Double-disk Synergy Test Research Articles

Genetic characterization of Escherichia coli and Klebsiella spp. from humans and poultry in Nigeria.

The emergence of antibiotic resistance in livestock, especially food-producing animals, is of major public health importance as a result of the possibility of these bacteria entering the food chain. In this study, the genetic characteristics of antibiotic-resistant Escherichia coli and Klebsiella spp. isolates from humans and poultry in Edo state, Nigeria, were investigated. In April 2017, 45 Klebsiella spp. and 46 E. coli isolates were obtained from urine, clinical wounds, nasal and chicken faecal samples. Isolates were recovered and identified as previously described. Species identification was achieved by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and ribosomal multilocus sequence typing. Antimicrobial susceptibility testing was carried out using the Kirby-Bauer method for 12 antibiotics. A double disc synergy test was used to screen for extended-spectrum beta-lactamse (ESBL) production. Whole genome sequencing was performed for strain characterization of the isolates. Thirteen Klebsiella spp. isolates yielded positive results by the ESBL phenotypic test and harboured ESBL genes. Of the 46 E. coli isolates, 21 human and 13 poultry isolates were resistant to at least one of the tested antibiotics. Four human E. coli isolates harboured ESBL genes and revealed positive results when applying ESBL double disc synergy tests. ESBL genes in the Klebsiella spp. and E. coli isolates include bla CTX-M-15 and bla SHV-28. Whole genome-based core gene multilocus sequence typing of the Klebsiella spp. and E. coli isolates revealed a close relatedness among the isolates. An integrated 'One Health' surveillance system is required to monitor transmission of antimicrobial resistance in Nigeria.

Extended-Spectrum Beta-lactamase and Metallo-beta-lactamase Production among Escherichia coli and Klebsiella pneumoniae Strains from Urine of Pregnant Women in Afikpo, Ebonyi State, Nigeria

Aim: The aim of this study was to determine the occurrence of Extended-spectrum beta-lactamase (ESBL) and Metallo-beta-lactamase (MBL) among Escherichia coli and Klebsiella pneumoniae strains from pregnant women attending Mater Misericordia Hospital Afikpo, Ebonyi state, Nigeria.
 Study Design: This is a laboratory based prospective study carried out on pregnant women suspected of having urinary tract infection and was requested to undergo diagnosis at microbiology laboratory of the hospital.
 Place and Duration of Study: The study was conducted in the Department of Science Laboratory Technology, Akanu Ibiam Federal Polytechnic, Unwana, Afikpo, Ebonyi State, Nigeria from October, 2022 to January, 2023.
 Methodology: Clean-catch midstream urine samples were collected from 206 pregnant women suspected of having urinary tract infection and were requested to undergo medical diagnosis at microbiology laboratory of the hospital. The urine samples were processed following standard microbiological procedure. Antimicrobial susceptibility testing was determined using the disc diffusion method, while ESBL phenotypes were determine by the Double-Disc Synergy Test (DDST). Disc potentiation test was performed to check for MBL production.
 Results: Out of the 206 urine samples processed, 24 (11.7 %) E. coli and 12 (5.8 %) K. pneumoniae were isolated. The antimicrobial susceptibility of the isolates recorded a 100 % resistance with Amoxicillin/Clavulanic acid and Cotrimoxazole. The Gram-negative isolates showed a high sensitivity of 100 % to Netilmicin, Meropenem and Ofloxacin. Overall, 35 (97.2 %) multidrug resistance (MDR) was observed of the bacteria isolates. A total of 9 (37.5 %) E. coli and 4 (33.3 %) K. pneumoniae was found positive for ESBL production whereas, 5 (20.8 %) E. coli and 2 (16.7 %) K. pneumoniae were MBL positive.
 Conclusion: The level of drug resistance in this study underscores the need for regular surveillance for effective management of urinary tract infection in pregnancy.

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.

