Optical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak. - Related Documents




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492701.0000Optical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak. The global spread of antibiotic resistance among Enterobacteriaceae is largely due to multidrug resistance plasmids that can transfer between different bacterial strains and species. Horizontal gene transfer of resistance plasmids can complicate hospital outbreaks and cause problems in epidemiological tracing, since tracing is usually based on bacterial clonality. We have developed a method, based on optical DNA mapping combined with Cas9-assisted identification of resistance genes, which is used here to characterize plasmids during an extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae outbreak at a Swedish neonatal intensive care unit. The outbreak included 17 neonates initially colonized with ESBL-producing Klebsiella pneumoniae (ESBL-KP), some of which were found to carry additional ESBL-producing Escherichia coli (ESBL-EC) in follow-up samples. We demonstrate that all ESBL-KP isolates contained two plasmids with the bla(CTX-M-15) gene located on the smaller one (~80 kbp). The same ESBL-KP clone was present in follow-up samples for up to 2 years in some patients, and the plasmid carrying the bla(CTX-M-15) gene was stable throughout this time period. However, extensive genetic rearrangements within the second plasmid were observed in the optical DNA maps for several of the ESBL-KP isolates. Optical mapping also demonstrated that even though other bacterial clones and species carrying bla(CTX-M) group 1 genes were found in some neonates, no transfer of resistance plasmids had occurred. The data instead pointed toward unrelated acquisition of ESBL-producing Enterobacteriaceae (EPE). In addition to revealing important information about the specific outbreak, the method presented is a promising tool for surveillance and infection control in clinical settings.IMPORTANCE This study presents how a novel method, based on visualizing single plasmids using sequence-specific fluorescent labeling, could be used to analyze the genetic dynamics of an outbreak of resistant bacteria in a neonatal intensive care unit at a Swedish hospital. Plasmids are a central reason for the rapid global spread of bacterial resistance to antibiotics. In a single experimental procedure, this method replaces many traditional plasmid analysis techniques that together provide limited details and are slow to perform. The method is much faster than long-read whole-genome sequencing and offers direct genetic comparison of patient samples. We could conclude that no transfer of resistance plasmids had occurred between different bacteria during the outbreak and that secondary cases of ESBL-producing Enterobacteriaceae carriage were instead likely due to influx of new strains. We believe that the method offers potential in improving surveillance and infection control of resistant bacteria in hospitals.201931289171
492610.9999Complete Assembly of Escherichia coli Sequence Type 131 Genomes Using Long Reads Demonstrates Antibiotic Resistance Gene Variation within Diverse Plasmid and Chromosomal Contexts. The incidence of infections caused by extraintestinal Escherichia coli (ExPEC) is rising globally, which is a major public health concern. ExPEC strains that are resistant to antimicrobials have been associated with excess mortality, prolonged hospital stays, and higher health care costs. E. coli sequence type 131 (ST131) is a major ExPEC clonal group worldwide, with variable plasmid composition, and has an array of genes enabling antimicrobial resistance (AMR). ST131 isolates frequently encode the AMR genes bla(CTX-M-14), bla(CTX-M-15), and bla(CTX-M-27), which are often rearranged, amplified, and translocated by mobile genetic elements (MGEs). Short DNA reads do not fully resolve the architecture of repetitive elements on plasmids to allow MGE structures encoding bla(CTX-M) genes to be fully determined. Here, we performed long-read sequencing to decipher the genome structures of six E. coli ST131 isolates from six patients. Most long-read assemblies generated entire chromosomes and plasmids as single contigs, in contrast to more fragmented assemblies created with short reads alone. The long-read assemblies highlighted diverse accessory genomes with bla(CTX-M-15), bla(CTX-M-14), and bla(CTX-M-27) genes identified in three, one, and one isolates, respectively. One sample had no bla(CTX-M) gene. Two samples had chromosomal bla(CTX-M-14) and bla(CTX-M-15) genes, and the latter was at three distinct locations, likely transposed by the adjacent MGEs: ISEcp1, IS903B, and Tn2 This study showed that AMR genes exist in multiple different chromosomal and plasmid contexts, even between closely related isolates within a clonal group such as E. coli ST131.IMPORTANCE Drug-resistant bacteria are a major cause of illness worldwide, and a specific subtype called Escherichia coli ST131 causes a significant number of these infections. ST131 bacteria become resistant to treatments by modifying their DNA and by transferring genes among one another via large packages of genes called plasmids, like a game of pass-the-parcel. Tackling infections more effectively requires a better understanding of what plasmids are being exchanged and their exact contents. To achieve this, we applied new high-resolution DNA sequencing technology to six ST131 samples from infected patients and compared the output to that of an existing approach. A combination of methods shows that drug resistance genes on plasmids are highly mobile because they can jump into ST131's chromosomes. We found that the plasmids are very elastic and undergo extensive rearrangements even in closely related samples. This application of DNA sequencing technologies illustrates at a new level the highly dynamic nature of ST131 genomes.201931068432
