Extended-spectrum Beta-lactamase Enzymes Research Articles

Molecular characterization of carbapenemase and extended spectrum beta-lactamase producing Acinetobacter baumannii isolates causing surgical site infections in Ethiopia

BackgroundAcinetobacter baumannii is an opportunistic pathogen that can cause a variety of nosocomial infections in humans. This study aimed to molecularly characterize extended-spectrum beta-lactamase (ESBL) producing and carbapenem-resistant Acinetobacter species isolated from surgical site infections (SSI).MethodsA multicentre cross-sectional study was performed among SSI patients at four hospitals located in Northern, Southern, Southwest, and Central parts of Ethiopia. The isolates were identified by microbiological methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Antibiotic susceptibility was determined using disk diffusion. The presence of phenotypic ESBL and carbapenemase production was detected by employing standard microbiological tests, including combined disk diffusion (CDT). ESBL and carbapenem resistance determinants genes were studied by polymerase chain reaction (PCR) and sequencing.ResultsA total of 8.7% Acinetobacter species were identified from 493 culture-positive isolates out of 752 SSI wounds. The species identified by MALDI-TOF MS were 88.4% A. baumannii, 4.7% Acinetobacter pittii, 4.7% Acinetobacter soli, and 2.3% Acinetobacter lactucae. Of all isolates 93% were positive for ESBL enzymes according to the CDT. Using whole genome sequencing 62.8% of the A. baumannii harbored one or more beta-lactamase genes, and 46.5% harbored one or more carbapenemase producing genes. The distribution of beta-lactamases among Acinetobacter species by hospitals was 53.8%, 64.3%, 75%, and 75% at JUSH, TASH, DTCSH, and HUCSH respectively. Among ESBL genes, blaCTX−M alleles were detected in 21.4% of isolates; of these 83.3% were blaCTX−M−15. The predominant carbapenemase gene of blaOXA type was detected in 24 carbapenem-resistant A. baumannii followed by blaNDM alleles carried in 12 A. baumannii with blaNDM−1 as the most common.ConclusionsThe frequency of Acinetobacter species that produce metallobetalactamases (MBLs) and ESBLs that were found in this study is extremely scary and calls for strict infection prevention and control procedures in health facilities helps to set effective antibiotics stewardship.

Prevalence of blaCTX-M and blaTEM Genes in Cefotaxime-Resistant Escherichia coli Recovered from Tertiary Care at Central Nepal: A Descriptive Cross-Sectional Study.

Urinary tract infections (UTIs) are highly prevalent globally, and various antibiotics are employed for their treatment. However, the emergence of drug-resistant uropathogens towards these antibiotics causes a high rate of morbidity and mortality. This study was conducted at the Microbiology Laboratory of Grande International Hospital from November 2021 to May 2022 and aimed to assess the prevalence of UTI caused by Escherichia coli and their antibiotic susceptibility pattern with a focus on extended-spectrum beta-lactamases (ESBLs) and the prevalence of two genes (blaCTX-M and blaTEM) in cephalosporin-resistant E. coli. Altogether, 1050 urine samples were processed to obtain 165 isolates of E. coli. The isolates were identified by colony morphology and biochemical characteristics. Antimicrobial susceptibility tests (ASTs) were determined by the Kirby-Bauer disk diffusion method, and their ESBL enzymes were estimated by the combined disk method (CDM). Two ESBL genes (blaCTX-M and blaTEM) were investigated by polymerase chain reaction (PCR) in cefotaxime-resistant E. coli. Among the 1050 urine samples that were processed, 335 (31.9%) were culture-positive with 165 (49.2%) identified as E. coli. The age group ≥60 years (30.3%) had greater susceptibility to bacterial infections. AST revealed that meropenem was highly effective (95.7% susceptibility), while ampicillin showed the least sensitivity (42.4%). Among the E. coli isolates, 86 were multidrug resistant (MDR) and 10 were extensively drug resistant (XDR). Of these, 46 MDR (96%) and 2 XDR (4%) were ESBL producers. The prevalence of ESBL genes (blaCTX-M and blaTEM) was 49.3% and 54.8%, respectively. The overall accuracy of CDM as compared to PCR for the detection of the blaCTX-M gene was 55.26%. The prevalence of MDR E. coli harboring the blaCTX-M and blaTEM genes underscores the imperative role of ESBL testing in accurately identifying both beta-lactamase producers and nonproducers.