Phenotypic and molecular characterization of enteropathogenic Escherichia coli and Salmonella spp. causing childhood diarrhoea in Awka, South-Eastern Nigeria

BackgroundDiarrhoea is a major cause of childhood disease in the developing countries. This experimental study investigated the prevalence of ESBL and MBL genes in enteropathogenic strains of Escherichia coli and Salmonella spp. isolated from diarrheagenic children in Awka, Nigeria.MethodsTwo hundred stool samples were collected from diarrhea patients in three paediatric hospitals within Awka metropolis, Nigeria. All E. coli and Salmonella spp. isolated through standard bacteriological methods were subjected to antibiotic-susceptibility testing. Double disc synergy and imipenem-EDTA combined disc tests were used to phenotypically confirm the presence of ESBL and MBL respectively. PCR amplification of β-lactamase genes was done.ResultsThe prevalence of E. coli and Salmonella species in this study were 54% and 24.5% respectively. The organisms were highly resistant to metronidazole, cefuroxime and ceftazidime, and also showed a high sensitivity to nitrofurantoin and gentamicin. ESBL production was recorded in E. coli (49%) and Salmonella spp. (51.1%) while 27 isolates of E. coli (25%) and 7 isolates of Salmonella spp. were confirmed MBL positive by the combined disk diffusion technique. Eleven E. coli and 4 Salmonella spp. co-harbored both ESBL and MBL production. The most prevalent MBL gene in this study is the blaVIM gene (18.8%) which mediate MBL production in Gram negative bacteria; and this was followed by blaSHV (12.5%), blaTEM and blaCTX-M (6.3% each) for E. coli isolates. Salmonella spp. was recorded to have blaVIM (28.8%), blaSHV (28.8%), blaTEM (14.3%) and blaCTX-M (14.3%) genes.ConclusionsThis study reveals the prevalence of enteropathogenic E. coli and Salmonella strains bacteriologically recovered from diarrheic children in Awka, Nigeria, and which were found to be multiple resistant to clinically-relevant antibiotics because they co-express ESBL and MBL genes which mediate multidrug resistance in Gram negative bacteria.

Isolation and Speciation of Acinetobacter Species with Special Reference to Antibiotic Resistance in Tertiary Care Hospital

Acinetobacter can survive longer in the environment and is also known for developing resistance against agents like disinfectants and nutritional deprivation. Very restricted information about Acinetobacter is available because of their confused taxonomic status. The isolation and identification of resistance pattern help in the selection and search for new antibiotics, reducing the morbidity and mortality of patients. The present study was conducted to find the different types of resistant mechanisms in Acinetobacter species and their antimicrobial susceptibility pattern from various clinical samples. Gram's stain and biochemical reactions identified Acinetobacter isolated from various clinical samples. Antibiotic susceptibility testing was done, and their resistant pattern was observed. Phenotypic methods for drug resistance were carried out -Detection of Extended Spectrum Beta Lactamase (ESBL) by Double disc synergy test (DDST), detection of Metallo Beta Lactamase (MBL) by Imipenem with EDTA, and detection of Carbapenemase production by Modified Hodge test (MHT). The results showed that out of 150 isolates of Acinetobacter species, Acinetobacter baumannii 138 (92%) was the most common species isolated, followed by Acinetobacter lwoffii 10 (7%) and Acinetobacter hemolytic 2 (1%). Of these, 22 (15%) isolates showed ESBL production by Double disc synergy test, 80 (53%) isolates were MBL producers, and 11 (7%) were Carbapenemase producers by Modified Hodge test. The study observed that ESBL production in Acinetobacter was 22(15%) and MBL 80(53%). It demonstrated that most of the Acinetobacter isolated were found to be Multi-Drug Resistant (95%). It brings out the need for active surveillance combined to eradicate the curtail of this organism in hospital settings.