157220.9999Phenotypic and Genomic Characterization of AmpC-Producing Klebsiella pneumoniae From Korea. The prevalence of multidrug-resistant gram-negative bacteria has continuously increased over the past few years; bacterial strains producing AmpC β-lactamases and/or extended-spectrum β-lactamases (ESBLs) are of particular concern. We combined high-resolution whole genome sequencing and phenotypic data to elucidate the mechanisms of resistance to cephamycin and β-lactamase in Korean Klebsiella pneumoniae strains, in which no AmpC-encoding genes were detected by PCR. We identified several genes that alone or in combination can potentially explain the resistance phenotype. We showed that different mechanisms could explain the resistance phenotype, emphasizing the limitations of the PCR and the importance of distinguishing closely-related gene variants.201829611388
569730.9999In Silico Analysis of Extended-Spectrum β-Lactamases in Bacteria. The growing bacterial resistance to available β-lactam antibiotics is a very serious public health problem, especially due to the production of a wide range of β-lactamases. At present, clinically important bacteria are increasingly acquiring new elements of resistance to carbapenems and polymyxins, including extended-spectrum β-lactamases (ESBLs), carbapenemases and phosphoethanolamine transferases of the MCR type. These bacterial enzymes limit therapeutic options in human and veterinary medicine. It must be emphasized that there is a real risk of losing the ability to treat serious and life-threatening infections. The present study aimed to design specific oligonucleotides for rapid PCR detection of ESBL-encoding genes and in silico analysis of selected ESBL enzymes. A total of 58 primers were designed to detect 49 types of different ESBL genes. After comparing the amino acid sequences of ESBLs (CTX-M, SHV and TEM), phylogenetic trees were created based on the presence of conserved amino acids and homologous motifs. This study indicates that the proposed primers should be able to specifically detect more than 99.8% of all described ESBL enzymes. The results suggest that the in silico tested primers could be used for PCR to detect the presence of ESBL genes in various bacteria, as well as to monitor their spread.202134356733
191940.9998Combining Functional Genomics and Whole-Genome Sequencing to Detect Antibiotic Resistance Genes in Bacterial Strains Co-Occurring Simultaneously in a Brazilian Hospital. (1) Background: The rise of multi-antibiotic resistant bacteria represents an emergent threat to human health. Here, we investigate antibiotic resistance mechanisms in bacteria of several species isolated from an intensive care unit in Brazil. (2) Methods: We used whole-genome analysis to identify antibiotic resistance genes (ARGs) and plasmids in 34 strains of Gram-negative and Gram-positive bacteria, providing the first genomic description of Morganella morganii and Ralstonia mannitolilytica clinical isolates from South America. (3) Results: We identified a high abundance of beta-lactamase genes in resistant organisms, including seven extended-spectrum beta-lactamases (OXA-1, OXA-10, CTX-M-1, KPC, TEM, HYDRO, BLP) shared between organisms from different species. Additionally, we identified several ARG-carrying plasmids indicating the potential for a fast transmission of resistance mechanism between bacterial strains. Furthermore, we uncovered two pairs of (near) identical plasmids exhibiting multi-drug resistance. Finally, since many highly resistant strains carry several different ARGs, we used functional genomics to investigate which of them were indeed functional. In this sense, for three bacterial strains (Escherichia coli, Klebsiella pneumoniae, and M. morganii), we identified six beta-lactamase genes out of 15 predicted in silico as those mainly responsible for the resistance mechanisms observed, corroborating the existence of redundant resistance mechanisms in these organisms. (4) Conclusions: Systematic studies similar to the one presented here should help to prevent outbreaks of novel multidrug-resistant bacteria in healthcare facilities.202133920372
