Genetic Environment Analysis Research Articles

Molecular Epidemiology of Type F Clostridium perfringens Among Diarrheal Patients and Virulence-Resistance Dynamics - 11 Provinces, China, 2024.

Type F Clostridium perfringens (C. perfringens) represents a significant pathogen in human gastrointestinal diseases, primarily through its cpe gene encoding C. perfringens enterotoxin (CPE). This investigation examined the prevalence, antimicrobial resistance patterns, and genetic characteristics of Type F C. perfringens within the Chinese population. The study analyzed 2,068 stool samples collected from 11 provincial hospitals in 2024. Antimicrobial susceptibility testing was conducted following Clinical & Laboratory Standards Institute (CLSI) guidelines, while whole-genome sequencing provided detailed genetic profiles. Evolutionary relationships and clonal transmission patterns were investigated through phylogenetic and genetic environment analyses. The prevalence of Type F C. perfringens was 2.38%, with isolates predominantly identified in human clinical samples and higher detection rates in gastroenterology departments. Notably, 47.1% of isolates demonstrated high resistance to metronidazole, while all exhibited intermediate resistance to erythromycin. Phylogenetic analysis revealed high similarity among isolates from patients within the same province (single-nucleotide polymorphism (SNPs)<100), and genetic environment analysis indicated potential horizontal gene transfer between animal and human strains. This investigation predominantly identified Type F C. perfringens in human clinical cases, with sporadic detection in pets and food products. These findings highlight the emergence of Type F C. perfringens outbreaks among diarrheal patients, emphasizing the necessity for targeted interventions as virulence factors increase.

Antimicrobial Resistance Genes in Clinical Escherichia coli Strains from Livestock and Poultry in Shandong Province, China During 2015-2020.

Antimicrobial resistant (AMR) Escherichia coli (E. coli) isolated from animals may lead to antibiotic treatment failure and economic losses to farmers. The co-existence of antimicrobial resistant genes (ARGs) in the same isolate presents a major challenge for the prevention and control of infection in multidrug-resistant (MDR) Gram-negative organisms. There have been a lot of studies on the antibiotic resistance of E. coli in livestock and poultry, but few of them have focused on clinical pathogens. Objective: The aim of this study was to explore the genetic characteristics, co-occurrence, and correlations between ARGs of E. coli isolated from the pathological tissues of livestock and poultry in Shandong Province, East China during 2015-2020. Methods: A total of 158 E. coli strains were collected and subjected to antimicrobial susceptibility testing and sequencing by whole-genome Next Generation Sequencing (NGS). Results: MDR strains accounted for 46.20% of the 158 E. coli strains with the highest resistant rate of ciprofloxacin (71.52%). In addition, strains with blaNDM-5/mcr-1.1 and mcr-1.1/mcr-3.24 were found in chickens, while three strains with Tet(X4) were found in pigs. In addition, the most common serotypes detected were the O serotype (76/158) and H serotype (36/158). Moreover, seventy-one STs were found and the most common STs were ST10 (6.33%), ST155 (6.33%), and ST101 (5.69%). The genetic environment analysis of the phylogroups revealed that E. coli belonging to phylogroup B1, phylogroup A, and phylogroup C constituted 39.87%, 27.85%, and 15.19%, respectively. Through the correlation analysis, mcr genes were observed to have certain relationships with ARGS such as blaTEM, floR, catA/B, and oqx. Conclusions: This study demonstrates the high prevalence and gene diversity of MDR E. coli isolated from a clinic in Shandong Province, East China. We predicted the transmission risk of animal-borne Tet(X4)-bearing and mcr-harboring E. coli to public health and provided insight into the relationship of co-existence or co-transfer between mcr with ARGS. These relationships present a great challenge for the infection control of MDR Gram-negative organisms.

The prevalence of optrA-carrying Enterococci in the vaginal micro-ecology of pregnant women in late pregnancy.

