Β-lactamase Resistance Genes Research Articles

Evolution of ceftazidime/avibactam resistance and plasmid dynamics in OXA-48-producing Klebsiella spp. during long-term patient colonization.

To prospectively monitor the evolution of the resistome of OXA-48-producing Klebsiella species in a patient with long-term colonization, with a particular focus into the plasmid dynamics and the evolution of ceftazidime/avibactam resistance. All OXA-48-producing Klebsiella spp. isolates from a single patient admitted to a hospital during seven months were prospectively collected. MICs were determined through reference broth microdilution. Multilocus sequence types, SNPs analysis, resistance mechanisms, genetic context of β-lactamases and plasmid dynamics were determined by WGS and bioinformatic analysis. The impact of β-lactamase variant obtained after ceftazidime/avibactam exposure was determined via cloning experiments. Four isolates, two before (one OXA-48-producing K. pneumoniae and one CTX-M-15-like-producing K. pneumoniae) and two after treatment with ceftazidime/avibactam (one OXA-48- and CTX-M-15-like-producing K. pneumoniae and one OXA-48- and CTX-M-15-like-producing K. aerogenes) were collected. The plasmid dynamics analysis demonstrated that the IncL and IncFIIK plasmids, in which blaOXA-48 and blaCTX-M-15-like genes were located, respectively, exhibited a high degree of conservation indicating a potential for both intra- and interspecies transmission. The K. pneumoniae isolate obtained after treatment, which differed from the previous isolate by just six SNPs, exhibited resistance to ceftazidime/avibactam through P167S substitution in CTX-M-15, which is now designated CTX-M-273. Cloning experiments demonstrated enhanced resistance to ceftazidime/avibactam. The transfer of plasmid-borne β-lactamase resistance genes between intra- and interspecies bacterial populations enables the rapid diversification of the bacterial genome. The emergence of ceftazidime/avibactam resistance through the modification of CTX-M-enzymes represents a mechanism by which OXA-48-producing Enterobacterales may evolve toward ceftazidime/avibactam resistance in vivo.

Prevalence of ESBL resistance genes and fecal carriage of multidrug-resistant Pseudomonas aeruginosa isolates from patients with chronic kidney disease at the Laquintinie Hospital in Douala, Littoral Region, Cameroon.

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium capable of causing severe infections in immunocompromised patients such as those suffering from chronic kidney disease (CKD). This study aimed to determine the resistance profile of Pseudomonas aeruginosa, and the prevalence of extended-spectrum β-lactamase (ESBL) resistance genes in patients with chronic kidney disease. The prevalence of Pseudomonas aeruginosa was investigated in 458 patients, including 197 CKD patients and 261 patients suffering from gastrointestinal infections. The study was conducted at Laquintinie Hospital in Douala from January 2022 to December 2023. Stool samples were used to isolate Pseudomonas aeruginosa on Cetrimide agar. Antibiotic susceptibility testing was carried out using the Kirby-Bauer diffusion method. extended-spectrum β-lactamase (ESBL) resistance genes were detected by the polymerase chain reaction (PCR) method. The prevalence of fecal carriage of Pseudomonas aeruginosa was 9.17 % (n = 42/458), including twenty-nine (69.05 %) in patients with chronic kidney disease and thirteen (30.95 %) in patients without chronic kidney disease. The Pseudomonas aeruginosa isolates had a high rate of resistance to ceftazidime (72.41 %) in patients with CKD compared to patients without CKD (69.23 %). All isolates had a high resistance to ticarcillin (93.10 % and 92.31 %). The prevalence of multidrug-resistant (MDR) Pseudomonas aeruginosa isolates was 73.81 %. The MDR Pseudomonas aeruginosa were higher (70.97 % vs. 29.03 %) in patients suffering from CKD compared to patients without CKD. Up to 85.71 % of the Pseudomonas aeruginosa isolates harbored at least one ESBL gene. The blaTEM type gene (66.67 %) was the most frequently detected gene, followed by blaCTX-M (61.90 %) and blaSHV (47.62 %). ESBL resistance genes were more common in CKD patients (72.22 %) compared to patients without CKD (27.78 %). These results demonstrate that antibiotics belonging to the carbapenem and aminoglycoside classes could be used for Pseudomonas aeruginosa infection. This highlights the importance of regular surveillance of multidrug resistance and extended-spectrum β-lactamase production for Pseudomonas aeruginosa infections in patients with chronic kidney disease.

Carriage of antimicrobial resistance genes in Escherichia coli of bovine origin.

