ESBL Producers Research Articles

A Case Report on Perinephric Abscess Caused by Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae

Perinephric abscess is a rare life-threatening condition. It can complicate a urologic infection or occur secondary to hematogenous seeding. We report a case of a 49-year-old diabetic female who came to the outpatient department with complaints of right-sided abdominal pain for 2 days. It was associated with fever, headache, vomiting, and generalized weakness. Non-contrast computerized Tomography (NCCT) showed a subcapsular collection measuring 6.1× 5.7 × 4.4 cm noted adjacent to the lateral aspect of the right kidney. Following percutaneous nephrostomy, thick pus was drained from the abscess and sent for culture and sensitivity in the microbiology laboratory. Culture grew extended-spectrum beta-lactamase-producing (ESBL) Klebsiella pneumoniae which was sensitive to Carbapenems, Piperacillin Tazobactam, Fluoroquinolones, Netilmicin, Amikacin, and Tetracycline. The patient improved on treatment with the appropriate antibiotics. Perinephric abscesses should be considered as a differential diagnosis of fever with abdominal pain or flank pain since its non-specific nature can delay the diagnosis. Underlying diabetes mellitus (DM), urinary tract abnormalities, and immunodeficiency should be considered in a patient with a perinephric abscess. Ultrasonography (USG) and Computerized Tomography with contrast enhancement are crucial for diagnosis. The drainage of the abscess, either percutaneous or open, should be done. Klebsiella pneumoniae is a frequent cause of perinephric abscesses and mostly follows a complicated urinary tract infection. With high mortality rates, early diagnosis, and effective treatment of perirenal abscesses are required to improve the prognosis of patients.

ESBL PREVALANCE IN URINE SPECIMENSOF CANCER PATIENTS AT A TERTIARY CARE CANCER CENTRE IN KERALA

Objective: The objective of this study documents the prevalence of extended-spectrum beta-lactamase (ESBL)-producing multiple drug resistance uropathogens reported prospectively for a period of 3 months from February 2020 to April 2020 and its susceptibility to the commonly recommended antibiotics for urinary tract infections (UTI). Methods: Identification and characterization of the uro-pathogens from 200 samples were done by routine smear microscopy, culturing, Kirby–Bauer disc-diffusion assay, and double disc synergy test. Results: Thirty percent of samples were culture-positive pointing Escherichia coli, Klebsiella pneumoniae, Pseudomonas Aeruginosa, and Serratia marcescens (51.9%, 36.5%, 9.61%, and 1.92%, respectively). Almost all ESBL producers were found to be multi-drug resistant that includes E. coli (56.6%), K. pneumoniae (36.74%) and P. aeruginosa (6.66%) warranting prompt need of surveillance for effective clinical management. Conclusion: The current study pointed the emergence and incidence of ESBL producing Gram-negative bacilli that are multi-drug resistant and causing UTI among cancer patients.

Occurrence of extended-spectrum β-lactamases-producing Escherichia coli isolates over gradient pollution in an urban tropical estuary.

Bacterial resistance to antimicrobials is a global public health problem that surpasses the human context and can be increased by pollution. However, the lack of systematic monitoring of resistance in some aquatic matrices, such as tropical estuaries, makes it unknown whether its occurrence is associated with anthropogenic pollution in these environments. Therefore, we investigated the occurrence of extended-spectrum beta-lactamases (ESBLs) producing Escherichia coli as a resistance indicator for 12 consecutive months at three representative points of a pollution gradient in Guanabara Bay (GB), Brazil. Sixty-six E. coli strains were selected from 72 samples of GB waters in the presence of ceftriaxone (8 μg mL-1 ) and identified by MALDI-TOF MS. Of the 66, 55 (83.3%) strains were ESBL producers. They carried beta-lactamase/ESBL genes, with the predominance of blaCTX-M (54, 98.2%), especially the blaCTX-M-1,2 allele (49.1%). These strains were detected frequently (81.8%) from the point with the highest pollution levels. Furthermore, the marker for Class 1 integron, intI1 gene, was detected in 54.5% of ESBL producers. These data suggest an association between antimicrobial-resistant E. coli and sewage pollution in aquatic environments raising concerns about the possible risks of human exposure to these waters and fish consumption.