The colonization of Enterococcus in the female vagina leads to neonatal and pediatric enterococcal septicemia. Linezolid (LZD) is a kind of mainstream drug for treating multidrug-resistant Gram-positive infections. OptrA is the main LZD-resistance gene at Enterococci in human isolates. It is essential to explore the prevalence of optrA-carrying Enterococcus in vaginal secretions of late pregnant women and the drug resistance of optrA. From May to June 2023, this study recruited 340 volunteers in late pregnancy (35-40 weeks of pregnancy) to provide non-repetitive vaginal discharge samples. Luria-Bertani broth and florfenicol (10 µg/mL) were used to enrich bacteria. Enterococci was identified through time-of-flight mass spectrometry. Additionally, antimicrobial susceptibility, polymerase chain reaction, and next-generation sequencing assays were applied to this study. Fifty-four optrA-carrying Enterococcus strains were obtained, the proportion of the whole vagina of late pregnant women was 15.88% (54 out of 340), and Enterococcus faecalis account the highest proportion. All optrA-carrying Enterococcus were resistant to at least three drugs. Tetracycline, chloramphenicol, erythromycin, and LZD have higher bacterial resistance rates. Genetic environment analysis revealed that IS1216E, fexA, and erm(A) may synergistically exert multidrug resistance with optrA. It is necessary to strengthen the surveillance of optrA-carrying Enterococcus in pregnant women. This study provides scientific support for controlling hospital infections and managing antibiotic-resistant bacteria, and provides a scientific basis for rational clinical medication.IMPORTANCEThe disruption of cervicovaginal microbiota homeostasis is considered a key factor in causing imbalance in the microenvironment, leading to inflammation, transmission of infections, and illness. Enterococcus is considered a major cause of healthcare-related infections globally. It has resistance to multiple antimicrobial drugs, which pose significant challenges for clinical treatment. Therefore, it is crucial to assess the prevalence of optrA-carrying Enterococcus in vaginal secretions of late pregnant women and the drug resistance of optrA. This study detected 15.88% of optrA-carried Enterococci in 340 pregnant women. Furthermore, we found that optrA-carrying Enterococcus strains are highly resistant to tetracycline, chloramphenicol, erythromycin, and Linezolid. Additionally, genetic environment analysis revealed that IS1216E, fexA, and erm(A) may synergistically exert multidrug resistance with optrA. This study provides scientific support for controlling hospital infections and managing antibiotic-resistant bacteria and provides a scientific basis for rational clinical medication.

Prevalence and characteristics of ESBL-producing Salmonella in Weifang, China.

This study examined the prevalence and antibiotic resistance pattern of blaCTX-M extended-spectrum β-lactamase positive Salmonella species isolated from a hospital in Weifang. Salmonella strains were isolated from hospitalized patients from January 2018 to April 2023. Whole-genome sequencing was performed by Illumina platform. CTX-M-producing Salmonella were identified by Comprehensive Antibiotic Research Database (CARD). Strain susceptibility to six antimicrobial agents was assessed by BD Phoenix™ M50 System. MLST analysis confirmed sequence types and additionally, serotypes were determined by SeqSero2. Genetic environments of blaCTX-M genes were analyzed by Isfinder and BLASTn. Single nucleotide polymorphisms were used to construct a phylogenetic tree to analyze homology.A total of 34 CTX-M-producing Salmonella were detected. The most prevalent serotype was Salmonella enterica subsp. enterica 1,4,[5],12:i:- (14/34, 41.18%), belonging to ST34, followed by SalmonellaEnteritidis (10/34, 29.41%), belonging to ST11. The highest resistance rate was detected to ampicillin (97.06%), followed by ceftriaxone (94.12%) and ceftazidime (58.83%). In CTX-M-producing Salmonella five types of blaCTX-M genes were identified, the most prevalent was blaCTX-M-55 (47.06%, 16/34), followed by blaCTX-M-14, blaCTX-M-65, blaCTX-M-125, and blaCTX-M-27 at 26.47% (9/34), 11.77% (4/34), 8.82% (3/34), and 5.88% (2/34), respectively. Apart from blaCTX-M, 40 antibiotic resistance genes were also detected, conveying resistance to multiple drugs and the most frequent genes were namely, mcr-1.1, aph(6)-Id, aph(3″)-Ib, oqxAB, qnrB6, qnrS1. According to genetic environment analysis, the insertion sequence ISEcp1 was prevalent upstream of the blaCTX-M gene. Our study demonstrates that multiple resistance genes are carried by clinical isolates of Salmonella spp. however, the dominant ESBL genotype is CTX-M-55, that is associated with ISEcp1.