Phenotypic and genotypic detection of Beta-lactamase producing Pseudomonas aeruginosa isolated from Haryana, India

Antimicrobial resistance causes substantial risks to human health globally, and millions of people die worldwide due to multiple drug resistances. Beta-lactam drugs are common for curing infections, and resistance to these drugs cause serious threat to humans. The resistance is acquired by the gram-negative Pseudomonas aeruginosa by producing beta-lactamases such as metallo beta-lactamase (MBL), extended-spectrum beta-lactamase enzymes (ESBL), and AmpC β-lactamases. Hence, this study was intended to detect the occurrence of MBL, ESBL, and AmpC β-lactamases producing P. aeruginosa and to evaluate antibiotic sensitivity at the Pandit Bhagwat Dayal Sharma, Post Graduate Institute of Medical Sciences, Rohtak, Haryana, India. A total of 163 P. aeruginosa were isolated from the different samples of patients, such as urine, blood, sputum, pus, and pleural fluids. The P. aeruginosa was characterized morphologically, biochemically, and with matrix-assisted laser desorption ionization-time of flight mass spectrometry. Their antibiotic sensitivity was evaluated by the Kirby-Baur disc diffusion method. Antibiotic sensitivity tests of P. aeruginosa showed 163/163 were susceptible to Polymyxin-B, 78/163 and 65/163 were resistant against Ceftazidime (CAZ) and IMP antibiotics, respectively. The IMP, CAZ, and cefoxitin-resistant isolates were selected and further evaluated for ESBL, MBL, and AmpC enzyme production. In conclusion, the findings of this study indicated a significant presence of ESBL, MBL, and AmpC enzyme-producing P. aeruginosa among the patients. The ESBL prevalence was much higher in indoor patients than in outdoor patients. The total prevalence of MBL-producing strains in Imipenem-resistant P. aeruginosa (IRPA) was (46/62) 74.19%, which is an alarming signal. There was a higher prevalence of IRPA MBL-producing strains in indoor patients (36/46) 78.6% as compared to outdoor patients (10/16) 62.50%. Identification of bronchoalveolar lavage and sputum was also done using the Biofire Film Array, which revealed the resistant genes, including NDM (20 genes), CTX-M (17 genes), OXA-48-like (9 genes), VIM (5 genes), and IMP (2 genes). Antibiotics like cefotaxime and CAZ have less effect, but carbapenems and aminoglycosides are the best options for treating ESBL-producing P. aeruginosa. Drugs not recommended for treating this pathogen are penicillins and sulfonamides like co-trimoxazoles. Strict infection control measures, careful monitoring of antibiotic administration, and routine screening for ESBL-producing strains are advised before treating the patients.

Molecular identification of blaTEM and blaCTX-M genes in multidrug-resistant Escherichia coli found in milk samples from dairy cattle farms in Tulungagung, Indonesia.

Escherichia coli is an opportunistic bacteria that can grow easily, produce toxins, and resist antibiotics. The phenomenon of E. coli developing multidrug resistance is currently the subject of extensive research. The objective of this study was to molecularly identify blaTEM and blaCTX-M genes in multidrug-resistant E. coli found in milk samples from dairy cattle farms in Tulungagung, Indonesia. One hundred and ten milk samples were collected from 45 dairy cattle farms in Tulungagung, Indonesia. Indole, methyl red, Voges-Proskauer and in citrate tests and triple iron sugar agar tests were used to identify E. coli. Multidrug resistance was determined in isolates through antibiotic sensitivity tests using tetracycline, streptomycin, trimethoprim, chloramphenicol and aztreonam. Extended-spectrum beta lactamase enzyme production was confirmed by double-disc synergy test (DDST). Molecular identification was performed to confirm the blaTEM and blaCTX-M genes. One hundred and one (91.82%) E. coli strains were isolated from the samples. The antibiotic sensitivity test showed four (3.96%) multidrug-resistant (MDR) and one (0.99%) ESBL-positive E. coli by DDST confirmation. There were three (77.78%) blaTEM genes and one (0.99%) blaCTX-M gene discovered in the MDR E. coli isolates using PCR for molecular identification. The findings of the blaTEM and blaCTX-M genes encoding ESBL E. coli in dairy cattle milk in Tulungagung, Indonesia is concerning and argues for prompt action to stop the emergence of antibiotic resistance which has an impact on public health.

Pisciculture and Fish Parts as Non-clinical Source of CTX-M and TEM Extended Spectrum Beta-lactamases Producing Escherichia coli in Southeastern Nigeria

Background and Objectives: Antimicrobial agent use in pisciculture exerts a selective pressure, resulting in a reservoirs of extended spectrum beta-lactamase producing Escherichia coli in the aquatic environment. Here we screened pisciculture and fish parts as non-clinical source of CTX-M and TEM Extended Spectrum Beta-lactamases (ESBL) producing Escherichia coli in Southeastern Nigeria Methodology: A total of sixty fish parts [fish gills, body part and intestinal swab samples] were collected from pisciculture farm A, C, F and G within Abakaliki metropolis. The samples were bacteriologically analyzed using standard microbiological techniques for isolation and identification. ESBL enzymes were phenotypically detected in E. coli, isolates using the double disk diffusion technique. The presence of TEM and CTX-M genes was determined by polymerase chain reaction using specific primers. Results: Escherichia coli and Extended spectrum beta-lactamase producing Escherichia coli accounted for overall occurrence rate of 27(45.0 %) and 17(28.3 %) respectively. There was no statistically significant difference in the occurrence of Escherichia coli and ESBL-E. coli among the different samples P>0.05. The presence of TEM and CTX-M gene 80 % and 100 % respectively were confirmed among the isolates. Conclusion: Our findings reports the presence of ESBL gene in pisciculture and fish parts and it is important in understanding the mechanism of resistance operating in these common pathogens, which are also endemic in most pisciculture area. Strict rules and monitoring/surveillance of antimicrobial agent used in pisciculture activities combined with food safety training of farmer owners/breeders on various aspects of good hygiene practices are strongly recommended.

