blaCTX-M Genes Research Articles

Prevalence of Antibiotic-Resistant Pathogenic Bacteria and Level of Antibiotic Residues in Hospital Effluents in Selangor, Malaysia: Protocol for a Cross-sectional Study.

Antimicrobial resistance (AMR) has emerged as a major global public health challenge due to the overuse and misuse of antibiotics for humans and animals. Hospitals are among the major users of antibiotics, thereby having a large contribution to AMR. The aim of this study is to determine the prevalence of antibiotic-resistant pathogenic bacteria and the level of antibiotic residues in the hospital effluents in Selangor, Malaysia. A cross-sectional study will be performed in the state of Selangor, Malaysia. Tertiary hospitals will be identified based on the inclusion and exclusion criteria. The methods are divided into three phases: sample collection, microbiological analysis, and chemical analysis. Microbiological analyses will include the isolation of bacteria from hospital effluents by culturing on selective media. Antibiotic sensitivity testing will be performed on the isolated bacteria against ceftriaxone, ciprofloxacin, meropenem, vancomycin, colistin, and piperacillin/tazobactam. The identification of bacteria will be confirmed using 16S RNA polymerase chain reaction (PCR) and multiplex PCR will be performed to detect resistance genes (ermB, mecA, blaNDM-L, blaCTX-M, blaOXA-48, blaSHV, VanA, VanB, VanC1, mcr-1, mcr-2, mcr-3, Intl1, Intl2, and qnrA). Finally, the level of antibiotic residues will be measured using ultrahigh-performance liquid chromatography. The expected outcomes will be the prevalence of antibiotic-resistant Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter (ESKAPE) bacterial species from the hospital effluents, the occurrence of antibiotic resistance genes (ARGs) from the isolated ESKAPE bacteria, and the level of antibiotic residues that may be detected from the effluent. Sampling has been conducted in three hospitals. Data analysis from one hospital showed that as of July 2022, 80% (8/10) of E. faecium isolates were resistant to vancomycin and 10% (1/10) were resistant to ciprofloxacin. Further analysis will be conducted to determine if the isolates harbor any ARGs and effluent samples are being analyzed to detect antibiotic residues. Sampling activities will be resumed after being suspended due to the COVID-19 pandemic and are scheduled to end by December 2022. This study will provide the first baseline information to elucidate the current status of AMR of highly pathogenic bacteria present in hospital effluents in Malaysia. DERR1-10.2196/39022.

Characterization of plasmids carrying blaCTX-M genes among extra-intestinal Escherichia coli clinical isolates in Ethiopia

CTX-Ms are encoded by blaCTX-M genes and are widely distributed extended-spectrum β-lactamases (ESBLs). They are the most important antimicrobial resistance (AMR) mechanism to β-lactam antibiotics in the Enterobacteriaceae. However, the role of transmissible AMR plasmids in the dissemination of blaCTX-M genes has scarcely been studied in Africa where the burden of AMR is high and rapidly spreading. In this study, AMR plasmid transmissibility, replicon types and addiction systems were analysed in CTX-M-producing Escherichia coli clinical isolates in Ethiopia with a goal to provide molecular insight into mechanisms underlying such high prevalence and rapid dissemination. Of 100 CTX-Ms-producing isolates obtained from urine (84), pus (10) and blood (6) from four geographically distinct healthcare settings, 75% carried transmissible plasmids encoding for CTX-Ms, with CTX-M-15 being predominant (n = 51). Single IncF plasmids with the combination of F-FIA-FIB (n = 17) carried the bulk of blaCTX-M-15 genes. In addition, IncF plasmids were associated with multiple addiction systems, ISEcp1 and various resistance phenotypes for non-cephalosporin antibiotics. Moreover, IncF plasmid carriage is associated with the international pandemic E. coli ST131 lineage. Furthermore, several CTX-M encoding plasmids were associated with serum survival of the strains, but less so with biofilm formation. Hence, both horizontal gene transfer and clonal expansion may contribute to the rapid and widespread distribution of blaCTX-M genes among E. coli populations in Ethiopian clinical settings. This information is relevant for local epidemiology and surveillance, but also for global understanding of the successful dissemination of AMR gene carrying plasmids.