The present study aimed to search for the presence of the plasmid-mediated antimicrobial resistance genes in 106 Escherichia coli (E. coli) isolates from a total of 240 fresh fecal samples collected from 12 private cattle farms in Bingol province of East Turkey from November 2021 to January 2022. In those colistin-resistant E. coli (mcr-1 to -9), the major carbapenemase (blaOXA-48, blaNDM-1, blaIMP, blaVIM, and blaKPC), β-lactamase (blaTEM-1, blaCTX-M and blaSHV-1) and OXA-48 like β- lactamase (blaOXA-162, blaOXA-163, blaOXA-181, blaOXA-204 and blaOXA-232) resistance genes were searched for determined a multiplex polymerase chain reaction (PCR) method and Next-generation sequencing (NGS) - PCR Amplicons with Nanopore Technology. Only the mcr-4 gene was found in one isolate and the remaining genes (mcr-1-9) were not shown in all E. coli isolates from cattle. The minimal inhibitory concentration (MIC) to colistin was detected in mcr-4 positive E. coli isolates using broth microdilution. We assessed the antimicrobial susceptibilities of mcr-4 positive E. coli isolates using the Kirby-Bauer disk diffusion method. E coli isolate was detected as negative for carbapenemase and OXA-48 like β-lactamase resistance genes and positive for β-lactamase. In addition, E. coli isolates carrying mcr-4 were more resistant to colistin. Antimicrobial susceptibility testing using the disk diffusion assay indicated that all 106 E. coli isolates (100%) were sensitive to AMK, 105 E. coli isolates (99.1%) exhibited sensitivity to imipenem, meropenem and doripenem, and 1 E. coli isolate (0.9%) had intermediate resistance to imipenem, meropenem and doripenem; It was observed that all strains (100%) were resistant to cefotaxime. E. coli isolates are resistant to ampicillin (95.3%), amoxicillin/clavulanic acid (95.3%), cefepime (14.2%), cefixime (19.8%), cephalexin (74.5%), gentamicin (42.5%), kanamycin (37.7%), streptomycin (69.8%), tetracycline (80.2%), ciprofloxacin (60.4%), norfloxacin (13.2%), chloramphenicol (59.4%) and trimethoprim/sulfamethoxazole (68.9%). When we investigated the sequence in the Blast database, the genome of the E. coli isolate indicated high similarity with the mcr-4 sequences. To our knowledge, this is the first report investigating on the mcr-4 gene in E. coli identified from cattle in Turkey. Our results highlighted that cattle might be a potential risk in transmitting mcr genes.

AmpC β-lactamases detected in Southeast Asian Escherichia coli and Klebsiella pneumoniae.

AmpC β-lactamases are neglected compared with ESBL as a cause of third-generation cephalosporin (3GC) resistance in Enterobacterales in low- and middle-income countries and the burden is unknown. The aim of this study was to investigate the presence of AmpC β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in clinical specimens from three clinical research laboratories in Southeast Asia. Stored clinical isolates of E. coli and K. pneumoniae resistant to ceftriaxone or ceftazidime or cefpodoxime and ESBL confirmation test negative were screened using MASTDISCS AmpC, ESBL and Carbapenemase Detection Set-D72C. Short-read WGS was performed to identify ampC genes. Of 126 isolates collected between 2010 and 2020, 31 (24.6%) and 16 (12.7%) were phenotypically AmpC and inducible AmpC positive by MASTDISCS testing, respectively. All inducible AmpC isolates were ceftriaxone susceptible and 97.7% of AmpC/inducible AmpC isolates tested against cefoxitin were resistant. Through WGS, 17 and eight different STs were detected for the AmpC/inducible AmpC E. coli and K. pneumoniae isolates, respectively. Twelve different β-lactamase resistance genes were detected, with bla CMY-2 most commonly in AmpC-positive isolates (20/31; 64.5%; 15 chromosomal, five plasmid). All inducible AmpC-positive isolates had the bla DHA-1 gene (seven chromosomal, nine plasmid). Though uncommon, AmpC and inducible AmpC β-lactamases in E. coli and K. pneumoniae are an important cause of infection in Southeast Asia. With current testing methods, these infections may be going undetected, resulting in patients receiving suboptimal treatment.

Genotypic characterization of β-lactamase resistant Salmonella species isolated from different quail houses and their susceptibility to extracts of thyme and chitosan, and Saccharomyces Cerevisiae

ABSTRACT This study aimed to identify Salmonella species in two systems for quail raising (deep litter system and cages system). Determination of antibiotic susceptibility, β-lactamase resistance genes profile and susceptibility to chitosan, thyme extract and S. cerevisiae was examined. Fifteen Salmonella isolates were isolated from eighty cloacal swabs examined, with a higher incidence from those of deep litter systems than cage housing systems. Regarding the environmental samples, Salmonella was higher in the deep litter system than in cage housing. Serological identification of 27 salmonella isolates typed them into 8 Salmonella spp. including S. Tsevie, S. Infantis, S. Kentucky, S. Typhimurium, S. Larochelle, S. Molade, S. Enteritidis and S. Labadi. The profile of β-lactamase-resistance genes revealed the presence of blaTEM and blaSHV genes in S. Typhimurium and S. Infantis; blaTEM and blaCTX-M1 genes in S. Enteritidis and S. Molade, while S. Kentucky and S. Tsevie were positive for the blaCTX-M1 gene only. S. Larochelle was negative for all of them. Thyme and chitosan extract showed that S. cerevisiae had the highest inhibitory activity against the tested strains. In conclusion, farms with superior biosecurity and hygiene practices will reduce the risk of pathogens and eliminate the need for prophylactic antibiotics, thereby preventing antibiotic resistance.