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.

A Molecular Investigation of Extended Spectrum Beta-Lactamase genes in Escherichia coli and Klebsiella spp. in raw cow Milk

Objective: Raw milk is an important source of nutrients. Therefore, today, there is a great demand for raw milk consumption. The positive side of milk consumption on growth and development cannot be ignored, but unfortunately, pathogens in raw milk are always potential public health risks for transmission pathogens. Bacteria such as Enterobacteriaceae in normal flora can cause serious problems due to their extended-spectrum beta-lactamase (ESBL) production. These bacteria and their resistance genes have been reported in raw milk. In this matter, the aim of the study is to determine the status of blaCTX-M-1, blaCTX-M-2, blaTEM, and blaSHV genes responsible for the production of ESBL enzyme in Escherichia coli and Klebsiella spp. strains to identify risk factors in raw milk consumption and to gain an understanding of the epidemiology of these resistant strains. 
 Materials and methods: A total of different 50 raw milk samples were collected and subjected to phenotypic microbiological analysis and Real-time PCR targeting blaCTX-M-1, blaCTX-M-2, blaTEM, and blaSHV genes. In the phenotypic analyses, suspicious isolates were identified by classical microbiological methods and antibiotic resistance profiles were revealed. 
 Results: These results indicated that raw milk is a potential reservoir for ESBL producing E. Coli, Klebsiella spp. strains are obviously significant. And It was determined that CTX-M based ESBL genes are predominant in ESBL production. The present study revealed that raw milk is epidemiologically involved in the transmission of ESBL genes. Raw milk could be distributed to ESBL genes widely with which is consumed in Şanlı Urfa.

Molecular surveillance reveals widespread colonisation by carbapenemase and extended spectrum beta-lactamase producing organisms in neonatal units in Kenya and Nigeria

ObjectivesNeonatal sepsis, a major cause of death amongst infants in sub-Saharan Africa, is often gut derived. Gut colonisation by Enterobacteriaceae producing extended spectrum beta-lactamase (ESBL) or carbapenemase enzymes can lead to antimicrobial-resistant (AMR) or untreatable infections. We sought to explore the rates of colonisation by ESBL or carbapenemase producers in two neonatal units (NNUs) in West and East Africa.MethodsStool and rectal swab samples were taken at multiple timepoints from newborns admitted to the NNUs at the University College Hospital, Ibadan, Nigeria and the Jaramogi Oginga Odinga Teaching and Referral Hospital, Kisumu, western Kenya. Samples were tested for ESBL and carbapenemase genes using a previously validated qPCR assay. Kaplan–Meier survival analysis was used to examine colonisation rates at both sites.ResultsIn total 119 stool and rectal swab samples were taken from 42 infants admitted to the two NNUs. Colonisation with ESBL (37 infants, 89%) was more common than with carbapenemase producers (26, 62.4%; P = 0.093). Median survival time before colonisation with ESBL organisms was 7 days and with carbapenemase producers 16 days (P = 0.035). The majority of ESBL genes detected belonged to the CTX-M-1 (36/38; 95%), and CTX-M-9 (2/36; 5%) groups, and the most prevalent carbapenemase was blaNDM (27/29, 93%).ConclusionsGut colonisation of neonates by AMR organisms was common and occurred rapidly in NNUs in Kenya and Nigeria. Active surveillance of colonisation will improve the understanding of AMR in these settings and guide infection control and antibiotic prescribing practice to improve clinical outcomes.

Phenotypic detection of extended spectrum beta-lactamases (ESBLs) in Gram-negative bacilli isolated from deferent clinical source

This study aimed to the detection of extended spectrum beta-lactamases by phenotypic and genotypic methods in nosocomial Gram-negative isolates. A number of 194 samples from hospitalized patients (urine, burn swab, surgical swab, and sputum) were collected These patients were at Al-Hussein and Al-Nasiriyah teaching hospitals in Al-Nasiriyah city. ESBLs enzymes were detected phenotypically by ceftazidime (30 μg) and cefotaxime (30 μg) (Screening test) and Double Disk Synergy Test (DDST) (Confirming test). The results showed that out of 109 isolates 89 (81.65%) were Gram negative bacilli. A total of 68 Gram-negative were β-lactamases producer. Moreover, the results of screening test recorded that 55 (80.88%) of isolates were resistant to cefotaxime and ceftazidime, while 17 (25%) isolates produced ESBLs by (DDST).