Isolation of Hv-CRKP with co-production of three carbapenemases (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1) and a virulence plasmid: a study from a Chinese tertiary hospital.

The worldwide dissemination of K. pneumoniae isolates is a significant public health concern, as these organisms possess a unique capacity to acquire genetic elements encoding both resistance and hypervirulence. This study aims to investigate the epidemiological, resistance, and virulence characteristics of K. pneumoniae isolates that carry both virulence plasmids and blaOXA-48-like genes in a tertiary hospital in China. A total of 217 clinical isolates of carbapenem-resistant K. pneumoniae (CRKP) were collected between April 2020 and March 2022. The antimicrobial susceptibility test was conducted to evaluate the drug resistance profile. All isolates were screened for the presence of genes encoding carbapenemases (blaKPC, blaNDM, blaIMP, blaVIM, and blaOXA-48-like), ESBLs genes (blaCTX-M, blaSHV, blaTEM), and virulence plasmid pLVPK-borne genes (rmpA, rmpA2, iucA, iroB, and peg344) using polymerase chain reaction (PCR) amplification. Clonal lineages were assigned using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The plasmid incompatibility groups were identified using PCR-based replicon typing (PBRT). The transferability of carbapenemase-encoding plasmids and pLVPK-like virulence plasmids was assessed via conjugation. The plasmid location of rmpA2 was determined using S1-Pulsed Field Gel Electrophoresis (S1-PFGE) and southern blotting hybridization. The virulence potential of the isolates was assessed using the string test, capsular serotyping, serum killing assay and a Galleria mellonella larval infection model. Of the 217 CRKP clinical isolates collected, 23% were identified as carrying blaOXA-48-like genes. All blaOXA-48-like isolates exhibited resistance to commonly used clinical antimicrobial agents, except for ceftazidime/avibactam, colistin, tigecycline, trimethoprim-sulfamethOXAzole, polymyxin B, and nitrofurantoin. The main common OXA-48-like carbapenemase enzymes were found to be blaOXA-181 and blaOXA-232. MLST and PFGE fingerprinting analysis revealed clonal transmission and plasmid transmission. OXA-48-like producing CRKP isolates mainly clustered in K64 ST11 and K47 ST15. Results of the string Test, serum killing assay (in vitro) and Galleria mellonella infection model (in vivo) indicated hypervirulence. PBRT showed that the blaOXA-181 and blaOXA-232 producing hypervirulent carbapenem-resistant Klebsiella pneumoniae (Hv-CRKP) were mainly carried on ColE-type, IncF, and IncX3. Eight clinical isolates of hv-CRKP were identified as carrying three carbapenem-resistant genes (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1). Moreover, Southern blotting hybridization revealed that all eight isolates had a pLVPK-like virulent plasmid (138.9-216.9 kb) with an uneven number and size of plasmid. In our investigation, we have observed the emergence of hv-CRKP carrying blaOXA-48-like genes, which identified two genetic relationships: clonal transmission and plasmid transmission. PBRT analysis showed that these genes were mainly carried on ColE-type, IncF, and IncX3 plasmids. These isolates have been shown to be hypervirulent in vitro and in vivo. Additionally, eight clinical isolates of hv-CRKP were identified as carrying three carbapenem-resistant genes (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1) and carrying a pLVPK-like virulent plasmid. Hence, our findings highlight the need for further investigation and active surveillance of hypervirulent OXA-48-like producing Hv-CRKP isolates to control their transmission.

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.

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.

ESBL-producing Vibrio vulnificus and V. alginolyticus harbour a plasmid encoding ISEc9 upstream of blaCTX-M-55 and qnrS2 isolated from imported seafood.