Coexistence and genomics characterization of mcr-1 and extended-spectrum-β-lactamase-producing Escherichia coli, an emerging extensively drug-resistant bacteria from sheep in China

The emergence of pathogens harboring multiple resistance genes poses a great threat to global public health. However, the coexistence of mobile resistance genes that provide resistance to both third-generation cephalosporins and colistin in sheep-origin Escherichia coli has not been previously investigated in China. This study is the first to characterize five E. coli isolates from sheep in Shaanxi province that harbor both Extended-Spectrum β-Lactamase (ESBL) and mcr-1 resistance genes. The isolates were identified and characterized by Illumina sequencing, nanopore sequencing, bioinformatic analysis, conjugation experiments, and antimicrobial susceptibility testing. Genetic analysis revealed that blaCTX-M-55 gene, mediated by the IS26, was located on the IncFIB-IncFIC plasmid, while the mcr-1 gene was located on the IncI2(Delta) plasmid. Notably, two copies of blaCTX-M-55 gene were also identified on the chromosome of one isolate (SX45), facilitated by the ISEcp1 insertion sequence. Additionally, the plasmid pSX23–2 was identified as a complex plasmid derived through homologous recombination of pMG337 from E. coli (MK878890) and pZY-1 from Citrobacter freundii (CP055248). Data mining of publicly available databases revealed that isolates carrying both blaCTX-M-55 and mcr-1 genes have been found in humans, animals, and the environment, indicating the widespread presence of these critical resistance genes across different niches. Antimicrobial susceptibility testing showed that the five isolates were resistant to a nearly all tested antibiotics, except meropenem. Conjugative transfer experiments demonstrated that the IncFIB-IncFIC and IncI2(Delta) plasmids carrying mcr-1 and blaCTX-M-55 were capable of transferring between different sequence types (STs) of sheep-origin E. coli, including ST10, ST162, and ST457. This finding suggests the potential for wide dissemination of these resistance markers among diverse E. coli strains. Overall, the characterization of these ESBL and mcr-1 co-harboring isolates enhances our understanding of the spread of these resistance genes in sheep-origin E. coli. Global surveillance of these isolates, particularly within the One Health framework, is essential to monitor and mitigate the risks posed by the dissemination of these resistance genes across various settings.

Drug-resistant Pantoea agglomerans Causing Bacteremia at a Tertiary Care Hospital in Kolkata, India: First Report of Carbapenem Resistance Mediated by OXA-181.

The spread of antibiotic resistance (ABR) in uncommon human pathogens endangers global public health, escalating morbidity, death, and healthcare expenditures. Pantoea agglomerans, a member of the Erwiniaceae family that rarely infects humans, is emerging as a drug-resistant nosocomial pathogen. Seven P. agglomerans isolates were recovered from bacteremia patients at a tertiary care hospital in Kolkata, West Bengal, between March 2022 and October 2022. The isolates were evaluated for phenotypic resistance, β-lactamase and plasmid-mediated quinolone resistance (PMQR) genes, plasmid profiling, and clonality assessment. All isolates were resistant to fluoroquinolones and third-generation cephalosporins, with four resistant to carbapenems. The following β-lactamases and PMQR genes were identified: blaOXA-1 (n = 1), blaTEM (n = 1), blaCTX-M-1 (n = 2), blaNDM (n = 5), blaOXA-181 (n = 1), qnrB (n = 2), and qnrS (n = 4). Six isolates carried up to seven plasmids ranging in size from 2kb to > 212kb. IncFI, FII, HI, and X3 plasmid types were detected in three isolates, while the rest remained untypable. Four different genetic patterns were noted. Four isolates were clonally related, with three being clonal. The swap of environmental isolates to human pathogens exacerbates the ABR dilemma, periling patient care and outcomes. This is the first report in India of a carbapenem-resistant P. agglomerans blood isolate carrying blaOXA-181. In-depth genomic research of drug-resistant microbes adapted to the environment-human interfaces might underpin the source-route-containment of ABR.

Chromosomally located blaCMH in Enterobacter cloacae complex across human-bird-environment interfaces: A one-health perspective

To improve the understanding of antimicrobial resistance (AMR) in migratory birds derived-Enterobacter cloacae (E. cloacae) complex and its spread at the human-bird-environment interface, we isolated 11 strains of third-generation cephalosporin-resistant E. cloacae from 1003 specimens from 29 migratory bird species over two years in Chongming, Shanghai, China. The comprehensive analysis incorporated second- and third-generation sequencing techniques and extensive bioinformatic analysis. Four human-associated E. cloacae sequence types (STs), including ST432, ST412, ST1, and ST639, were found in migratory birds. We confirmed that the blaCMH-4 and blaCMH-6 genes were the major genotypes of the β-lactamase resistance genes in E. cloacae found in migratory birds. In addition, a thorough genomic analysis was performed on a global collection of 398 E. cloacae isolates carrying the blaCMH gene from 46 different countries. China had the highest proportion with 19.10 % (76/398), followed by Singapore with 18.34 % (73/398), Nigeria with 15.83 % (63/398), and the USA with 14.07 % (56/398). The first transmission of E. cloacae carrying blaCMH-4 and blaCMH-6 was defined around 1894 and 1549, respectively. Time-based phylogenetic analysis revealed that host jumps among humans, birds, and the environment led to the emergence of modern strains with ESBL- and carbapenem-resistant genes from about 2004 to 2016. The detection rate of insertion sequences (IS) of E. cloacae carrying blaCMH from human sources is higher than that from migratory bird sources, which is related to the different genetic environments caused by antibiotic selective pressure. It is crucial to have a comprehensive understanding of the characteristics exhibited by blaCMH producing E. cloacae in different ecological environments. Our results contribute to the effective monitoring and implementation of proactive strategies to reduce the spread of multidrug-resistant E. cloacae.

