Β-lactamase Resistance Genes Research Articles

Effects of Wastewater Discharge on Antibiotic Resistance Genes and Microbial Community in a Coastal Area

Wastewater treatment plants (WWTPs) are important sources of antibiotic resistance genes (ARGs) in aquatic environments. Mobile genetic elements (MGEs) and microbial communities are key factors that affect the proliferation of ARGs. To reveal the effects of WWTPs effluent discharge on the ARGs and microbial community in a coastal area, the structure and distribution of ARGs, MGEs, and microbial community in Shangyu (SY) and Jiaxing (JX) effluent receiving areas (ERAs) and the offshore area of Hangzhou Bay (HB) were investigated via high-throughput quantitative PCR and 16S rRNA high-throughput sequencing. The results showed that multidrug resistance genes were the most abundant ARGs across all the sampling sites. The diversity and abundance of ARGs and MGEs in the ERAs were much higher than those in the HB. Additionally, the diversities of the microbial community in the JX-ERA were higher than those in the SY-ERA and HB. PCoA showed that the distribution of ARGs, MGEs, and microbial communities in the ERAs and HB were significantly different, indicating that the long-term wastewater discharge could alter the distribution of ARGs, MGEs, and microbial communities in the coastal area. The co-occurrence pattern among ARGs, MGEs, and microbial communities revealed that 12 bacterial genera, such as Psychrobacter, Pseudomonas, Sulfitobacter, Pseudoalteromonas, and Bacillus, showed strong positive correlations with ARGs and MGEs. Most potential hosts carried multidrug and β-lactamase resistance genes.

First report from supermarket chicken meat and genomic characterization of colistin resistance mediated by mcr-1.1 in ESBL-producing, multidrug-resistant Salmonella Minnesota

Plasmid-borne colistin resistance is considered one of the most complex public health concerns worldwide. Several studies reported the presence of the mcr-1.1 harboring Salmonella from the foodstuffs worldwide; still, there is a knowledge gap about the occurrence of these isolates in the Middle East. In this study, we report an mcr-1.1-mediated colistin resistance in two multidrug-resistant (MDR) S. Minnesota (denoted as Sal_2 and Sal_10), with both being also extended-spectrum β-lactamase (ESBL) producing. These isolates have been recovered from two independent samples out of 315 chilled chicken meat tested from retail supermarkets in the United Arab Emirates (UAE). Based on whole-genome sequencing (WGS) analysis, both isolates belonged to the same Sequence Type (ST) ST548. They shared the same genes encoding resistance to the following antimicrobials: polymyxin (mcr-1.1), phenicol (floR), quinolone (qnrB19), aminoglycoside (aac(6′)-Iaa), tetracycline (tet(A)), and sulfonamide (sul2). However, the isolates featured different patterns of β-lactamase resistance genes, which included blaCTX-M-55 (ESBL-β-lactamase) and blaCMY-2 (AmpC-β-lactamase) in the isolate Sal_2, and blaTEM-215 (ESBL-β-lactamase) in the isolate Sal_10. WGS analysis inferred that both S. Minnesota isolates in this study carry an IncX4 plasmid harboring the mcr-1.1 variant. To understand the possible origin of the two mcr-1.1 carrying S. Minnesota isolated from retail chicken meat in this study, we conducted a phylogenomic analysis using available genomes of S. enterica, which harbored mcr-1.1 gene (n = 240, from the Middle East and Asian countries) deposited in the NCBI database. We found that Sal_2 and Sal_10 independently clustered together with other isolates detected in China, mainly from the chicken origin and to a lesser extent from human clinical origin. The finding of mcr-producing colistin-resistant strains in retail chicken meat warrants a more comprehensive One Health investigations involving strains from animals, retail food chains, and human clinical isolates at the national level in the UAE.

Companion Animals as Potential Reservoirs of Antibiotic Resistant Diarrheagenic Escherichia coli in Shandong, China

Antibiotic resistance genes of Escherichia coli (E. coli) from companion animals were still poorly understood. Here, we investigated the extended-spectrum β-lactamases (ESBLs) resistance genes of E. coli from companion animals in Shandong, China. A total of 79 isolates (80.6%) were recovered from 98 healthy or diarrheal companion animals in 2021, among which ESBLs-producing isolates accounted for 43.0% (34/79), and more than half of ESBL E. coli (ESBL-EC) strains (n = 19) were isolated from healthy companion animals. Diarrheagenic E. coli isolates (45.6%, n = 36) were represented by enterotoxigenic (ETEC) (32.9%), enteropathogenic (EPEC) (10.1%) and enteroinvasive (EIEC) (2.6%), 20 isolates of which were from healthy pets. Among tested antibiotics, resistance to tetracycline (64.6%) was the most commonly observed, followed by doxycycline (59.5%) and ampicillin (53.2%). Notably, all isolates were susceptible to meropenem. The multidrug-resistant (MDR) rate was 49.4%, 20 isolates of which were ESBLs producers; moreover, 23.4%, 16.4% of ESBL-EC strains were resistant to 5 or more, 7 or more antibiotics, respectively. Among the 5 β-lactamase resistance genes, the most frequent gene was blaCTX-M (60.76%), followed by blaSHV (40.51%). The plasmid-mediated quinolone resistance (PMQR) gene aac(6’)-Ib-cr was detected in 35 isolates. Additionally, ESBL-associated genes (i.e., blaCTX-M, blaSHV) were found in 76.5% ESBL-EC strains, with six isolates carrying blaCTX-M and blaSHV. The marker gene of high-pathogenicity island gene irp2 (encoding iron capture systems) was the most frequency virulence gene. Our results showed that ESBL-EC were widespread in healthy or diarrhea companion animals, especially healthy pets, which may be a potential reservoir of antibiotic resistance, therefore, enhancing a risk to public and animal health.