In recent years, trade liberalisation has led to the spread of antibiotic-resistant bacteria (ARB) in food products. Because ARB has reportedly been found in imported foods, the spread of plasmid-mediated ARB through food products is a concern. Here, we report the complete genome sequences of ESBL-producing Vibrio vulnificus and V. alginolyticus strains harbouring a plasmid isolated from imported seafood. First, V. vulnificus and V. alginolyticus were isolated from purchased frozen and thawed Litopenaeus vannamei shrimp, and genome extraction and sequencing were performed. Hybrid genome assemblies were performed using Unicycler and annotated using DFAST. Then genome analysis was performed using BRIG. Plasmid comparisons showed that the plasmids carried by both Vibrios are remarkably similar and encode the same antibiotic-resistance genes. The 270-310kb region specific to both Vibrios were isolated in this study and encodes the antibiotic-resistance genes blaCTX-M and qnr. Furthermore, the mobile genetic factors ISEc9, ISVch4, and ISVpa4 are located upstream and downstream of these genes. This is the first report of ESBL-producing V. vulnificus and V. alginolyticus harbouring a common plasmid encoding ISEc9 upstream of blaCTX-M-55 and qnrS2 isolated from imported seafood.

Antibiotic use in infants within the first year of life is associated with the appearance of antibiotic-resistant genes in their feces.

Antibiotic resistance, an increasing challenge, is not only a national threat but also a global threat. Carriage of resistance genes is not limited to adults alone, various microbiota niches present in the body system of children have been found to harbor bacteria carrying resistant genes, especially, their gut microbiota. This study aims to identify selected antibiotic-resistant genes from the fecal samples of infants and the association of antibiotics use with the occurrence of resistant genes in the infant's gut. A total number of 172 metagenomic DNA samples previously extracted from stool samples of 28 Nigerian babies longitudinally within their first year of life were screened for the presence of ESBL genes (blaSHV, blaTEM, and blaCTX-M), PMQR genes (qnrA, qnrB, qnrS, qepA), ribosomal protection protein tetracycline resistance gene, (RPP) β-lactamase (blaZ), macrolide (ermA, ermB, mefA/E), aminoglycoside modifying enzymes gentR (aac(6')/aph(2″)) and dfrA genes by PCR. Nineteen (19) of the 28 babies used antibiotics during the study. The association between antibiotic use by the babies within the first year of life and occurrence of resistant genes were analyzed by Spearman rank correlation. One hundred and twenty-two (122) samples (71%) out of the 172 isolates had antibiotic-resistance genes. PMQR genes were absent in all the samples. Three isolates had blaTEM gene, nine isolates had blaSHV gene, six isolates had blaCTX-M gene and 19 isolates had dfrA gene, 31 samples had tet gene, 29 samples had mef gene, 27 samples had ermB gene, four samples had ermA gene, 13 samples had blaZ gene and 16 samples had aac gene. The babies whose samples had resistant genes used antibiotics in the same months the samples were collected. Interestingly, the 11 babies whose samples had the dfrA gene all used antibiotics in the same months their samples were collected but none of them used trimethoprim/sulfamethoxazole antibiotic. The overall correlation matrix of the babies showed a strong association between antibiotic use (AU) and antibiotic use presence of resistance genes (AUPRG) with a coefficient of 0.89. Antibiotic-resistant genes are present in the gut of infants and their occurrence is strongly connected with antibiotic use by infants.

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.
 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.
 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).
 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.
 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.

The highly diverse plasmid population found in Escherichia coli colonizing travellers to Laos and its role in antimicrobial resistance gene carriage.

Increased colonization by antimicrobial-resistant organisms is closely associated with international travel. This study investigated the diversity of mobile genetic elements involved with antimicrobial resistance (AMR) gene carriage in extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli that colonized travellers to Laos. Long-read sequencing was used to reconstruct complete plasmid sequences from 48 isolates obtained from the daily stool samples of 23 travellers over a 3 week period. This method revealed a collection of 105 distinct plasmids, 38.1 % (n=40) of which carried AMR genes. The plasmids in this population were diverse, mostly unreported and included 38 replicon types, with F-type plasmids (n=23) the most prevalent amongst those carrying AMR genes. Fine-scale analysis of all plasmids identified numerous AMR gene contexts and emphasized the importance of IS elements, specifically members of the IS6/IS26 family, in the evolution of complex multidrug resistance regions. We found a concerning convergence of ESBL and colistin resistance determinants, with three plasmids from two different F-type lineages carrying bla CTX-M and mcr genes. The extensive diversity seen here highlights the worrying probability that stable new vehicles for AMR will evolve in E. coli populations that can disseminate internationally through travel networks.