668. Presence of the Narrow-Spectrum OXA-1 Beta-Lactamase Enzyme is not Associated with Elevated Ceftolozane-Tazobactam MIC Values among ESBL-Producing Escherichia coli Clinical Isolates (CANWARD, 2011-2018)

Abstract Background Beta-lactam-beta-lactamase inhibitor combinations have been proposed as an alternative to carbapenems for the treatment of infections caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli as an antimicrobial stewardship initiative. However, in a recent randomized trial evaluating patients with bacteremia, presence of the narrow spectrum OXA-1 beta-lactamase enzyme among ESBL-producing Enterobacterales was associated with higher piperacillin-tazobactam MICs, and in turn excess mortality among patients treated with piperacillin-tazobactam relative to meropenem. The purpose of this study was to determine whether the in vitro activity of ceftolozane-tazobactam versus ESBL-producing E. coli is similarly compromised by the presence of OXA-1. Methods E. coli clinical isolates were obtained from patients evaluated at hospitals across Canada (January 2011 to December 2018) as part of an ongoing national surveillance study (CANWARD). ESBL production was confirmed using the Clinical and Laboratory Standards Institute phenotypic method. Susceptibility testing was carried out using custom broth microdilution panels, and all isolates underwent whole genome sequencing for beta-lactamase gene detection. Results In total, 485 ESBL-producing E. coli identified as part of the CANWARD study were included. The majority of isolates (91.3%; 443/485) harbored a CTX-M ESBL enzyme. OXA-1 was present in 39.6% (192/485) of isolates. OXA-1 was detected in 62.5% (187/299) of isolates with a CTX-M-15 ESBL enzyme versus only 2.7% (5/186) of isolates with other ESBL enzyme types. Ceftolozane-tazobactam MIC50 and MIC90 values were identical (0.25 µg/mL and 1 µg/mL, respectively) for the isolate subsets with and without OXA-1. Overall, 97.4% and 96.9% of isolates with and without OXA-1 remained susceptible (CLSI breakpoint) to ceftolozane-tazobactam. Conclusion The presence of OXA-1 among ESBL-producing E. coli clinical isolates was not associated with an elevation in MIC values for ceftolozane-tazobactam. These data support further evaluation of ceftolozane-tazobactam as an alternative to carbapenems for the treatment of infections caused by ESBL-producing E. coli, as is being done with the MERINO-3 trial. Disclosures George Zhanel, PhD, Merck: Grant/Research Support.

229. Molecular and Clinical Epidemiology of Carbapenemase-Producing E. coli Isolates from Different Global Regions (CRACKLE-2)

Abstract Background Carbapenemase-producing (CP) Escherichia coli (CPEc) are a global public health threat. We describe the epidemiology and outcomes of patients with CPEc isolates obtained in CRACKLE 2, a prospective cohort study of hospitalized patients with positive cultures for CP Enterobacteriaceae. Methods In CRACKLE-2, patients with CPEc were enrolled from 26 hospitals in 6 countries (ClinicalTrials.gov NCT03646227). Clinical data were collected, and bacterial isolates underwent whole genome sequencing (WGS). Here, we included unique patients with CPEc by WGS (n=114). The primary outcome was desirability of outcome ranking (DOOR) at 30 days after index culture. Chi squared tests with alpha = 0.05 were used to evaluate differences in culture source and outcomes between metallo-beta-lactamase (MBL) and non-MBL isolates. Results Of 114 CPEc isolates, 57 (50%) represented infection (Table 1). Isolates primarily arose from urine (34%) and blood (21%). Compared to non-MBL isolates, isolates containing MBL were more often from urine (41% vs 29%) and less frequently from blood (6% vs 32%); p=0.02. We observed strong regional variations in CP (Figure 1) and MBL (p < 0.0001). Sequence type (ST) 167 was more common among MBL than non-MBL isolates (31% vs 2%, p< 0.0001); non-MBL isolates were more often ST410 and ST131 (17% and 20%). Extended-spectrum beta-lactamases (ESBL) were present in 52% of isolates; most commonly, CTX-M-15 in both MBL (33%) and non-MBL isolates (34%). Phylogenetic analysis of the isolates and corresponding region, bacterial characteristics, and DOOR outcomes are in Figure 1. Death at 30 days occurred in 18 (16%) of patients, more commonly among non-MBL than MBL CPEc (23% vs 6%; p=0.01). The probability of a better DOOR outcome for a randomly selected MBL was 58% [95% CI: 48.2, 67.4], indicating no significant difference between the groups. Figure 1:Phylogenetic population structures of Carbapenemase-producing Escherichia coli (CPEc) isolates Legend: Infection = categorization as infection or colonization. ST = sequence type. BlaCarb = Carbapenemase gene. BlaESBL = acquired ESBL enzymes. DOOR = desirability of outcome ranking. DOOR rankings: 1 = Alive without events; DOOR 2 = Alive with 1 event; DOOR 3 = Alive with 2 or 3 events; DOOR 4 = dead. Conclusion Emergence of carbapenem resistance with important geographic variations was observed in E coli including among high-risk clones such as ST131. Mortality was higher among non-MBL isolates, which were more frequently from blood, but these findings may be confounded by region. Disclosures Vance G. Fowler, Jr, MD, MHS, Affinergy: Grant/Research Support|Affinergy: Honoraria|Affinium: Honoraria|Amphliphi Biosciences: Honoraria|ArcBio: Stocks/Bonds|Basilea: Grant/Research Support|Basilea: Honoraria|Bayer: Honoraria|C3J: Honoraria|Cerexa/Forest/Actavis/Allergan: Grant/Research Support|Contrafect: Grant/Research Support|Contrafect: Honoraria|Cubist/Merck: Grant/Research Support|Debiopharm: Grant/Research Support|Deep Blue: Grant/Research Support|Destiny: Honoraria|Genentech: Grant/Research Support|Genentech: Honoraria|Integrated Biotherapeutics: Honoraria|Janssen: Grant/Research Support|Janssen: Honoraria|Karius: Grant/Research Support|Medicines Co.: Honoraria|MedImmune: Grant/Research Support|MedImmune: Honoraria|NIH: Grant/Research Support|Novartis: Grant/Research Support|Novartis: Honoraria|Pfizer: Grant/Research Support|Regeneron: Grant/Research Support|Regeneron: Honoraria|Sepsis diagnostics: Sepsis diagnostics patent pending|UpToDate: Royalties|Valanbio: Stocks/Bonds Cesar A. Arias, MD, PhD, Entasis Phramceuticals: Grant/Research Support|MeMed Diagnostics: Grant/Research Support|Merck: Grant/Research Support David Paterson, MBBS, Accelerate: Honoraria|bioMerieux: Honoraria|Entasis: Advisor/Consultant|Janssen-Cilag: Grant/Research Support|MSD: Advisor/Consultant|MSD: Grant/Research Support|MSD: Honoraria|Pfizer: Grant/Research Support|Pfizer: Honoraria|PPD: Grant/Research Support|Shionogi: Grant/Research Support|VenatoRx: Advisor/Consultant Karen M. Ordonez, MD, AstraZeneca: Expert Testimony|Biomerieux: Expert Testimony|Farma de Colombia: Expert Testimony|MSD: Expert Testimony|Pfizer: Expert Testimony Souha S. Kanj, Pr, MSD, Pfizer, Gilead, Menarini, Astellas: Advisor/Consultant|MSD, Pfizer, Gilead, Menarini, Astellas: Honoraria Robert Bonomo, MD, Cystic Fibrosis Foundation: Grant/Research Support|Merck: Grant/Research Support|NIH: Grant/Research Support|VA: Grant/Research Support|VenatoRx: Grant/Research Support|Wockhardt: Grant/Research Support David van Duin, MD, PhD, Achaogen: Advisor/Consultant|Allergan: Advisor/Consultant|Astellas: Advisor/Consultant|MedImmune: Advisor/Consultant|Melinta: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|NeuMedicine: Advisor/Consultant|Pfizer: Advisor/Consultant|Qpex: Advisor/Consultant|Roche: Advisor/Consultant|Sanofi-Pasteur: Advisor/Consultant|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support|T2 Biosystems: Advisor/Consultant|Tetraphase: Advisor/Consultant.