Phenotypic and Genetic Characterization of Klebsiella pneumoniae Isolates from Wild Animals in Central Italy.

Simple SummaryKlebsiella pneumoniae is an opportunistic bacterium known as a nosocomial pathogen, but it is able to colonize a multitude of ecological niches, both free-living and host-associated. For this reason, the presence of antimicrobial resistance genes in wildlife is an indicator that resistant bacteria of human- or livestock-origin are widespread in the environment. Wild animals can act as efficient reservoirs and epidemiological links between humans, livestock, and natural environments. Despite the renewal of interest in epidemiology, there is a critical lack of an unbiased ecological approaches to defining reservoirs and sources of infections. The aims of the present study were to investigate the occurrence of K. pneumoniae in wildlife, to characterize antimicrobial features through phenotypic and whole genome sequencing approaches, and to identify similar available sequences that could suggest a transmission event.Despite Klebsiella pneumoniae being widely recognized as a nosocomial pathogen, there is a critical lack in defining its reservoirs and sources of infections. Most studies on risk factors have focused on multidrug-resistant (MDR) isolates and clinically-oriented questions. Over a two-year period, we sampled 131 wild animals including mammal and bird species from three regions of Central Italy. All typical colonies isolated from the analytical portions were confirmed by real-time PCR and identified by MALDI-TOF mass spectrometry (MALDI-TOF MS). All confirmed K. pneumoniae isolates were tested for antimicrobial susceptibility to 29 antimicrobials and subjected to whole genome sequencing. Typical colonies were detected in 17 samples (13%), which were identified as K. pneumoniae (n = 16) and as K. quasipneumoniae (n = 1) by MALDI-TOF MS. The antimicrobial susceptibility profile showed that all the isolates were resistant to β-lactams (ceftobiprole, cloxacillin, cefazolin) and tetracycline; resistance to ertapenem and trimethoprim was observed and nine out of 16 K. pneumoniae isolates (56.2%) were classified as MDR. Genomic characterization allowed the detection of fluoroquinolone resistance-associated efflux pumps, fosfomycin and β-lactamase resistance genes, and virulence genes in the overall dataset. The cluster analysis of two isolates detected from wild boar with available clinical genomes showed the closest similarity. This study highlights the link between humans, domestic animals, and wildlife, showing that the current knowledge on this ecological context is lacking and that the potential health risks are underestimated.

Multidrug-Resistant and Extended-Spectrum β-Lactamase (ESBL) - Producing Enterobacterales Isolated from Carriage Samples among HIV Infected Women in Yaoundé, Cameroon.

The exacerbation of antimicrobial resistance (AMR) is a major public health threat worldwide. In sub-Saharan Africa, there is a scarcity of data regarding multidrug-resistant (resistance to at least one antibiotic of three or more families of antibiotics) as well as extended spectrum β-lactamase-producing Enterobacterales (ESBL-PE), isolated among clinical and asymptomatically healthy patients, especially in women living with HIV (WLHIV) despite their immunocompromised status. The overarching aim of this study was set to determine the prevalence and characterize genotypically multi-drug resistant Enterobacterales (MDR-E) and ESBL- PE isolated from vaginal swabs of WLHIV attending the Yaoundé Central Hospital, Yaoundé, Cameroon. A cross-sectional study was conducted among WLHIV during a four-month periods from 1 February to 31 May 2021. A total of 175 WLHIV, of childbearing age and under antiretroviral treatment were contacted. One hundred and twenty participants (120) were recruited and vaginal swabs were collected from them. After culture on Eosine-Methylen Blue (EMB) agar, the identification of Enterobacterales was performed using API 20E kit. A double-screening of ESBL-PE was performed using a combined disc diffusion method and ROSCO Diagnostica kits. An antibiotic susceptibility test was carried out by disc diffusion as per the Kirby-Bauer method and the β-lactamase resistance genes, blaCTX-M, blaCTX-M-group1-2-9, blaTEM were molecularly characterized using a conventional Polymerase Chain Reaction (PCR). Overall, 30.83% (37/120) of the included WLHIV were colonized with Enterobacterales and the prevalence of vaginal carriage of MDR Enterobacterales among them was 62.16% (23/37). Among MDR-E isolates, the most prevalent species were E. coli (56.0%; 14/25) and K. pneumoniae (20.0%; 5/25). High rates of resistance to trimethoprim-sulfamethoxazole (96.0%; 24/25), amoxicillin-clavulanic acid (88.0%; 22/25) and gentamicin (72%; 18/25) were observed. The resistance mechanisms detected among these isolates were ESBL (48.0%; 12/25), ESBL+ porin loss (8.0%; 2/25), ESBL+AmpC (24%; 6/25), with blaCTX-M, blaCTX-M-group-1,2,9 being identified at 48.0% (12/25) for each of them and blaTEM at 72.0% (18/25). Our findings confirm the high-prevalence of MDR as well as ESBL-PE isolated in WLHIV, and suggest that a real time monitoring system of antimicrobial resistant bacteria coupled with the reinforcement of infection prevention control (IPC) strategies are needed to sustainably contain these life-threatening pathogens especially in the most vulnerable populations.