MALDI-TOF MS Approaches for the Identification of the Susceptibility of Extended-Spectrum β-Lactamases in Escherichia coli.

The increase in multidrug-resistant microorganisms that produce extended-spectrum β-lactamases (ESBLs) and carbapenemases is a serious problem worldwide. Recently, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has been used for the rapid detection of antibiotic-resistant bacteria. The objective of this study was to establish a method to detect ESBL-producing Escherichia coli by monitoring the hydrolyzation of cefotaxime (CTX) using MALDI-TOF MS. According to the ratio of the peak intensity of CTX and hydrolyzed-CTX-related compounds, the ESBL-producing strains could be clearly distinguished after 15 min of incubation. Moreover, the minimum inhibitory concentration (MIC) values for E. coli were 8 μg/mL and lower than 4 μg/mL, which could be distinguished after 30 min and 60 min of incubation, respectively. The enzymatic activity was determined using the difference in the signal intensity of the hydrolyzed CTX at 370 Da for the ESBL-producing strains incubated with or without clavulanate. The ESBL-producing strains with low enzymatic activity or blaCTX-M genes could be detected by monitoring the hydrolyzed CTX. These results show that this method can rapidly detect high-sensitivity ESBL-producing E. coli.

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.

Extended-Spectrum β-Lactamase-Producing Escherichia coli and Virulence Genes in Pediatric Patients with Health-Care Urinary Tract Infections.

Healthcare-associated urinary tract infection (UTI) represents a significant health problem, especially in infants and young children. The most common pathogen associated with this infection is Escherichia coli (E. coli). The present study aimed to detect the frequency of virulence genes among clinical isolates of E. coli isolated from healthcare-associated urinary tract infections in children and the correlation between these virulence genes and the presence of the blaCTX gene. The study included one hundred clinical isolates of E. coli isolated from healthcareassociated urinary tract infections in children in intensive care units. The isolates were subjected to antibiotics sensitivity by disc diffusion method and detection of extended-spectrum beta-lactamase by double disc diffusion method. In addition, multiplex polymerase chain reaction (PCR) was used to detect some virulence genes, and PCR was used to detect the blaCTX-M gene. E. coli producing ESBL by double discs method was identified in 74 isolates. blaCTX-M gene detection by PCR was identified among 38 isolates representing 51.4% of ESBL-producing E. coli. There was a significant association between ESBL and blaCTX-M Gene, P = 0.0001. The frequency of the studied virulence genes by multiplex PCR in the isolated E. coli was 66% for the Fim gene, 75% for the Aer gene, 68% for the FliC gene, 53% for each of IucD gene and Usp gene, 40% for pap gene, 35% for each of AFA and ironN genes and 17% for sfa gene. None of the isolated E. coli had the Cdt gene. There was a significant association between the presence of the FimH gene (P = 0.0001), Pap gene (P = 0.05), sfa (P = 0.026), Afa gene (P = 0.018), and aer gene (P = 0.035) and the presence of the blaCTX-M gene in the isolated E. coli. The present study highlights the presence of virulence genes and blaCTX-M gene in uropathogenic E. coli isolated from pediatric patients with healthcare-associated urinary tract infections. There was an association between the blaCTX-M gene and virulence genes FimH, pap, sfa, Afa, and aer. Various distributions of the studied genes with a high frequency of fimbria are flic genes. Moreover, the ESBL had high frequency in E. coli with the presence of blaCTX-M in about one-third of the isolates.

Phenotypic and genotypic characterization of pathogenic Escherichia coli identified in resistance mapping of β-lactam drug-resistant isolates from seafood along Tuticorin coast.