Emergence of aztreonam/avibactam and tigecycline-resistant Pseudomonas putida group Co-producing blaIMP-1, blaAFM-4 and blaOXA-1041 with a novel sequence type ST268 in Southwestern China

ObjectivesThe emergence of carbapenem-resistant Pseudomonas putida (CRPP) has raised public awareness. This study investigated two strains from the Pseudomonas putida group that were resistant to carbapenem, tigecycline, and aztreonam-avibactam (ATM−AVI), with a focus on their microbial and genomic characteristics. MethodsWe assessed the antibiotic resistance profile using broth dilution, disk diffusion, and E-test methods. Efflux pump phenotype testing and real-time quantitative PCR were employed to evaluate efflux pump activity in tigecycline resistance, while polymerase chain reaction was utilized to detect common carbapenem genes. Additionally, whole-genome sequencing was performed to analyze genomic characteristics. The transferability of blaIMP-1 and blaAFM-4 was assessed through a conjugation experiment. Furthermore, growth kinetics and biofilm formation were examined using growth curves and crystal violet staining. ResultsBoth strains demonstrated resistance to carbapenem, tigecycline, and ATM-AVI. Notably, NMP can restore sensitivity to tigecycline. Subsequent analysis revealed that they co-produced blaIMP-1, blaAFM-4, tmexCD-toprJ, and blaOXA-1041, belonging to a novel sequence type ST268. Although they were closely related on the phylogenetic tree, they exhibited different levels of virulence. Genetic environment analysis indicated variations compared to prior studies, particularly regarding the blaIMP-1 and blaAFM-4 genes, which showed limited horizontal transferability. Moreover, it was observed that temperature exerted a specific influence on their biological factors. ConclusionWe initially identified two P. putida ST268 strains co-producing blaIMP-1, blaAFM-4, blaOXA-1041, and tmexCD-toprJ. The resistance to tigecycline and ATM-AVI can be attributed to the presence of multiple drug resistance determinants. These findings underscore the significance of P. putida as a reservoir for novel antibiotic resistance genes. Therefore, it is imperative to develop alternative antibiotic therapies and establish effective monitoring of bacterial resistance.

RATA: A novel class A carbapenemase with broad geographic distribution and potential for global spread

Carbapenem resistance's global proliferation poses a significant public health challenge. The primary resistance mechanism is carbapenemase production. In this study, we discovered a novel carbapenemase, RATA, located on the chromosome of Riemerella anatipestifer isolates. This enzyme shares ≤52 % amino acid sequence identity with other known β-lactamases. Antimicrobial susceptibility tests and kinetic assays demonstrated that RATA could hydrolyze not only penicillins and extended-spectrum cephalosporins but also monobactams, cephamycins, and carbapenems. Furthermore, its activity was readily inhibited by β-lactamase inhibitors. Bioinformatic analysis revealed 46 blaRATA-like genes encoding 27 variants in the NCBI database, involving 21 different species, including pathogens, host-associated bacteria, and environmental isolates. Notably, blaRATA-positive strains were globally distributed and primarily collected from marine environments. Concurrently, taxonomic analysis and GC content analysis indicated that blaRATA orthologue genes were predominantly located on the chromosomes of Flavobacteriaceae and shared a similar GC content as Flavobacteriaceae. Although no explicit mobile genetic elements were identified by genetic environment analysis, blaRATA-2 possessed the ability of horizontal transfer in R. anatipestifer via natural transformation. This work's data suggest that RATA is a new chromosome-encoded class A carbapenemase, and Flavobacteriaceae from marine environments could be the primary reservoir of the blaRATA gene.