569650.9998Co-introduction of plasmids harbouring the carbapenemase genes, bla(NDM-1) and bla(OXA-232), increases fitness and virulence of bacterial host. BACKGROUND: Bacterial isolates with multiple plasmids harbouring different carbapenemase genes have emerged and been identified repeatedly, despite a general notion that plasmids confer fitness cost in bacterial host. In this study, we investigated the effects of plasmids with carbapenemase genes on the fitness and virulence of bacteria. METHODS: Different plasmids harbouring the carbapenemase genes, bla(NDM-1) and bla(OXA-232), were isolated from a carbapenem-resistant K. pneumoniae strain. Each plasmid was conjugated into the Escherichia coli strain DH5α, and a transconjugant with both plasmids was also obtained by transformation. Their in vitro competitive ability, biofilm formation, serum resistance, survival ability within macrophage and fruit fly, and fly killing ability were evaluated. RESULTS: The transconjugants with a single plasmid showed identical phenotypes to the plasmid-free strain, except that they decreased fly survival after infection. However, significantly increased fitness, virulence and biofilm production were observed consistently for the transconjugant with both plasmids, harbouring bla(NDM-1) and bla(OXA-232). CONCLUSIONS: Our data indicate that bacteria carrying multiple plasmids encoding different carbapenemases may have increased fitness and virulence, emphasizing the need for diverse strategies to combat antimicrobial resistance.202031900177
157960.9998Inverse Association between the Existence of CRISPR/Cas Systems with Antibiotic Resistance, Extended Spectrum β-Lactamase and Carbapenemase Production in Multidrug, Extensive Drug and Pandrug-Resistant Klebsiella pneumoniae. Antimicrobial resistance, with the production of extended-spectrum β-lactamases (ESBL) and carbapenemases, is common in the opportunistic pathogen, Klebsiella pneumoniae. This organism has a genome that can contain clustered regularly interspaced short palindromic repeats (CRISPRs), which operate as a defense mechanism against external invaders such as plasmids and viruses. This study aims to determine the association of the CRISPR/Cas systems with antibiotic resistance in K. pneumoniae isolates from Iraqi patients. A total of 100 K. pneumoniae isolates were collected and characterized according to their susceptibility to different antimicrobial agents. The CRISPR/Cas systems were detected via PCR. The phenotypic detection of ESBLs and carbapenemases was performed. The production of ESBL was detected in 71% of the isolates. Carbapenem-resistance was detected in 15% of the isolates, while only 14% were susceptible to all antimicrobial agents. Furthermore, the bacteria were classified into multidrug (77%), extensively drug-resistant (11.0%) and pandrug-resistant (4.0%). There was an inverse association between the presence of the CRISPR/Cas systems and antibiotic resistance, as resistance was higher in the absence of the CRISPR/Cas system. Multidrug resistance in ESBL-producing and carbapenem-resistant K. pneumoniae occurred more frequently in strains negative for the CRISPR/Cas system. Thus, we conclude that genes for exogenous antibiotic resistance can be acquired in the absence of the CRISPR/Cas modules that can protect the bacteria against acquiring foreign DNA.202337370299
182570.9998Free online genome analyses reveal multiple strains in the beginning of a hospital outbreak of Enterobacter hormaechei carrying bla (OXA-436) carbapenemase gene. Free online tools for bacterial genome analyses are available for local infection surveillance at hospitals. The tools do not require bioinformatic expertise and provide rapid actionable results. Within half a year carbapenemase producing Enterobacter cloacae was reported in clinical samples from three patients who had been hospitalized at the same ward. The aim of this outbreak investigation was to characterize and compare genomes of the isolated bacteria in order to determine molecular evidence of hospital transmission. The three isolates and two isolates reported as susceptible to carbapenems were locally analyzed by whole genome sequencing (WGS). Draft genome assembly, species identification, phylogenetic analyses, typing, resistance gene determination, and plasmid analyses were carried out using free online tools from the Center for Genomic Epidemiology (CGE). Genome analyses identified all three suspected outbreak isolates as E. hormaechei carrying bla (OXA-436) gene. Two of the suspected outbreak isolates were closely related, while one was substantially different from them. Horizontal transfer of plasmid may have taken place in the ward. Detailed knowledge on the genomic composition of bacteria in suspected hospital outbreaks can be obtained by free online tools and may reveal transfer of resistance genes between different strains in addition to dissemination of specific clones.202236003132