Phenotypic and genotypic detection of extended spectrum beta lactamase enzyme in Klebsiella pneumoniae.

Klebsiella species are ubiquitous in nature and can be found in the natural environment and on mucosal surfaces of mammals and it is an important multidrug-resistant pathogen affecting humans and is a major source for hospital acquired infections. The aim of this study is to investigate the prevalence of ESBL enzyme among Klebsiella pneumoniae isolates by phenotypic methods from different hospital wards and detection of ESBL resistance genes such as TEM and SHV in Sulaimani city/ Kurdistan-Iraq. Klebsiella pneumoniae isolates were collected from different clinical samples from different hospitals, the isolates were identified by standard technique. Screening of ESBLs was undertaken by using double disk diffusion and standard disk diffusion methods. Real-time PCR was used for genotypic detection of TEM and SHV genes according to the standard protocol. Out of 54 Klebsiella pneumoniae isolates; 28 were ESBL positive, The pattern of antimicrobial susceptibility testing showed that the most resistant antibiotic are AMP (100%), AMC (100%) followed by CAZ (83.33%), CTX (75.9%), CPM (74%), ATM (70.37%). Both TEM and SHV genes were detected among 28 (51.85%) ESBL positive by using Real-time PCR method. SHV gene was detected in most of the isolates of ESBL producers of Klebsiella pneumoniae.

Colonization dynamics of extended-spectrum beta-lactamase-producing Enterobacterales in the gut of Malawian adults

Drug-resistant bacteria of the order Enterobacterales which produce extended-spectrum beta-lactamase enzymes (ESBL-Enterobacterales, ESBL-E) are global priority pathogens. Antimicrobial stewardship interventions proposed to curb their spread include shorter courses of antimicrobials to reduce selection pressure but individual-level acquisition and selection dynamics are poorly understood. We sampled stool of 425 adults (aged 16–76 years) in Blantyre, Malawi, over 6 months and used multistate modelling and whole-genome sequencing to understand colonization dynamics of ESBL-E. Models suggest a prolonged effect of antimicrobials such that truncating an antimicrobial course at 2 days has a limited effect in reducing colonization. Genomic analysis shows largely indistinguishable diversity of healthcare-associated and community-acquired isolates, hence some apparent acquisition of ESBL-E during hospitalization may instead represent selection from a patient’s microbiota by antimicrobial exposure. Our approach could help guide stewardship protocols; interventions that aim to review and truncate courses of unneeded antimicrobials may be of limited use in preventing ESBL-E colonization.