Epidemiological characteristics of human- and chicken-derived CTX-M-type extended-spectrum β-lactamase-producing Escherichia coli from China

Bacterial resistance to β-lactams is mainly attributed to CTX-M-type extended-spectrum β-lactamases (ESBLs). However, the predominant sequence type (ST) of blaCTX-M-carrying Escherichia coli (blaCTX-M-Ec) in chickens, an important food animal, in China and its contribution to human β-lactam resistance are not investigated. In this study, approximately 1808 chicken-derived strains collected from 10 provinces from 2012 to 2020 were screened for blaCTX-M-Ec, and 222 blaCTX-M-Ec were identified. Antimicrobial susceptibility tests, whole genome sequencing and conjugation experiment were performed. All quality-controlled 136 chicken-derived blaCTX-M-Ec and 1193 human-derived blaCTX-M-Ec genomes were downloaded from NCBI and EnteroBase to comprehensively analyze the prevalence of blaCTX-M-Ec in China. blaCTX-M-55 (153/358, 42.7% in chicken isolates; 312/1193, 26.2% in human isolates) and blaCTX-M-14 (92/358, 25.7% in chicken isolates; 450/1193, 37.7% in human isolates) were dominant in blaCTX-M-Ec. The STs of blaCTX-M-Ec were diverse and scattered, with ST155 (n = 21) and ST152 (n = 120) being the most abundant in chicken- and human-derived isolates, respectively. Few examples indicated that chicken- and human-derived blaCTX-M-Ec have 10 or less core genome single nucleotide polymorphisms (cgSNPs). Genetic environment analysis indicated that ISEcp1, IS26 and IS903B were closely associated with blaCTX-M transfer. The almost identical pc61–55 and pM-64–1161 indicated the possibility of plasmid-mediated transmission of blaCTX-M between humans and chickens. Although the genomes of most blaCTX-M-Ec isolated from chickens and humans were quite different, the prevalence and genetic environment of blaCTX-M variants in both hosts were convergent. CTX-M-mediated resistance is more likely to spread through horizontal gene transmission than bacterial clones.

Study on the Genome and Mechanism of Tigecycline Resistance of a Clinical Chryseobacterium indologenes Strain.

Purpose: Chryseobacterium indologenes is a clinically relevant microorganism that has been on the rise, with multidrug-resistant (MDR) strains being reported. C. indologenes carrying tet(X2) has been demonstrated to be resistant to the antibiotic tigecycline, yet, sensitive to all other members of the tetracycline family. This inconsistency in resistance prompts an inquiry into the contribution of tet(X2) to tigecycline resistance in C. indologenes. Materials and Methods: In this study, we report on a comprehensive analysis of the genomic mechanisms underlying tigecycline resistance in a MDR C. indologenes strain (CI3125) that was resistant to tigecycline but sensitive to tetracycline, doxycycline, and minocycline. We used whole-genome sequencing, quantitative reverse transcription PCR, Western blot, antibiotic-degrading tests, and efflux pump inhibiting tests to reveal the mechanism of tigecycline resistance in C. indologenes and elucidate the inconsistency in the antibiotic resistance mechanism for the tetracycline family. Results: Our findings demonstrate that CI3125 carries 60 antibiotic resistance genes distributed on 6 different genetic islands (GIs), with the potential for horizontal transfer. Notably, the tet(X2) gene is located on GI06 of CI3125. Genetic environment analysis of tet(X2) showed that all tet(X2) genes in Flavobacterium and Bacteroides share a conservative and functional ribosome-binding site upstream. Contrary to expectation, our RT-qPCR showed that tet(X2) was not transcribed in CI3125, and Western blot suggested the absence of tet(X2) protein in CI3125. Rather, we demonstrate that minimum inhibitory concentration values for tigecycline decreased two- to eight-folds in the presence of five different efflux pump inhibitors [1-(1-naphthyl- methyl)-piperazine, phenyl-arginine-β-naphthylamide, verapamil, reserpine, and carbonyl cyanide 3-chlorophenylhydrazone]. This finding provides evidence for the involvement of efflux pumps in tigecycline resistance, which is likely to be a universal mechanism among C. indologenes. Our study proposes that the inconsistency in resistance to the tetracycline family in CI3125 may be ascribed to the silence of tet(X2) and the functions of efflux pumps for tigecycline. Conclusions: Overall, our results highlight the importance of genomic approaches in understanding the underlying mechanisms of antibiotic resistance in clinically relevant microorganisms. While tet(X2) in CI3125 is silent, our findings suggest that it may be horizontally spread through GIs. Hence, our findings have significant implications for the management of C. indologenes infections in clinical settings.