570080.9998Gram-negative bacterial colonization in the gut: Isolation, characterization, and identification of resistance mechanisms. BACKGROUND: The gut microbiome is made up of a diverse range of bacteria, especially gram-negative bacteria, and is crucial for human health and illness. There is a great deal of interest in the dynamic interactions between gram-negative bacteria and their host environment, especially considering antibiotic resistance. This work aims to isolate gram-negative bacteria that exist in the gut, identify their species, and use resistance-associated gene analysis to define their resistance mechanisms. METHODS: Samples were collected from all patients who had a stool culture at a tertiary care center in Lebanon. Each type of bacteria that was identified from the stool samples was subjected to critical evaluations, and all discovered strains underwent antimicrobial susceptibility testing. Polymerase chain reaction was used to profile the genes for Carbapenem-resistant Enterobacteriaceae (CRE), Extended-spectrum beta-lactamase (ESBL), and that of Pseudomonas aeruginosa strains. RESULTS: Escherichia coli, Klebsiella species, and Pseudomonas aeruginosa turned out to be the predominant microbiota members. Escherichia coli strains had a high frequency of extended-spectrum beta-lactamase genes, with the most discovered gene being bla CTX-M. Additionally, a considerable percentage of isolates had carbapenemase-resistant Enterobacteriaceae genes, suggesting the rise of multidrug-resistant strains. Multidrug resistance genes, such as bla mexR, bla mexB, and bla mexA, were found in strains of Pseudomonas aeruginosa, highlighting the possible difficulties in treating infections brought on by these bacteria. CONCLUSION: The findings highlight the critical importance of effective surveillance and response measures to maintain the effectiveness of antibiotics considering the introduction of multidrug resistance genes in Pseudomonas aeruginosa and ESBL and CRE genes in Escherichia coli.202439216133
192090.9998Exploring the resistome, virulome, and mobilome of multidrug-resistant Klebsiella pneumoniae isolates: deciphering the molecular basis of carbapenem resistance. BACKGROUND: Klebsiella pneumoniae, a notorious pathogen for causing nosocomial infections has become a major cause of neonatal septicemia, leading to high morbidity and mortality worldwide. This opportunistic bacterium has become highly resistant to antibiotics due to the widespread acquisition of genes encoding a variety of enzymes such as extended-spectrum beta-lactamases (ESBLs) and carbapenemases. We collected Klebsiella pneumoniae isolates from a local tertiary care hospital from February 2019-February 2021. To gain molecular insight into the resistome, virulome, and genetic environment of significant genes of multidrug-resistant K. pneumoniae isolates, we performed the short-read whole-genome sequencing of 10 K. pneumoniae isolates recovered from adult patients, neonates, and hospital tap water samples. RESULTS: The draft genomes of the isolates varied in size, ranging from 5.48 to 5.96 Mbp suggesting the genome plasticity of this pathogen. Various genes conferring resistance to different classes of antibiotics e.g., aminoglycosides, quinolones, sulfonamides, tetracycline, and trimethoprim were identified in all sequenced isolates. The highest resistance was observed towards carbapenems, which has been putatively linked to the presence of both class B and class D carbapenemases, bla(NDM,) and bla(OXA), respectively. Moreover, the biocide resistance gene qacEdelta1 was found in 6/10 of the sequenced strains. The sequenced isolates exhibited a broad range of sequence types and capsular types. The significant antibiotic resistance genes (ARGs) were bracketed by a variety of mobile genetic elements (MGEs). Various spontaneous mutations in genes other than the acquired antibiotic-resistance genes were observed, which play an indirect role in making these bugs resistant to antibiotics. Loss or deficiency of outer membrane porins, combined with ESBL production, played a significant role in carbapenem resistance in our sequenced isolates. Phylogenetic analysis revealed that the study isolates exhibited evolutionary relationships with strains from China, India, and the USA suggesting a shared evolutionary history and potential dissemination of similar genes amongst the isolates of different origins. CONCLUSIONS: This study provides valuable insight into the presence of multiple mechanisms of carbapenem resistance in K. pneumoniae strains including the acquisition of multiple antibiotic-resistance genes through mobile genetic elements. Identification of rich mobilome yielded insightful information regarding the crucial role of insertion sequences, transposons, and integrons in shaping the genome of bacteria for the transmission of various resistance-associated genes. Multi-drug resistant isolates that had the fewest resistance genes exhibited a significant number of mutations. K. pneumoniae isolate from water source displayed comparable antibiotic resistance determinants to clinical isolates and the highest number of virulence-associated genes suggesting the possible interplay of ARGs amongst bacteria from different sources.202438664636