Utility of the MBT STAR-Cepha kit in detecting extended-spectrum β-lactamase producers in clinical urine samples and positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Urinary tract infection (UTI) and bloodstream infection (BSI) caused by extended-spectrum β-lactamase (ESBL)-producing bacteria are important healthcare problems. Appropriate infection management requires the direct detection of organisms in clinical specimens. We assessed the capability of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based MBT STAR-Cepha kit to detect ESBL producers in clinical urine and blood samples. Over a 1-year period, 90 urine samples and 55 positive monomicrobial (Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, or Proteus mirabilis) blood cultures were collected from patients with UTI or BSI at Hamamatsu University Hospital. β-lactamase activity in these samples was directly detected using the MBT STAR-Cepha kit, and the results were compared with those of antimicrobial susceptibility testing and polymerase chain reaction detection assay for the isolates. In receiver operating characteristic curve analysis, the kit assay showed low accuracy in detecting ESBL producers in urine samples (area under the curve [AUC], 0.69). Meanwhile, the AUC for detecting all ESBL-producing bacteria in positive blood cultures was 0.81. The kit assay detected cefotaxime (CTX) resistance (mainly in CTX-M-type ESBL producers) with high accuracy in positive blood cultures; however, it did not accurately detect ESBL producers in urine samples and CTX-susceptible isolates with other ESBL-associated genes (e.g., TEM and SHV types) in positive blood cultures. MBT STAR-Cepha testing can accurately discriminate CTX-resistant ESBL producers in BSI cases and thus can contribute to effective infection management. The results suggest that different sample types, antibiotic resistance profiles, and resistance genes can affect the kit performance.

Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolated from Food-Producing Animals in Tamaulipas, Mexico.

Extended-spectrum β-lactamase (ESBL)-producing E. coli has become an important global problem for the public health sector. This study aims to investigate the E. coli antimicrobial resistance profile among living food-producing animals in Tamaulipas, Mexico. A total of 200 fecal samples were collected from bovines, pigs, chickens and sheep. A total of 5.0% of the strains were phenotypically confirmed as ESBL producers. A high percentage of phenotypic antimicrobial resistance was observed against gentamicin (93.3%), tetracycline (86.6%) and streptomycin (83.3%). The gentamicin-resistant strains showed MDR, distributed among 27 resistance patterns to different antimicrobials. The antimicrobial resistance gene tet(A) was detected in 73.3% of isolates, aadA1 in 60.0% and sul2 in 43.3% of strains. The blaCTX-M gene was found in 23.3% of strains. The virulence gene hlyA was detected in 43.3% of isolates; stx1 and stx2 were not detected in any strain. The phylotyping indicated that the isolates belonged to groups A (33.3%), B1 (16.6%), B2 (40.0%) and D (10.0%). These results show that food-producing animals might be a reservoir of ESBL-producing bacteria and may play a role in their spread.

A computational odyssey: uncovering classical β-lactamase inhibitors in dry fruits

In the antibacterial arsenal, β-lactams have held a prominent position, but increasing resistance due to unauthorized use and genetic factors requires new strategies. Combining β-lactamase inhibitors with broad-spectrum β-lactams proves effective in combating this resistance. ESBL producers demand new inhibitors, leading to the exploration of plant-derived secondary metabolites for potent β-lactam antibiotics or alternative inhibitors. Using virtual screening, molecular docking, ADMET analysis, and molecular dynamic simulation, this study actively analyzed the inhibitory activity of figs, cashews, walnuts, and peanuts against SHV-1, NDM-1, KPC-2, and OXA-48 β-lactamases. Using AutoDock Vina, the docking affinities of various compounds for target enzymes were initially screened, revealing 12 bioactive compounds with higher affinities for the target enzymes compared to Avibactam and Tazobactam. Top-scoring metabolites, including Oleanolic acid, Protocatechuic acid, and Tannin, were subjected to MD simulation studies to further analyze the stability of the docked complexes using WebGro. The simulation coordinates, in terms of RMSD, RMSF, SASA, Rg, and hydrogen bonds formed, showed that these phytocompounds are stable enough to retain in the active sites at various orientations. The PCA and FEL analysis also showed the stability of the dynamic motion of Cα residues of phytochemical-bound enzymes. The pharmacokinetic analysis of the top phytochemicals was performed to analyze their bioavailability and toxicity. This study provides new insights into the therapeutic potential of phytochemicals of selected dry fruits and contributes to future experimental studies to identify βL inhibitors from plants. Communicated by Ramaswamy H. Sarma

Extended-Spectrum β-Lactamase and Carbapenemase-Producing Gram-Negative Bacteria and Associated Factors Among Patients Suspected of Community and Hospital-Acquired Urinary Tract Infections at Ayder Comprehensive Specialized Hospital, Tigrai, Ethiopia.

