Plasmid Replicon Research Articles

Genomic characterization of a carbapenem-resistant Citrobacter freundii clinical isolate from China carrying blaNDM-5 on a novel IncC-IncFIB-IncX3 plasmid

Citrobacter freundii is one of the important pathogens that can cause nosocomial infections. The advent of carbapenem-resistant C. freundii complicates clinical treatment. Here, we reported the genome sequence of a carbapenem-resistant C. freundii strain carrying a novel IncC-IncFIB-IncX3 plasmid in China. The genome sequence of C. freundii CRNMS1 was obtained using the Illumina NovaSeq 6000 platform and the long-read Nanopore sequencer. Multilocus sequence typing was identified using MLST (v.2.23.0). The identification of antimicrobial resistance genes (ARGs) and plasmid replicons was performed using the resfinder and plasmidfinder of ABRicate (v.1.0.1). Circular comparisons of plasmids were performed using the BLAST Ring Image Generator (BRIG). CRNMS1 belongs to ST116 in the C. freundii MLST scheme. Thirteen ARGs were predicted in all, including blaNDM-5, which was located in a plasmid. The plasmid pblaNDM5-S1, which carried the blaNDM-5 gene, was discovered to be a novel plasmid including three plasmid replicons (IncC, IncFIB, and IncX3) as well as seven ARGs (sul1, sul2, floR, dfrA17, aadA5, qnrA1, and blaNDM-5). A total of 38 blaNDM-5-bearing C. freundii strains can be retrieved from the NCBI database. Phylogenetic analysis revealed a worldwide distribution of C. freundii strains carrying the blaNDM-5 gene, with China having the highest prevalence (39%, 15/38). However, they were distantly related to CRNMS1 with SNP differences >2545. In summary, we reported a novel IncC-IncFIB-IncX3 plasmid carrying blaNDM-5 in a carbapenem-resistant C. freundii strain in China. The development of such hybrid plasmids facilitates the transmission of ARGs.

Antimicrobial resistance and genomic characteristics of Salmonella from broilers in Shandong Province

IntroductionThe emergence of multidrug-resistant (MDR) strains of Salmonella, which is a genus of important zoonotic pathogens, has aroused great public health concern worldwide.MethodsIn this study, 167 strains of Salmonella were isolated from 947 samples from broiler farms, slaughterhouses, and markets in Shandong Province. Antibiotic sensitivity testing was performed, and 70 strains of Salmonella were screened out by whole-genome sequencing (WGS) to evaluate serotypes, antimicrobial resistance genes (ARGs), the prevalence of class 1 integrons, the plasmid carriage rate, and phylogenetic characteristics and for multilocus sequence typing (MLST).ResultsThe results showed that the 167 isolates showed the highest resistance to ampicillin (AMP, 87.4%), sulfamethoxazole (SF, 87.4%), compound sulfamethoxazole (SXT, 81.4%), nalidixic acid (NAL, 80.2%), and amoxicillin/clavulanic acid (A/C, 77.8%). All the strains were sensitive to meropenem (MEM), and 91.0% of the isolates were MDR strains. We screened a total of 45 ARGs, with the highest detection rate observed for the tetracycline (TET) resistance gene tet (A) (81.4%). A total of 21 types of plasmid replicons were detected in Salmonella, of which IncX1 was the most common (74.3%), and 62.9% of the isolates carried a class 1 integron. In addition, a total of 11 different serotypes were detected, with S. enteritidis as the predominant serovar., followed by S. infantis and S. Newport. Twelve different sequence types (STs) were detected, among which ST11 was the main type. There was a strong correspondence between serotypes and STs. We also found that S. Indiana and S. Kentucky had extremely high rates of resistance to ciprofloxacin (CIP) and third-generation cephalosporins. System-wide genome analysis showed the occurrence of long-distance transmission across fields.ConclusionIn conclusion, the detection of multidrug resistance and isolates carrying multidrug resistance genes is the main problem, and emergency strategies should be implemented to address this issue.

Serotype Distribution, Antimicrobial Resistance, Virulence Genes, and Genetic Diversity of Salmonella spp. Isolated from small-scale Leafy Green Vegetable Supply Chains in South Africa