Evaluation of canine raw food products for the presence of extended-spectrum beta-lactamase- and carbapenemase-producing bacteria of the order Enterobacterales.

To assess the potential contamination of commercial raw dog food products with bacteria of the Enterobacterales order that produce extended spectrum beta-lactamase (ESBL) and carbapenemase enzymes, determine risk factors for contamination, and understand isolate genetic diversity. A total of 200 canine raw food products. Products were cultured on selective chromogenic agar following enrichment steps. Whole-genome sequencing was performed for isolates that were confirmed to produce an ESBL. Isolates were characterized by antimicrobial resistance genes, and multilocus sequences typing, and compared to other isolates in the NCBI database for clonality. Preservation method and protein sources were assessed as potential risk factors for contamination with ESBL and carbapenemase-producing bacteria of the Enterobacterales order. No carbapenemase-producing Enterobacterales (CPE) were identified, but ESBL-producing Enterobacterales bacteria were isolated from 20/200 products (10.0%; 95% CI, 7.3 to 16.5%), all of which were frozen. Pork-derived protein source products were 8.1 times (P = .001; 95% CI, 2.53 to 26.2) more likely to carry ESBL-producing Enterobacterales bacteria than other protein sources. WGS analysis confirmed the presence of ESBL genes in a total of 25 distinct isolates (19 Escherichia coli, 5 Klebsiella pneumoniae, and 1 Citrobacter braakii). Genes encoding CTX-M type ESBL enzymes were the most common (24/25 isolates, 96.0%) with blaCTX-M-27 being the most common allele (8/25, 32.0%). Frozen, raw food products may serve as a route of transmission of ESBL-producing Enterobacterales bacteria to companion animals. Veterinarians should advise owners about the risks of raw food diets, including potential exposure to antimicrobial-resistant bacteria.

Prevalence of ESBL genes in Klebsiella pneumoniae from individuals with community-acquired urinary tract infection in rural communities of Enugu State, Nigeria

Background: The emergence and spread of extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae is now a major challenge in treating urinary tract infections. This study was conducted to determine the Prevalence of ESBL genes in Klebsiella pneumoniae and the influence of these genes on the antimicrobial susceptibility patterns of the isolates, obtained from community acquired urinary tract infection in Enugu state rural communities. Method: A total of 735 clean catch mid-stream urine samples were collected from February, 2021 to June, 2021. The urine samples were cultured and Klebsiella pneumoniae were identified morphologically, biochemically, and then typed down, using PCR, Gel electrophoresis, and Sanger Sequencing by Genewiz. Antibiotic susceptibility testing was done by modified Kirby-Bauer disc diffusion method and interpretation was done following Clinical and Laboratory Standard Institute (CLSI) guidelines. ESBL screening was done by the phenotypic method, using the standard disk diffusion method, whereas the phenotypic confirmation of ESBL producers was done by the double-disk synergy method, and interpreted according to CLSI guidelines. Multiplex PCR was used to detect the genes for SHV and CTX-M while conventional linear PCR was done for TEM and blas GES type ESBL genes. Results: A total of 77 isolates were identified as Klebsiella pneumoniae, and the prevalence of positive ESBL in them were 10(12.9%). Of the 77 isolates that were tested for antibiogram, 29 isolates were multidrug-resistance (MDR). Out of the MDR isolates, 10 were ESBL positive, whereas 19 were not. Of this 29 MDR isolates, 6 were extensively-drug resistant (XDR). Of the 6 XDR isolates, 3 do not possess ESBL enzymes, whereas 3 of them do. Age range of 31-40 contributed the highest prevalence of ESBL genes (60%), whereas age range of 10-20, 21-30, and 51-70 did not produce any ESBL gene. bla TEM gene was the most prevalent ESBL resistant genes with a prevalence of 10 (100%), followed by SHV 9(90%), whereas bla GES gene and bla CTX-M have the least prevalence of 4 (40%) each. Conclusion: Higher prevalence of MDR, XDR, and ESBL-producing Klebsiella pneumoniae were observed, thus the need for public health intervention for effective prevention and control of antimicrobial resistance, and proper treatment of UTIs.

The Status of Extended-spectrum β-lactamase and AmpC β-lactamase production in uropathogenic isolates of Escherichia coli and Klebsiella pneumonia in Bangladesh