Little is known about bacteria that produce extended-spectrum beta-lactamases (ESBLs) and carbapenemase in patients with urinary tract infections (UTIs) in Tigrai, Ethiopia. The aim of this study was to describe the magnitude of ESBL- and carbapenemase -producing gram-negative bacteria among patients suspected of community- and hospital-acquired UTIs at a referral hospital in Tigrai, Ethiopia. A cross-sectional study was conducted at Ayder Comprehensive Specialized hospital from January 2020 to June 2020. A 10-20 mL sample of morning mid-stream and catheter urine was collected from consenting participants. Urine samples were cultured on cysteine lactose electrolyte deficient medium and MacConkey agar, and bacteria were identified using standard microbiological protocols. The Kirby-Bauer disk diffusion method was used for antimicrobial susceptibility testing. The combination disk and modified Hodge tests were used detect ESBL and carbapenemase production, respectively. The data was entered into EPI 3.1 software and analyzed using SPSS version 21. Overall, 67 gram-negative bacteria were recovered from 64 participants. Escherichia coli was the predominant isolate (68.6%), followed by Klebsiella pneumoniae (22.4%), while ESBL production was found in both Escherichia coli and Klebsiella pneumoniae (52.2% and 86.7%, respectively). Isolates recovered from patients with hospital-acquired UTIs were more likely to produce ESBLs (AOR= 16.2; 95% CI: 2.95-89.5). Carbapenemase was produced by 4.3% of E. coli and 20% of Klebsiella pneumoniae isolates. High resistance rates were found against tetracycline (84.8%), ampicillin (78.3%), amoxicillin/clavulanic acid (58.7%) for Escherichia coli isolates and against ampicillin (93.3%), sulphamethexazole trimethoprim (93.3%), cefotaxime (86.6%), and ceftazidime (86.6%), and tetracycline (73.3%) for Klebsiella pneumoniae. Most UTIs were caused by ESBL-producing bacteria, especially those that were related to healthcare. Microbiological-based therapy for patients with UTIs is essential at our study site due to high rates of ESBL and significant carbapenemase production with concomitant high rates of drug resistance to several antibiotics.

Prevalence of the blaCTX-M and blaTEM genes among extended-spectrum beta lactamase-producing Escherichia coli isolated from broiler chickens in Indonesia.

Infections of humans and animals by multidrug resistant bacteria are increasing because of the inappropriate use of antibiotics. Disease management may be more challenging if Escherichia coli produce extended-spectrum beta-lactamase (ESBL), which could cause resistance to aztreonam and third-generation cephalosporins. This study was aimed at determining the prevalence of the blaCTX-M and blaTEM genes among ESBL-producing E. coli isolated from broiler chickens in Indonesia. A total of 115 broiler cloacal swab samples were obtained from 22 farms and studied for the presence of E. coli. The isolates were identified using approved standard methods and were purified on eosin methylene blue agar media. The E. coli isolates were subjected to sensitivity testing using beta-lactam antibiotics, and ESBL production was confirmed by a double-disc synergy test. The presence of the blaCTX-M and blaTEM genes was identified using a PCR. It was found that 99/115 (86.1%) of the isolated E. coli were resistant to beta-lactam antibiotics and 34/115 (29.6%) of them were phenotypically detected to be ESBL producers. Of the 34 isolates that were confirmed ESBL producers, 32/34 (94.1%) of them harboured the blaCTX-M and 13/34 (38.2%) the blaTEM genes. The blaCTX-M and blaTEM genes were detected together in 12/34 (35.3%) isolates. This study discovered that broiler chickens are possible reservoirs of ESBL-producing E. coli that may infect humans. Thus, a committed public health education campaign is recommended in order to mitigate the potential threat to human health.

Virulence factors and antimicrobial resistance of Escherichia coli isolated from commercialized fresh cheese in the south of Espírito Santo.