Salmonella have been implicated in foodborne disease outbreaks globally and is a pressing concern in the South African small-scale sector due to inadequate hygiene standards and limited regulatory oversight, leading to a higher risk of foodborne diseases. By investigating irrigation water and leafy green vegetables produced by small-scale growers and sold through unregulated supply chains, this study was able to determine the presence, serotype distribution, virulence gene profiles, antibiotic resistance, and genetic diversity of Salmonella isolated from these sources. From 426 samples, 21 Salmonella-positive samples were identified, providing 53 Salmonella isolates. Of these, six different Salmonella serotypes and sequence types (STs) were identified, including Salmonella II 42:r: ST1208 (33.96%; n = 18), Salmonella Enteritidis: ST11 (22.64%; n = 12), Salmonella II 42:z29: ST4395 (16.98%; n = 9), Salmonella Havana: ST1524 (15.09%; n = 8), Salmonella Typhimurium: ST19 (9.43%; n = 5), and Salmonella IIIb 47:i:z: ST7890 (1.89%; n = 1). A total of 92.45% of the isolates were found to be multidrug-resistant, showing high rates of resistance to aztreonam (88.68%; n = 47), ceftazidime (86.79%; n = 46), nalidixic acid (77.36%; n = 41), cefotaxime (75.47%; n = 40), cefepime (71.70%; n = 38), and streptomycin (69.81%; n = 37). All isolates possessed the aac(6')-Iaa antimicrobial resistance gene, with a range of between 9 and 256 virulence genes. Eleven cluster patterns were observed from Enterobacterial Repetitive Intergenic Consensus sequence analyses, demonstrating high diversity among the Salmonella spp., with water and fresh produce isolates clustering, suggesting water as a potential contamination source. Plasmid replicon types were identified in 41.51% (n = 22) of the isolates, including Col(pHAD28) in Salmonella Havana (5.66%; n = 3), Col156 in Salmonella II 42:z29:- (1.89%; n = 1) and both IncFIB(S) and IncFII(S) in Salmonella Enteritidis (22.64; n = 12), Salmonella Typhimurium (9.43%; n = 5), and Salmonella Havana (1.89%; n = 1). This study highlights the presence of multidrug-resistant and multivirulent Salmonella spp. in the small-scale leafy green vegetable supply chains, underscoring the need for the development of a “fit-for-purpose” food safety management system within this system.

Genetic and functional enrichments associated with Enterococcus faecalis isolated from the urinary tract.

Urinary tract infection (UTI) is a global health issue that imposes a substantial burden on healthcare systems. Women are disproportionately affected by UTI, with >60% of women experiencing at least one UTI in their lifetime. UTIs can recur, particularly in postmenopausal women, leading to diminished quality of life and potentially life-threatening complications. Understanding how pathogens colonize and survive in the urinary tract is necessary to identify new therapeutic targets that are urgently needed due to rising rates of antimicrobial resistance. How Enterococcus faecalis, a bacterium commonly associated with UTI, adapts to the urinary tract remains understudied. Here, we generated a collection of high-quality closed genome assemblies of clinical urinary E. faecalis isolated from the urine of postmenopausal women that we used alongside detailed clinical metadata to perform a robust comparative genomic investigation of genetic factors that may be involved in E. faecalis survival in the urinary tract.

Prevalence and Transmission of Extended-Spectrum Cephalosporin (ESC) Resistance Genes in Escherichia coli Isolated from Poultry Production Systems and Slaughterhouses in Denmark.

The emergence of extended-spectrum cephalosporin (ESC)-resistant Escherichia coli is a global concern. This study aimed to assess the prevalence and transmission of ESC-resistant E. coli in the Danish broiler production system. Samples from two vertically integrated Production Systems (1 and 2) and two slaughterhouses (A and B) were analyzed (n = 943) for the occurrence of ESC-resistant E. coli from 2015 to 2018. ESC-resistant E. coli isolates were whole-genome sequenced (WGS) for characterization of the multi-locus sequence type (MLST), antibiotic resistance genes, virulence genes, and plasmid replicon types. An ad hoc core genome (cg) MLST based on 2513 alleles was used to examine the genetic relatedness among isolates. The prevalence of ESC-resistant E. coli in the conventional Production System 1 was 2.7%, while in Production System 2 the prevalence was 26.7% and 56.5% for samples from the conventional and organic production, respectively. The overall prevalence of ESC-resistant E. coli in broiler thigh and fecal samples ranged from 19.3% in Slaughterhouse A to 22.4% in Slaughterhouse B. In total, 162 ESC-resistant E. coli were isolated and shown to belong to 16 different sequence types (STs). The most prevalent STs were ST2040 (n = 85) and ST429 (n = 22). Seven ESC resistance genes were detected: blaCMY-2 (n = 119), blaTEM-52B (n = 16), blaCTX-M-1 (n = 5), blaTEM-52C (n = 3), blaCTX-M-14 (n = 1), blaSHV-12 (n = 1), and up-regulation of ampC (n = 16), with an unknown resistance gene in one isolate (n = 1). The carriage of blaCMY-2 in 119 isolates was primarily associated with IncI1 (n = 87), and IncK plasmids (n = 31). Highly similar blaCMY-2 carrying E. coli isolates from ST429 were found in production systems as well as in slaughterhouses. In conclusion, findings from this study indicate that ESC-resistant E. coli are transferred vertically from farms in the production systems to slaughterhouses with the potential to enter the food supply.