FIRST DHA-PRODUCING KLEBSIELLA PNEUMONIAE ISOLATED FROM A URINARY TRACT INFECTION IN ROMANIA

Beta-lactamase production is a major antibiotic resistance determinant in Enterobacterales. A carbapenemresistant Klebsiella pneumoniae isolate was recovered from routine screening in a tertiary urology hospital. The strain was evaluated phenotypically using the interpretative reading of an extended disk diffusion antibiogram and for specific antibiotics, the minimum inhibitory concentration (MIC) was performed. Phenotypic tests were further performed such as the CIM, mCIM, rCIM, the double disk synergy test, Blue Carba Test, and NGTEST® CARBA-5 and carbapenemase production was not confirmed. The isolate was sequenced and multiple resistance genes were identified. This report signifies the first description of DHA-1 in a Klebsiella pneumoniae strain in Romania.

PREVALENCE RATE OF ESBL AMONG ENTEROBACTERIACEAE ISOLATED FROM UTI PATIENTS IN SULAIMANI PROVINCE

Background Extended-Spectrum β-Lactamase are enzymes that provide resistance against third-and fourth generation Cephalosporins and Monobactams, and they are distributed among the Enterobacteriaceae family.&#x0D; Objectives To describe the prevalence of Extended-Spectrum β-Lactamase among Enterobacteriaceae causing urinary tract infections in Sulaimani province.&#x0D; Patients and MethodsOne hundred bacterial isolates of Enterobacteriaceae from patients with urinary tract infections attending Smart Hospital (inpatients and outpatients). Urine samples were inoculated onto different culture media. Colony morphology, gram staining, and BD Phoenix™ system were used for bacterial identification. Antibiotic profile and Extended-Spectrum β-Lactamase were observed phenotypically by antibiotic profile results, double disk synergy test, and confirmed by combined disk test methods and BD Phoenix™ system.&#x0D; ResultsOut of one hundred isolates of Enterobacteriaceae, Escherichia coli was the commonest isolate (89), followed by Klebsiella pneumoniae (10) and one isolate of Proteus mirabilis. According to the antibiotic profile, the most effective antibiotic among all three isolates was Imipenem and Nitrofurantoin, while the most resistant antibiotic was Nalidixic acid and third generation Cephalosporin. The prevalence rate of Extended-Spectrum β-Lactamase -producing Enterobacteriaceae was 69% by the screening tests and 48% by the confirmatory tests.&#x0D; ConclusionIn this study, Extended-Spectrum β-Lactamase prevalence was shown to be at an alarming rate that must be considered. The high priority of public health justifies further investigation to properly establish annual surveillance systems that can aid in selecting an appropriate antibiotic upon ESBL detection.

Detection of plasmid-mediated AmpC β-lactamases in Klebsiella pneumoniae clinical isolates from Bushehr province, Iran.

Plasmid-mediated AmpC producers are considered an increasing concern. The aim of this study was to investigate the prevalence of plasmid-mediated AmpC β-lactamases (pAmpCs) in Klebsiella pneumoniae isolates. A total of 228 clinical isolates of K. pneumoniae were collected in Bushehr province, Iran, from December 2017 to February 2019. Cefoxitin disks were applied for screening AmpC-producing isolates. Furthermore, 3 phenotypic confirmatory tests including combine disk test (CDT), double disk synergy test (DDST) and modified three dimensional test (M3DT) were used. Finally, the presence of pAmpC genes was tested by multiplex PCR. We identified 18 pAmpC-KP isolates among the 228 isolates (7.9%): 12 DHA (66.6%) and 6 CMY (33.3%). In the present study only 47% of cefoxitin-resistant isolates were pAmpC producers. The sensitivity of CDT, DDST, and M3DT was 89%, 67% and 100% and the specificity was 90%, 90% and 85%, respectively. In addition, M3DT displayed a higher rate of efficiency (92%) than CDT (89%) and DDST (79%) in detecting plasmid-meditated AmpC producers. DHA was the most prevalent pAmpC beta-lactamase in this study. DDST and CDT tests proved inefficient to detect two and six pAmpC producers, respectively, while M3DT represented the best overall performance.