The ubiquity of pathogenic E. coli isolate possessing antimicrobial resistance was investigated in seafood samples procured from major seafood supply chain markets established for export and domestic consumption along Tuticorin coast. Out of 63 seafood samples examined, 29 (46%) were found to be contaminated by pathogenic E. coli harbouring one or more genes of virulent potential. Based on virulome profiling, 9.55% of isolates belonged to enterotoxigenic E. coli (ETEC), 8.08% to enteroaggregative E. coli (EAEC), 7.35% to enterohemorrhagic E. coli (EHEC), 2.20% to enteropathogenic E. coli (EPEC), and 2.20% to uropathogenic E. coli (UPEC). All the 34 virulome positive and haemolytic pathogenic E. coli have been serogrouped as O119, O76, O18, O134, O149, O120, O114, O25, O55, O127, O6, O78, O83, O17 and clinically significant O111, O121, O84, O26, O103, and O104 (non-O157 STEC) serotypes in this study. Multi-drug resistance (MDR) (≥ 3 antibiotic classes/sub-classes) was exhibited in 38.23% of the pathogenic E. coli, and 17.64% were extensive drug resistant (XDR). Extended spectrum of β-lactamase (ESBL) genotypes were confirmed in 32.35% isolates and 20.63% isolates harboured ampC gene. One sample (Penaeus semisulcatus) collected from landing centre (L1) harboured all ESBL genotypes blaCTX-M, blaSHV, blaTEM, and ampC genes. Hierarchical clustering of isolates revealed the separation of ESBL isolates into three clusters and non-ESBL isolates into three clusters based on phenotypic and genotypic variations. Based on dendrogram analysis on antibiotic efficacy pattern, carbapenems and β-lactam inhibitor drugs are the best available treatment for ESBL and non-ESBL infections. This study emphasizes the significance of comprehensive surveillance of pathogenic E. coli serogroups that pose serious threat to public health and compliance of AMR antimicrobial resistant genes in seafood that hinder seafood supply chain.

Prevalence of Extended-Spectrum β-Lactamases in Multidrug-Resistant Klebsiella pneumoniae Isolates in Jordanian Hospitals

The increase in the prevalence of infections caused by certain bacteria, such as Klebsiella pneumonia (K. pneumoniae), is a global health concern. Bacterial production of an enzyme called extended-spectrum beta-lactamase (ESBL) can generate resistance to antimicrobial therapeutics. Therefore, between 2012 and 2013, we investigated K. pneumoniae that produce ESBLs with the prevalence of individual genes including blaSHV, blaCTX-M, blaTEM, and blaOXA isolated from clinical samples. A total of 99 variable diagnostic samples including blood from hematological malignancies (n = 14) or other clinical sources including sputum, pus, urine, and wound (n = 85) were analyzed. All samples' bacterial type was confirmed and their susceptibility to antimicrobial agents was established. Polymerase chain reaction (PCR) amplification was carried out to ascertain presence of specific genes that included blaSHV, blaCTX-M, blaTEM, and blaOXA. Plasmid DNA profiles were determined to assess significance between resistance to antimicrobial agents and plasmid number. It was found that among non-hematologic malignancy isolates, the highest rate of resistance was 87.9% to imipenem, with lowest rate being 2% to ampicillin. However, in hematologic malignancy isolates, the highest microbial resistance was 92.9% to ampicillin with the lowest rate of resistance at 28.6% to imipenem. Among collected isolates, 45% were ESBL-producers with 50% occurrence in hematologic malignancy individuals that were ESBL-producers. Within ESBL-producing isolates from hematologic malignancy individuals, blaSHV was detected in 100%, blaCTX-M in 85.7%, and blaTEM and blaOXA-1 at 57.1% and 27.1%, respectively. In addition, blaSHV,blaCTX-M, and blaOXA were found in all non-hematological malignancy individuals with blaTEM detected in 55.5% of samples. Our findings indicate that ESBLs expressing blaSHV and blaCTX-M genes are significantly prevalent in K. pneumoniae isolates from hematologic malignancy individuals. Plasmid analysis indicated plasmids in isolates collected from hematological malignancy individuals. Furthermore, there was a correlation between resistance to antimicrobial agents and plasmids within two groups analyzed. This study indicates an increase in incidence of K. pneumoniae infections displaying ESBL phenotypes in Jordan.