1901100.9998Discerning the dissemination mechanisms of antibiotic resistance genes through whole genome sequencing of extended-spectrum beta-lactamase (ESBL)-producing E. coli isolated from veterinary clinics and farms in South Korea. Extended-spectrum beta-lactamase (ESBL)-producing bacteria are resistant to most beta-lactams, including third-generation cephalosporins, limiting the treatment methods against the infections they cause. In this study, we performed whole genome sequencing of ESBL-producing E. coli to determine the mechanisms underlying the dissemination of antibiotic resistance genes. We analyzed 141 ESBL-producing isolates which had been collected from 16 veterinary clinics and 16 farms in South Korea. Long- and short-read sequencing platforms were used to obtain high-quality assemblies. The results showed that bla(CTX-M) is the dominant ESBL gene type found in South Korea. The spread of bla(CTX-M) appears to have been facilitated by both clonal spread between different host species and conjugation. Most bla(CTX-M) genes were found associated with diverse mobile genetic elements that may contribute to the chromosomal integration of the genes. Diverse incompatibility groups of bla(CTX-M)-harboring plasmids were also observed, which allows their spread among a variety of bacteria. Comprehensive whole genome sequence analysis was useful for the identification of the most prevalent types of ESBL genes and their dissemination mechanisms. The results of this study suggest that the propagation of ESBL genes can occur through clonal spread and plasmid-mediated dissemination, and that suitable action plans should be developed to prevent further propagation of these genes.202438554973
1573110.9998Genomic Analysis of a Pan-Resistant Isolate of Klebsiella pneumoniae, United States 2016. Antimicrobial resistance is a threat to public health globally and leads to an estimated 23,000 deaths annually in the United States alone. Here, we report the genomic characterization of an unusual Klebsiella pneumoniae, nonsusceptible to all 26 antibiotics tested, that was isolated from a U.S. PATIENT: The isolate harbored four known beta-lactamase genes, including plasmid-mediated bla(NDM-1) and bla(CMY-6), as well as chromosomal bla(CTX-M-15) and bla(SHV-28), which accounted for resistance to all beta-lactams tested. In addition, sequence analysis identified mechanisms that could explain all other reported nonsusceptibility results, including nonsusceptibility to colistin, tigecycline, and chloramphenicol. Two plasmids, IncA/C2 and IncFIB, were closely related to mobile elements described previously and isolated from Gram-negative bacteria from China, Nepal, India, the United States, and Kenya, suggesting possible origins of the isolate and plasmids. This is one of the first K. pneumoniae isolates in the United States to have been reported to the Centers for Disease Control and Prevention (CDC) as nonsusceptible to all drugs tested, including all beta-lactams, colistin, and tigecycline.IMPORTANCE Antimicrobial resistance is a major public health threat worldwide. Bacteria that are nonsusceptible or resistant to all antimicrobials available are of major concern to patients and the public because of lack of treatment options and potential for spread. A Klebsiella pneumoniae strain that was nonsusceptible to all tested antibiotics was isolated from a U.S. PATIENT: Mechanisms that could explain all observed phenotypic antimicrobial resistance phenotypes, including resistance to colistin and beta-lactams, were identified through whole-genome sequencing. The large variety of resistance determinants identified demonstrates the usefulness of whole-genome sequencing for detecting these genes in an outbreak response. Sequencing of isolates with rare and unusual phenotypes can provide information on how these extremely resistant isolates develop, including whether resistance is acquired on mobile elements or accumulated through chromosomal mutations. Moreover, this provides further insight into not only detecting these highly resistant organisms but also preventing their spread.201829615503
1867120.9998Plasmid diversity of Serratia marcescens and Klebsiella pneumoniae isolates involved in two carbapenem-resistant Enterobacteriaceae outbreaks in a Swiss hospital. This study investigates two distinct carbapenemase-producing Enterobacteriaceae outbreaks involving patients and contaminated sink traps at the University Hospital of Lausanne. It focuses on the diversity and transmission dynamics of plasmids carrying carbapenemase genes. Between 2022 and 2023, 57 carbapenem-resistant Klebsiella pneumoniae and Serratia marcescens isolates were collected and analyzed. Core-genome MLST confirmed genetic similarity among isolates, linking the outbreaks to sink trap contamination. DNA extraction, sequencing (MinION/Illumina MiSeq), and assembly were performed, followed by ARG screening and plasmid typing. Plasmids were annotated, clustered, and compared using core SNP distances and structural analyses. Known plasmids were identified through PLSDB database matching. Eight MLST types were identified in K. pneumoniae and one (ST356) in S. marcescens. Analysis of 52 bla-carrying plasmids revealed 22 plasmid clusters, including 6 bla(NDM-1) clusters in K. pneumoniae and 4 bla(KPC-2) clusters in S. marcescens. Plasmids showed close relatedness within and across patient and environmental isolates, with core SNP distances ranging from 0 to 18. Some bla(NDM-1) plasmids in K. pneumoniae clustered tightly, suggesting persistence and potential