Epidemiology of extended-spectrum beta-lactamase-producingEscherichia coli at the human-animal-environment interface in a farming community of central Uganda.

Extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-Ec) represents a significant global public health concern. The epidemiology of ESBL-Ec in Uganda is not well understood although it is harbored by humans, animals, and the environment. This study explains the epidemiology of ESBL-Ec using a one health approach in selected farming households in Wakiso district, Uganda. Environmental, human, and animal samples were collected from 104 households. Additional data were obtained using observation checklists and through interviews with household members using a semi-structured questionnaire. Surface swabs, soil, water, human and animal fecal samples were introduced onto ESBL chromogenic agar. The isolates were identified using biochemical tests and double-disk synergy tests. To assess associations, prevalence ratios (PRs) were computed using a generalized linear model (GLM) analysis with modified Poisson and a log link with robust standard errors in R software. Approximately 83% (86/104) households had at least one positive ESBL-Ec isolate. The overall prevalence of ESBL-Ec at the human-animal-environment interface was approximately 25.0% (95% CI: 22.7-28.3). Specifically, humans, animals and the environment had an ESBL-Ec prevalence of 35.4%, 55.4%, and 9.2% respectively. Having visitors (adj PR = 1.19, 95% CI: 1.04-1.36), utilizing veterinary services (adj PR = 1.39, 95% CI: 1.20-1.61) and using animal waste for gardening (adj PR = 1.29, 95% CI: 1.05-1.60) were positively associated with household ESBL-Ec contamination. Covering the drinking water container with a lid (adj PR = 0.84 95% CI: 0.73-0.96) was associated with absence of ESBL-Ec in a household. There is wider dissemination of ESBL-Ec in the environment, humans, and animals, indicating poor infection prevention and control (IPC) measures in the area. Improved collaborative one health mitigation strategies such as safe water chain, farm biosecurity, household and facility-based IPC measures are recommended to reduce the burden of antimicrobial resistance at community level.

Distribution of Class A Extended-Spectrum β-Lactamases Among Pseudomonas aeruginosa Clinical Strains Isolated from Ardabil Hospitals

Background: Currently, the emergence of extended-spectrum β-lactamase (ESBL)-producing bacteria is becoming a major threat to patients in the hospital and community. Such enzymes have been recently detected in Pseudomonas aeruginosa, but there is no epidemiological data on the prevalence of ESBL-producing clinical isolates in the hospitals of Ardabil City (Iran). Objectives: This study aimed to determine the phenotypic and genotypic prevalence of class A ESBL-producing P. aeruginosa strains in Ardabil City. Methods: A total of 120 clinical isolates of P. aeruginosa collected from Ardabil hospitals were used in this study. Phenotypic detection of class A ESBL-producing P. aeruginosa isolates was performed using a double-disk synergy test. In addition, the detection of class A ESBL-encoding genes, including Pseudomonas extended resistant (PER), Vietnamese extended-spectrum β-lactamase (VEB), temoniera (TEM), sulfhydryl variable (SHV), cefotaximase (CTX-M), guyana extended-spectrum β-lactamase (GES), and Pseudomonas-specific enzyme (PSE), was performed using the polymerase chain reaction (PCR). Results: The prevalence of class A ESBL-producing P. aeruginosa strains was 8.3% (10 out of 120) based on the double-disk synergy test. However, 40% (48 out of 120) of these isolates were found to carry genes encoding class A ESBLs based on PCR. Among 48 class A ESBL-positive strains, the prevalence of PSE, TEM, VEB, CTX-M, and PER genes were 64.6% (31/48), 25% (12/48), 4.2% (2/48), 4.2% (2/48), and 2% (1/48), respectively. However, the frequency of other class A ESBL genes (SHV and GES genes) was 0%. Conclusions: Our results confirmed the presence of class A ESBL-producing P. aeruginosa strains in the hospital environment of Ardabil. On the other hand, the use of molecular tests can be a more precise and reliable method than phenotypic ones to identify these resistant strains and prevent the emergence of antibiotic resistance and ensuing treatment failure.