High Levels of Multi Drug Resistant Escherichia coli Pathovars in Preharvest Environmental Samples: A Ticking Time Bomb for Fresh Produce Related Disease Outbreak

The preharvest environment provides a suitable platform for the emergence and dissemination of multidrug resistant pathogenic bacteria. This could be detrimental to public health and may lead to fresh produce related disease outbreaks when transferred to the food web. This study evaluated the occurrence and antibiogram fingerprints of the pathogenic strains of Escherichia coli (E. coli) recovered from irrigation water and agricultural soil samples in fresh produce farms in the Eastern Cape Province, South Africa. E. coli was isolated, pathotyped and screened for antibiogram fingerprints using standard cultural and molecular methods. An antibiogram-based K-means cluster analysis and neighbour-joining dendrogram was used to elucidate the relatedness of the isolates. Of the confirmed isolates, 37% harboured at least one of the screened virulence genes (VGs) that encode for intestinal and extraintestinal E. coli pathovars. The most prevalent pathovar/associated VG in all the samples was Enterohemorrhagic E. coli/stx2. Of all the antimicrobials tested, E. coli pathovars conferred the highest resistance against ampicillin with a median single antimicrobial resistance (AMR) index of 0.23 (IQR: 0.10–0.38, 95% CI: 0.16–0.27) for pathovars from irrigation water and 0.01 (IQR: 0.01–0.03, 95% CI: 0.01–0.03) for pathovars from agricultural soil. About 60 and 20% of the pathovars from irrigation water and agricultural soil were multi drug resistant with a median multiple AMR index of 0.41 (IQR: 0.25–0.52, 95% CI: 0.31–0.47) and 0.38 (IQR: 0.23–0.66, 95% CI: 0.19–0.69) respectively. The most prevalent resistance genes detected were sulI, catII, tetA and AmpC across all sample types. The blaTEM was the most prevalent extended-spectrum β-lactamase resistance gene detected. A positive correlation between the phenotypic and genotypic AMR profiles of the pathovars in irrigation water samples, r = 0.6, p ≤ 0.01 and agricultural soil samples, r = 0.8, p ≤ 0.01 was recorded. The antibiogram based dendrogram generated 11 and 7 clades from irrigation water and agricultural soil samples respectively, indicating that the isolates are highly diversified. The findings revealed the occurrence of highly diversified multidrug resistant E. coli pathovars in the preharvest environment, indicating potential public health threats.

Correlation between Identification of β-Lactamase Resistance Genes and Antimicrobial Susceptibility Profiles in Gram-Negative Bacteria: a Laboratory Data Analysis.

ABSTRACTWe reported the frequency of resistance gene detection in Gram-negative blood culture isolates and correlated these findings with corresponding antibiograms. Data were obtained from 1045 isolates tested on the GenMark Dx ePlex Blood Culture Identification Gram-Negative Panels at the Mount Sinai Hospital Clinical Microbiology Laboratory in New York from March 2019 to February 2021. Susceptibilities were performed using Vitek 2 (bioMérieux Clinical Diagnostics) or Microscan (Beckman Coulter Inc.). blaCTX-M was detected in 26.4% Klebsiella pneumoniae, 23.5% Escherichia coli, and 16.4% Proteus mirabilis isolates. As would be expected, both blaCTX-M and blaCTX-M negative isolates were likely to be susceptible to newer agents while blaCTX-M positive isolates were more likely to be resistant to earlier generations of beta-lactam antibiotics. 3/204 blaCTX-M-positive isolates were found to be ceftriaxone-susceptible. Conversely, 2.8% ceftriaxone nonsusceptible strains were negative for all β-lactamase genes on the ePlex BCID-GN panel, including blaCTX-M. The prevalence of CTX-M-producing Enterobacterales remains high in the United States. A small number of blaCTX-M-positive isolates were susceptible to ceftriaxone, and a small number of ceftriaxone nonsusceptible isolates were negative for blaCTX-M. Further studies are needed to determine the optimal management when an isolate is phenotypically susceptible to ceftriaxone, but blaCTX-M is detected.IMPORTANCE There is limited literature on corresponding results obtained from rapid molecular diagnostics with the antibiotic susceptibility profile. We reported a correlation between the results obtained from ePlex and the antibiograms against a large collection of Gram-negative bacteria. We reported that there can be a discrepancy in a small number of cases, but the clinical significance of that is unknown.