Diversity of Bacterial Clones and Plasmids of NDM-1 Producing Escherichia coli Clinical Isolates in Central Greece.

The objective of the present study was to genetically characterize ten NDM-1 producing Escherichia coli isolates, recovered from patients in a hospital in Central Greece during the period 2017 to 2021.The isolates were studied by whole genome sequencing to obtain multi-locus sequencing typing (MLST), identification of blaNDM1-environment, resistome and plasmid content. MLST analysis showed the presence of eight sequence types: ST46* (two isolates), ST46, ST744, ST998, ST410, ST224, ST4380, ST683 and ST12 (one isolate each). Apart of the presence of blaNDM-1, the isolates carried a combination of various to β-lactams encoding resistance genes: blaTEM-1B, blaCTX-15, blaOXA-1, blaVIM-1, blaSHV-5, blaOXA-16, blaOXA-10 and blaVEB-1. Additionally, plurality of resistance genes to aminoglycosides, macrolides, rifamycin, phenicols, sulfonamides and tetracycline was detected. The presence of multiple replicons was observed, with predominance of IncFII and IncFIB. Analysis of blaNDM-1 genetic environment of the isolates showed that seven had 100% identity with the pS-3002cz plasmid (Accession Number KJ 958927), two with the pB-3002cz plasmid (Accession Number KJ958926) and one with the pEc19397-131 plasmid (Accession Number MG878866). Τhis latter plasmid was derived by the fusion of two, previously identified, plasmids, pAMPD2 and pLK75 (Accession Numbers CP078058 and KJ440076, respectively). The diversity of clones and plasmids of NDM-1 producing E. coli isolated from patients in Greece indicates a continuous horizontal gene transfer.

Molecular characteristics of ciprofloxacin-cefotaxime-azithromycin co-resistant Salmonella enterica Serovar Thompson in foodborne diseases in Hunan Province

Objective: To investigate the molecular characteristics of ciprofloxacin-cefotaxime-azithromycin co-resistant Salmonella enterica serovar Thompson (S. Thompson) isolates from sporadic cases of foodborne diseases and aquatic foods in Hunan province. Methods: Ciprofloxacin-cefotaxime-azithromycin co-resistant S. Thompson isolates were selected from samples, and broth microdilution method was used to determine the resistance to 11 antibiotics of these isolates in vitro. Whole genome sequencing was used for investigating antimicrobial resistance gene patterns and phylogenetic relationships of strains. Results: Nine ciprofloxacin-cefotaxime-azithromycin co-resistant isolates were recovered from 19 S. Thompson isolates. Among nine ciprofloxacin-cefotaxime-azithromycin co-resistant isolates, eight of them harbored IncC plasmids, simultaneously carrying plasmid-mediated quinolone resistance (PMQR) genes qepA and qnrS1, β-lactamase resistance gene blaCMY-2, azithromycin resistance gene mph(A), and one isolate harbored IncR plasmid, and carried PMQR genes qnrB4 and aac(6')-Ib-cr, blaOXA-10 and mph(A). Genetic environment analysis showed that qnrS1, qepA, mph(A) and blaCMY-2 genes might be integrated on genomes of strains by ISKra4, IS91, IS6100 and ISEcp1, respectively. Phylogenetic core genome comparisons demonstrated that ciprofloxacin-cefotaxime-azithromycin co-resistant isolates from patients and aquatic foods were genetically similar and clustered together. Conclusion: Ciprofloxacin-cefotaxime-azithromycin co-resistant S. Thompson isolates have been isolated from both human and aquatic food samples, suggesting that the spread of multidrug resistant Salmonella between human and aquatic animals.