Background: The rapid dissemination of antibiotic resistance as extended-spectrum beta-lactamases (ESBLs) and AmpC β-lactamases in bacteria became a major public health concern worldwide. Hence, this study was carried out to evaluate the status of uropathogenic isolates of Bangladesh especially Escherichia coli and Klebsiella pneumoniae in terms of ESBL and AmpC β-lactamases enzymes production. Methods: A laboratory-based cross-sectional survey was conducted with 553 urine samples at the Department of Microbiology, Popular Diagnostic Centre Ltd. Dhaka, Bangladesh for a period of 2.5 years from July 2017 to December 2019. Pathogens were obtained upon culture, and screened for ESBL production following the cephalosporin disk diffusion method and cephalosporin-clavulanate combination disc test (confirmatory) method. For AmpC β-lactamases enzymes, cefoxitin disc diffusion, and AmpC disc test (confirmatory) methods were performed. The positive isolates were further genotyped for the presence of blaTEM, blaSHV, blaOXA, and blaCTX-M β-lactamases genes in case of ESBL and blaFOX, blaCMY, and blaDHA genes for AmpC β-lactamases by polymerase chain reaction. Results: Of all urine samples, uropathogens were detected among 28.57% (158, n=553) samples. Among them, 46.20% (73) were E. coli and 20.25% (32) were K. pneumoniae. The prevalence of ESBL and AmpC β-lactamases production in E. coli was 34.24% (25) and 17.80% (13) respectively whereas it was 43.75% (14) and 21.87% (7) in Klebsiella pneumoniae isolates respectively. These resistance properties were further confirmed by phenotypic disc test. In case of ESBLs, most typical isolates possessed two or more resistance genes. Overall, the most prevalent gene was blaCTX-M (84.61%) followed by blaTEM (64.10%), blaOXA (46.15%) and blaSHV (25.64%). In case of AmpC β-lactamases FOX gene was most prevalent (45%) followed by CMY (20%) and DHA 3 (15%). Conclusion: The high prevalence of ESBL and AmpC β-lactamases production in our study emphasizes the judicious use of antibiotics in controlling urinary tract infections in Bangladesh.

Detection of extended spectrum beta-lactamase (ESBL)–production in Salmonella Typhimurium isolated from poultry birds in Nasarawa State, Nigeria

Salmonella Typhimurium is an important zoonotic food borne pathogen and the second most widely studied pathogen in relation to antimicrobial resistance studies in Nigeria. Its resistance to β-lactam antibiotics is on the rise globally due to persistent exposure and production of Extended Spectrum Beta-lactamase enzymes (ESBLs) which is barely reported to occur among food producing animals in Nigeria. This poses a threat to the successful therapy of salmonellosis infections and raises concern on food safety. Further complicating these challenges is the lack of strict laws mitigating antibiotic usage in Nigeria. The study was conducted in the Microbiology laboratory of Nasarawa State University, Keffi, Nigeria. A total of 300 samples each of poultry droppings, flesh, feeds, human hand swabs and stool were randomly collected following ethical approval and screened for ESBL- producing S. Typhimurium using standard Microbiological methods of isolation and identification. Antibiotic susceptibility testing, phenotypic ESBL detection and interpretation were carried out as described by the Clinical Laboratory and Standard Institute (CLSI) guideline. The bla genes were identified using polymerase chain reaction (PCR) method. A total of 106 S.Typhimurium were isolated with the following resistance profile; Ampicillin 99(93.4%), Augmentin 104(98.1%), Ceftriaxone 74(69.8%), Ceftazidime 50(47.2%), Imipenem 1(1.0%). The antibiotic resistance of S. Typhimurium in the study is significant (P = 0.01). The Multiple Antibiotic Resistance (MAR) Index was > 0.2. Out of 106 isolates, 32 distinct antimicrobial resistant phenotypes were observed and 62 (81.6%) were confirmed ESBL producers having 19 distinct phenotypes. The bla genes were identified among 38.7% (24/62) isolates; 100% blaSHV (24/24), 87.5 %(21/24)blaTEM with 54.2% (13/24) co-carriage of blaTEM-SHV. The blaCTX-M was not detected. Poultry is therefore an important reservoir and source of transmission of ESBL- producing S.Typhimurium, which is a major public health concern. The high MAR index suggests frequent antibiotic usage within our study area, thus calls for antibiotic stewardship in animal production in Nigeria.

Bacteriological profile of purulent otitis media at the Avicenna Military Hospital in Marrakech

Chronic suppurative otitis media (CSOM) is a perforated tympanic membrane with persistent drainage from the middle ear. It is an avoidable cause of hearing loss, especially in developing countries. We conducted a prospective study over 8 years (1 January 2014 - 1 January 2022), Including 250 patients consulting the Oto-Rhino-Laryngology Department of the Avicenna Military Hospital in Marrakesh, in collaboration with the Laboratory of Microbiology. It included all non-redundant bacterial species isolated from pus of purulent chronic ear infections sent to the microbiology laboratory. The cytobacteriological analysis was carried out according to the conventional techniques and / or automated method of BD PhoenixTM. The antibiotic sensitivity study was carried using the diffusion method in agar medium according to the recommendations of the CA-SFM. There were 147 men and 103 women with a sex ratio of 1.4. The mean age of the patients was 34 years (8 to 82 years). The second and fourth decades represent the most affected age groups. The bacteriological profile was dominated by Pseudomonas aeruginosa (40%), followed by Staphylococcus aureus (32%) and Proteus mirabilis (13%). These different isolates represented low antibiotic resistance levels but all remained sensitive to fluoroquinolones. Five strains of enterobacteria produced derepressed cephalosporinase, and nine strains of Enterobacteria carrying extended spectrum Beta lactamase enzymes. No strain of Pseudomonas resistant to ceftazidime was isolated. Gram positive cocci accounted for 38% of the strains. In view of the bacteriological and the sensitivity profiles of the species found in chronic otitis, antibiotics based on fluoroquinolones are recommended.