Genomic characterization and cluster analysis of Carbapenem-resistant Klebsiella pneumoniae

To investigate the genomic features and perform cluster analysis of Carbapenem-resistant Klebsiella pneumoniae (CRKP) to provide an experimental basis for guiding the prevention and treatment of CRKP infections.A retrospective case-cohort study was conducted on 19 non-redundant CRKP strains isolated from the Tenth Affiliated Hospital of Southern Medical University between January and June 2023. Whole genome sequencing (WGS) and multilocus sequence typing (MLST) were performed to compare genomic features and analyze the resistance genes and homology of the strains.The results showed that the 19 CRKP strains were isolated from 8 different clinical departments, mainly from respiratory specimens. The whole genome sequencing revealed that the genomic lengths of CRKP ranged from 4.90 to 5.85 Mbp, with contigs N50 values>20 kb for each genome. The median overall GC content was 57.0% (50.4%-57.1%). Comparative genomic analysis identified three regions with high genomic variability. WGS detected 32 resistance genes across 11 categories. All 19 strains carried carbapenem resistance genes (blaKPC-2 and blaOXA-48), blaTEM-1B extended-spectrum β-lactamase resistance genes, qnrS1 quinolone resistance gene, and fosA fosfomycin resistance gene, with each strain carrying only one carbapenemase gene. The detection rate of blaKPC-2 was 94.7% (18/19). MLST identified three sequence types: ST11, ST437 and ST147, with ST11 being predominant (89.5%, 17/19). Clustering analysis based on acquired resistance genes revealed three clonal transmission patterns among strains 72 and 90, and strains 88, 84, 66 and 79.In conclusion, CRKP strains carry multiple resistance genes, and clustering analysis indicating that nosocomial clonal transmission is closely related to acquired resistance genes. The ST11-blaKPC-2 type strain is the predominant clone. Strengthened surveillance and effective control strategies are necessary to reduce nosocomial transmission of CRKP.

Effect of subinhibitory concentrations on the spreading of the ampicillin resistance gene blaCMY-2 in an activated sludge microcosm

ABSTRACT As the problem of multi-resistant bacteria grows a better understanding of the spread of antibiotic resistance genes is of utmost importance for society. Wastewater treatment plants contain subinhibitory concentrations of antibiotics and are thought to be hotspots for antibiotic resistance gene propagation. Here we evaluate the influence of sub-minimum inhibitory concentrations of antibiotics on the spread of resistance genes within the bacterial community in activated sludge laboratory-scale sequencing batch reactors. The mixed communities were fed two different ampicillin concentrations (500 and 5000 µg/L) and the reactors were run and monitored for 30 days. During the experiment the β-lactamase resistance gene bla CMY-2 was monitored via qPCR and DNA samples were taken to monitor the effect of ampicillin on the microbial community. The relative copy number of bla CMY-2 in the reactor fed with the sub-minimum inhibitory concentration of 500 µg/L ampicillin was spread out over a wider range of values than the control and 5000 µg/L ampicillin reactors indicating more variability of gene number in the 500 µg/L reactor. This result emphasises the problem of sub-minimum inhibitory concentrations of antibiotics in wastewater. High-throughput sequencing showed that continuous exposure to ampicillin caused a shift from a Bacteroidetes to Proteobacteria in the bacterial community. The combined use of qPCR and high-throughput sequencing showed that ampicillin stimulates the spread of resistance genes and leads to the propagation of microbial populations which are resistant to it.

Molecular characterization of superbugs K. pneumoniae harboring extended-spectrum β-lactamase (ESBL) and carbapenemase resistance genes among hospitalized patients in southwestern Iran, Western Asia

BackgroundDetection of K. pneumoniae superbugs carrying Extended-spectrum β-lactamase (ESBL) and Carbapenemase resistance genes among hospitalized patients is crucial for infection control and prevention. The aim of this molecular study was to investigate the spread of ESBL and Carbapenemase-producing K. pneumoniae in two hospitals located in Southwest Iran. MethodsOne hundred clinical isolates of K. pneumoniae were randomly collected from two hospitals over a period of five months, from November 2023. The isolates were confirmed using biochemical and genotypic tests. According to the CLSI 2022 guidelines, K. pneumoniae isolates that exhibited resistance to at least one of the three indicator cephalosporins or carbapenems were selected for evaluation of ESBL and carbapenemase production. This was done using a combination disk confirmatory test and the modified carbapenem inactivation method (mCIM). Finally, the presence of ESBLs and carbapenemase resistance encoding genes was assessed using PCR and specific primers. ResultsOut of the 100 isolates, the percentage of antibiotic resistance was cefoxitin (29 %), cefixime (28 %), ceftazidime (26 %), cefotaxime (24 %), cefepime (22 %), ceftriaxone (21 %), imipenem (20 %), and meropenem (17 %). Additionally, thirty isolated strains were found to be multidrug-resistant. Out of these, twenty-seven strains demonstrated a potential for ESBLs, twenty strains for Carbapenemase, and seventeen strains for both ESBLs and Carbapenemase production. Moreover, the occurrence of ESBLs and carbapenemase genes was as follows: blaSHV (25 %), blaTEM (23 %), blaCTX-M (20 %), blaOXA-48 (17 %), and blaVIM (13 %). It is important to mention that we did not detect the blaIMP and blaKPC. resistant genes among clinical isolates. ConclusionBased on the results, the existence of this type of resistance in hospital centers needs to be reevaluated in terms of empirical antibiotic prescribing. Additionally, it is recommended that infection control measures should be taken for public health. Also, it's suggested that hospital-acquired infections caused by superbug K. pneumoniae resistant strains should be addressed.