Phenotypic and molecular characterization of β-lactamase and plasmid-mediated quinolone resistance genes in Klebsiella oxytoca isolated from slaughtered pigs in Thailand.

Background and Aim:Over recent years, antimicrobial-resistant Klebsiella species in humans, animals, food animals, food products, and agricultural environments have been the center of attention due to its role in the evolution of antimicrobial resistance. The emergence of resistance to fluoroquinolones and cephalosporins of third and higher generations in Klebsiella oxytoca has not received much attention in animal husbandry compared to that in Klebsiella pneumoniae. Reports on K. oxytoca are limited in the study area. Therefore, we investigated the antimicrobial susceptibility and resistance genes in K. oxytoca isolated from slaughtered pigs in Thailand.Materials and Methods:Microbiological examination was conducted on 384 Klebsiella spp. isolates recovered from slaughtered pigs in ten provinces of Thailand. Seventy-two K. oxytoca isolates (18.75%) were examined for antimicrobial-resistant genes (β-lactamase [blaTEM, blaCTX-M, and blaSHV]) and fluoroquinolone-resistant genes (qnrA, qnrB, qnrC, qnrD, qnrS, oqxAB, aac(6’)-Ib-cr, and qepA).Results:The most common genotype was blaCTX-M (58/72, 80.55%), followed by blaTEM with blaCTX-M (7/72, 9.72%) and blaTEM (6/72, 8.33%). The most common blaCTX-M group was blaCTX-M-1 (19/58, 32.76%), followed by blaCTX-M-9 (1/58, 1.72%). Plasmid-mediated quinolone resistance genes were identified in 13 (18.05%) isolates: qnrS (16.70%) and qnrB (1.4%). All 13 isolates had qnrS transferable to an Escherichia coli recipient, whereas qnrB was not detected in any transconjugants. Either blaCTX-M or blaTEM harbored by one K. oxytoca strain was transferable to an E. coli recipient. Analysis of antimicrobial susceptibility revealed that more than 90% of the blaCTX-M-carrying K. oxytoca isolates were susceptible to chloramphenicol, trimethoprim, ceftazidime, cefepime, cefotaxime, amoxicillin-clavulanic acid, piperacillin–tazobactam, and fosfomycin. All K. oxytoca isolates (13) harboring qnr were susceptible to carbapenem and ceftriaxone; however, 43 (74.13%) of the K. oxytoca isolates harboring blaCTX-M exhibited extended-spectrum β-lactamase activity. Most of the K. oxytoca isolates from pigs were highly resistant to ampicillin, azithromycin, and gentamicin.Conclusion:To prevent further transmission of Klebsiella spp. Between food animals and humans, strict control of antibiotic use in clinical and livestock settings is necessary along with routine disinfection of the livestock environment and efforts to increase awareness of antimicrobial resistance transmission.

Molecular Detection of Integrons, Colistin and β-lactamase Resistant Genes in Salmonella enterica Serovars Enteritidis and Typhimurium Isolated from Chickens and Rats Inhabiting Poultry Farms.

The rapid growth of multidrug-resistant Salmonella is a global public health concern. The aim of this study was to detect integrons, colistin and β-lactamase resistance genes in Salmonella enteritidis and typhimurium. A total of 63 isolates of S. enteritidis (n = 18) and S. typhimurium (n = 45) from fecal samples of layers and rats at chicken farms were screened for antibiotic resistant genes. Conventional PCR was performed for the detection of integrons (classes 1, 2, and 3), colistin (mcr-1-5) and β-lactamase (blaCTX-M, blaCTX-M-1, blaCTX-M-2, blaCTX-M-9, blaCTX-M-15, blaTEM, blaSHV, and blaOXA) resistant genes. Of these isolates, 77% and 27% of S. typhimurium and S. enteritidis harboured the mcr-4 encoded gene for colistin, respectively. The prevalence of class 1 integrons for S. typhimurium and S. enteritidis was 100% for each serovar, while for class 2 integrons of S. typhimurium and S. enteritidis it was 49% and 33% respectively, while class 3 integron genes was not detected. Our study also detected high levels of β-lactamase encoding genes (bla gene), namely blaCTX-M, blaCTX-M-1, blaCTX-M-9 and blaTEM from both S. typhimurium and S. enteritidis. This, to our knowledge, is the first report of mcr-4 resistance gene detection in Salmonella serovars in South Africa. This study also highlights the importance of controlling rats at poultry farms in order to reduce the risk of transmission of antibiotic resistance to chickens and eventually to humans.