Prevalence, antibiotic resistance, virulence genes and molecular characteristics of Salmonella isolated from ducks and wild geese in China

Salmonella is a major foodborne pathogen and the cause of significant morbidity and mortality via consumption of contaminated meat and meat-products. The prevalence of Salmonella in ducks and wild geese in China are poorly characterized and these sources represent a potential pool that could be transferred to farm-reared fowl. In this study, we isolated 335 (18.3%) Salmonella from 1830 samples and identified 24 serotypes and most prevalent were Salmonella Indiana, Salmonella Kentucky and Salmonella Typhimurium. Whole genome sequencing revealed the presence of the dominant sequence types ST17, ST198 and ST19 for these three serotypes, respectively. In addition, these isolates were most likely clonally spread across different regions while S. Kentucky also crossed the species barrier. The majority of the Salmonella isolates possessed β-lactam and fluoroquinolone resistance and these were consistent with antibiotic resistance gene profiles. We also identified 8 plasmid replicon types and all isolates possessed virulence genes and the numbers were greatest for S. Enteritidis and S. Typhimurium isolates. This study provides novel insights concerning the epidemiology of Salmonella in ducks and wild geese and provides basic data for public health screening and management.

Molecular characterization of IncFII plasmid carrying blaNDM-5 in a Salmonella enterica serovar Typhimurium ST34 clinical isolate in China.

In this study, an IncFII plasmid pIncFII-NDM5 carrying blaNDM-5 was found in carbapenem-resistant Salmonella enterica serovar Typhimurium (S. enterica serovar Typhimurium), which has conjugative transferability and carried blaNDM-5, bleMBL, mph(A), and blaTEM-1 four resistance genes that can mediate resistance to multiple antibiotics including cephalosporins, beta-lactamase inhibitor combinations, carbapenems, and macrolides. Phylogenetic analysis showed that 1104-65 and 1104-75 were closely related to other S. enterica serovar Typhimurium in this area. The above-mentioned S. enterica serovar Typhimurium chromosome carries blaCTX-M-55, qnrS1, and tet(A) genes, so the antibiotic resistance of isolates will be further enhanced after obtaining the pIncFII_NDM5-like plasmid. Meanwhile, we discovered a novel genetic structure of blaNDM-5 mediated by the IS26 composite transposon, which will expand our understanding of the emergence and spread of carbapenem-resistance genes. Altogether, the presence of the IncFII plasmid pIncFII-NDM5 further underscores the need for vigilant surveillance and appropriate infection control measures to mitigate the impact of carbapenem-resistant S. enterica serovar Typhimurium in clinical settings.

Prevalence and Molecular Characterization of Shiga Toxin-Producing Escherichia coli from Food and Clinical Samples.

Shiga toxin-producing Escherichia coli (STEC) is one of the most prominent food-borne pathogens in humans. The current study aims to detect and to analyze the virulence factors, antibiotic resistance, and plasmid profiles for forty-six STEC strains, isolated from clinical and food strains. Pulsed-field gel electrophoresis (PFGE) was used to determine the genetic relatedness between different serotypes and sources of samples. The clinical samples were found to be resistant to Nb (100%), Tet (100%), Amp (20%), SXT (15%), and Kan (15%) antibiotics. In contrast, the food strains were found to be resistant to Nb (100%), Tet (33%), Amp (16.6%), and SXT (16.6%) antibiotics. The PFGE typing of the forty-six isolates was grouped into more than ten clusters, each with a similarity between 30% and 70%. Most of the isolates were found positive for more than five virulence genes (eae, hlyA, stx1, stx2, stx2f, stx2c, stx2e, stx2, nelB, pagC, sen, toxB, irp, efa, and efa1). All the isolates carried different sizes of the plasmids. The isolates were analyzed for plasmid replicon type by PCR, and 72.5% of the clinical isolates were found to contain X replicon-type plasmid, 50% of the clinical isolates contained FIB replicon-type plasmid, and 17.5% of the clinical isolates contained Y replicon-type plasmid. Three clinical isolates contained both I1 and Hi1 replicon-type plasmid. Only two food isolates contained B/O and W replicon-type plasmid. These results indicate that STEC strains have diverse clonal populations among food and clinical strains that are resistant to several antimicrobials. In conclusion, our findings indicate that food isolates of STEC strains harbor virulence, antimicrobial resistance, plasmid replicon typing determinants like those of other STEC strains from clinical strains. These results suggest that these strains are unique and may contribute to the virulence of the isolates. Therefore, surveillance and characterization of STEC strains can provide useful information about the prevalence of STEC in food and clinical sources. Furthermore, it will help to identify STEC serotypes that are highly pathogenic to humans and may emerge as a threat to public health.