Performance evaluation of the Streck ARM-DⓇ Kit, β-Lactamase for molecular detection of acquired β-lactamase genes

ObjectivesDespite clinical relevance, commercially available molecular tools for accurate β-lactamase detection are limited. In this study, we evaluated the performance of the ARM-DⓇ Kit, β-Lactamase, a commercially available multiplex PCR assay designed to detect nine β-lactamase genes, including the five major plasmid-mediated carbapenemases, ESBL and AmpC genes circulating in the United States. MethodsA diverse collection of 113 Gram-negative isolates, including 42 with multiple β-lactamases genes, was selected from the U.S. Centers for Disease Control and Prevention (CDC) & Food and Drug Administration (FDA) Antimicrobial Resistance Isolate Bank, to represent the most frequently detected bacterial species carrying plasmid-mediated β-lactam resistance genes. ResultsResults were compared with whole genome sequence data. Of 164 β-lactamase gene targets with 49 unique variants, all were detected correctly without any cross-reactivity. The sensitivity and specificity were 100% (164/164) and 99.9% (852/853), respectively. ConclusionThe ARM-DⓇ Kit, β-Lactamase detected a wide range of β-lactamase genotypes at a low upfront cost. The Streck assay represents a suitable, comprehensive tool for the detection of key β-lactamase resistance genes of public health concern in the United States.

Analysis of the molecular epidemiological characteristics of carbapenem-resistant Klebsiella pneumoniae in a hospital in Hunan Province

To examine the molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae (CRKP) and investigate the horizontal transmission of blaKPC and blaNDM genes for the prevention and treatment of CRKP. A total of 49 clinically isolated CRKP strains were retrospectively analyzed from January to December 2022 at The First Hospital of Hunan University of Chinese Medicine. Phenotypic screening was performed using modified carbapenem inactivation assay (mCIM) and EDTA-carbapenem inactivation assay (eCIM). Polymerase chain reaction (PCR) was utilized to identify carbapenem resistance genes, β-lactamase resistance genes, and virulence genes, while multi-locus sequence analysis (MLST) was employed to assess the homology of CRKP strains. Conjugation experiments were conducted to infer the horizontal transmission mechanism of blaKPC and blaNDM genes. The results showed that the study included 49 CRKP strains, with 44 carrying blaKPC and 8 carrying blaNDM, Three strains were identified as blaKPC+blaNDM-CRKP. In this study, 28 out of 49 CRKP strains (57.2%) were found to carry virulence genes. Additionally, one CRKP strain tested positive in the string test and was found to carry both Aerobactin and rmpA virulence genes. MLST results revealed a total of 5 ST types, with ST11 being predominant (41/49, 83.7%). Successful conjugation was observed in all 3 blaKPC-CRKP strains, while only 1 out of 3 blaNDM-CRKP strains showed successful conjugation. The transconjugant exhibited significantly reduced susceptibility to imipenem and cephalosporin antibiotics. In conclusion, the resistance mechanism of CRKP in this study is primarily attributed to the production of KPC enzymes, along with the presence of multiple β-lactamase resistance genes. Additionally, there is a local prevalence of hv-CRKP and blaKPC+blaNDM-CRKP. blaKPC and blaNDM can be horizontally transmitted through plasmids, with varying efficiency among different strains.

Colistin-, cefepime-, and levofloxacin-resistant Salmonella enterica serovars isolated from Egyptian chicken carcasses

ObjectivesThe emergence of multidrug-resistant (MDR) Salmonella strains, especially resistant ones toward critically important antimicrobial classes such as fluoroquinolones and third- and fourth-generation cephalosporins, is a growing public health concern. The current study, therefore, aimed to determine the prevalence, and existence of virulence genes (invA, stn, and spvC genes), antimicrobial resistance profiles, and the presence of β-lactamase resistance genes (blaOXA, blaCTX-M1, blaSHV, and blaTEM) in Salmonella strains isolated from native chicken carcasses in Egypt marketed in Mansoura, Egypt, as well as spotlight the risk of isolated MDR, colistin-, cefepime-, and levofloxacin-resistant Salmonella enterica serovars to public health.MethodsOne hundred fifty freshly dressed native chicken carcasses were collected from different poultry shops in Mansoura City, Egypt between July 2022 and November 2022. Salmonella isolation was performed using standard bacteriological techniques, including pre-enrichment in buffered peptone water (BPW), selective enrichment in Rappaport Vassiliadis broth (RVS), and cultivating on the surface of xylose-lysine-desoxycholate (XLD) agar. All suspected Salmonella colonies were subjected to biochemical tests, serological identification using slide agglutination test, and Polymerase Chain Reaction (PCR) targeting the invasion A gene (invA; Salmonella marker gene). Afterward, all molecularly verified isolates were screened for the presence of virulence genes (stn and spvC). The antimicrobial susceptibility testing for isolated Salmonella strains towards the 16 antimicrobial agents tested was analyzed by Kirby–Bauer disc diffusion method, except for colistin, in which the minimum inhibition concentration (MIC) was determined by broth microdilution technique. Furthermore, 82 cefotaxime-resistant Salmonella isolates were tested using multiplex PCR targeting the β-lactamase resistance genes, including blaOXA, blaCTX-M1, blaSHV, and blaTEM genes.ResultsSalmonella enterica species were molecularly confirmed via the invA Salmonella marker gene in 18% (27/150) of the freshly dressed native chicken carcasses. Twelve Salmonella serotypes were identified among 129 confirmed Salmonella isolates with the most predominant serotypes were S. Kentucky, S. Enteritidis, S. Typhimurium, and S. Molade with an incidence of 19.4% (25/129), 17.1% (22/129), 17.1% (22/129), and 10.9% (14/129), respectively. All the identified Salmonella isolates (n = 129) were positive for both invA and stn genes, while only 31.8% (41/129) of isolates were positive for the spvC gene. One hundred twenty-one (93.8%) of the 129 Salmonella-verified isolates were resistant to at least three antibiotics. Interestingly, 3.9%, 14.7%, and 75.2% of isolates were categorized into pan-drug-resistant, extensively drug-resistant, and multidrug-resistant, respectively. The average MAR index for the 129 isolates tested was 0.505. Exactly, 82.2%, 82.2%, 63.6%, 51.9%, 50.4%, 48.8%, 11.6%, and 10.1% of isolated Salmonella strains were resistant to cefepime, colistin, cefotaxime, ceftazidime/clavulanic acid, levofloxacin, ciprofloxacin, azithromycin, and meropenem, respectively. Thirty-one out (37.8%) of the 82 cefotaxime-resistant Salmonella isolates were β-lactamase producers with the blaTEM as the most predominant β-lactamase resistance gene, followed by blaCTX-M1 and blaOXA genes, which were detected in 21, 16, and 14 isolates respectively).ConclusionThe high prevalence of MDR-, colistin-, cefepime-, and levofloxacin-resistant Salmonella serovars among Salmonella isolates from native chicken is alarming as these antimicrobials are critically important in treating severe salmonellosis cases and boost the urgent need for controlling antibiotic usage in veterinary and human medicine to protect public health.Graphical

