extended-spectrum β-lactamase (ESBL)-producing Enterobacterales are a major public health problem, and wastewater from municipal wastewater treatment plants (WWTPs) is a potential means of spreading them into the environment and community. Our objective was to isolate ESBL-producing E. coli and other Enterobacterales from wastewater after treatment at Croatia's largest WWTP and to characterize these isolates by phenotypic and genotypic testing. Of the 200 bacterial isolates, 140 were confirmed as Enterobacterales by MALDI-TOF MS, with Escherichia coli and Klebsiella spp. predominating (69% and 7%, respectively). All 140 enterobacterial isolates were multidrug-resistant (MDR) and produced ESBLs. The most prevalent ESBL genes among the isolates tested were blaCTX-M-15 (60%), blaTEM-116 (44%), and blaCTX-M-3 (13%). Most isolates (94%) carried more than one ESBL gene in addition to blaCTX-M. Genes encoding plasmid-mediated AmpC, most notably blaEBC, were detected in 22% of isolates, whereas genes encoding carbapenemases (blaOXA-48, blaNDM-1, blaVIM-1) were less represented (10%). In E. coli, 9 different sequence types (ST) were found, with the emerging high-risk clones ST361 (serotype A-O9:H30) and pandemic ST131 (serotype B2–O25:H4) predominating (32% and 15%, respectively). Other high-risk E. coli clones included ST405 (3%), ST410 (3%), CC10 (3%), ST10 (3%), and ST38 (2%), and emerging clones included ST1193 (2%) and ST635 (2%). Whole-genome sequencing of three representative E. coli from two dominant clone groups (ST361 and ST131) and one extensively drug-resistant K. oxytoca revealed the presence of multiple plasmids and resistance genes to several other antibiotic classes, as well as association of the blaCTX-M-15 gene with transposons and insertion sequences. Our findings indicate that treated municipal wastewater contributes to the spread of emerging and pandemic MDR E. coli clones and other enterobacterial strains of clinical importance into the aquatic environment, with the risk of reintroduction into humans.

Presence of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Food-Producing and Companion Animals and Wildlife on Small-Holder Farms of Floreana Island, Galápagos Islands.

Background: Antimicrobial resistance (AR) has led to increasing human and animal morbidity and mortality and negative consequences for the environment. AR among Escherichia coli (EC) is on the rise, with serious concerns about extended-spectrum β-lactamase-producing E. coli (ESBL-EC). In the Galápagos Islands, where antimicrobials are available without a prescription, growing demands for food production can drive antimicrobial use. Food producing animals are at the interface of wildlife and environmental health on the smallest human-inhabited Galápagos Island, Floreana. We sought to determine if ESBL-EC were present in Floreana Island farm animal species and nearby wildlife and the relatedness of ESBL-EC isolates identified. Materials and Methods: During July 4-5, 2022, we visited 8 multispecies farms, representing 75% of food-producing animal production on Floreana, and collected 227 fecal samples from farm animals and wildlife. Each sample was plated on MacConkey agar supplemented with cefotaxime (4 μg/mL). Results: ESBL-EC was isolated from 20 (9%) fecal samples collected from pigs (N = 10), chickens (N = 6), wildlife (N = 3), and dog (N = 1). All ESBL-EC isolates were from samples taken at three (38%) of the eight farms. Fifteen (75%) of the ESBL-EC isolates were from a single farm. All ESBL-EC isolates were multidrug resistant. The most prevalent ESBL genes belonged to the blaCTX-M group. Among the typeable isolates from the farm with the largest proportion of ESBL-EC isolates (N = 14), we observed nine unique pulsed-field gel electrophoresis (PFGE) patterns, with identical patterns present across pig and chicken isolates. PFGE patterns in the three farms with ESBL-EC isolates were different. Conclusions: These results lend support for future routine AR monitoring activities at the livestock-wildlife interface in Galápagos to characterize potential interspecies transmission of AR bacteria and AR genes in this unique protected ecosystem, and the related human, animal, and environmental health impacts, and to formulate interventions to reduce AR spread in this setting.