Identification of oral anaerobic bacteria and the beta-lactamase resistance genes from Iranian patients with periodontitis

ObjectivesThe dysbiosis of bacteria and horizontal transfer of antibiotic resistance genes (ARGs) could be highly problematic particularly in the oral environment. Here, we aimed to identify the anaerobic species from patients with periodontitis and to screen the isolates for the β-lactamase resistance genes, blaTEM, cfxA, its variants, and mobA. MethodsThe 129 samples from periodontal pockets were subjected to anaerobic culture, followed by 16S rRNA gene sequencing, PCR assays for the cfxA, blaTEM, and mobA. The minimum inhibitory concentration (MIC) of amoxicillin, ampicillin, amoxicillin/clavulanate, ampicillin/sulbactam, and cefixime was determined against CfxA producing isolates using MIC Test Strips. ResultsThe species with frequency higher than 10% were Lactobacillus spp. (26.3%), Streptococcus spp. (18.8%), Leptotrichia wadei (14%) and Veillonella spp. (11.4%). The blaTEM was not found in any of the isolates whereas cfxA was found in 12.5% of isolates including V. parvula, V. rogosae, Prevotella nigrescens and Campylobacter concisus. Of CfxA variants, CfxA2 (90%) was the most frequent one. Among the CfxA producing isolates, the resistance to ampicillin and amoxicillin was observed only in two isolates of P. nigrescens and V. rogosae. ConclusionsThis study showed that various anaerobes species may be involved in the development of periodontitis. Of them, Prevotella and Veillonella species were found to commonly carry cfxA even though they are susceptible to beta-lactams and its combination.

The Genomic Characterization of KPC-Producing Klebsiella pneumoniae from the ICU of a Teaching Hospital in Shanghai, China.

PurposeThis study retrospectively analyzed the genome characteristics of blaKPC-2 in multidrug-resistant Klebsiella pneumoniae collected from the ICU of a teaching hospital in Shanghai, China.MethodsFrom February 2018 to December 2019, 36 strains of multidrug-resistant Klebsiella pneumoniae were collected from the bronchoalveolar lavage fluid of critically ill patients. The genome of all isolates was obtained through the Illumina sequence, and single nucleotide polymorphisms of the blaKPC-2 gene were analyzed to explore blaKPC-2’s evolutionary characteristics. Different strains’ genetic relationships and homology were studied by constructing an evolutionary tree on a single copy orthologue. Pacbio combined Illumina sequence was conducted to evaluate the structure and potential mobility of drug-resistant plasmids of the strain KP-s26.ResultsThe distribution of resistance and virulence genes had little difference, but most strains had significant differences in the plasmid-encoded region. Most strains (31/36) carried the carbapenemase gene blaKPC-2, with no single nucleotide polymorphism in different strains. Extended-spectrum β-lactamase resistance genes, such as blaCTX-M and blaSHV, were found in the isolates, but no metallo-β-lactamases were detected. All strains with blaKPC-2 coexisted with chromosomal-associated fosfomycin resistance genes fosA6, and the coexistence of blaKPC-2 and blaCTX variants (blaCTX-M-15, blaCTX-M-65, and blaCTX-M-27) was also detected in 29/31 strains. The isolate KP-s26 carried five circular plasmids. pA and pB were conjugate plasmids, as they carried drug resistance genes and contained a complete IV secretion system.ConclusionThe blaKPC-2 carbapenemase gene is relatively conservative in the process of evolution; drug-resistant plasmids containing conjugated transfer elements contribute to the spreading of drug resistance. The coexistence of blaKPC-2 with fosA6 or blaCTX-M variants was associated with increased fosfomycin resistance and broad-spectrum β-lactam resistance, respectively.Clinical Trials RegistrationClinical Trials.gov Identifier: NCT03950544

Characterization and Comparative Genomics Analysis of lncFII Multi-Resistance Plasmids Carrying bla CTX - M and Type1 Integrons From Escherichia coli.

This research aimed to investigate the presence and transferability of the extended-spectrum β-lactamase resistance genes to identify the genetic context of multi-drug resistant (MDR) loci in two Escherichia coli plasmids from livestock and poultry breeding environment. MICs were determined by broth microdilution. A total of 137 E. coli resistant to extended-spectrum β-lactam antibiotics were screened for the presence of the ESBL genes by PCR. Only two E. coli out of 206 strains produced carbapenemases, including strain 11011 that produced enzyme A, and strain 417957 that produced enzyme B. The genes were blaKPC and blaNDM, respectively. The plasmids containing blaCTX–M were conjugatable, and the plasmids containing carbapenem resistance gene were not conjugatable. Six extended-spectrum β-lactamase resistance genes were detected in this research, including blaTEM, blaCTX–M, blaSHV, blaOAX–1, blaKPC, and blaNDM, and the detection rates were 94.89% (130/137), 92.7% (127/137), 24.81% (34/137), 20.43% (28/137), 0.72% (1/137), and 0.72% (1/137), respectively. Two conjugative lncFII multi-resistance plasmids carrying blaCTX–M, p11011-fosA and p417957-CTXM, were sequenced and analyzed. Both conjugative plasmids were larger than 100 kb and contained three accessory modules, including MDR region. The MDR region of the two plasmids contained many antibiotic resistance genes, including blaCTX–M, mph (A), dfrA17, aadA5, sul1, etc. After transfer, both the transconjugants displayed elevated MICs of the respective antimicrobial agents. A large number of resistance genes clusters in specific regions may contribute to the MDR profile of the strains. The presence of mobile genetic elements at the boundaries can possibly facilitate transfer among Enterobacteriaceae through inter-replicon gene transfer. Our study provides beta-lactam resistance profile of bacteria, reveals the prevalence of β-lactamase resistance genes in livestock and poultry breeding environment in Zhejiang Province, and enriches the research on IncFII plasmids containing blaCTX–M.