Serovar and sequence type distribution and phenotypic and genotypic antimicrobial resistance of Salmonella originating from pet animals in Chongqing, China.

A total of 334 Salmonella isolates were recovered from 6,223 pet rectal samples collected at 50 pet clinics, 42 pet shops, 7 residential areas, and 4 plazas. Forty serovars were identified that included all strains except for one isolate that did not cluster via self-agglutination, with Salmonella Typhimurium monophasic variant, Salmonella Kentucky, Salmonella Enteritidis, Salmonella Pomona, and Salmonella Give being the predominant serovars. Fifty-one sequence types were identified among the isolates, and ST198, ST11, ST19, ST451, ST34, and ST155 were the most common. The top four dominant antimicrobials to which isolates were resistant were sulfisoxazole, ampicillin, doxycycline, and tetracycline, and 217 isolates exhibited multidrug resistance. The prevalence of β-lactamase genes in Salmonella isolates was 59.6%, and among these isolates, 185 harbored blaTEM, followed by blaCTX-M (66) and blaOXA (10). Moreover, six PMQR genes, namely, including qnrA (4.8%), qnrB (4.2%), qnrD (0.9%), qnrS (18.9%), aac(6')-Ib-cr (16.5%), and oqxB (1.5%), were detected. QRDR mutations (76.6%) were very common in Salmonella isolates, with the most frequent mutation in parC (T57S) (47.3%). Furthermore, we detected six tetracycline resistance genes in 176 isolates, namely, tet(A) (39.5%), tet(B) (8.1%), tet(M) (7.7%), tet(D) (5.4%), tet(J) (3.3%), and tet(C) (1.8%), and three sulfonamide resistance genes in 303 isolates, namely, sul1 (84.4%), sul2 (31.1%), and sul3 (4.2%). Finally, we found 86 isolates simultaneously harboring four types of resistance genes that cotransferred 2-7 resistance genes to recipient bacteria. The frequent occurrence of antimicrobial resistance, particularly in dogs and cats, suggests that antibiotic misuse may be driving multidrug-resistant Salmonella among pets.IMPORTANCEPet-associated human salmonellosis has been reported for many years, and antimicrobial resistance in pet-associated Salmonella has become a serious public health problem and has attracted increasing attention. There are no reports of Salmonella from pets and their antimicrobial resistance in Chongqing, China. In this study, we investigated the prevalence, serovar diversity, sequence types, and antimicrobial resistance of Salmonella strains isolated from pet fecal samples in Chongqing. In addition, β-lactamase, QRDR, PMQR, tetracycline and sulfonamide resistance genes, and mutations in QRDRs in Salmonella isolates were examined. Our findings demonstrated the diversity of serovars and sequence types of Salmonella isolates. The isolates were widely resistant to antimicrobials, notably with a high proportion of multidrug-resistant strains, which highlights the potential direct or indirect transmission of multidrug-resistant Salmonella from pets to humans. Furthermore, resistance genes were widely prevalent in the isolates, and most of the resistance genes were spread horizontally between strains.

β-Lactamase and Macrolide Resistance Gene Carriage in Escherichia coli Isolates Among Children Discharged From Inpatient Care in Western Kenya: A Cross-sectional Study.