Molecular and clinical characterization of hypervirulent Klebsiella pneumoniae isolates from individuals with urinary tract infections.

Despite being a significant public health concern, hypervirulent Klebsiella pneumoniae (hvKP) has rarely been investigated in urinary tract infections (UTIs). To investigate the molecular and clinical characterization of hvKP in UTIs, we collected K. pneumoniae strains and clinical data from patients with UTIs. HvKP was confirmed by virulence-related genes and the Galleria mellonella model and sequenced by next-generation sequencing. Our data showed that 30/121 isolates were hvKP [17 carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP), 12 hvKP, and 1 extended-spectrum β-lactamase-producing hvKP]; these had higher resistance to most antimicrobials and were more likely to cause complicated UTIs (cUTIs). Notably, the mucoid phenotype-regulating genes prmpA and prmpA2 were truncated in 3 and 19 hvKP, respectively. Eight serotypes were detected and divided into three groups: K64 (n = 17), K1/K2 (n = 6), and others (n = 7). Furthermore, 16/17 K64 hvKP isolates were CR-hvKP but with a lower mortality rate of G. mellonella as the truncated prmpA/prmpA2 incurred high fitness cost to the isolates. In addition, all K64 isolates belonged to ST11 with the same cluster, and in two of these strains (KP88 and KP92) bla KPC-2 gene was successfully transferred to EC600. Genetic environment analysis showed that IS26–tnpR–ISKpn27–bla KPC−2–ISKpn6 may be the core structure in the horizontal transfer of bla KPC-2. The highest mortality rate among the infected G. mellonella was observed in the K1/K2 group. In conclusion, hvKP had a higher resistance rate and was more likely to lead to cUTIs. Convergence of hypervirulence and carbapenem resistance in a transmissible ST11 clone of K64 K. pneumoniae was mediated by a plasmid in UTIs. Therefore, surveillance of hvKP in UTIs should be strengthened.

Molecular characterization of resistance determinants and mobile genetic elements of ESBL producing multidrug-resistant bacteria from freshwater lakes in Kashmir, India

BackgroundAntibiotic resistance conceded as a global concern is a phenomenon that emerged from the bacterial response to the extensive utilization of antimicrobials. The expansion of resistance determinants through horizontal transfer is linked with mobile genetic elements (MGEs) like transposons, insertion sequences, and integrons. Heavy metals also create consequential health hazards. Metal resistance gene in alliance with antibiotic resistance genes (ARGs) and MGEs is assisting bacteria to attain exalted quantity of resistance. MethodologyThe present work was carried out to study ARGs blaCTX-M, AmpC, qnrS, MGEs like ISecp1, TN3, TN21, and Int I by performing PCR and sequencing from Wular and Dal lakes of Kashmir; India. The genetic environment analysis of blaCTX-M-15 was carried out using PCR amplification, and sequencing approach followed by in-silico docking and mutational studies. Co-occurrence of ARGs and HMRGs was determined. Plasmid typing was done using PCR-based replicon typing (PBRT) and conjugation assay was also performed. ResultsOut of 201 isolates attained from 16 locations, 33 were ESBLs producers. 30 ESBL displaying isolates were perceived positive for CTX-M gene, followed by AmpC (17), qnrS (13), ISecp1 (15), TN3 (11), TN21 (11), Int I (18), and SulI (14). The genetic environment of blaCTX-M-15 was observed as (ISEcp1-blaCTX-M-15-orf477), classical promoter-10 TACAAT and -35 TTGAA was found at the 3′ region. The 3D structure of CTX-M-15 and ISEcp1 was generated and CTX-M-15-ISEcp1 (R299L) docking and mutation showed a reduction in hydrogen bonds. Co-occurrence of antibiotics and HMRGs (mer, sil, and ars) was found in 18, 14, and 8 isolates. PBRT analysis showed the presence of Inc. groups- B/O, F, I1, HI1, FIA, HI2, N, FIB, L/M. Molecular analysis of transconjugants showed the successful transfer of ARGs, MGEs, and HMRGs in the E. coli J53 AZR strain. ConclusionThis study highlights the occurrence of ESBL producing bacteria in the aquatic environment of Kashmir India that can serve as a reservoir of ARGs. It also discussed the molecular mechanisms of MGEs which can help in containing the spread of antibiotic resistance.