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.

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.

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.

Bacterial Pathogens and Antimicrobial Susceptibility Patterns of Urinary Tract Infections in Children during COVID-19 2019-2020: A Large Tertiary Care Center in Saudi Arabia.

One of the most prevalent bacterial infections in children is urinary tract infection (UTI), which has become a major concern with increasing resistance of the pathogens to the routinely used antimicrobial agents. The aim of the study is to determine the antimicrobial susceptibility patterns of pediatric UTI-causing pathogens, including ESBL-producing bacteria, in Saudi Arabia. This cross-sectional retrospective study was conducted to ascertain the frequency of isolation and the antimicrobial resistance pattern of uropathogens among children aged 0-15 years. The data from the urine cultures was collected during 2019-2020 at the King Fahad Medical City, a major tertiary hospital in Riyadh, Saudi Arabia. A total of 1022 urine samples from patients diagnosed with urinary tract infections (UTIs) were collected for this study. Microbial species present in the samples were cultured and identified using standard biochemical techniques. To assess the resistance of these strains to antimicrobial drugs, an in vitro method was employed, and the criteria set by the Clinical Laboratory Standard Institute (CLSI) were followed. In addition, a double-disc synergy test was conducted to identify strains of E. coli that produce extended-spectrum beta-lactamase (ESBL). The predominant pathogens were E. coli (58.6%), followed by Klebsiella sp. (23.9%). E. coli isolates were more sensitive to meropenem and ertapenem in 99.2% of cases, followed by amikacin (99%). Klebsiella sp. were sensitive to amikacin in 97.1% of cases, followed by meropenem and ertapenem (92.2% in both). The highest sensitivities of antimicrobials toward ESBL were for meropenem and ertapenem (100% in both), followed by amikacin (99%). Our study recommends using local antibiotic sensitivity data for empirical UTI treatment. Amikacin, ertapenem, and meropenem are effective intravenous options. Cephalosporin, cefuroxime, amoxicillin/clavulanic acid, and nitrofurantoin are suitable oral choices. No significant changes in antimicrobial susceptibility were observed during the COVID-19 pandemic. Further research is needed to assess potential pandemic-related alterations.

Molecular Detection of Carbapenem Resistance in Clinical Isolates of Klebsiella pneumoniae in Tertiary Care Hospital

Antibiotic resistance has become a serious global threat, mainly due to misuse, overuse of antibiotics and non-compliance with infection control protocol. Superbugs are multidrug-resistant (MDR) and extended drug-resistant (XDR) bacteria, mainly Klebsiella pneumoniae and Escherichia coli from the Enterobacteriaceae family, which cause opportunistic infections and raise death rates and hospital expenditures. The present study was conducted at a tertiary care teaching hospital to study the epidemiology and molecular detection of carbapenem-resistant K. pneumoniae isolated from various clinical specimens. 240 K. pneumoniae isolates were collected from January 2020 to December 2021 at the Bacteriology laboratory, Index Medical College and Hospital, Indore. All isolates were analyzed for carbapenem resistance by the conventional disc diffusion method. All carbapenem-resistant isolates were tested for carbapenemase production using the phenotypic double-disk synergy test (DDST) and modified Hodge test (MHT) as per 2020 CLSI guidelines. All isolates were negative by phenotypic methods, further confirmed by conventional PCR to detect the gene responsible for carbapenemase production. 240 isolates of K. pneumoniae were included during the study periods. Out of 240 isolates, 102 isolates were found resistant to carbapenem drugs. All 102 isolates were confirmed carbapenemase and MBL producers by MHT and DDST tests. Among 102, 60 isolates were found to be MBL producers negative by MHT and DDST tests. Sixty phenotypic negative carbapenem-resistant isolates were tested by conventional PCR. One or more carbapenemase genes were detected in 61.0% of isolates. The blaKPC was detected in 13/60 (21%) isolates, followed by blaNDM 10/60 (16%) isolates, followed by blaVIM in 6/60(10%), blaOXA-48 in 5/60 (8%) and blaIMP in 3/60(5%) isolates. K. pneumoniae produces carbapenemase, which enhances resistance to the carbapenem class of antibiotics. The simultaneous detection of these resistance genes expressed by Klebsiella pneumoniae might be managed by early detection and adhering to antibiotic policies that limit the use of antibiotics.