Antimicrobial resistance and whole genome sequencing of novel sequence types of Enterococcus faecalis, Enterococcus faecium, and Enterococcus durans isolated from livestock

The emergence of antimicrobial-resistant, livestock-associated Enterococcus faecalis represents a public health concern. Here, we report the isolation, molecular detection of virulence and antimicrobial resistance determinants, in addition to the phylogenetic analyses of 20 Enterococcus species using whole genome sequencing analysis of 15 Enterococcus faecalis strains including six strains of three novel sequence types, three Enterococcus faecium and two Enterococcus durans. All strains were isolated from food chain animals in South Africa. Enterococcus strains were isolated on bile aesculin azide agar, followed by identification using MALDI-TOF MS analysis. Antibiotic susceptibility testing was performed using the Kirby–Bauer disk diffusion method. The genomic DNA of the isolates was extracted and sequencing was performed using the Illumina MiSeq platform. Sequence reads were trimmed and de novo assembled. The assembled contigs were analyzed for antimicrobial resistance genes and chromosomal mutations, extra-chromosomal plasmids, and multi-locus sequence type (MLST). Multidrug antimicrobial resistance genes conferring resistance to aminoglycosides (ant(6)-Ia, aph(3′)-IIIa, sat4, and spw), lincosamides (lnu(B), lsa(A), and lsa(E)), macrolides (erm(B)), trimethoprim (dfrG) and tetracyclines (tet(L) and tet(M)) were identified. Plasmid replicons were detected in seven E. faecalis and three E. faecium isolates. The sequence type (ST) of each isolate was determined using the Enterococcus PubMLST database. Ten STs were identified in the collection, three of which (ST1240, ST1241, and ST1242) have not been previously reported and are described in the present study for the first time. To compare the sequenced strains to other previously sequenced E. faecalis strains, assembled sequences of E. faecalis from livestock were downloaded from the PubMLST database. Core genome-based phylogenetic analysis was performed using ParSNP. The detection of multiple drug-resistance in Enterococcus including E. faecalis and E. faecium highlights the significance of genomic surveillance to monitor the spread of antimicrobial resistance in food chain animals. In addition, the genome sequences of Enterococcus strains reported in the present study will serve as a reference point for future molecular epidemiological studies of livestock-associated and antibiotic-resistant E. faecalis in Africa. In addition, this study enables the in-depth analysis of E. faecalis genomic structure, as well as provides valuable information on the phenotypic and genotypic antimicrobial resistance, and the pathogenesis of livestock-associated E. faecalis and E. faecium.

Comparative genomic analysis of strong biofilm-forming Klebsiella pneumoniae isolates uncovers novel ISEcp1-mediated chromosomal integration of a full plasmid-like sequence

Background The goal of the current study was to elucidate the genomic background of biofilm formation in Klebsiella pneumoniae. Methods Clinical isolates were screened for biofilm formation using the crystal violet assay. Antimicrobial resistance (AMR) profiles were assessed by disk diffusion and broth microdilution tests. Biofilm formation was correlated to virulence and resistance genes screened by PCR. Draft genomes of three isolates that form strong biofilm were generated by Illumina sequencing. Results Only the siderophore-coding gene iutA was significantly associated with more pronounced biofilm formation. ST1399-KL43-O1/O2v1 and ST11-KL15-O4 were assigned to the multidrug-resistant strain K21 and the extensively drug-resistant strain K237, respectively. ST1999-KL38-O12 was assigned to K57. Correlated with CRISPR/Cas distribution, more plasmid replicons and prophage sequences were identified in K21 and K237 compared to K57. The acquired AMR genes (bla OXA-48, rmtF, aac(6′)-Ib and qnrB) and (bla NDM-1, bla CTX-M, aph(3′)-VI, qnrS, and aac(6′)-Ib-cr) were found in K237 and K21, respectively. The latter showed a novel ISEcp1-mediated chromosomal integration of replicon type IncM1 plasmid-like structure harboring bla CTX-M-14 and aph(3′)-VI that uniquely interrupted rcsC. The plasmid-mediated heavy metal resistance genes merACDEPRT and arsABCDR were spotted in K21, which also exclusively carried the acquired virulence genes mrkABCDF and the hypervirulence-associated genes iucABCD-iutA, and rmpA/A2. Pangenome analysis revealed NTUH-K2044 accessory genes most frequently shared with K21. Conclusions While less virulent to Galleria mellonella than ST1999 (K57), the strong biofilm former, multidrug-resistant, NDM-producer K. pneumoniae K21 (ST1399-KL43-O1/O2v1) carries a novel chromosomally integrated plasmid-like structure and hypervirulence-associated genes and represents a serious threat to countries in the area.