Carriage of ESBL-producing Klebsiella pneumoniae and Escherichia coli among children in rural Ghana: a cross-sectional study

BackgroundExtended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae (ESBL-KP) and Escherichia coli (ESBL-EC) present a high burden in both communities and healthcare sectors, leading to difficult-to-treat infections. Data on intestinal carriage of ESBL-KP and ESBL-EC in children is scarce, especially in sub-Saharan African countries. We provide data on faecal carriage, phenotypic resistance patterns, and gene variation of ESBL-EC and ESBL-KP among children in the Agogo region of Ghana.MethodsFrom July to December 2019, fresh stool samples were collected within 24 h from children < 5 years with and without diarrhoea attending the study hospital. The samples were screened for ESBL-EC and ESBL-KP on ESBL agar and confirmed using double-disk synergy testing. Bacterial identification and an antibiotic susceptibility profile were performed using the Vitek 2 compact system (bioMérieux, Inc.). ESBL genes, blaSHV, blaCTX-M, and blaTEM were identified by PCR and further sequencing.ResultsOf the 435 children recruited, stool carriage of ESBL-EC and ESBL-KP was 40.9% (n/N = 178/435) with no significant difference in prevalence between children with diarrhoea and non-diarrhoea. No association between ESBL carriage and the age of the children was found. All isolates were resistant to ampicillin and susceptible to meropenem and imipenem. Both ESBL-EC and ESBL-KP isolates showed over 70% resistance to tetracycline and sulfamethoxazole-trimethoprim. Multidrug resistance was observed in over 70% in both ESBL-EC and ESBL-KP isolates. The blaCTX-M-15 was the most prevalent ESBL gene detected. blaCTX-M-27, blaCTX-M-14, and blaCTX-M-14b were found in non-diarrhoea stools of children, whereas blaCTX-M-28 was found in both the diarrhoea and non-diarrhoea patient groups.ConclusionsThe carriage of ESBL-EC and ESBL-KP among children with and without diarrhoea in the Agogo community with a high prevalence of blaCTX-M-15 is noteworthy, highlighting the importance of both the population as a possible reservoir. This study reports for the first time the ESBL gene blaCTX-M-28 among the studied populations in Ghana.

Genotypic Determination of Extended Spectrum β-Lactamases and Carbapenemase Production in Clinical Isolates of Klebsiella pneumoniae in Southwest Nigeria

Introduction: Klebsiella pneumoniae is a major pathogen implicated in healthcare-associated infections. Extended-spectrum β-lactamase (ESBL) and carbapenemase-producing K. pneumoniae isolates are a public health concern. This study investigated the existence of some ESBL and carbapenemase genes among clinical isolates of K. pneumoniae in Southwest Nigeria and additionally determined their circulating clones. Materials and Methods: Various clinical samples from 420 patients from seven tertiary hospitals within Southwestern Nigeria were processed between February 2018 and July 2019. These samples were cultured on blood agar and MacConkey agar, and the isolated bacteria were identified by Microbact GNB 12E. All K. pneumoniae were confirmed by polymerase chain reaction (PCR) using the 16s rRNA gene. Antibiotic susceptibility testing (AST) was done on these isolates, and the PCR was used to evaluate the common ESBL-encoding genes and carbapenem resistance genes. Genotyping was performed using multi-locus sequencing typing (MLST). Results: The overall prevalence of K. pneumoniae in Southwestern Nigeria was 30.5%. The AST revealed high resistance rates to tetracyclines (67.2%), oxacillin (61.7%), ampicillin (60.2%), ciprofloxacin (58.6%), chloramphenicol (56.3%), and lowest resistance to meropenem (43.0%). All isolates were susceptible to polymyxin B. The most prevalent ESBL gene was the TEM gene (47.7%), followed by CTX-M (43.8%), SHV (39.8%), OXA (27.3%), CTX-M-15 (19.5%), CTX-M-2 (11.1%), and CTX-M-9 (10.9%). Among the carbapenemase genes studied, the VIM gene (43.0%) was most detected, followed by OXA-48 (28.9%), IMP (22.7%), NDM (17.2%), KPC (13.3%), CMY (11.7%), and FOX (9.4%). GIM and SPM genes were not detected. MLST identified six different sequence types (STs) in this study. The most dominant ST was ST307 (50%, 5/10), while ST258, ST11, ST147, ST15, and ST321 had (10%, 1/10) each. Conclusion: High antimicrobial resistance in K. pneumoniae is a clear and present danger for managing infections in Nigeria. Additionally, the dominance of a successful international ST307 clone highlights the importance of ensuring that genomic surveillance remains a priority in the hospital environment in Nigeria.