Multidrug resistance, biofilm formation and detection of blaCTX-M and blaVIM genes in E. coli and Salmonella isolates from chutney served at the street-food stalls of Bharatpur, Nepal

Antimicrobial resistance (AMR) amid the bacteria found in ready-to-eat foods is a grave concern today warranting an immediate intervention. The current study was undertaken to explore the status of AMR in E. coli and Salmonella species in ready-to-eat Chutney samples (n = 150) served at the street food stalls in Bharatpur, Nepal, with a major focus on detecting extended-spectrum β-lactamase (ESBL) and metallo β-lactamase (MBL) genes along with biofilm formation. Average viable counts, coliform counts, and Salmonella Shigella counts were 1.33 × 106±141481.4, 1.83 × 105±91303.6, and 1.24 × 105±63933.19 respectively. Out of 150 samples, 41 (27.33%) harbored E. coli, of which 7 were E. coli O157:H7; whereas Salmonella spp. were found in 31 (20.67%) samples. Bacterial contamination of Chutney by E. coli and Salmonella and ESBL-production were both found significantly affected by different sources of water used, personal hygiene and literacy rate of the vendors as well as by the type of cleaning materials used to wash knives and chopping boards (P < 0.05). Antibiotic susceptibility testing revealed that imipenem was the most effective drug against both types of bacterial isolates. Additionally, 14 (45.16%) Salmonella isolates and 27 (65.85%) E. coli isolates were found to be multi-drug resistant (MDR). Total ESBL (blaCTX-M) producers reported were 4 (12.90%) Salmonella spp. and 9 (21.95%) E. coli. Only 1 (3.23%) Salmonella spp. and 2 (4.88%) E. coli isolates were blaVIM gene carriers. Dissemination of knowledge of personal hygiene amongst the street vendors and consumer awareness regarding ready-to-eat foods are crucial factors that can be suggested to curtail the emergence and transmission of food-borne pathogens.

Enumeration, antimicrobial resistance and genomic characterization of extended-spectrum β-lactamases producing Escherichia coli from supermarket chicken meat in the United Arab Emirates

The occurrence and counts of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in retail chicken sold in the United Arab Emirates (UAE) were investigated in this study. Results indicated that 79.68 % of chicken carcasses (251/315) sampled from UAE supermarkets harbored ESBL-producing E. coli. About half (51.75 % [163/315]) of the tested samples had an ESBL-producing E. coli count range between ≥3 log10 and < 5 log10 CFU/g. The antimicrobial resistance profiles of a subset of 100 isolates showed high rates of non-susceptibility to clinically significant antibiotics, particularly ciprofloxacin (80 %) and cefepime (46 %). Moreover, 7 % of the isolates exhibited resistance to colistin, with PCR-based screening revealing the presence of the mcr-1 gene in all colistin-resistant isolates. Multiplex PCR screening identified blaCTX-M and blaTEM genes as the most frequently presented genes among the phenotypically confirmed ESBL-producing E. coli. Further whole-genome sequencing and bioinformatic analysis of 27 ESBL-producing E. coli isolates showed that the gene family blaCTX group 1 was the most prevalent, notably CTX-M-55 (55.55 % [15/27]), followed by CTX-M-15 (22.22 % [6/27]). The most common sequence types (STs) were ST359 and ST1011, with three evident clusters identified based on phylogenomic analysis, aligned with isolates from specific production companies. Analysis of plasmid incompatibility types revealed IncFIB, IncFII, Incl2, and IncX1 as the most commonly featured plasmids. The findings of this study indicate a noticeable prevalence and high counts of ESBL-producing E. coli in chicken sampled from supermarkets in the UAE. The high rates of antimicrobial resistance to clinically important antibiotics highlight the potential public health risk associated with consuming chicken contaminated with ESBL-producing E. coli. Overall, this study emphasizes the importance of continued antimicrobial resistance monitoring in the UAE food chain and calls for further exposure risk assessment of the consumption of ESBL-producing E. coli via chicken meat.

Serotype distribution, trend of multidrug resistance and prevalence of β-lactamase resistance genes in human Salmonella isolates from clinical specimens in Guizhou, China.