Genomics analysis of KPC-2 and NDM-5-producing Enterobacteriaceae in migratory birds from Qinghai Lake, China.

The study examined the epidemiological characteristics of carbapenem-resistant Enterobacteriaceae (CRE) isolated from migratory birds and surroundings in Qinghai Lake, China. We identified 69 (15.7%) CRE isolates from a total of 439 samples including 29 (6.6%) blaNDM-5 Escherichia coli and 40 (9.1%) blaKPC-2 Klebsiella pneumoniae. WGS analysis indicated that ST746, ST48, ST1011, and ST167 were the primary sequence types (ST) for blaNDM-5 E. coli, while all blaKPC-2K. pneumoniae were ST11 and harbored numerous antibiotic resistance gene types including blaCTX-M, qnrS, and rmtB. A phylogenetic tree based on core genomes revealed that blaNDM-5 E. coli was highly heterogeneous while the blaKPC-2K. pneumoniae was highly genetically similar within the group and to human Chinese isolates. IncX3, IncHI2, and IncFIB-HI2 plasmid replicon types were associated with blaNDM-5 spread, while IncFII-R and IncFII plasmids mediated blaKPC-2 spread. We also identified IncFII-R hybrid plasmids most likely formed by IS26-mediated integration of IncFII into IncR plasmid backbones. This also facilitated the persistence of IncFII-R plasmids and antibiotic resistance genes including blaKPC-2. In addition, all of the blaKPC-2K. pneumoniae isolates harbored a pLVKP-like virulence plasmid carrying a combination of two or more hypervirulence markers that included peg-344, iroB, iucA, rmpA, and rmpA2. This is the first description of ST11 K. pneumoniae that co-carried blaKPC-2- and pLVKP-like virulence plasmids from migratory birds. The blaKPC-2K. pneumoniae carried by migratory birds displayed high genetic relatedness to human isolates highlighting a high risk of transmission of these K. pneumoniae. KEY POINTS: • Multidrug resistance plasmids (blaKPC-2, bla436NDM-5, blaCTX-M, qnrS, and rmtB). • Co-occurrence of plasmid-mediated resistance and virulence genes. • High similarity between migratory bird genomes and humans.

Prevalence and molecular epidemiology of mcr-mediated colistin-resistance Escherichia coli from healthy poultry in France after national plan to reduce exposure to colistin in farm.

Within the 2007-2014 programme for the surveillance of antimicrobial resistance (AMR) in livestock in France, mcr-1 prevalence average in commensal Escherichia coli was found to be 5.9% in turkeys and 1.8% in broilers, indicating that mobile colistin resistance had spread in farm animals. In 2017, the French national Ecoantibio2 plan was established to tackle AMR in veterinary medicine, with the objective of a 50% reduction in exposure to colistin in farm animals within 5 years (from 2014-2015 to 2020). Our objective was to update data concerning the prevalence and molecular epidemiology of colistin resistance, in consideration of colistin sales in poultry production in France. Antimicrobial susceptibility of commensal E. coli isolated from broilers and turkeys at slaughterhouse was determined by broth micro-dilution. The mcr genes were screened by polymerase chain reaction (PCR). Whole genome sequencing (WGS) was used to investigate the genetic diversity of colistin-resistant isolates. Transformation experiments enabled identification of the mcr-bearing plasmid replicon types. The correlation between prevalence of colistin resistance and colistin usage data was explored statistically. In 2020, in France, the resistance prevalence to colistin in poultry production was 3% in turkeys and 1% in broilers, showing a significant highly positive correlation with a -68% decrease of poultry exposure to colistin since 2014. Only the mcr-1 gene was detected among the colistin-resistant E. coli. More than 80% of isolates are multi-drug resistant with 40% of isolates originating from turkeys and 44% originating from broilers co-resistant to the critically important antimicrobial ciprofloxacin. Most of the strains had no clonal relationship. The mcr gene was located in different plasmid types, carrying various other AMR genes. The decrease in colistin resistance among poultry in France can be considered a positive outcome of the national action plans for reduced colistin usage.

The Resistance and Virulence Characteristics of Salmonella Enteritidis Strain Isolated from Patients with Food Poisoning Based on the Whole-Genome Sequencing and Quantitative Proteomic Analysis.