Characterization of antibiotic resistance genes and mobile elements in extended-spectrum β-lactamase-producing Escherichia coli strains isolated from hospitalized patients in Guangdong, China.

This study aimed to investigate the high-resolution phenotypic and genotypic characterization of extended-spectrum β-lactamase (ESBL)-producing E. coli strains isolated from hospitalized patients to explore the resistance genes and mobile genetic elements (MGEs) involved in horizontal dissemination. Between May and September 2021, a total of 216 ESBL-producing E. coli isolates were recovered from multiple departments. The identification of strains was performed using MALDI-TOF mass spectrometry and PCR, while antibiotic susceptibility testing was carried out using the Vitek 2 COMPACT system to determine resistance patterns, while PCR was used to detect different resistance genes and MGEs. In addition, a conjugation assay was performed to investigate the horizontal gene transfer of resistance genes. Selected isolates underwent whole-genome sequencing using the Illumina MiSeq platform. A total of 216 out of 409 E. coli isolates recovered from a tertiary hospital were observed to be ESBL-producing, giving a carriage rate of 52.8%, as determined by phenotypic screening. The most frequent sources of ESBL-producing E. coli isolates were urine (129/216, 59.72%,) and blood (50/216, 23.14%). The most prevalent ESBL genes identified were blaCTX-M (60.18%), blaTEM (40.27%), and blaSHV (18.05%). Three E. coli isolates were found to carry the genes blaNDM, mcr-1, and fosA3 genes. The most prevalent MGEs were IS26 (95.37%), Int (87.03%), and IncFIB (76.85%). Whole-genome sequencing analysis of eight MDR E. coli strains revealed that these isolates belonged to eight different sequence types (STs) and serotypes and were found to harbor multiple plasmid replicons and virulence factors. This study highlights a high incidence of antibiotic resistance genes and MGEs associated with the dissemination of ESBLs and other resistance genes.

Co-Harboring of Beta-Lactamases and mcr-1 Genes in Escherichia coli and Klebsiella pneumoniae from Healthy Carriers and Backyard Animals in Rural Communities in Ecuador.

Few studies have addressed drug resistance of Enterobacterales in rural communities in developing countries. This study aimed to determine the coexistence of extended-spectrum β-lactamase (ESBL) and carbapenemase genes in Escherichia coli and Klebsiella pneumoniae strains carrying the mcr-1 gene in rural communities in Ecuador from healthy humans and their backyard animals. Sixty-two strains, thirty E. coli and thirty-two K. pneumoniae strains carrying the mcr-1 gene were selected from a previous study. PCR were performed for the presence of ESBLs and carbapenemase genes. The strains were further characterized, and the genetic relationship was studied with multi-locus sequencing typing (MLST) of seven housekeeping genes. Fifty-nine of the sixty-two mcr-1 isolates (95%) harbored at least on β-lactam resistance gene. The most prevalent ESBL genes were the blaTEM genes (present in in 80% of the E. coli strains) and the blaSHV gene (present in 84% of the K. pneumoniae strains). MSLT analysis revealed 28 different sequence types (ST); 15 for E. coli and 12 for K. pneumoniae, with most ST never described in humans and animals. The coexistence of mcr-1 and β-lactams resistant genes in E. coli and K. pneumoniae strains is alarming and threatens the efficacy of last-resort antibiotics. Our findings highlight backyard animals as a reservoir of mcr-1/β-lactams resistant genes.