Emergence of Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae Coharboring a blaNDM-1-Carrying Virulent Plasmid and a blaKPC-2-Carrying Plasmid in an Egyptian Hospital.

ABSTRACTThe emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in Egyptian hospitals has been reported. However, the genetic basis and analysis of the plasmids associated with carbapenem-resistant hypervirulent K. pneumoniae (CR-HvKP) in Egypt have not been presented. Therefore, we attempted to decipher the plasmid sequences that are responsible for transferring the determinants of carbapenem resistance, particularly blaNDM-1 and blaKPC-2. Out of 34 K. pneumoniae isolates collected from two tertiary hospitals in Egypt, 31 were CRKP. Whole-genome sequencing revealed that our isolates were related to 13 different sequence types (STs). The most prevalent ST was ST101, followed by ST383 and ST11. Among the CRKP isolates, one isolate named EBSI036 has been reassessed by Nanopore sequencing. Genetic environment analysis showed that EBSI036 carried 20 antibiotic resistance genes and was identified as a CR-HvKP strain: it harbored four plasmids, namely, pEBSI036-1-NDM-VIR, pEBSI036-2-KPC, pEBSI036-3, and pEBSI036-4. The two carbapenemase genes blaNDM-1 and blaKPC-2 were located on plasmids pEBSI036-1-NDM-VIR and pEBSI036-2-KPC, respectively. The IncFIB:IncHI1B hybrid plasmid pEBSI036-1-NDM-VIR also carried some virulence factors, including the regulator of the mucoid phenotype (rmpA), the regulator of mucoid phenotype 2 (rmpA2), and aerobactin (iucABCD and iutA). Thus, we set out in this study to analyze in depth the genetic basis of the pEBSI036-1-NDM-VIR and pEBSI036-2-KPC plasmids. We report a high-risk clone ST11 KL47 serotype of a CR-HvKP strain isolated from the blood of a 60-year-old hospitalized female patient from the intensive care unit (ICU) in a tertiary care hospital in Egypt, which showed the cohabitation of a novel hybrid plasmid coharboring the blaNDM-1 and virulence genes and a blaKPC-2-carrying plasmid.IMPORTANCE CRKP has been registered in the critical priority tier by the World Health Organization and has become a significant menace to public health. The emergence of CR-HvKP is of great concern in terms of both disease and treatment. In-depth analysis of the carbapenemase-encoding and virulence plasmids may provide insight into ongoing recombination and evolution of virulence and multidrug resistance in K. pneumoniae. Thus, this study serves to alert contagious disease clinicians to the presence of hypervirulence in CRKP isolates in Egyptian hospitals.

Characterization of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolates from Jurong Lake, Singapore with Whole-Genome-Sequencing.