cross-contamination routes. The findings highlight sink traps as critical reservoirs for carbapenem-resistant Enterobacteriaceae and plasmids, promoting resistance gene spread across species. The observed plasmid diversity indicates transmission can occur independently of bacterial clonal spread, challenging traditional outbreak definitions. IMPORTANCE: This research is critical in addressing the growing threat of antibiotic resistance, driven by the spread of resistance genes through plasmids. Plasmids, which can transfer between different bacteria, play a major role in spreading multidrug resistance, posing a serious challenge to healthcare systems worldwide. By highlighting how plasmids can move independently of bacterial spread, this study reveals the complexity of resistance transmission. It also underscores the importance of environmental reservoirs, such as hospital sink traps, in harboring and spreading resistant bacteria. These findings emphasize the need for better monitoring of plasmids and targeted infection control measures to prevent the spread of resistance genes and protect the effectiveness of current antibiotics.202540396774
1902130.9998Large-scale analysis of putative plasmids in clinical multidrug-resistant Escherichia coli isolates from Vietnamese patients. INTRODUCTION: In the past decades, extended-spectrum beta-lactamase (ESBL)-producing and carbapenem-resistant (CR) Escherichia coli isolates have been detected in Vietnamese hospitals. The transfer of antimicrobial resistance (AMR) genes carried on plasmids is mainly responsible for the emergence of multidrug-resistant E. coli strains and the spread of AMR genes through horizontal gene transfer. Therefore, it is important to thoroughly study the characteristics of AMR gene-harboring plasmids in clinical multidrug-resistant bacterial isolates. METHODS: The profiles of plasmid assemblies were determined by analyzing previously published whole-genome sequencing data of 751 multidrug-resistant E. coli isolates from Vietnamese hospitals in order to identify the risk of AMR gene horizontal transfer and dissemination. RESULTS: The number of putative plasmids in isolates was independent of the sequencing coverage. These putative plasmids originated from various bacterial species, but mostly from the Escherichia genus, particularly E. coli species. Many different AMR genes were detected in plasmid contigs of the studied isolates, and their number was higher in CR isolates than in ESBL-producing isolates. Similarly, the bla(KPC-2), bla(NDM-5), bla(OXA-1), bla(OXA-48), and bla(OXA-181) β-lactamase genes, associated with resistance to carbapenems, were more frequent in CR strains. Sequence similarity network and genome annotation analyses revealed high conservation of the β-lactamase gene clusters in plasmid contigs that carried the same AMR genes. DISCUSSION: Our study provides evidence of horizontal gene transfer in multidrug-resistant E. coli isolates via conjugative plasmids, thus rapidly accelerating the emergence of resistant bacteria. Besides reducing antibiotic misuse, prevention of plasmid transmission also is essential to limit antibiotic resistance.202337323902
1578140.9998Association of CRISPR/Cas System with the Drug Resistance in Klebsiella pneumoniae. BACKGROUND: Klebsiella pneumoniae is a common opportunistic pathogen and its production of extended-spectrum β-lactamases (ESBL) and carbapenemases leads to drug resistance. Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated genes (Cas) are widespread in the genome of many bacteria and are a defense mechanism against foreign invaders such as plasmids and viruses. PURPOSE: To investigate the prevalence of the CRISPR/Cas system in wild type strains of K. pneumoniae in the hospital and its association with drug resistance. MATERIALS AND METHODS: A total of 136 strains were collected and characterized their susceptibility to antimicrobial agents. The prevalence of CRISPR/Cas system was detected by PCR and DNA sequencing was analyzed by CRISPRFinder. The statistical analysis of the results was performed by SPSS. RESULTS: We found that 50/136 (37%) isolates produced ESBL and 30/136 (22%) isolates were resistant to carbapenems. These isolates were liable to be multidrug resistant against β-lactams, quinolones, and aminoglycosides. Among the carbapenem-resistant isolates, blaKPC was the main drug resistance-associated gene and different types of ESBL and AmpC genes were present. Resistance to β-lactams, quinolones, aminoglycosides, tetracyclines, and β-lactams/enzyme inhibitor were higher in absence of the CRISPR/Cas system. Eighteen spacers within the CRISPR arrays matched with the genomes of plasmids or phages, some of which carried drug resistance genes. CONCLUSION: ESBL-producing and carbapenem-resistant K. pneumoniae are more likely to develop multidrug resistance and show an inverse correlation between drug resistance and CRISPR/Cas system. Absence of CRISPR/Cas modules allow for the acquisition of external drug resistance genes.202032606841