Cheeses are dairy products that can potentiallycontaina diverse range of harmful bacteria that could be consumed by humans, including the enteric pathogen Escherichia coli. This study aimed to characterize the presence of total coliforms, assess the antimicrobial susceptibility profiles of the main commercial antimicrobial classes and biocides, and evaluate the ability of 50 E. coli isolates obtained from fresh cheese sold in the southern region of Espírito Santo, Brazil, to produce biofilms. The counts of total coliforms + E. coli obtained averages of (A) 7.22 × 106CFU/g, (B) 9.35 × 107CFU/g, and (C) 1.16 × 106CFU/g for different brands. All isolates were capable of forming biofilms, with 8%, 76%, and 16% of these isolates presenting high, moderate, and low adherence in biofilm formation, respectively. Most strains showed inhibition halos for the biocides chlorhexidine digluconate 2% (16mm ± 4.34), iodopovidone 10% (7.14mm ± 0.36), and sodium hypochlorite 2% (7.12mm ± 0.33). Out of the 50 strains, 21 (42%) were resistant to at least one of the antimicrobials. Regarding the multiple resistance index, 3 (6%) strains were resistant to 3 or more antimicrobial classes. Furthermore, 2 (4%) were extended-spectrum beta-lactamases producers. Resistance to ampicillin and amoxicillin was observed in 20% and 40% of the strains, respectively. In contrast, gentamicin was the most effective antimicrobial, with a sensitivity rate of 100%. The findings indicate that E. coli present in fresh cheese may possess unique physiological characteristics that could be associated with their persistence, virulence, and multidrug resistance. These results raise significant public health concerns since contaminated food can pose risks to consumers' health, emphasizing the importance of reinforcing hygienic-sanitary controls at all stages of production.

Commonly Consumed Vegetables as a Potential Source of Multidrug-Resistant and β-Lactamase-Producing Bacteria in Debre Berhan Town, Ethiopia.

Recently, antibiotic resistance of bacteria contained in foods such as vegetables has become a public health problem. In Ethiopia, the diversity of bacterial contamination and level of antibiotic resistance in vegetables are poorly understood. Local analysis of vegetable contamination and its contribution to the spread of antibiotic resistance are therefore essential for One Health interventions. Therefore, the aim of this study was to investigate the level of bacterial contamination of commonly consumed vegetables and their antimicrobial resistance patterns. A cross-sectional research was conducted in Debre Berhan town from February to August 2022. Questionnaires were used to collect data on sociodemographic variables, hygiene practices, and market hygiene. Six carefully selected vegetables (30 each, 180 in total) were purchased at a local market. Bacterial isolation and identification, multidrug-resistant (MDR) screening and confirmation, extended-spectrum β-lactamase (ESBL) screening and confirmation, and antibiotic susceptibility tests were performed using standard operating procedure. The data were analysed statistically using SPSS software version 25. The contamination rate of vegetables was 119 (66.1%). Of the 176 bacteria isolates, E. coli (26.1%; 46/176), S. aureus (18.8%; 176), S. epidermidis (10.8%; 19/176), Klebsiella spp. (9.1%; 16/179) and Acinetobacter spp. (6.8%; 12/176) were the most frequently detected isolates. Of the 180 samples tested, (66.1%; 119/180) were contaminated with at least one type of bacteria. Lettuce (22.7%; 40/176), spinach (18.6%; 33/176), and cabbage (19.2%; 32/176) were the most contaminated vegetables. Of the 176 bacteria isolates, (64.8%; 114/176) were MDR, and (18.5%; 23/124) isolates were ESBL producers. The kind of vegetables, vendor/seller finger-nail status, medium of display, market type, and not cleaned before to display were all significantly associated with bacterial contamination. This study found that commonly consumed vegetables are contaminated with antibiotic-resistant bacteria. Vegetables were also notable for the incidence of multidrug-resistant, extended β-lactamase-resistant, and methicillin-resistant bacterial isolates. Therefore, we urge local health authorities to develop and implement effective control strategies to reduce vegetable contamination.

Food wastes as a potential hotspot of antibiotic resistance: synergistic expression of multidrug resistance and ESBL genes confer antibiotic resistance to microbial communities.

This study investigated antibiotic resistance (ABR) and extended-spectrum ß-lactamases (ESBL) patterns in bacterial isolates collected from the dairy, hotel, meat, and canteen food waste samples. A total of 144 bacterial strains were collected and screened for resistance against 9 standard antibiotics belonging to three generations and ESBL production. The ABR profile of the bacterial isolates was observed against all four major antibiotic groups (aminoglycosides, β-lactams, quinolone, and others), where resistance against cefotaxime (> 70%) and methicillin (> 50%) was high. Though the ABR pattern of strains from dairy waste (> 50%) was high against first-generation antibiotics, the strains from meat waste (> 50%) showed considerable resistance against second- and third-generation antibiotics. ESBL-producing isolates were screened (> 60%, n = 144) through primary identification tests (combined disk test and double disk synergy tests) and further confirmed through Hexa G-minus 23 and 24 and MIC E-stripe following CLSI guidelines. Genes conferring ESBL resistance blaCTX-M, blaSHV, blaOXA, blaTEM, blaKPC genes and multidrug resistance (MDR) mexF gene were detected in the selected isolates with ABR and ESBL traits. Isolates with multidrug ABR and ESBL phenotype were further genotypically identified through 16s rRNA gene sequencing. The synergy of ABR was detected through the co-expression of ESBL and MDR in isolates with a high occurrence of ABR and ESBL. The results demonstrate the significance of food waste as a natural reservoir of ABR and ESBL-producing pathogens, highlighting the importance of resistance monitoring and its interventions.