The molecular study for evaluation the antibiotic resistance of Escherichia coli and Klebsiella pneumoniae bacteria isolated from urinary tract infection patients

Urinary tract infection is a bacterial infection that often affects the bladder and thus the urinary system. E. coli is one of the leading uropathogenic bacteria that cause urinary tract infections. Uropathogenic E. coli is highly effective and successful in causing urinary tract infections through biofilm formation and urothelial cell invasion mechanisms. Other organisms that cause urinary tract infections include members of the Enterobacteriaceae family, streptococci and staphylococci species and perch. In addition, K.penumoniae is another important gram-negative bacterium that causes urinary tract infections. With the PCR technique, unseen bacterial species can be detected using standard clinical microbiology methods. In this study, the antibiotic resistance of E. coli and K. penumoniae bacteria causing urinary tract infection was analyzed by PCR technique. As a result of the experiments conducted within the scope of our study, it was found that bla SHV, one of the virulence factors of E. coli isolates, and bla CTX-M, one of the genes that produce ESBL, were related that both these virulence factors can be found at the same time in ESBL positive and negative isolates. It appeared that bla CTX-M gene is not detected in any of the ESBL negative isolates. It demonstrated that the bla CTX-M gene was more dominant in the development of resistance to β-lactam group antibiotics. Also, the results of the experiments conducted within the scope of our study, the frequency percentage of β-lactamase resistance genes (bla TEM, bla SHV and bla CTX-M) increased in K. pneumoniae compared to E. coli isolates. Moreover, phenotypic and genotypic methods are needed to detect the presence of different gene products associated with resistance in E. coli and K. pneumoniae isolates.

Genotypic and Phenotypic Analyses of Antibiotic Resistance in Indonesian Indigenous Lactobacillus Probiotics

Abstract Background and Objective: In the authors’ previous study, four unique Lactobacillus strains (Lactobacillus plantarum Dad-13, Lactobacillus plantarum Mut-7, Lactobacillus plantarum T-3 and Lactobacillus paracasei SNP-2) from Indonesian fermented foods and healthy feces have been studied as probiotic agents. In the current study, antibiotic resistance phenotypes of the highlighted Lactobacillus plantarum and Lactobacillus paracasei against eight antibiotics (amoxicillin, tetracycline, erythromycin, clindamycin, chloramphenicol, streptomycin, kanamycin, ciprofloxacin) and antibiotic resistance genes of these strains were investigated. Material and Methods: The bacterial antibiotic susceptibility to eight antibiotics was assessed using disk diffusion method. Genome sequencing was carried out using NovaSeq 6000 sequencing platform. Genome was annotated using Rapid Annotation using Subsystem Technology v.2.0. Each group of the predicted products of resistance genes was further aligned using multiple sequence comparison by log-expectation and their functions were verified using comprehensive antibiotic resistance database 2020. Results and Conclusion: All strains showed resistance to aminoglycoside and ciprofloxacin but sensitive to amoxicillin, clindamycin and erythromycin. Resistance to chloramphenicol and tetracycline varied within the strains. Two strains were sensitive and others were intermediate resistance to chloramphenicol. One strain was resistant to tetracycline, while the other three strains demonstrated intermediate resistance to the antibiotic. Genome sequence of the four strains verified the presence of the tetracycline, β-lactamase and ciprofloxacin resistance genes as well as multidrug resistance efflux systems. Occurrence of the resistance genes was correlated to the phenotype results, except for amoxicillin and aminoglycosides. Rapid Annotation using Subsystem Technology annotation showed that all Lactobacillus strains did not include transposable elements, gene transfer agents and plasmid linked functions; thus, horizontal transfer of the antibiotic resistance genes unlikely occurred. Conflict of interest: The authors declare no conflict of interest.

Whole Genome Sequencing of Extended-Spectrum- and AmpC- β-Lactamase-Positive Enterobacterales Isolated From Spinach Production in Gauteng Province, South Africa.