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.

Prevalence of extended spectrum Beta-Lactamase producing Klebsiella species from patients' specimens in a tertiary teaching hospital in Ile-Ife, Southwest Nigeria.

This study determined the prevalence of ESBL genes amongst Klebsiella species isolated from patients' specimens attending a Tertiary Teaching Hospital, in Ile-Ife, Southwest Nigeria. A cross sectional study of presumptive isolates of Klebsiella (n=180) were collected, after ethical approval in the microbiology laboratory. Isolates were identified to species level by conventional biochemical tests and MicrobactTM 24E Identification Kits. Antibiotic susceptibility testing was performed by the Kirby Bauer's disk diffusion method. ESBL production was detected by the double disc synergy and ESBL genes by the multiplex PCR protocol. The Klebsiella species identified were Klebsiella pneumoniae 95.6%, Klebsiella oxytoca 3.3%, Klebsiella ornithinolytica 0.6% and Klebsiella terrigena 0.6%. The prevalence of ESBL genes among the Klebsiella isolates was 47.2%, and the most common ESBL gene was blaSHV (38.9%), also 75% of the study isolates had MAR index greater than 0.2. The study establishes the prevalence of Extended Spectrum Beta-Lactamase producing Klebsiella sp in the hospital and identified the most prevalent ESBL genes circulating as blaSHV followed by blaTEM and blaCTX-M in this environment. This underscores the need for regular and continuous surveillance of antimicrobial resistance trend as a strong component of the antibiotics stewardship and infection prevention and control programs in the hospital.

Intestinal Carriage of Extended-Spectrum β-lactamase and Carbapenemase-Producing Enterobacteriaceae in Hemodialysis Patients

Background: Gastrointestinal colonization with resistant pathogens is significant because they could be easily transmitted to other hosts or spread to different microbiota through mobile genetic elements. Objectives: This study assessed the prevalence of fecal carriage of extended-spectrum β-lactamase-producing and carbapenemase-producing Enterobacteriaceae (ESBL-E and CPE, respectively) among hemodialysis patients and the factors affecting their emergence in a hospital in Tehran. Methods: From January 2018 to May 2019, 150 hemodialysis patients referred to a hospital in Tehran were sampled in this study. Stool samples of the patients were diluted in saline and cultured on MacConkey agar plates containing cefotaxime, ceftazidime, imipenem, and meropenem discs. The clinical data were analyzed to identify the risk factors using a logistic regression model. Results: The colonization rate of ESBL-E was 48.6%, while only 2% of patients were identified as the carriers of CPE (3 of 150). A higher prevalence rate was obtained for intestinal carriage of ESBL-E among hemodialysis patients aged 18 to 42 years using multivariate analysis. The prevalence rate of multidrug-resistant isolates was 73.8%. The blaCTX-M1 gene was identified as the most prevalent ESBL gene. Among carbapenemase-encoding genes, blaKPC and blaoxa-48 were found in 12 and two isolates, respectively. Conclusions: These results demonstrated a high prevalence rate of ESBLs among hemodialysis patients, although this rate was low for carbapenemases. Therefore, more control measures should be taken in hospitals to prevent the spread of antibiotic resistance genes in healthcare settings.

Synergistic Combination of AS101 and Azidothymidine against Clinical Isolates of Carbapenem-Resistant Klebsiella pneumoniae.