Molecular characterization and antibiotic resistance profile of ESBL-producing Escherichia coli isolated from healthy cow raw milk in smallholder dairy farms in Bangladesh.

The emergence of antimicrobial-resistant bacteria, such as Escherichia coli in milk, is a serious public health concern as milk is considered a complete food and an important part of daily human diet worldwide, including in Bangladesh. However, there have been no reports on the molecular characterization and antibiotic resistance profile of extended-spectrum beta-lactamase (ESBL)-producing E. coli from milk of healthy cows in Bangladesh. Therefore, this study aimed to detect and characterize ESBL-producing E. coli (ESBL-Ec) in milk samples from healthy cows in smallholder dairy farms in Mymensingh district, Bangladesh, and assess the potential risk of consuming this milk. A total of 100 milk samples were collected from apparently healthy cows on smallholder dairy farms. Escherichia coli was isolated from the collected samples using standard methods. The detection of ESBL-Ec was performed phenotypically using cultural methods and genotypically by ESBL genetic determinants using multiplex polymerase chain reaction. Antimicrobial susceptibility testing of the ESBL-Ec isolates was performed using the disk diffusion method with 15 common antimicrobials. In this study, out of the 100 samples tested, 70 (70%) were found to be positive for E. coli. Among these, 41 (58.6%) strains were identified as ESBL-producing, both phenotypically and genotypically, with the presence of blaCTX-M, blaTEM, and blaSHV individually or combined (blaCTX-M plus blaTEM plus blaSHV). The antibiogram of these ESBL-positive isolates revealed high resistance against commonly used antibiotics, such as ampicillin, cefotaxime, and gentamicin (100%), azithromycin (88%), oxytetracycline (27%), nalidixic acid, cotrimoxazole/trimethoprim (24%), and streptomycin (22%). In addition, one isolate showed resistance to 4th generation of cephalosporin (cefepime). Most importantly, extensive multidrug resistance was found in many ESBL-Ec isolates. However, the isolates were highly sensitive to drugs such as ceftriaxone (100%) and imipenem (100%). This is the first study to detect ESBL-Ec in raw milk from healthy cows on smallholder dairy farms in Bangladesh. More than 58% of the E. coli isolated from raw milk of healthy cows tested positive for ESBL production and showed resistance to most commonly used antimicrobials which may be alarming for human health. A limitation of our study is that we had a small size of sample collected from one district in Bangladesh. Therefore, a larger sample size covering a wider geographic area, and using multi-locus sequence typing and whole genome sequencing could provide a more comprehensive understanding of the prevalence and characteristics of ESBL-Ec in Bangladesh.

Evaluation of presence of clone ST131 and biofilm formation in ESBL producing and non-producing Escherichia coli strains.

Escherichia coli ST131 is a pandemic clone associated with multidrug resistance, starting with beta-lactamase production and fluoroquinolone resistance in the first place, leading to significant systemic infections. Clones that develop due to the frequency of antimicrobial resistance and the rate of spread in our country are important issues that need to be investigated. This study aims to investigate the incidence of ST131which is a "high-risk pandemic clone E. coli" in ESBL-producing and non-ESBL-producing strains, as well as their biofilm-forming abilities and antibiotic resistance rates. A total of 86 E. coli isolates were used in the study. Bacterial identifications were performed by conventional and automated methods. The double disc synergy method was used to demonstrate the presence of ESBL. Molecular studies in all E. coli strains were performed by real-time PCR method. 86 strains were studied, of which 83.72% were urine, 6.98% were wound, 4.65% were blood, and 2.33% were tracheal aspirate and sputum. 79.07% of these strains were ESBL-positive. 58.1% of the strains were female, whereas 41.9% were male patients, and the average age was 46.2. Out of 86 strains, 38.72% were ST131 positive, the H30 subclone was detected in 27.27% of them, and the H30-Rx subclone was detected in all of the H30 subclone positive strains. The presence of the ESBL resistance gene was detected at the rate of TEM 41.86%, SHV 37.21%, CTX-M 36.04%, and OXA 4.65%. Most commonly SHV gene (54.54%) was seen in ST131 clone-positive samples. Finally, while it was found that 48.83% of the strains formed biofilm by any method, biofilm formation was detected in 69.7% of the samples that were positive for the ST131 clone. Our study can reveal the dramatic prevalence of the ESBL-producing E. coli strains along with the high-risk ST131 clone, the dominance of the H30Rx subclone of this risky clone, as well as the importance of the influence of resistance mechanisms along with resistance and biofilm.