Salmonella, one of the major causes of foodborne infections, can cause bacterial foodborne illness. We investigated the serotype distribution, multidrug resistance (MDR), and β-lactamase resistance genes of human Salmonella isolates collected from clinical specimens in Guizhou, China, between 2013 and 2018. A total of 363 Salmonella isolates were collected from clinical specimens at 17 surveillance hospitals. Twenty-four serotypes were identified by sliding agglutination test. S. Enteritidis (33.9%), Salmonella 4,[5],12:i:- (24.0%), S. Typhimurium (16.3%), S. London (6.3%), and S. Derby (3.9%) were the top five serotypes. In 2018, the most common serotype changed from S. Enteritidis to S. Typhimurium. Among the 363 Salmonella isolates, 97.5% of isolates were resistant to at least one class of antimicrobial agents. For cephalosporins, ceftriaxone had the highest resistance rate of 10.5%, and cefepime and cefoxitin were 8.0% and 2.2%, respectively. Three hundred and one (82.9%) Salmonella isolates showed MDR. Salmonella 4,[5],12:i:- had the highest MDR rate with 94.2%, followed by S. London (91.3%) and S. Typhimurium (88.1%). Multidrug resistance rates of Salmonella isolates in Guizhou from 2013 to 2017 increased from 75.8% to 86.7%. Sixteen isolates (4.4%) showed extensive drug resistance. One hundred thirty-four antimicrobial resistance patterns were found. Two hundred and forty-one (66.4%) isolates carried at least one β-lactamase resistance gene. The blaTEM gene (61.2%) was the most prevalent resistant gene in all Salmonella isolates, followed by the blaCTX-M gene (6.1%) and blaOXA-1 gene (4.1%). Our findings showed that the MDR rate of Salmonella isolates from Guizhou province increased year by year. Therefore, systematic and long-term surveillance on MDR Salmonella isolates from clinical patients should be further strengthened.

Bacterial Assessment of Stethoscopes Used by Healthcare Workers at a Tertiary Care Government Hospital in Bharatpur, Nepal

The transmission of healthcare-associated infections (HCAIs) in healthcare settings is a serious challenge in the medical fraternity. Medical devices, such as stethoscopes used by healthcare workers (HCWs), are likely to harbor a considerable number of pathogenic microbes, which may result in the transmission of HCAIs. This study sought to investigate bacterial contamination of stethoscopes used by HCWs at Bharatpur Hospital, Nepal. During the study period of 3 months from December 2019 to February 2020, a total of 87 stethoscopes were examined; bacterial pathogens were isolated and identified by culture and biochemical tests, and their susceptibilities against different antibiotics were determined using standard protocols of the Clinical and Laboratory Standards Institute (CLSI). The disc diffusion method was used primarily to screen for extended-spectrum beta-lactamase (ESBL)- and metallo-beta-lactamase (MBL)-producing isolates, followed by their confirmation using cephalosporin/clavulanate combination discs and the disc potentiation methods, respectively. In addition, molecular detection of blaCTX-M and blaVIM genes was performed using conventional polymerase chain reaction (PCR). Of the 87 stethoscopes examined, more than a quarter (28.7%) were colonized with different pathogenic bacteria. Bacterial contamination of stethoscopes was found to be significantly associated with various factors, such as disinfecting routine, method of disinfection, and department of the hospital (p < 0.05). A higher rate of bacterial contamination was observed on the diaphragm of the stethoscope (12.64%) and among HCWs who overlooked hand hygiene practices (45.45%). The prevalence of methicillin-resistant S. aureus (MRSA) was 44.44%, and approximately half of the Gram-negative isolates (47%) were multidrug resistant (MDR). Imipenem (81.25%) and chloramphenicol (83.33%) were found to be the most effective antibiotics for Gram-negative and Gram-positive bacteria, respectively. Phenotypic screening showed that 43.75% of isolates were ESBL producers, and 18.75% were MBL producers, but blaCTX-M and blaVIM genes were detected in only 31.25% and 6.25% of isolates, respectively. The results of the study call for effective stethoscope disinfection practices along with the judicious use of antibiotics by HCWs in order to minimize cross-contamination, emergence of resistance, and spread of nosocomial infections in clinical settings.