Owing to the over usage of carbapenems, carbapenem resistance has become a vital threat worldwide, and, thus, the World Health Organization announced the carbapenem-resistant Enterobacteriaceae (CRE) as the critical priority for antibiotic development in 2017. In the current situation, combination therapy would be one solution against CRE. Azidothymidine (AZT), a thymidine analog, has demonstrated its synergistically antibacterial activities with other antibiotics. The unexpected antimicrobial activity of the immunomodulator ammonium trichloro(dioxoethylene-o,o’)tellurate (AS101) has been reported against carbapenem-resistant Klebsiella pneumoniae (CRKP). Here, we sought to investigate the synergistic activity between AS101 and AZT against 12 CRKP clinical isolates. According to the gene detection results, the blaOXA-1 (7/12, 58.3%), blaDHA (7/12, 58.3%), and blaKPC (7/12, 58.3%) genes were the most prevalent ESBL, AmpC, and carbapenemase genes, respectively. The checkerboard analysis demonstrated the remarkable synergism between AS101 and AZT, with the observable decrease in the MIC value for two agents and the fractional inhibitory concentration (FIC) index ≤0.5 in all strains. Hence, the combination of AS101 and azidothymidine could be a potential treatment option against CRKP for drug development.

Evaluation of efflux pumps overexpression and β-lactamase genes among colistin resistant Pseudomonas aeruginosa

Pseudomonas aeruginosa (PA) is one of the major factors in severe opportunistic and hospital-acquired infections. Structural mechanisms such as resistance–nodulation–division (RND) efflux pumps and drug resistance genes such as extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) are factors causing drug resistance. Colistin is the prominent therapeutic strategy with an excellent inhibitory role for combating multidrug-resistant (MDR) and extensively drug-resistant (XDR) isolates. Wide usage of colistin and genetics interactions leads to the emergence of colistin-resistant (CoR) PA isolates which is a dangerous occurrence in XDR-PA isolates. The current study investigates the presence and expression of PA-related RND genes and harboring the antibiotic resistance genes such as ESBL and MBL genes in CoR-PA strains.70 clinical isolates were obtained from different clinical specimens during 2019–2020. The antimicrobial susceptibility testing was performed by the both Kirby-Bauer disk agar diffusion method and the minimum inhibitory concentration (MIC). Presence of blaTEM-1, blaSHV, blaCTX-M, blaIMP, blaVIM and blaNDM was measured in all CoR-PA isolates. Besides, screening of mcr-1, aadA, aadB and efflux pump genes (mexA, mexD, mexE and mexY) was conducted for CoR-PA isolates. To determine the expression level of efflux pump genes, Quantitative real-time PCR (qRT-PCR) reactions were performed.According to the standard antimicrobial susceptibility methods 39 isolates, (55.7%) and 31 isolates (44.3%) were considered as MDR-PA and XDR-PA strains, respectively. 12 isolates (17.1%) displayed resistance to colistin using MIC assessment. All CoR-PA isolates were negative for mcr-1 gene. The blaSHV (n = 11, 91.6%) and blaNDM (n = 7, 58.3%) were the most prevalent ESBL and MBL genes among CoR-PA, respectively. PCR results showed all CoR-PA isolates were positive for all 4 efflux pumps genes. On the other hand, overexpression of MexA, MexD, MexE and MexY genes was observed in 5 (41.6%), 5 (41.6%), 5 (41.6%) and 6 (50%) CoR-PA isolates by qRT-PCR, respectively. 9 (75%) CoR-PA isolates overexpressed at least one of the four efflux pumps and overexpression of altogether 4 efflux pumps was shown in 2 (16.6%) CoR-PA isolates. Overexpression of MexA, MexD, MexE and MexY genes was associated to the resistance toward imipenem, ciprofloxacin, ceftazidime and aminoglycosides, respectively.The current research demonstrated the co-existence of CoR-PA with ESBL and MBL genes besides overexpression of efflux pumps. The distribution of such isolates can lead to a burden on the healthcare system by the incidence of the most crucial situation named pan-drug resistant PA strains.

On-Farm Anaerobic Digestion of Dairy Manure Reduces the Abundance of Antibiotic Resistance-Associated Gene Targets and the Potential for Plasmid Transfer.