1896150.9998Difference analysis and characteristics of incompatibility group plasmid replicons in gram-negative bacteria with different antimicrobial phenotypes in Henan, China. BACKGROUND: Multi-drug-resistant organisms (MDROs) in gram-negative bacteria have caused a global epidemic, especially the bacterial resistance to carbapenem agents. Plasmid is the common vehicle for carrying antimicrobial resistance genes (ARGs), and the transmission of plasmids is also one of the important reasons for the emergence of MDROs. Different incompatibility group plasmid replicons are highly correlated with the acquisition, dissemination, and evolution of resistance genes. Based on this, the study aims to identify relevant characteristics of various plasmids and provide a theoretical foundation for clinical anti-infection treatment. METHODS: 330 gram-negative strains with different antimicrobial phenotypes from a tertiary hospital in Henan Province were included in this study to clarify the difference in incompatibility group plasmid replicons. Additionally, we combined the information from the PLSDB database to elaborate on the potential association between different plasmid replicons and ARGs. The VITEK mass spectrometer was used for species identification, and the VITEK-compact 2 automatic microbial system was used for the antimicrobial susceptibility test (AST). PCR-based replicon typing (PBRT) detected the plasmid profiles, and thirty-three different plasmid replicons were determined. All the carbapenem-resistant organisms (CROs) were tested for the carbapenemase genes. RESULTS: 21 plasmid replicon types were detected in this experiment, with the highest prevalence of IncFII, IncFIB, IncR, and IncFIA. Notably, the detection rate of IncX3 plasmids in CROs is higher, which is different in strains with other antimicrobial phenotypes. The number of plasmid replicons they carried increased with the strain resistance increase. Enterobacterales took a higher number of plasmid replicons than other gram-negative bacteria. The same strain tends to have more than one plasmid replicon type. IncF-type plasmids tend to be associated with MDROs. Combined with PLSDB database analysis, IncFII and IncX3 are critical platforms for taking bla(KPC-2) and bla(NDM). CONCLUSIONS: MDROs tend to carry more complex plasmid replicons compared with non-MDROs. The plasmid replicons that are predominantly prevalent and associated with ARGs differ in various species. The wide distribution of IncF-type plasmids and their close association with MDROs should deserve our attention. Further investigation into the critical role of plasmids in the carriage, evolution, and transmission of ARGs is needed.202438373913
1918160.9998Molecular Detection of Class 1 Integron-Associated Gene Cassettes in KPC-2-Producing Klebsiella pneumoniae Clones by Whole-Genome Sequencing. The dissemination of antimicrobial resistance genes and the bacterium that harbor them have increasingly become a public concern, especially in low- and middle-income countries. The present study used whole-genome sequencing to analyze 10 KPC-2-producing Klebsiella pneumoniae isolates obtained from clinical specimens originated from Brazilian hospitals. The study documents a relevant "snapshot" of the presence of class 1 integrons in 90% of the strains presenting different gene cassettes (dfrA30, dfrA15, dfrA12, dfrA14, aadA1, aadA2, and aac(6')Iq), associated or not with transposons. Two strains presented nonclassical integron (lacking the normal 3'conserved segment). In general, most strains showed a complex resistome, characterizing them as highly resistant. Integrons, a genetically stable and efficient system, confer to bacteria as highly adaptive and low cost evolution potential to bacteria, even more serious when associated with high-risk clones, indicating an urgent need for control and prevention strategies to avoid the spread of resistance determinants in Brazil. Despite this, although the class 1 integron identified in the KPC-2-producing K. pneumoniae clones is important, our findings suggest that other elements probably have a greater impact on the spread of antimicrobial resistance, since many of these important genes were not related to this cassette.201931074706
1916170.9998Species Diversity of Environmental GIM-1-Producing Bacteria Collected during a Long-Term Outbreak. Reports of outbreaks concerning carbapenemase-producing Gram-negative bacteria in which the main source of transmission is the hospital environment are increasing. This study describes the results of environmental sampling in a protracted polyspecies metallo-beta-lactamase GIM-1 outbreak driven by plasmids and bacterial clones of Enterobacter cloacae and Pseudomonas aeruginosa in a tertiary care center. Environmental sampling targeting wet locations (especially sinks) was carried out on a surgical intensive care unit and on a medical ward on several occasions in 2012 and 2013. We were able to demonstrate 43 blaGIM-1-carrying bacteria (mainly nonfermenters but also Enterobacteriaceae) that were either related or unrelated to clinical strains in 30 sinks and one hair washbasin. GIM-1 was found in 12 different species, some of which are described here as carriers of GIM-1. Forty out of 43 bacteria displayed resistance to carbapenems and, in addition, to various