This paper explores the drug resistance, genome and proteome expression characteristics of Salmonella from a food poisoning event. A multidrug-resistant Salmonella Enteritidis strain, labeled as 27A, was isolated and identified from a food poisoning patient. Antimicrobial susceptibility testing determined the resistance of 27A strain to 14 antibiotics. Then, WGS analysis and comparative genomics analysis were performed on 27A, and the functional annotation of resistance genes, virulence genes were performed based on VFDB, ARDB, COG, CARD, GO, KEGG, and CAZY databases. Meanwhile, based on iTRAQ technology, quantitative proteomic analysis was conducted on 27A to analyze the functions and interactions of differentially expressed proteins related to bacterial resistance and pathogenicity. Strain 27A belonged to ST11 S. Enteritidis and was resistant to levofloxacin, ciprofloxacin, ampicillin, piperacillin, and ampicillin/sulbactam. There were 33 drug resistance genes, 384 virulence genes and 2 plasmid replicon, IncFIB(S) and IncFII(S), annotated by WGS. Proteomic analysis revealed significant changes in virulence and drug proteins, which were mainly involved in bacterial pathogenicity and metabolic processes. PPI prediction showed the relationship between virulence proteins and T3SS proteins, and PagN cooperated with proteins related to T3SS to jointly mediate the invasion of 27A strain on the human body. Phylogenetic analysis indicated that S. Enteritidis has potential transmission in humans, food, and animals. This study comprehensively analyzed the drug resistance and virulence phenotypes of S. Enteritidis 27A using genomic and proteomic approaches. These helps reveal the drug resistance and virulence mechanisms of S. Enteritidis, and provides important information for the source tracing and the prevention of related diseases, which lays a foundation for research on food safety, public health monitoring, and the drug resistance and pathogenicity of S. Enteritidis.

Genomic epidemiological investigation of an outbreak of Serratia marcescens neurosurgical site infections associated with contaminated haircutting toolkits in a hospital barber shop

Nine surgical site infections caused by Serratia marcescens were diagnosed in neurosurgical patients in a 3500-bed hospital between 2nd February and 6th April 2022. To trace the source of infections caused by S.marcescens to expedite termination of the outbreak and prevent future epidemics. A review of all surgical procedures and cultures yielding S.marcescens since February 2022 was conducted. Samples were collected from patients and environmental sources. S.marcescens isolates were characterized by antibiotic susceptibility testing. Whole-genome sequencing (WGS) was used to investigate genetic relationships. Resistance genes, virulence genes and plasmid replicons were identified. S.marcescens was isolated from patients' puncture fluid, cerebrospinal fluid and other secretions, and was also cultured from the barbers' haircutting tools, including leather knives, slicker scrapers and razors. In total, 15 isolates were obtained from patients and eight isolates were obtained from haircutting tools. All isolates exhibited identical antibiotic resistance patterns. WGS revealed close clustering among the 23 isolates which differed significantly from previous strains. Three resistance genes and nine virulence-associated genes were detected in all isolates, and 19 of 23 isolates harboured an MOBP-type plasmid. The results confirmed an outbreak of S.marcescens, which was traced to contaminated haircutting tools in the hospital barber shop. The outbreak ended after extensive reinforcement of infection control procedures and re-education of the barbers. These results highlight the risk of postoperative infections related to pre-operative skin preparation, and demonstrate the value of next-generation sequencing tools to expedite outbreak investigations.

Genomic Analysis of Multidrug-Resistant Escherichia coli Strains Isolated in Tamaulipas, Mexico.

The global spread of antimicrobial resistance genes (ARGs) is a major public health concern. Mobile genetic elements (MGEs) are the main drivers of this spread by horizontal gene transfer (HGT). Escherichia coli is widespread in various environments and serves as an indicator for monitoring antimicrobial resistance (AMR). Therefore, the objective of this work was to evaluate the whole genome of multidrug-resistant E. coli strains isolated from human clinical, animal, and environmental sources. Four E. coli strains previously isolated from human urine (n = 2), retail meat (n = 1), and water from the Rio Grande River (n = 1) collected in northern Tamaulipas, Mexico, were analyzed. E. coli strains were evaluated for antimicrobial susceptibility, followed by whole genome sequencing and bioinformatic analysis. Several ARGs were detected, including blaCTX-M-15, blaOXA-1, blaTEM-1B, blaCMY-2, qnrB, catB3, sul2, and sul3. Additionally, plasmid replicons (IncFIA, IncFIB, IncFII, IncY, IncR, and Col) and intact prophages were also found. Insertion sequences (ISs) were structurally linked with resistance and virulence genes. Finally, these findings indicate that E. coli strains have a large repertoire of resistance determinants, highlighting a high pathogenic potential and the need to monitor them.

The origin and evolution of IncF33 plasmids based on large-scale data sets.