The increasing occurrence of multidrug-resistant (MDR) extended-spectrum β-lactamase- (ESBL) and/or AmpC β-lactamase- (AmpC) producing Enterobacterales in irrigation water and associated irrigated fresh produce represents risks related to the environment, food safety, and public health. In South Africa, information about the presence of ESBL/AmpC-producing Enterobacterales from non-clinical sources is limited, particularly in the water–plant-food interface. This study aimed to characterize 19 selected MDR ESBL/AmpC-producing Escherichia coli (n=3), Klebsiella pneumoniae (n=5), Serratia fonticola (n=10), and Salmonella enterica (n=1) isolates from spinach and associated irrigation water samples from two commercial spinach production systems within South Africa, using whole genome sequencing (WGS). Antibiotic resistance genes potentially encoding resistance to eight different classes were present, with blaCTX-M-15 being the dominant ESBL encoding gene and blaACT-types being the dominant AmpC encoding gene detected. A greater number of resistance genes across more antibiotic classes were seen in all the K. pneumoniae strains, compared to the other genera tested. From one farm, blaCTX-M-15-positive K. pneumoniae strains of the same sequence type 985 (ST 985) were present in spinach at harvest and retail samples after processing, suggesting successful persistence of these MDR strains. In addition, ESBL-producing K. pneumoniae ST15, an emerging high-risk clone causing nosocomical outbreaks worldwide, was isolated from irrigation water. Known resistance plasmid replicon types of Enterobacterales including IncFIB, IncFIA, IncFII, IncB/O, and IncHI1B were observed in all strains following analysis with PlasmidFinder. However, blaCTX-M-15 was the only β-lactamase resistance gene associated with plasmids (IncFII and IncFIB) in K. pneumoniae (n=4) strains. In one E. coli and five K. pneumoniae strains, integron In191 was observed. Relevant similarities to human pathogens were predicted with PathogenFinder for all 19 strains, with a confidence of 0.635–0.721 in S. fonticola, 0.852–0.931 in E. coli, 0.796–0.899 in K. pneumoniae, and 0.939 in the S. enterica strain. The presence of MDR ESBL/AmpC-producing E. coli, K. pneumoniae, S. fonticola, and S. enterica with similarities to human pathogens in the agricultural production systems reflects environmental and food contamination mediated by anthropogenic activities, contributing to the spread of antibiotic resistance genes.

Molecular Surveillance and Assessment of Ceftolozane/Tazobactam Resistance with Common β-Lactam Antibiotics and β-Lactamase Genes

Ceftolozane/tazobactam (c/t) is a potent β-lactam antibiotic which combines the fifth generation cephalosporin ceftolozane and tazobactam, a β-lactamase inhibitor. The c/t combination therapy was approved in 2014 for the treatment of multidrug resistant (MDR) Enterobacteriaceae, especially intra-abdominal and urinary tract infections. The aim of this study is to assess c/t activity and to examine the association of c/t resistance with four common β-lactamase resistance genes found in clinical Enterobacteriaceae isolates collected from mainly urinary tract infections in an agricultural region in California (USA) between 2013-2020. We tested 993 Extended Spectrum β-lactamases (ESBL) producing Enterobacteriaceae isolates (885 E. coli, 94 K. pneumoniae, 14 other) for c/t susceptibility by Kirby-Bauer disk diffusion and screened using PCR for four common resistance genes with β-lactamase activity(blaTEM, blaOXA, blaSHV, and blaCTX-M) for 855 of the isolates. We also investigated co-resistance of c/t and nine other β-lactam antibiotics. We found that most isolates were susceptible to c/t (58.3%), while 38.5% showed intermediate resistance, and 3.2% were resistant. We also found that K. pneumoniae isolates were more resistant to c/t than E. coli isolates, and that c/t may be a good alternative to carbapenems, in that that some carbapenem resistant isolates were susceptible to c/t. Genotypic analysis showed blaSHV and blaCTX-M are independently associated with elevated c/t resistance. Although c/t demonstrated strong activity against Enterobacteriaceae, the high percentage of isolates with intermediate susceptibility emphasizes the need for close monitoring and continued surveillance for c/t resistance among ESBLs

High frequency of multidrug-resistant (MDR) Klebsiella pneumoniae harboring several \u03b2-lactamase and integron genes collected from several hospitals in the north of Iran

BackgroundKlebsiella pneumoniae is one of the leading causes of hospital outbreaks worldwide. Also, antibiotic-resistant K. pneumoniae is progressively being involved in invasive infections with high morbidity and mortality. The aim of the current study was to determine antimicrobial susceptibility patterns and the incidence of resistance genes (integron types and β-lactamase-encoded genes) among clinical isolates of K. pneumoniae.MethodsIn this cross-sectional study, a total of 100 clinical samples were obtained from hospitalized patients in three teaching hospitals in the north of Iran, from November 2018 and October 2019. Antimicrobial susceptibility testing was performed using disk agar diffusion test in line with CLSI recommendations. For colistin, minimum inhibitory concentration (MIC) was determined using broth microdilution. Based on antibiogram, multi-drug resistant (MDR) and extensive-drug resistant (XDR) strains were detected. Finally, integron types and β-lactamase resistance genes were identified using polymerase chain reaction technique.ResultsThe most and least clinical samples were related to the urine and bronchoalveolar lavage, respectively. Based on the antibiogram results, amikacin and gentamicin exhibited good activity against K. pneumoniae strains in vitro. The high resistance rate (93%) to ampicillin/sulbactam predicts the limited efficacy of this antibiotic, in the hospitals studied. Among all the 100 isolates, the frequency of MDR and XDR phenotypes were 58% and 13%, respectively, while no pan-drug resistant (PDR) strains were found. In the MDR K. pneumoniae strains, the prevalence of blaSHV, blaTEM, blaCTX-M-15, blaKPC, blaOXA-48, blaNDM β-lactamase genes were 91.4%, 82.7%, 79.3%, 29.3%, 36.2% and 6.9%, respectively, however 91.4% of the isolates were carrying intI gene. Class II and III integrons were not detected in any isolates.ConclusionThe MDR K. pneumoniae is becoming a serious problem in hospitals, with many strains developing resistance to most available antimicrobials. Our results indicate co-presence of a series of β-lactamase and integron types on the MDR strains recovered from hospitalized patients. The increasing rate of these isolates emphasizes the importance of choosing an appropriate antimicrobial regimen based on antibiotic susceptibility pattern.