Monitoring of the Evolution of Antibiotic Resistance in Urinary Tract Infection at the Mohamed VI University Hospital in Marrakech

Introduction: Urinary tract infections are a public health problem due to the excess mortality and the additional costs they entail. Objective: To monitor the evolution of antibiotic resistance of uropathogenic germs over the last three years in patients treated at the CHU Med VI of MARRAKECH. Materials and Methods: Descriptive study carried out over a period of 3 years (from January 2018 to December 2020) including all urinary tract infections retained as meeting the criteria for microbiological and clinical infections in patients treated at the Mohammed VI University Hospital in Marrakech. Results: Out of a total of 37,394 ECBUs, the prevalence of UTI was 11% (n = 4275). The average age was 36 years, with a standard deviation of 25.9 years. The sex ratio M / F was 0.95. Uropathogenic germs were represented mainly by Enterobacteriaceae 78%. E. coli accounted for 66%. Enterobacteriaceae were resistant to third generation cephalosporins in 22% of cases, decreased sensitivity to Carbapenems in 7.2%, resistant to ciprofloxacin in 31%, to Cotrimoxazole in 42%, to Gentamicin in 17.5%, to Amikacin in 2%. Extended spectrum beta-lactamase production was 21%. Over the past three years, 895 multi-resistant bacteria have been identified, i.e. 23%. Klebsiella spp accounted for 41%. Conclusion: The results testify to the increased resistance of uropathogenic bacteria. Updated local studies are important to understand the state of play of bacterial ecology and resistance profiles in order to avoid treatment failures.

Highly sensitive detection of antimicrobial resistance genes in hospital wastewater using the multiplex hybrid capture target enrichment.

Wastewater can be useful in monitoring the spread of antimicrobial resistance (AMR) within a hospital. The abundance of antibiotic resistance genes (ARGs) in hospital effluent was assessed using metagenomic sequencing (mDNA-seq) and hybrid capture (xHYB). mDNA-seq analysis and subsequent xHYB targeted enrichment were conducted on two effluent samples per month from November 2018 to May 2021. Reads per kilobase per million (RPKM) values were calculated for all 1,272 ARGs in the constructed database. The monthly numbers of patients with presumed extended-spectrum β-lactamase (ESBL)-producing and metallo-β-lactamase (MBL)-producing bacteria, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant enterococci (VRE) were compared with the monthly RPKM values of blaCTX-M, blaIMP, mecA, vanA, and vanB by xHYB. The average RPKM value for all ARGs detected by xHYB was significantly higher than that of mDNA-seq (665, 225, and 328, respectively, and P < 0.05). The average number of patients with ESBL producers and RPKM values of blaCTX-M-1 genes in 2020 were significantly higher than that in 2019 (17 and 13 patients per month and 921 vs 232 per month, respectively, both P < 0.05). The average numbers of patients with MBL-producers, MRSA, and VRE were 1, 28, and 0 per month, respectively, while the average RPKM values of blaIMP, mecA, vanA, and vanB were 6,163, 6, 0, and 126 per month, respectively. Monitoring ARGs in hospital effluent using xHYB was found to be more useful than conventional mDNA-seq in detecting ARGs including blaCTX-M, blaIMP, and vanB, which are important for infection control.IMPORTANCEEnvironmental ARGs play a crucial role in the emergence and spread of AMR that constitutes a significant global health threat. One major source of ARGs is effluent from healthcare facilities, where patients are frequently administered antimicrobials. Culture-independent methods, including metagenomics, can detect environmental ARGs carried by non-culturable bacteria and extracellular ARGs. mDNA-seq is one of the most comprehensive methods for environmental ARG surveillance; however, its sensitivity is insufficient for wastewater surveillance. This study demonstrates that xHYB appropriately monitors ARGs in hospital effluent for sensitive identification of nosocomial AMR dissemination. Correlations were observed between the numbers of inpatients with antibiotic-resistant bacteria and the ARG RPKM values in hospital effluent over time. ARG surveillance in hospital effluent using the highly sensitive and specific xHYB method could improve our understanding of the emergence and spread of AMR within a hospital.