To comprehensively understand the formation and evolution of IncF33 plasmids, a global collection of whole-genome sequences of 863 strains positive for IncF33 plasmid replicons was analyzed. The results showed that the IncF33 plasmids were mainly identified in Enterobacterales, of which Escherichia coli (86.44%) was the dominant host, followed by Salmonella (8.57%) and Klebsiella pneumoniae (3.48%). Salmonella ST11 and K. pneumoniae ST11 were common and mostly from humans. IncF33 plasmids were found worldwide but prevalent in Chinese farm animals, predominantly carrying antibiotic resistance genes such as blaCTX-M-55, fosA3, blaTEM-1B, rmtB, and floR. Comparative genomics analysis of 103 complete IncF33 plasmid sequences showed highly similar backbones except for 16 lacking partial backbone fragments. Variable regions were diverse, containing various antibiotic resistance genes, insertion sequences, or other plasmid fragments. They can be roughly divided into two sublines based on the production of different CTX-M enzymes. Similar IncF33 plasmids from different countries were identified. Some early IncF33 plasmids lacked part of their leader regions, which showed over 99% homology with early F2:A-:B- plasmids, indicating that leader regions of IncF33 likely came from F2:A-:B- plasmids. In addition, IncF33 plasmids cointegrating with other types of plasmids to form new cointegrate plasmids are increasing, making them more efficient in their dissemination and persistence in Enterobacterales, which could pose a significant threat to global public health. IMPORTANCE Plasmids that capture multiple antibiotic resistance genes are spreading widely, leading to the emergence and prevalence of multidrug-resistant bacteria. IncF33 plasmids are a newly emerged plasmid type highly prevalent in animal-source Enterobacterales in China, and they are important vectors for transmitting several clinically important antibiotic resistance genes. The study revealed that the IncF33 plasmid is mainly prevalent in China animal-derived Escherichia coli and has the potential for cointegration and intercontinental dissemination. Therefore, it is crucial to enhance surveillance and control measures to limit the spread of IncF33 plasmids and their associated antibiotic resistance genes.

High Prevalence of Carbapenem-Resistant Enterobacterales Colonization Among Intensive Care Unit Patients in a Tertiary Hospital, China.

Intestinal colonization with carbapenem-resistant Enterobacterales (CRE) has been shown as a significant risk factor for subsequent CRE infections, especially in intensive care units (ICUs). The aim of this study was to determine the prevalence of intestinal CRE colonization among ICU patients in a Chinese tertiary hospital. Fecal sample screenings for CRE were performed on ICU patients weekly. Antibiotic-susceptibility profile of CRE strains was determined using the Vitek-2 analysis system and broth microdilution method. The carbapenemases of all isolates were determined by phenotypes and genotypes. Clonal relatedness was analyzed by pulsed-field gel electrophoresis (PFGE). Whole-genome sequencing was used to identify the multilocus sequence type (ST), plasmid replicons, and insertion sequences (ISs) of isolates. The overall colonization rate of CRE was 40.4% (82/203). A total of 84 CRE strains were detected, mostly with Klebsiella pneumoniae (92.9%). Antibiotic susceptibility testing profile revealed that 84 CRE strains were resistant to most antibiotics except for tigecycline and colistin. The carbapenemase-encoding genes including blaKPC-2, blaNDM-1, and blaIMP-4 were detected, and blaKPC-2 was the predominant genotype (90.8%). A total of 9 STs were identified among 84 CRE strains, and ST11 was the most common type (83.3%). A variety of mobile genetic elements, including plasmids and ISs, were detected via online tool prediction. PFGE analysis of the 78 K. pneumoniae strains showed 8 different pulsotypes, and pulsotype A was highly prevalent. This study found that the prevalence of CRE colonization was alarmingly high in the ICU, and that effective infection control measures are urgently needed to prevent the dissemination of CRE.

Genomic and clinical characterization of Klebsiella pneumoniae carrying the pks island.

The pks island and its production of the bacterial secondary metabolite genotoxin, colibactin, have attracted increasing attention. However, genomic articles focusing on pks islands in Klebsiella pneumoniae, as well as comparative genomic studies of mobile genetic elements, such as prophages, plasmids, and insertion sequences, are lacking. In this study, a large-scale analysis was conducted to understand the prevalence and evolution of pks islands, differences in mobile genetic elements between pks-negative and pks-positive K. pneumoniae, and clinical characteristics of infection caused by pks-positive K. pneumoniae. The genomes of 2,709 K. pneumoniae were downloaded from public databases, among which, 1,422 were from NCBI and 1,287 were from the China National GeneBank DataBase (CNGBdb). Screening for virulence and resistance genes, phylogenetic tree construction, and pan-genome analysis were performed. Differences in mobile genetic elements between pks-positive and pks-negative strains were compared. The clinical characteristics of 157 pks-positive and 157 pks-negative K. pneumoniae infected patients were investigated. Of 2,709 K. pneumoniae genomes, 245 pks-positive genomes were screened. The four siderophores, type VI secretion system, and nutritional factor genes were present in at least 77.9% (191/245), 66.9% (164/245), and 63.3% (155/245) of pks-positive strains, respectively. The number and fragment length of prophage were lower in pks-positive strains than in pks-negative strains (p < 0.05). The prevalence of the IS6 family was higher in pks-negative strains than in pks-positive strains, and the prevalence of multiple plasmid replicon types differed between the pks-positive and pks-negative strains (p < 0.05). The detection rate of pks-positive K. pneumoniae in abscess samples was higher than that of pks-negative K. pneumoniae (p < 0.05). The pks-positive strains had abundant virulence genes. There were differences in the distribution of mobile genetic elements between pks-positive and pks-negative isolates. Further analysis of the evolutionary pattern of pks island and epidemiological surveillance in different populations are needed.