Resistance genomics and molecular epidemiology of high-risk clones of ESBL-producing Pseudomonas aeruginosa in young children.

The emergence of multidrug-resistant Pseudomonas aeruginosa poses a global threat, but the distribution and resistance profiling are unclear, especially in young children. Infections due to P. aeruginosa are common, associated with high mortality, and increasingly β-lactam drug resistant. We studied the molecular epidemiology and antibiotic resistance mechanisms in 294 clinicalisolates of P. aeruginosa from a pediatric hospital in China. Non-duplicate isolates were recovered from clinical cases and were identified using an API-20 kit followed by antimicrobial susceptibility testing using the VITEK®2 compact system (BioMerieux, France) and also by broth dilution method. In addition, a double-disc synergy test for the ESBL/E-test for MBL was performed. The presence of beta-lactamases, plasmid types, and sequence types was determined by PCR and sequencing. Fifty-six percent (n = 164) of the isolates were resistant to piperacillin-tazobactam, followed by cefepime (40%; n = 117), ceftazidime (39%; n = 115), imipenem (36%; n = 106), meropenem (33%; n = 97), and ciprofloxacin (32%; n = 94). Forty-two percent (n = 126) of the isolates were positive for ESBL according to the double-disc synergy test. The blaCTX-M-15 cephalosporinase was observed in 32% (n = 40/126), while 26% (n = 33/126) werepositive for blaNDM-1 carbapenemase. Aminoglycoside resistance gene aac(3)IIIawas observed in 16% (n = 20/126), and glycylcyclines resistance gene tet(A) was observed in 12% (n = 15/126) of the isolates. A total of 23 sequence types were detected, including ST1963 (12%; n = 16), followed by ST381 (11%; n = 14), ST234 (10%; n = 13), ST145 (58%; n = 10), ST304 (57%; n = 9), ST663 (5%; n = 7), and a novel strain. In ESBL-producing P. aeruginosa, 12 different Incompatibility groups (Inc) were observed, the most common being IncFI, IncFIS, and IncA/C. The MOBP was the most common plasmid type, followed by MOBH, MOBF, and MOBQ. Our data suggest that the spread of antibiotic resistance is likely due toclonal spread and dissemination of different clinical strains of P. aeruginosa harbouring different plasmids. This is a growing threat in hospitals particularly in young children which needs robust prevention strategies.

Cross-Sectional Study for Detection and Risk Factor Analysis of ESBL-Producing Avian Pathogenic Escherichia coli Associated with Backyard Chickens in Pakistan.