The present study investigated the impact of on-farm anaerobic digestion on the abundance of enteric bacteria, antibiotic resistance-associated gene targets, and the horizontal transfer potential of extended-spectrum β-lactamase (ESBL) genes. Samples of raw and digested manure were obtained from six commercial dairy farms in Ontario, Canada. Digestion significantly abated populations of viable coliforms in all six farms. Conjugative transfer of plasmids carrying β-lactamase genes from manure bacteria enriched overnight with buffered peptone containing 4 mg/liter cefotaxime into a β-lactam-sensitive green fluorescent protein (GFP)-labeled Escherichia coli recipient strain was evaluated in patch matings. Digestion significantly decreased the frequency of the horizontal transfer of ESBL genes. Twenty-five transconjugants were sequenced, revealing six distinct plasmids, ranging in size from 40 to 180 kb. A variety of ESBL genes were identified: blaCTX-M-1, blaCTX-M-14, blaCTX-M-15, blaCTX-M-27, blaCTX-M-55, and blaPER-1. blaCTX-M-15 was the most prevalent ESBL gene detected on plasmids harbored by transconjugants. Various mobile genetic elements were found located proximal to resistance genes. Ten gene targets, including sul1, str(A), str(B), erm(B), erm(F), intI1, aadA, incW, blaPSE, and blaOXA-20, were quantified by quantitative PCR on a subset of 18 raw and 18 digested samples. Most targets were significantly more abundant in raw manure; however, erm(B) and erm(F) targets were more abundant in digested samples. Overall, on-farm digestion of dairy manure abated coliform bacteria, a number of antibiotic resistance-associated gene targets, and the potential for in vitro conjugation of plasmids conferring resistance to extended-spectrum β-lactams and other classes of antibiotics into E. coli CV601. IMPORTANCE Using livestock manure for fertilization can entrain antibiotic-resistant bacteria into soil. Manure on some dairy farms is anaerobically digested before being land applied. Recommending the widespread implementation of the practice should be founded on understanding the impact of this treatment on various endpoints of human health concern. Although lab-scale anaerobic treatments have shown potential for reducing the abundance of antibiotic resistance genes, there are very few data from commercial farms. Anaerobic digestion of manure on six dairy farms efficiently abated coliform bacteria, E. coli, and a majority of antibiotic resistance-associated gene targets. In addition, the conjugation potential of plasmids carrying ESBL genes into introduced E. coli strain CV601 was reduced. Overall, anaerobic digestion abated coliform bacteria, the genes that they carry, and the potential for ESBL-carrying plasmid transfer.

High prevalence of blaCTX-M-15 and nosocomial transmission of hypervirulent epidemic clones of Klebsiella pneumoniae at a tertiary hospital in Ethiopia

BackgroundGenomic epidemiology of antibiotic resistance is not sufficiently studied in low-income countries.ObjectivesTo determine prevalence of ESBL production, and resistome and virulome profiles, of Klebsiella pneumoniae isolated at Jimma Medical Center, Ethiopia.MethodsStrains isolated from patients with suspected infections between June and November 2016 were characterized by MALDI-TOF for species identification and disc diffusion for antimicrobial susceptibility testing. All K. pneumoniae isolates were characterized by double disc diffusion for ESBL production and all ESBL-producing strains (ESBL-KP) were subjected to WGS on the Illumina (HiSeq 2500) platform. DNA was extracted by automated systems (MagNA Pure 96). Genome assembly was performed using SPAdes (v. 3.9) and draft genomes were used for analysing molecular features of the strains. Maximum likelihood trees were generated using FastTree/2.1.8 based on SNPs in shared genomic regions to identify transmission clusters.ResultsOf the 146 K. pneumoniae strains isolated, 76% were ESBL-KP; 93% of the ESBL-KP strains showed resistance to multiple antimicrobial classes. blaCTX-M-15 (84.4%) was the most prevalent ESBL gene. Resistance genes for aminoglycosides and/or fluoroquinolones [aac(6′)-Ib-cr (65.1%)], phenicols [catB3 (28.4%)], sulphonamides [sul1 (61.2%) and sul2 (60.5%)], trimethoprim [dfrA27 (32.1%)], macrolides [mph(A) (12.8%)] and rifampicin [arr2/arr3 (39.4%)] were prevalent. Plasmids of the IncF and IncR families were prevalent among ST218, ST147, ST15 and ST39. KL64 and KL57 capsular types and O1 and O2 LPSs were prevalent. A high-risk clone, ST218-KL57 encoding rmpA1/rmpA2 and iutA, was detected. Phylogenetic analysis showed a cluster of clonally related strains from different units of the hospital.ConclusionsPrevalence of ESBL-KP was high and blaCTX-M-15 was the predominant ESBL gene. ESBL genes had spread through both clonal and polyclonal expansion of high-risk and hypervirulent clones. Nosocomial transmission of MDR strains between different units of the hospital was observed.