Prevalence, Antimicrobial Susceptibility, and Molecular Characterization of Escherichia coli Isolated From Raw Milk in Dairy Herds in Northern China.

Escherichia coli is a common bacterium in the intestines of animals, and it is also the major important cause of toxic mastitis, which is an acute or peracute disease that causes a higher incidence of death and culling of cattle. The purpose of this study was to investigate E. coli strains isolated from the raw milk of dairy cattle in Northern China, and the antibacterial susceptibility of these strains and essential virulence genes. From May to September 2015, 195 raw milk samples were collected from 195 dairy farms located in Northern China. Among the samples, 67 (34.4%) samples were positive for E. coli. About 67 E. coli strains were isolated from these 67 samples. The prevalence of Shiga toxin-producing E. coli (STEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), and enteroinvasive E. coli (EIEC) were 9, 6, 4.5, and 1.5%, respectively. Among the virulence genes detected, stx1 was the most prevalent (6/67, 9%) gene, followed by eae (3/67, 4.5%), and estB (2/67, 3%). Moreover, the strains exhibited different resistance levels to ampicillin (46.3%), amoxicillin-clavulanic acid (16.4%), trimethoprim-sulfamethoxazole (13.4%), tetracycline (13.4%), cefoxitin (11.9%), chloramphenicol (7.5%), kanamycin (7.5%), streptomycin (6.0%), tobramycin (4.5%), azithromycin (4.5%), and ciprofloxacin (1.5%). All of the E. coli isolates were susceptible to gentamicin. The prevalence of β-lactamase-encoding genes was 34.3% in 67 E. coli isolates and 45% in 40 β-lactam-resistance E. coli isolates. The overall prevalence of blaSHV, blaTEM, blaCMY, and blaCTX-M genes were 1.5, 20.9, 10.4, and 1.5%, respectively. Nine non-pathogenic E. coli isolates also carried β-lactamase resistance genes, which may transfer to other pathogenic E. coli and pose a threat to the farm’s mastitis management projects. Our results showed that most of E. coli were multidrug resistant and possessed multiple virulence genes, which may have a huge potential hazard with public health, and antibiotic resistance of E. coli was prevalent in dairy herds in Northern China, and ampicillin should be used cautiously for mastitis caused by E. coli in Northern China.

Imipenem-Relebactam Susceptibility and Genotypic Characteristics of Carbapenem-Resistant Enterobacterales Identified during Population-Based Surveillance.

Laboratories submit all carbapenem-resistant Enterobacter, Escherichia coli, and Klebsiella species to the Alameda County Public Health Department (ACPHD). ACPHD evaluated 75 isolates submitted during 9 months for susceptibility to imipenem-relebactam (I-R) and, using whole-genome sequencing, identified β-lactamase genes. Of 60 (80%) isolates susceptible to I-R, 8 (13%) had detectable carbapenemase genes, including 4 KPC, two NDM, and two OXA-48-like; we described the relationship between the presence of β-lactamase resistance genes and susceptibility to I-R.

High Prevalence of ESBL and Plasmid-Mediated Quinolone Resistance Genes in Salmonella enterica Isolated from Retail Meats and Slaughterhouses in Egypt

The emergence and spread of multidrug-resistant Salmonella enterica (S. enterica) to humans through food of animal origin are considered a major global public health concern. Currently, little is known about the prevalence of important antimicrobial resistance genes in S. enterica from retail food in Africa. Therefore, the screening and characterization of the extended-spectrum β-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes in S. enterica isolated from retail meats and slaughterhouses in Egypt were done by using PCR and DNA sequencing techniques. Twenty-eight out of thirty-four (82.4%) non-duplicate S. enterica isolates showed multidrug-resistance phenotypes to at least three classes of antimicrobials, and fourteen (41.2%) exhibited an ESBL-resistance phenotype and harbored at least one ESBL-encoding gene. The identified β-lactamase-encoding genes included blaCTX-M-1, blaCTX-M-3, blaCTX-M-13, blaCTX-M-14, blaCTX-M-15, and blaSHV-12 (ESBL types); blaCMY-2 (AmpC type); and blaTEM-1 and blaOXA-1 (narrow-spectrum types). PMQR genes (included qnrA, qnrB, qnrS, and aac(6′)-Ib-cr) were identified in 23 (67.6%) isolates. The presence of ESBL- and PMQR-producing S. enterica with a high prevalence rate in retail meats and slaughterhouses is considered a major threat to public health as these strains with resistance genes could be transmitted to humans through the food chain.