The increasing incidence of extended-spectrum β-lactamase (ESBL)-producing Escherichia (E.) coli in backyard chicken farming in Pakistan is of serious concern. This study aimed to assess the prevalence, antimicrobial resistance patterns and risk factors associated with ESBL avian pathogenic E. coli (APEC) isolated from backyard chickens in the Jhang district, Punjab, Pakistan. In total, 320 cloacal swabs were collected from four breeds of backyard chicken (Aseel, Golden, Misri and Necked Neck). ESBL E. coli were phenotypically identified using double disc synergy test (DDST) and corresponding genes were confirmed by multiplex polymerase chain reaction (mPCR). Out of the 320 samples, 164 (51.3%) were confirmed as E. coli, while 74 (45.1%) were characterized as ESBL E. coli. The frequency of isolation of ESBL E. coli was highest in Aseel chickens (35.1%). Of the 164 confirmed E. coli, 95.1%, 78.6%, 76.8%, 71.3%, 70.1%, 68.9%, 60.4% and 57.3% were resistant against tylosin, doxycycline, cefotaxime, enrofloxacin, colistin, trimethoprim/sulfamethoxazole, chloramphenicol and gentamicin, respectively. The ESBL gene types detected and their corresponding proportions were blaCTX-M (54.1 %, 40/74), blaTEM, (12.2%, 9/74) and co-existence (blaCTX-M and blaTEM) were shown in 33.8% (25/74). The blaCTX-M gene sequence showed homology to blaCTX-M-15 from clinical isolates. The mean multiple antibiotic resistance index (MARI) was found to be higher among ESBL E. coli (0.25) when compared to non-ESBL E. coli (0.17). Both free-range husbandry management system (p = 0.02, OR: 30.00, 95% CI = 1.47-611.79) and high antimicrobial usage in the last 6 months (p = 0.01, OR: 25.17, 95% CI = 1.81-348.71) were found significantly associated with isolation of ESBL-producing E. coli in the tested samples using binary logistic regression analysis. This study confirmed the potential of backyard chickens as a reservoir for ESBL E. coli in the Jhang district, Punjab, Pakistan.

Molecular Detection of Extended Spectrum Beta-Lactamase Gene in Clinical Isolates of Klebsiella Species in Jos North Central of Nigeria

Aims: The aim of the study was to determine the antimicrobial susceptibility of Klebsiella species, prevalence of Extended Spectrum Beta-Lactamases (ESBLs) and the gene coding ESBL production in Klebsiella Species obtained from clinical specimens of patients attending Plateau State Specialist and Jos University Teaching Hospitals both in Jos North Central of Nigeria.&#x0D; Study Design: This was hospital based study conducted in the Department of Medical Microbiology unit of Plateau State Specialist and Jos University Teaching Hospital (JUTH) during the periods of January to April 2020.&#x0D; Methodology: Out of 156 non-motile, oxidase negative gram negative organisms collected, 137 isolates were identified to be Klebsiella species using oxoid Microbact 24E kit for Enterobacteriaceae identification and Microsoft for bacteria identification. Antimicrobial Susceptibility Testing (AST) and ESBL screening was done by Kirby-Bauer disk diffusion method using oxoid Single disk antibiotics which include Ceftazidime (30µg), Cefotaxime (30µg), Imipenem (10µg), Ciprofloxacin (5µg), Gentamicin (10µg), Ampicillin (10 µg), Aztreonam (30 µg), and Azithromycin (15 µg) on Mueller Hinton Agar (oxoid).Break point was determined by CLSI AST guidelines. The isolates that showed resistance or reduced susceptibility to Ceftazidime and Cefotaxime are suspected to be ESBL positive Klebsiella. Phenotypic confirmation of ESBL positive isolates was carried out using Double Disk Synergy Test (DDST) with Ceftazidime 30µg (oxoid), Cefotaxime 30µg (oxoid) and Amoxicillin Clavulanic acid 20/10ug (Oxoid).Genotypic detection of ESBL gene was done using Polymerase Chain Reaction (PCR).&#x0D; Results: Antimicrobial Susceptibility Test showed Imipenem to be highly sensitive to ESBL positive Klebsiella pneumonia with 100% sensitivity. Gentamicin, Ampicillin, and Azithromycin showed reduced susceptibility. Genotypic results showed presence of bla TEM, bla SHV genes at 868bp and 972bp band size with no occurrence of bla CTX-M gene. There was multiple occurrence of gene in some of the isolates tested while some recorded no ESBL gene. The ESBL prevalence of 15.3% was recorded in this study.&#x0D; Conclusions: The prevalence of 15.3% recorded in this study is low compared to other studies from some other states within the country. Efforts should be put in place to ensure non-proliferation of this ESBL Klebsiella strain as it creates not only a therapeutic challenge but health problem.