Emergence of High Prevalence of Extended-Spectrum Beta-Lactamase and Carbapenemase-Producing Enterobacteriaceae Species among Patients in Northwestern Ethiopia Region.

Background World Health Organization identified some Enterobacteriaceae as superbugs because of their high production and spread of extended-spectrum beta-lactamases (ESBL) and carbapenemases. Moreover, their resistance against commonly prescribed antibiotics left few choices of drugs to treat infection. This study is aimed at determining the magnitude of ESBL and carbapenemase-producing Enterobacteriaceae pathogens and their antimicrobial resistance pattern. Materials and Methods A hospital-based cross-sectional study was carried out from February to April 2019 in the Northwestern Ethiopia region. A total of 384 patients presumptive for bacterial infections were conveniently enrolled in the study. Specimens were collected and processed following standard bacteriological procedures. Drug susceptibility tests were performed using disk diffusion technique. ESBL and carbapenemase enzymes were tested by double disk diffusion and modified carbapenem inhibition methods, respectively. The data obtained were analyzed using SPSS version 22 software, and descriptive statistics were summarized in tables and graphs. Results Out of 384 clinical specimens processed 100 (26%) were culture positive for Enterobacteriaceae. The proportion of Enterobacteriaceae infection was relatively higher among in-patients 86 (32.6%) than out-patients 14 (11.7%). Overall, Escherichia coli 35 (9.1%) was the leading isolate followed by Klebsiella pneumoniae 31 (8.1%). Klebsiella pneumoniae 15 (15.6%) was the most frequent isolate from bloodstream infection and is the leading isolate from intensive care unit patients 15 (38.3%). Overall, 44 (44%) of Enterobacteriaceae were extended-spectrum beta-lactamase producers. Among them, Citrobacter spp. was the leading one 4 (80%) followed by Enterobacter cloacae 6 (60%) and K. pneumoniae 18 (58.1%). Furthermore, 6 (6%) of Enterobacteriaceae were carbapenemase-producers, in which 5 (50%) of E. cloacae and 3 (9.7%) of K. pneumoniae had highest percentage. Conclusions. ESBL and carbapenemase-producing isolates of Enterobacteriaceae are alarmingly spreading in the study area. Thus, improving the infection prevention strategy and further screening at the national level is recommended to develop the optimal use of antibiotics.

1216. Presence of the Narrow-Spectrum OXA-1 Beta-lactamase Enzyme Is Associated with Elevated Piperacillin-Tazobactam MIC Values Among ESBL-producing Escherichia coli Clinical Isolates (CANWARD, 2007-2018)

BackgroundThe clinical outcome of patients with bacteremia due to an extended-spectrum beta-lactamase (ESBL)-producing member of the family Enterobacteriaceae who are treated with piperacillin-tazobactam appears to depend, at least in part, on the piperacillin-tazobactam MIC. The purpose of this study was to determine whether there is any association between the MIC of piperacillin-tazobactam and presence of the narrow spectrum OXA-1 beta-lactamase enzyme among ESBL-producing Escherichia coli.Methods E. coli clinical isolates were obtained from patients evaluated at hospitals across Canada (January 2007 to December 2018) as part of an ongoing national surveillance study (CANWARD). ESBL production was confirmed using the Clinical and Laboratory Standards Institute phenotypic method. Susceptibility testing was carried out using custom broth microdilution panels, and all isolates underwent whole genome sequencing for beta-lactamase gene detection.ResultsIn total, 671 ESBL-producing E. coli were identified as part of the CANWARD study. The majority of isolates (92.0%; 617/671) harbored a CTX-M ESBL enzyme. CTX-M-15 (62.3%; 418/671), CTX-M-27 (13.9%; 93/671), and CTX-M-14 (13.4%; 90/671) were the most common variants identified. The narrow spectrum OXA-1 beta-lactamase enzyme was present in 42.6% (286/671) of isolates. OXA-1 was detected in 66.3% (277/418) of isolates with a CTX-M-15 ESBL enzyme versus only 3.6% (9/253) of isolates with other ESBL enzyme types. The piperacillin-tazobactam MIC50 and MIC90 values were 8 µg/mL and 32 µg/mL for isolates that possessed the OXA-1 enzyme versus 2 μg/mL and 8 µg/mL for those that did not. The percentage of ESBL-producing E. coli isolates that were inhibited by a piperacillin-tazobactam MIC of ≤8 μg/mL was 68.5% for isolates that were OXA-1 positive and 93.8% for isolates that were OXA-1 negative.ConclusionThe MIC50 and MIC90 values of piperacillin-tazobactam among ESBL-producing E. coli were higher for the subset of isolates that harbored a narrow spectrum OXA-1 beta-lactamase enzyme relative to the subset that did not. This association was primarily observed among ESBL-producers with the CTX-M-15 enzyme variant. OXA-1 was infrequently detected among isolates with other ESBL enzyme types.Disclosures George Zhanel, PhD, AVIR (Grant/Research Support)Iterum (Grant/Research Support)Merck (Grant/Research Support)Pfizer (Grant/Research Support)Sandoz (Grant/Research Support)Sunovion (Grant/Research Support)Venatorx (Grant/Research Support)Verity (Grant/Research Support)