Antimicrobial resistance (AMR) is a global threat to infectious disease control, particularly among recently hospitalized children. We sought to determine the prevalence and mitigating factors of resistance in enteric Escherichia coli among children discharged from health facilities in western Kenya. Between June 2016 and November 2019, children aged 1 to 59 months were enrolled at the point of discharge from the hospital. E coli was isolated by microbiological culture from rectal swabs at baseline. β-Lactamases and macrolide resistance-conferring genes were detected by polymerase chain reaction. A modified Poisson regression model was used to assess the predictors mph(A) and CTX-M-type extended-spectrum β-lactamase (ESBL). Of the 238 children whose E coli isolates were tested, 91 (38.2%) and 109 (45.8%) had detectable CTX-M-type ESBL and mph(A) genes, respectively. Antibiotic treatment during hospitalization (adjusted prevalence ratio [aPR], 2.47; 95% CI, 1.12-5.43; P = .025), length of hospitalization (aPR, 1.42; 95% CI, 1.00-2.01; P = .052), and the practice of open defecation (aPR, 2.47; 95% CI, 1.40-4.36; P = .002) were independent predictors for CTX-M-type ESBL and mph(A) genes. Pneumococcal vaccination was associated with a 43% lower likelihood of CTX-M-type ESBL (aPR, 0.57; 95% CI, .38-.85; P = .005), while measles vaccination was associated with a 32% lower likelihood of mph(A) genes (aPR, 0.68; 95% CI, .49-.93; P = .017) in E coli isolates. Among children discharged from the hospital, history of vaccination, shorter hospital stay, lack of in-hospital antibiotic exposure, and improved sanitation were associated with a lower likelihood of AMR genes. To mitigate the continued spread of AMR, AMR control programs should consider strategies beyond antimicrobial stewardship, including improvements in sanitation, increased vaccine coverage, and the development of novel vaccines.

Genomic analysis of multidrug-resistant Escherichia coli from Urban Environmental water sources in Accra, Ghana, Provides Insights into public health implications.

Wastewater discharge into the environment in resource-poor countries poses a threat to public health. Studies in this area within these countries are limited, and the use of high-throughput whole-genome sequencing technologies is lacking. Therefore, understanding of environmental impacts is inadequate. The present study investigated the antibiotic resistance profiles and diversity of beta-lactamases in Escherichia coli strains isolated from environmental water sources in Accra, Ghana. Microbiological analyses were conducted on wastewater samples from three hospitals, a sewage and wastewater treatment plant, and water samples from two urban surface water bodies. Confirmed isolates (N = 57) were selected for phenotypic antibiotic resistance profiles. Multi-drug-resistant isolates (n = 25) were genome sequenced using Illumina MiSeq sequencing technology and screened for sequence types, antibiotic resistance, virulence and beta-lactamase genes, and mobile genetic elements. Isolates were frequently resistant to ampicillin (63%), meropenem (47%), azithromycin (46%), and sulfamethoxazole-trimethoprim (42%). Twenty different sequence types (STs) were identified, including clinically relevant ones such as ST167 and ST21. Five isolates were assigned to novel STs: ST14531 (n = 2), ST14536, ST14537, and ST14538. The isolates belonged to phylogroups A (52%), B1 (44%), and B2 (4%) and carried β-lactamase (TEM-1B, TEM-1C, CTX-M-15, and blaDHA-1) and carbapenemase (OXA-1, OXA-181) resistance genes. Dominant plasmid replicons included Col440I (10.2%) and IncFIB (AP001918) (6.8%). Polluted urban environments in Accra are reservoirs for antibiotic-resistant bacteria, posing a substantial public health risk. The findings underscore the need for targeted public health interventions to mitigate the spread of antibiotic-resistant bacteria and protect public health.

Emergence of multidrug-resistant Bacillus spp. derived from animal feed, food and human diarrhea in South-Eastern Bangladesh

BackgroundAntimicrobial resistance poses a huge risk to human health worldwide, while Bangladesh is confronting the most severe challenge between the food supply and the huge consumption of antibiotics annually. More importantly, probiotics containing Bacillus spp. are claimed to be an alternative to antimicrobial stewardship programs. However, their antibiotic resistance remains elusive. Thus, we employed the antimicrobial susceptibility test and PCR to assess the prevalence of resistance, including multidrug resistance (MDR) and resito-genotyping of isolated Bacillus spp.ResultsThe phenotypic profile showed that Bacillus spp. were 100% sensitive to gentamicin (2 µg/mL), whereas lowered sensitivity to levofloxacin (67.8%, 0.5–1 µg/mL), ciprofloxacin (62.3%, 0.5–1 µg/mL), clindamycin (52.2%, 0.25–0.5 µg/mL), amoxicillin-clavulanic acid (37.6%, 0.06 µg/mL), azithromycin (33.4%, 1–2 µg/mL), tetracycline (25.6%, 2–4 µg/mL), nitrofurantoin (21.1%, 16–32 µg/mL), co-trimoxazole (19.2%, 2 µg/mL), and erythromycin (18.8%, 0.25–0.5 µg/mL). The strains were completely resistant to penicillin, amoxicillin-clavulanic acid, cefixime, ceftriaxone, vancomycin, and co-trimoxazole, and a species-specific trend was seen in both phenotypic and genotypic resistance patterns. Genotypic resistance indicated prevalence of the bla1 (71.5%), tetA (33%), erm1 (27%), blaTEM (13.1%), blaCTX-M-1/blaCTX-M-2 /sul1 (10.1%), blaSHV (9.6%), and qnrS (4.1%) genes. The β-lactamase resistance gene bla1 was found in all penicillin-resistant (MIC ≥ 32 µg/mL) Bacillus spp. One hundred ninety-one isolates (89.6%) were MDR, with 100% from diarrhea, 90.3% from food, and 88.7% from animal feed.ConclusionBased on the MIC value and profile analysis of antibiotic resistance genes, this is the first study that Bacillus spp. antimicrobial susceptibilities have been identified in Bangladesh, and our study will shed light on the adverse effects of feed-borne Bacillus spp. emerging from animal feed to the food chain. A comprehensive investigation is urgently needed by policymakers on tolerance limits and harmful effects in the animal industry.