non-beta-lactam antibiotics. Colistin resistance was observed in two E. cloacae isolates with MICs above 256 mg/liter. The blaGIM-1 gene was harbored in 12 different class 1 integrons, some without the typical 3' end. The blaGIM-1 gene was localized on plasmids in five isolates. In vitro plasmid transfer by conjugation was successful in one isolate. The environment, with putatively multispecies biofilms, seems to be an important biological niche for multidrug-resistant bacteria and resistance genes. Biofilms may serve as a "melting pot" for horizontal gene transfer, for dissemination into new species, and as a reservoir to propagate future hospital outbreaks. IMPORTANCE: In Gram-negative bacteria, resistance to the clinically relevant broad-spectrum carbapenem antibiotics is a major public health concern. Major reservoirs for these resistant organisms are not only the gastrointestinal tracts of animals and humans but also the (hospital) environment. Due to the difficulty in eradicating biofilm formation in the latter, a sustained dissemination of multidrug-resistant bacteria from the environment can occur. In addition, horizontal transfer of resistance genes on mobile genetic elements within biofilms adds to the total "resistance gene pool" in the environment. To gain insight into the transmission pathways of a rare and locally restricted carbapenemases resistance gene (blaGIM-1), we analyzed the genetic background of the blaGIM-1 gene in environmental bacteria during a long-term polyspecies outbreak in a German hospital.201627060121
4938180.9998Optical maps of plasmids as a proxy for clonal spread of MDR bacteria: a case study of an outbreak in a rural Ethiopian hospital. OBJECTIVES: MDR bacteria have become a prevailing health threat worldwide. We here aimed to use optical DNA mapping (ODM) as a rapid method to trace nosocomial spread of bacterial clones and gene elements. We believe that this method has the potential to be a tool of pivotal importance for MDR control. METHODS: Twenty-four Escherichia coli samples of ST410 from three different wards were collected at an Ethiopian hospital and their plasmids were analysed by ODM. Plasmids were specifically digested with Cas9 targeting the antibiotic resistance genes, stained by competitive binding and confined in nanochannels for imaging. The resulting intensity profiles (barcodes) for each plasmid were compared to identify potential clonal spread of resistant bacteria. RESULTS: ODM demonstrated that a large fraction of the patients carried bacteria with a plasmid of the same origin, carrying the ESBL gene blaCTX-M-15, suggesting clonal spread. The results correlate perfectly with core genome (cg)MLST data, where bacteria with the same plasmid also had very similar cgMLST profiles. CONCLUSIONS: ODM is a rapid discriminatory method for identifying plasmids and antibiotic resistance genes. Long-range deletions/insertions, which are challenging for short-read next-generation sequencing, can be easily identified and used to trace bacterial clonal spread. We propose that plasmid typing can be a useful tool to identify clonal spread of MDR bacteria. Furthermore, the simplicity of the method enables possible future application in low- and middle-income countries.202032653928
4956190.9998Rapid Identification of Plasmid Replicon Type and Coexisting Plasmid-Borne Antimicrobial Resistance Genes by S1-Pulsed-Field Gel Electrophoresis-Droplet Digital Polymerase Chain Reaction. Bacterial drug resistance is a significant food safety problem and public health threat. Plasmids carrying drug resistance genes may result in the rapid spread of resistance among different bacteria, hosts, and environments; therefore, antibiotic resistance monitoring and continuing research into the mechanisms of drug resistance are urgently needed. Southern blotting with probes for antibiotic resistance genes and even next-generation sequencing have been used previously to detect plasmid-borne resistance genes, but these approaches are complex and time-consuming. The next-generation sequencing requires strict laboratory conditions and bioinformatics analysis ability. In this study, we developed a simplified and sensitive method to detect plasmid-borne antimicrobial resistance genes and plasmid replicon types. Salmonella strains carrying plasmids of three different replicon types that contained mcr-1 and two ESBL-producing genes were used to verify the new method. The plasmids harbored by the Salmonella strains were separated by S1 nuclease treatment and pulsed-field gel electrophoresis (PFGE), then recovered and used as the templates for droplet digital polymerase chain reaction (ddPCR) to identify target genes. The target genes were present in significantly higher copy numbers on the plasmids than the background noise. These results were consistent with the plasmid sequencing results. This S1-PFGE-ddPCR method was less time-consuming to perform than Southern blot and complete plasmid sequencing. Therefore, this method represents a time-saving alternative for detecting plasmid-borne genes, and is likely to be a valuable tool for detecting coexisting plasmid-borne drug resistance genes.202133661029