Antimicrobial resistant Escherichia coli prevalence in freshwaters in Belgium and human exposure risk assessment

The objective of this work was to evaluate the antimicrobial resistant (AR) E. coli prevalence in recreational waters in Belgium and to assess the exposure risk for bathers. Nine stations were sampled during the 2021 bathing season. A total of 912 E. coli strains were isolated and tested by the disk diffusion method in accordance with EUCAST recommendations, including Extended-Spectrum Beta-Lactamase (ESBL) production. AR E. coli were counted at each bathing sites, 24% of strains were resistant to at least one antibiotic and 6% were Multi-Drug Resistant (MDR). A Multiple Antibiotic Resistance (MAR) index was calculated to compare the bathing sites. The Lesse river had the highest MAR index as well as the highest E. coli absolute abundance and the largest number of ESBL-producing E. coli. Conversely, the 3 lakes showed lower E. coli contamination levels and AR rates. A human health risk assessment of exposure to AR E. coli, based on the calculation of measured prevalence, was performed considering four different dose-response model scenarios. The human health risk (Pd) ranged from 10−9 to 0.183 (children). The exposure probabilities were low, except for scenario 3 (E. coli O157:H7), which is the most severe.

Frequency of Extended-Spectrum Beta-Lactamases-Producing Escherichia coli Among Out- and In-patients in Rafsanjan City, Iran

Background: The frequency of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strains among clinical isolates has been steadily increasing, leading to limited treatment options. Objectives: This study aimed to assess the antibiotic sensitivity of E. coli and the frequency of ESBL isolates among both out-patients and in-patients. Methods: A total of 390 E. coli isolates were received at the Ali-Ebn-e-Abitaleb Hospital laboratory in Rafsanjan. The antibiogram, as well as the phenotypic and genotypic detection of ESBL isolates, were conducted using Kirby-Bauer, combination disk confirmatory, and polymerase chain reaction tests, respectively. Results: Of all the E. coli isolates, 45.6% exhibited ESBL production. Among these isolates, 46.1% were obtained from hospital wards, while 42.5% were from outpatients. Meropenem and imipenem displayed sensitivities of 97.2% and 93.3%, respectively, whereas amikacin and nitrofurantoin showed sensitivities of 89.7% and 85.6%, respectively, for all isolates. Notably, ceftriaxone, cefotaxime, cefixime, ampicillin, co-trimoxazole, and nalidixic acid demonstrated high resistance rates, surpassing 50%. ESBL-producing isolates were more frequently observed in blood samples (65%) and wounds (60%) compared to other tested isolates. Approximately 8.6% of isolates carried a single type of ESBL gene, while 38.5% carried multiple ESBL genes. Conclusions: The data indicate a prevalence of ESBL-producing E. coli isolates among both out-patients and in-patients, with some of them acquiring two or three types of ESBL enzymes. As a result, their ability to hydrolyze antibiotics has increased, leading to their higher occurrence in clinical samples.

In Vitro Activity of Omadacycline and Comparator Antibiotics against Extended-Spectrum Beta-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae Urinary Isolates.

Limited oral antibiotic options exist for urinary tract infections (UTI) caused by ESBL-producing Enterobacterales. The aim of the study was to evaluate in vitro activity of omadacycline and comparator antibiotics against clinical ESBL-producing and non-ESBL-producing E. coli and K. pneumoniae urinary isolates. 102 isolates each of E. coli and K. pneumoniae were collected from clinical urine specimens in 2019. By design, an equal number of each species were included that tested positive and negative for ESBL production. Omadacycline MICs were determined using gradient test strips and compared to MICs of comparator antibiotics as determined by an automated broth microdilution system. Isolates were considered susceptible to omadacycline if the MIC was ≤4 µg/mL for each species. 54.9% of all ESBL-producing isolates were susceptible to omadacycline, but better susceptibility was observed for ESBL-producing E. coli (74.5%). Omadacycline MICs were 2-4 fold lower for E. coli and K. pneumoniae strains not producing ESBL. The omadacycline MIC 50 and 90 values were 4 and 16 µg/mL, respectively, for all isolates studied. 74.5% of all isolates were considered susceptible to omadacycline. MICs were generally lower for E. coli strains with MIC 50 and 90 values of 4 and 8 µg/mL, respectively (87.3% susceptible), compared with K. pneumoniae. Overall, the most active agents were omadacycline and nitrofurantoin, while other comparator antibiotics were less active. Omadacycline represents a promising oral antibiotic for treating UTI caused by ESBL-producing E. coli, particularly when resistance limits other oral options. Prospective, controlled clinical trials are needed to validate these in vitro results.