Antimicrobial resistance profiles of Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae strains isolated from broiler chickens

Globally, the spread of multidrug-resistant Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae from food to humans poses a severe threat to public health. The aim of this study was to assess the co-occurrence of colistin and β-lactamase resistance genes in E. coli, K. pneumoniae, and P. aeruginosa strains isolated from faeces of abattoir broiler chickens. The E. coli, P. aeruginosa and K. pneumoniae isolates were successfully detected from faecal samples by polymerase chain reaction (PCR) at infection rates of 60.7%, 22.5% and 16.7% respectively. The isolates displayed the highest levels of antibiotic resistance (AR) against ampicillin (82.3%) and amoxicillin-clavulanic acid (74.2%) for E. coli, followed by cefoxitin (70.6%) for K. pneumoniae, whilst P. aeruginosa displayed 26.1% antibiotic resistance (AR) against both ampicillin and colistin sulphate. The colistin mcr-1 gene was harboured by 46.8%, 47.1% and 21.7%, E. coli, K. pneumonia and P. aeruginosa isolates respectively. Ten out of 62 (16.1%), 6/17 (35.3%), 4/23 (17.4%) isolates were phenotypically classified as ESBL E. coli, K. pneumoniae, and P. aeruginosa respectively. The ESBL-E. coli isolates respectively possessed blaCTX-M (60%), blaTEM (20%) and blaCTX-M-9 (10%) genes. The ESBL-K. pneumoniae harboured, blaCTX-M (50%), blaOXA (33%), blaCARB (17%), and blaCTX-M-9 (17%) genes respectively, whilst, P. aeruginosa isolates respectively carried blaTEM (75%), blaCTX-M (50%), blaOXA (25%) and blaCARB (25%) genes. Molecular analysis identified the blaCTX-Mβ-lactamase-encoding genes collectively from E. coli, P. aeruginosa, K. pneumoniae isolates. Colistin and β-lactamase genes were present in only 16.7%, 6.9%, and 2.9% of E. coli, K. pneumoniae, and P. aeruginosa isolates, respectively. A total of 17, 7 and 3 isolates for E. coli, K. pneumoniae and P. aeruginosa respectively carried both colistin and β-lactamase antibiotics resistant genes. This is a public health threat that points to a challenge in the treatment of infections caused by these zoonotic bacteria. Data generated from this study will contribute to formulation of new strategies for combating spread of E. coli, K. pneumoniae, and P. aeruginosa isolates as well as prevention of their AR development.

Multi-drug resistant (MDR) Gram-negative pathogenic bacteria isolated from poultry in the Noakhali region of Bangladesh.

Rapidly increasing antibiotic-resistant bacterial strains in Bangladesh's food and farm animals stem from the excessive and inappropriate use of antibiotics. To assess the prevalence of multi-drug resistant (MDR) Gram-negative bacteria in poultry chicks, we sought to isolate and identify strains carrying antimicrobial resistance genes. Isolation and identification involved biochemical tests, 16S rRNA sequencing, and PCR screening of species-specific genes. MDR patterns were evaluated using CLSI guidelines with seventeen antibiotics across twelve classes. Targeted gene sequences were amplified for the detection of Extended-spectrum β-Lactamase (ESBL), carbapenem, tetracycline, sulfonamide, and colistin resistance genes. Common isolates, such as Escherichia coli, Klebsiella pneumoniae, Proteus penneri, and Enterobacter hormaechei, exhibited average Multiple Antimicrobial Resistance (MAR) indices of 0.66, 0.76, 0.8, 0.84, and 0.81, 0.76, 0.84, 0.41 for broiler and layer chicken, respectively. Providencia stuartii and Salmonella enterica, exclusive to broiler samples, had MAR indices of 0.82 and 0.84, respectively. Additional isolates Morganella morganii, Aeromonas spp., and Wohlfahrtiimonas chitiniclastica were found in layers (Average MAR indices: 0.73, 0.71, and 0.91). Notably, M. morganii, E. hormaechei and W. chitiniclastica were identified for the first time in Bangladeshi poultry chicken, although their evolution is yet to be understood. In this study, Pan-drug resistance was observed in one P. stuartii (broiler) and one Aeromonas spp. (layer) with a MAR index 1, while all isolates exhibited MAR indices >0.2, indicating MDR. Antimicrobial resistance (AMR) gene screening identified blaTEM, blaSHV, tetA, and sul1 in a majority of the MDR strains. Interestingly, E. coli (lactose positive and negative) and E. hormaechei were exclusively found to possess the tetB gene. In addition, E. coli (lactose negative), Klebsiella pneumoniae, Enterobacter hormaechei, M. morganii, and P. stuartii were observed to carry the colistin-resistant mcr-1 gene, whereas sul2 was detected in E. coli (lactose positive and negative), E. hormaechei, P. stuartii, and P. penneri. These findings emphasize the health risk of our consumers of both broiler and layer chickens as they have turned into a potent reservoir of various AMR gene carrying MDR and Pan-drug resistant bacteria.