Comparative genomics of ESBL-producing Escherichia coli (ESBL-Ec) reveals a similar distribution of the 10 most prevalent ESBL-Ec clones and ESBL genes among human community faecal and extra-intestinal infection isolates in the Netherlands (2014-17).

IntroductionThe human gut microbiota is an important reservoir of ESBL-producing Escherichia coli (ESBL-Ec). Community surveillance studies of ESBL-Ec to monitor circulating clones and ESBL genes are logistically challenging and costly.ObjectivesTo evaluate if isolates obtained in routine clinical practice can be used as an alternative to monitor the distribution of clones and ESBL genes circulating in the community.MethodsWGS was performed on 451 Dutch ESBL-Ec isolates (2014–17), including 162 community faeces and 289 urine and blood isolates. We compared proportions of 10 most frequently identified STs, PopPUNK-based sequence clusters (SCs) and ESBL gene subtypes and the degree of similarity using Czekanowski’s proportional similarity index (PSI).ResultsNine out of 10 most prevalent STs and SCs and 8/10 most prevalent ESBL genes in clinical ESBL-Ec were also the most common types in community faeces. The proportions of ST131 (39% versus 23%) and SC131 (40% versus 25%) were higher in clinical isolates than in community faeces (P < 0.01). Within ST131, H30Rx (C2) subclade was more prevalent among clinical isolates (55% versus 26%, P < 0.01). The proportion of ESBL gene blaCTX-M-1 was lower in clinical isolates (5% versus 18%, P < 0.01). Czekanowski’s PSI confirmed that the differences in ESBL-Ec from community faeces and clinical isolates were limited.ConclusionsDistributions of the 10 most prevalent clones and ESBL genes from ESBL-Ec community gut colonization and extra-intestinal infection overlapped in majority, indicating that isolates from routine clinical practice could be used to monitor ESBL-Ec clones and ESBL genes in the community.

Broiler Farms and Carcasses Are an Important Reservoir of Multi-Drug Resistant Escherichia coli in Ecuador.

Antimicrobial resistance (AMR) is a major health threat for public and animal health in the twenty-first century. In Ecuador, antibiotics have been used by the poultry industry for decades resulting in the presence of multi-drug resistant (MDR) bacteria in the poultry meat production chain, with the consequent risk for public health. This study evaluated the prevalence of ESBL/AmpC and mcr genes in third-generation cephalosporin-resistant Escherichia coli (3GC-R E. coli) isolated from broiler farms (animal component), broiler carcasses (food component), and human enteritis (human component) in Quito-Ecuador. Samples were collected weekly from November 2017 to November 2018. For the animal, food, and human components, 133, 335, and 302 samples were analyzed, respectively. Profiles of antimicrobial resistance were analyzed by an automated microdilution system. Resistance genes were studied by PCR and Sanger sequencing. From all samples, 122 (91.7%), 258 (77%), and 146 (48.3%) samples were positive for 3GC-R E. coli in the animal, food, and human components, respectively. Most of the isolates (472/526, 89.7%) presented MDR phenotypes. The ESBL blaCTX-M-55, blaCTX-M-3, blaCTX-M-15, blaCTX-M-65, blaCTX-M-27, and blaCTX-M-14 were the most prevalent ESBL genes while blaCMY-2 was the only AmpC detected gene. The mcr-1 gene was found in 20 (16.4%), 26 (10.1%), and 3 (2.1%) of isolates from animal, food, and human components, respectively. The implication of poultry products in the prevalence of ESBL/AmpC and mcr genes in 3GC-R must be considered in the surveillance of antimicrobial resistance.