Antibacterial Efficacy of N-(4-methylpyridin-2-yl) Thiophene-2-Carboxamide Analogues against Extended-Spectrum-β-Lactamase Producing Clinical Strain of Escherichia coli ST 131.

Development in the fields of natural-product-derived and synthetic small molecules is in stark contrast to the ongoing demand for novel antimicrobials to treat life-threatening infections caused by extended-spectrum β-lactamase producing Escherichia coli (ESBL E. coli). Therefore, there is an interest in the antibacterial activities of synthesized N-(4-methylpyridin-2-yl) thiophene-2-carboxamides (4a-h) against ESBL-producing E. coli ST131 strains. A blood sample was obtained from a suspected septicemia patient and processed in the Bactec Alert system. The isolate's identification and antibacterial profile were determined using the VITEK 2® compact system. Multi-locus sequence typing of E. coli was conducted by identifying housekeeping genes, while ESBL phenotype detection was performed according to CLSI guidelines. Additionally, PCR was carried out to detect the blaCTX-M gene molecularly. Moreover, molecular docking studies of synthesized compounds (4a-h) demonstrated the binding pocket residues involved in the active site of the β-lactamase receptor of E. coli. The result confirmed the detection of E. coli ST131 from septicemia patients. The isolates were identified as ESBL producers carrying the blaCTX-M gene, which provided resistance against cephalosporins and beta-lactam inhibitors but sensitivity to carbapenems. Among the compounds tested, 4a and 4c exhibited high activity and demonstrated the best fit and interactions with the binding pocket of the β-lactamase enzyme. Interestingly, the maximum of the docking confirmations binds at a similar pocket region, further strengthening the importance of binding residues. Hence, the in vitro and molecular docking studies reflect the promising antibacterial effects of 4a and 4c compounds.

Biofilm formation and antibiotic resistance profiles of water-borne pathogens

Water sources (surface water, drinking water, rivers, and ponds) are significant reservoirs for transmitting antibiotic-resistant bacteria. In addition, these waters are an important public health problem because they are suitable environments for transferring antibiotic resistance genes between bacterial species. Our study aimed to assess the prevalence of Extended-spectrum beta-lactamase (ESBL) producing isolates in water samples, the susceptibility of the isolates to the specified antibiotics, the determination of biofilm ability, antibiotic resistance genes, and the molecular typing of the isolates. For this purpose, Polymerase chain reaction (PCR) and Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analyses were used. Out of 70 isolates, 15 (21%) were ESBL producing, and sent for the MALDI-TOF analysis, where Escherichia coli, Acinetobacter calcoaceticus, Enterobacter bugandensis, Acinetobacter pittii, Pseudomonas aeruginosa, Acinetobacter junii, Pseudomonas oleovorans, and Enterobacter ludwigigii were identified. Moreover, colistin resistance genes (mcr 1/2/6, mcr 4, mcr 5, mcr 3/7, and mcr 8), ESBL-encoding genes (blaSHV, blaTEM, and blaCTX-M) and carbapenemase genes (blaNDM, blaOXA-48, and blaKPC) using molecular analysis (PCR) were confirmed. The colistin resistance gene was detected at 80% (12/15) in the isolates obtained. The distribution of these isolates according to resistance genes was found as mcr 1/2/6 4 (20%), mcr 3/7 3 (13%), and mcr 5 (40%). Additionally, the isolates harbored blaSHV(6.6%) and blaTEM (6.6%) genes. However, blaNDM, blaOXA-48, blaKPC, and blaCTX-M genes were not detected in any isolates. According to the Congo red agar method, seven (46.6%) isolates showed negative biofilm ability, and eight (53.3%) showed moderate biofilm ability. However, the microplate method detected weak biofilm in 53.3% of the isolates. In conclusion, this study provides evidence for the existence of multidrug-resistant bacteria that co-exist with mcr and ESBL genes in water sources. These bacteria can migrate to other environments and pose increasing threats to public health.