Phenotypic and molecular characterization of ESBL producing Escherichia coli and Klebsiella pneumoniae among Lebanese patients.

Antimicrobial resistance is a major public health issue worldwide and became one of the principal international healthcare crises of the 21st century. The production of ESBLs is one of the resistance mechanisms in Enterobacteriaceae, and they are increasingly detected in Escherichia coli and Klebsiella pneumoniae globally. Therefore, the aim of this study was to determine the phenotypic and molecular characteristics of ESBL-producing E. coli and K. pneumoniae among Lebanese patients. A total of 152 ESBL-producing E. coli and K. pneumoniae were obtained from Geitaoui Hospital in Beirut between September 2019 and October 2020 from various clinical samples. The phenotype of ESBL producers was confirmed by a double-disc synergy test and antibiotic susceptibility was determined using the disc diffusion method. Genotypically, multiplex PCR was used to detect the ESBL genes (blaTEM, blaCTX-M and blaSHV). All strains were confirmed to be ESBL producers (121 isolates were E. coli and 31 isolates were K. pneumoniae). All isolates showed resistance to cefotaxime, cefuroxime, ampicillin and piperacillin. On the other hand, they showed a low susceptibility rate to trimethoprim/sulfamethoxazole and ciprofloxacin. Almost all the isolates were susceptible to ertapenem, imipenem and amikacin. In our study, ESBL genes were detected among 48 (39.67%) E. coli isolates and 8 (58.06%) K. pneumoniae isolates, and the most prevalent gene was blaTEM (25%), followed by blaCTX-M (19.08%) and blaSHV (16.45%). Imipenem and ertapenem are the most effective drugs to treat ESBL producers. However, antibiotic stewardship programs must be implemented immediately to combat antibiotic resistance.

Prevalence of extended-spectrum beta-lactamase-producing Enterobacteriaceae among clinical isolates in Turaif general hospital, northern borders- Saudi Arabia.

Enterobacteriaceae that produce extended-spectrum beta-lactamase (ESBL) are quickly spreading, posing a threat to world healthcare. 138 gram-negative bacteria were collected from different samples (stool, urine, wound, blood, tracheal aspirate, catheter tip, vaginal swab, sputum, and tracheal aspirate) from hospitalized patients. Samples were subcultured and identified in accordance with their biochemical reactions and culture characteristics. Against all the isolated Enterobacteriaceae, an antimicrobial susceptibility test was performed. VITEK®2 system, phenotypic confirmation, and Double-Disk Synergy Test (DDST) had been utilized to identify the ESBLs. Of the 138 samples studied, the prevalence of ESBL-producing infections among the clinical samples of the present study was 26.8 % (n = 37). E. coli was the commonest ESΒL producer at 51.4% (n = 19) followed by K. pneumoniae at 27% (n = 10). The potential risk factors for the ESBL development that produces bacteria were as follows, patients with the presence of indwelling devices, previous history of hospital admission, and usage of antibiotics. ESBL is statistically (p ≤ 0.05) higher among the patients with indwelling devices, ICU admission, who had a previous hospital admission in the last 6 months as well as who was given antibiotics (quinolones and/or cephalosporins) in the last 6 months. One hundred thirty-two (95.7%) of ESBL isolates were resistant to amoxicillin, while the lowest resistance was for fosfomycin (15.2%). ESBL-producing Enterobacteriaceae are highly prevalent in Turaif General Hospital setting with some potential risk factors. A strict policy to be made available on the usage of antimicrobials in hospitals and clinics should be established.