High Prevalence of blaCTX-M-15 Gene among Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolates Causing Extraintestinal Infections in Bangladesh

The emergence of multidrug-resistant (MDR) Escherichia coli (E. coli) clonal lineages with high virulence potential is alarming. Lack of sufficient data on molecular epidemiology of such pathogens from countries with high infection burden, such as Bangladesh, hinders management and infection control measures. In this study, we assessed the population structure, virulence potential and antimicrobial susceptibility of clinical E. coli isolates from Dhaka, Bangladesh. A high prevalence of MDR (69%) and extended-spectrum β-lactamase production (ESBL) (51%) was found. Most E. coli isolates were susceptible to amikacin (95%), meropenem (94%) and nitrofurantoin (89%) antibiotics. A high prevalence of ST131 (22%) and ST95 (9%) followed by ST69 (4%) and ST73 (3%) was observed. Phylogroups B2 (46%), B1 (16%), D (10%) and F (9%) were prominent. blaCTX-M-15 (52%) and blaNDM-1 (5%) were the most prevalent ESBL and carbapenem resistance genes, respectively. Moreover, the predominant pathotype identified was extraintestinal pathogenic E. coli (ExPEC) (41%) followed by enteric pathogens (11%). In conclusion, our results suggest the transmission of clonal E. coli groups amidst diverse E. coli population that are associated with high virulence potential and MDR phenotype. This is of high concern and mandates more efforts towards molecular surveillance of antimicrobial resistance (AMR) in clinically significant pathogens.

Occurrence of emerging sulfonamide resistance (sul1 and sul2) associated with mobile integrons-integrase (intI1 and intI2) in riverine systems

Global use of antibiotics has exceedingly enhanced in agricultural, veterinary and prophylactic human use in recent days. Hence, these antibiotics can easily be found in the environment. This study revealed the occurrence of emerging MDR and ESBL producing strains, pollution profile, and factors integrons (intI1 and intI2) and environmental factors associated, in the riverine systems under different ecological and geo-climatic zones were investigated. The samples were collected based on anthropogenic intervention such as discharge of domestic wastes, industrial wastes, hospital, and municipal wastes. Among 160bacterial morphotypes, 121 (75.62%) exhibited MDR trait with maximum resistance towards lincosamide (CD = 71.3%), beta-lactams (P = 70.6%; AMX = 66.3%), cephalosporin (CZ = 60.6%; CXM = 34.4%), sulfonamide (COT = 50.6%; TR = 43.8%) followed by macrolide (E = 29.4%), tetracycline (TET = 18.8%), aminoglycosides (S = 18.8%; GEN = 6.3%), fluoroquinolones (NX = 18.1%; OF = 4.4%) and carbapenem (IPM = 5.0%). IntI1 gene was detected in 73 (60.3%) of isolates, whereas intI2 was found in 11 (9.09%) isolates. Eight (6.61%) isolates carried both integron genes (intI1 and intI2). sul1 and dfrA1 genes were detected in 53 (72.6%) and 63 (86.3%) isolates, respectively. A total of 103 (85.1%) were found ESBL positive with the presence of ESBL genes in 100 (97.08%) isolates. In riverine systems most prevalent ESBL gene blaTEM (93.0%) was detected alone as well as in combination with bla genes. The data can be utilized for public awareness and regulation of guidelines by local governing bodies as an alarming threat to look-out against the prevalent resistance in environment thereby assisting in risk management during epidemics. This study is a comprehensive investigation of emerging antibiotic pollutants and its resistance in bacteria associated with factors integrons-integrase responsible for its dissemination. It may also assist in global surveillance of antibiotic resistance and policies to curtail unnecessary antibiotic use.