Virulence genes and plasmid replicon profiles of selected β-lactamase-producing Acinetobacter baumannii from orthopaedic patients and the environment in a tertiary referral hospital in Tanzania, East Africa

Acinetobacter baumannii has emerged as an important nosocomial pathogen due to its high resistance to multi-drugs and disinfectants plus its ability to survive in hospital environments. Rectal swabs were collected for screening β-lactamases-producing Acinetobacter baumannii among hospitalized orthopedic patients at a tertiary referral hospital in Tanzania. Swabs were also taken from patients' caretakers, healthcare workers, and the neighboring inanimate environment. A total of 26 confirmed β-lactamases producing Acinetobacter baumannii were isolated, of which 4 representative isolates (two from patients and two from hospital environment) underwent whole-genome sequencing (WGS) to detect sequence types (ST), β-lactamases genes, plasmid replicon types, and virulence genes. All four isolates harbored multiple β-lactamases genes including blaADC-25(3), blaOXA(4), blaCTX-M-15(2) and blaNDM-1(2). Furthermore, isolates harbored virulence genes encoding outer membrane protein (ompA), curli protein (csg), siderophore biosynthesis systems (enterobactin [entABCDEFS, fepABCDG, fes]; yersiniabactin [ybtAEPQSTUX, irp1, irp2, fyuA] and aerobactin [iucABCD, iutA]), transport secretion system type II (T2SS) and type III (T3SS), E. coli common pilus (ecpRABCDE operon), type 1 fimbriae (fim), arylsulfatase (aslA) and adhesions (fedC). Only isolates from patients harbored 4 plasmid replicons each, with the most common plasmid replicons being IncFIA_1; IncY_1 and IncFIB(AP001918)_1. Admitted orthopedic patients and the hospital environment act as a reservoir of multiple β-lactamases producing Acinetobacter baumannii (including those against carbapenems like blaOXA and blaNDM-1) endowed with virulence genes, highlighting the necessity to routinely screening of orthopedic patients with open fractures on admission as well as reinforcing infection prevention and control measures to reduce the dissemination of nosocomial infection within the hospital environment.

Detection of clinically relevant carbapenemase encoding genes in carbapenem-resistant Enterobacter cloacae complex and Klebsiella pneumoniae isolated from farmed freshwater fish.

The present study was aimed to detect clinically relevant carbapenemase encoding genes in carbapenem-resistant Enterobacter cloacae complex (CR-ECC), Klebsiella pneumoniae (CR-KP), and Serratia plymuthica (CR-SP) isolated from farmed freshwater fish. Out of 243 spatially diverse freshwater fish samples analysed, 5.3% were contaminated with CR-ECC, 1.6% with CR-KP, and 0.4% with CR-SP. The CR-ECC was further identified as E. asburiae (38.5%), E. mori (23.1%), E. cloacae (15.4%), E. hormaechei (15.4%), and E. kobei (7.7%) by 16S rRNA gene sequencing. The CR-ECC were resistant to carbapenems and cefoxitin, whereas CR-KP and CR-SP were multi-drug resistant (MDR). The CR-ECC harboured the carbapenemase gene blaIMI alone or in combination with blaTEM, blaEBC, blaCIT, blaACC, and tet(E). Whereas, CR-KP harboured carbapenemase gene, blaNDM-5 along with blaOXA-48, blaSHV, blaOXA-1, blaCTX-M-15, tet(A), sul1, and qnrB. No carbapenemase-encoding genes were detected in CR-SP. The MLST analysis showed that CR-KP belonged to ST231 and ST1561 lineages, while CR-ECC did not show exact match with any reported STs. The plasmid replicons predominantly detected were IncF and IncI1. Broth mating assays of CR-KP and CR-ECC with recipient Escherichia coli J53 indicated that blaNDM-5 was transferable but not blaIMI. This study highlights the low-level contamination of carbapenem-resistant Enterobacterales (CRE) harbouring clinically relevant carbapenemase-encoding genes in farmed freshwater fish from India. The CR-ECC of fish origin did not show the potential to spread carbapenem resistance.