Comparison of Horizontal blaCTX-M Gene Transfer via Conjugation among Extended Spectrum β-Lactamases Producing Escherichia coli Isolates from Patients with Urinary Tract Infection, Their Animals, and Environment.

The dissemination of the extended spectrum β-lactamases (ESBL) producing E. coli poses a significant public health problem. Understanding the efficiency and frequency of horizontal gene transfer via conjugation of ESBL producing E. coli is imperative towards devising prevention and control measures. This study compared the frequencies and efficiencies of horizontal blaCTX-M gene transfer via conjugation among Escherichia coli isolates from urine and gastrointestinal tract (GIT) of patients with urinary tract infection (UTI), their animals and environment. Horizontal blaCTX-M gene transfer via conjugation by a broth mating experiment was performed using 50 confirmed ESBL producing E. coli isolates as donors and Escherichia coli J53 (F-, met, pro, Azr), as the recipient. The transconjugants were detected and their frequencies and efficiencies of conjugation were measured and compared between ESBL producing E. coli isolates multi-sourced from urine, GIT, animals and environment. Antimicrobial susceptibility testing of all resulting transconjugants was performed. DNA was extracted from all transconjugants to confirm the presence and the acquisition of blaCTX-M gene. Out of 50 ESBL producing E. coli isolates harboring blaCTX-M gene, 37 (74.0%) successfully exercised horizontal gene transfer through conjugation. All transconjugants were confirmed phenotypically and genotypically by PCR. Of note, all of the isolates from environment 100.0% (7/7) performed conjugation, exhibiting the highest transfer efficiency, followed by isolates from urine and animals, with the conjugation transfer efficiency of 77.8% (14/18) and 76.1% (10/13), respectively. The isolates from the environment conjugated with a significant more efficiency than those from the GIT [Two-sample test of proportions; p-value = 0.0119]. The overall conjugation transfer frequencies ranged from 0.4 × 10-14 - 5.5 × 10-11 per donor cells with the highest median conjugation transfer frequency observed among isolates from animal (3.23 × 10-12 [IQR: 0.70 × 10-12 - 7.22 × 10-12]) followed by that of isolates from the environment (1.60 × 10-12 [IQR: 0.30 × 10-12 - 5.0 × 10-12]). ESBL producing E. coli from human, animals and environment exercises horizontal blaCTX-M gene transfer efficiently with the highest occurrence among isolates from the environment and animals. The antimicrobial resistance control and prevention strategies should be widened up to explore strategies to prevent horizontal AMR gene transfer.

Epidemiology of Gram-Negative Bloodstream Infections in the United States: Results From a Cohort of 24 Hospitals.

To address knowledge gaps in management of Gram-negative bloodstream infection, the Antibiotic Stewardship Implementation Collaborative was established consisting of programs from 24 academic and community hospitals across the United States. A retrospective cohort study was conducted of unique adult patients with Gram-negative bloodstream infection hospitalized at participating hospitals from January to December 2019. Patient level and microbiologic data were collected via electronic medical record review with a standardized data collection form and data dictionary. Data analysis was largely descriptive. The Pearson χ2 test to compare categorical variables and the Wilcoxon rank sum test for continuous variables were used. In total, 4851 bacterial isolates from 3710 eligible unique patients were included in the cohort. Most common source of infection was the urinary tract (47.9%). Source control was achieved in 84% of cases. Escherichia coli (2471, 51.0%) was the most common Gram-negative organism recovered. Antibiogram combining isolates from all participating centers with species-level susceptibilities and source specific antibiograms for isolates from urinary, respiratory, and intraabdominal source were created. Northeast sites contributed the most extended spectrum beta-lactamase (ESBL) producing organisms (73%), but West sites had the highest percentage of ESBL producers of total isolates (16%). A statistically significant difference in percentage of ESBL-producing organisms in Whites vs. non-Whites (14.6 % and 9.5 %, respectively, P<0.01) was observed. While the present study was conducted pre-pandemic, it highlights the need for stewardship data collaboratives to enhance our understanding of the antimicrobial resistance patterns.