Background: The fast-spreading of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli (ESBL-producing E. coli) and ESBL genes has become a big challenge to public health. The risk of spreading ESBL genes and pathogens in the environment and community has raised public health concern. The characterizing and whole-genome sequencing studies of ESBL-producing bacteria from reservoir water in Singapore is still limited. Materials and methods: The reservoir water sample was taken from two randomly selected sampling points of the Chinese Garden (Jurong river reservoir), which is a popular reservoir park in Singapore. The bacteria of the water sample were collected with 0.45 µm filter membranes and enriched before processing with ESBL-producing E. coli screening. The collected ESBL positive isolates were further characterized by both phenotypic tests including disc diffusion and microdilution Minimum Inhibitory Concentration (MIC) test, and also genotypic test as whole-genome sequencing analysis. Besides, to investigate the transferability of the resistance gene, a conjugation test was performed with the J53 E. coli strain as the gene receptor. Result: Nine ESBL-producing E. coli isolates were collected and confirmed as ESBL-producing with both phenotypic and genotypic tests. A potential pathogen as ST131 clade A isolate was identified, and all isolates were determined to harbor a blaCTX-M gene. Among them, strain J1E4 was resistant to polymyxin E and confirmed to harboring a conjugatable mcr-1 gene. Further genetic environment analysis has reflected a conversed gene cluster formed by insert sequence (IS), blaCTX-M-15, and WbuC family cupin-fold metalloprotein, which may potentially jump from the plasmids to the chromosome. Conclusion: The first time we reported the whole genome sequencing (WGS) data of ESBL-producing E. coli including potential pathogen (ST131) present in reservoir water in Singapore. The ESBL-producing E. coli from reservoir water also carrying conjugatable colistin resistance genes which may become a risk to human health.

First report of the multiresistance gene cfr in Pasteurella multocida strains of avian origin from China

ObjectivesThe aim of this study was to investigate the presence and genetic environment of the multiresistance gene cfr gene in Pasteurella multocida of avian origin from China. MethodsA total of 113 P. multocida isolates were collected from sick poultries (ducks, chickens and geese) from 2003 to 2016 in Southern China and were screened for the presence of the cfr gene by PCR. The cfr-carrying P. multocida strains were subjected to antimicrobial susceptibility testing, S1 nuclease PFGE and Southern blot hybridisation, conjugative transfer and analysis of genetic environment of the cfr gene. ResultsAmong 113 P. multocida isolates, strains FJ6671 and FJ6683 from Muscovy duck harboured the cfr gene and presented a multiresistant phenotype. The cfr gene in the two strains was located on an ∼40-kb conjugative plasmid in different genetic environments, including ISApl12–cfr–IS26 and IS26–cfr–IS256. ConclusionsThese results demonstrate plasmid-carried cfr in P. multocida and suggest that transposition and homologous recombination mediated by IS26, ISApl1 and IS256 might have played an important role in transfer of the cfr gene in P. multocida. To the best of our knowledge, this is the first report of the cfr gene in P. multocida. Active and ongoing surveillance of cfr in P. multocida is urgently warranted.

Prevalence and Distribution Characteristics of blaKPC-2 and blaNDM-1 Genes in Klebsiella pneumoniae.

BackgroundCarbapenem-resistant Klebsiella pneumoniae infections have caused major concern and posed a global threat to public health. As blaKPC-2 and blaNDM-1 genes are the most widely reported carbapenem resistant genes in K. pneumonia, it is crucial to study the prevalence and geographical distribution of these two genes for further understanding of their transmission mode and mechanism.PurposeHere, we investigated the prevalence and distribution of blaKPC-2 and blaNDM-1 genes in carbapenem-resistant K. pneumoniae strains from a tertiary hospital and from 1579 genomes available in the NCBI database, and further analyzed the possible core structure of blaKPC-2 or blaNDM-1 genes among global genome data.Materials and MethodsK. pneumoniae strains from a tertiary hospital in China during 2013–2018 were collected and their antimicrobial susceptibility testing for 28 antibiotics was determined. Whole-genome sequencing of carbapenem-resistant K. pneumoniae strains was used to investigate the genetic characterization. The phylogenetic relationships of these strains were investigated through pan-genome analysis. The epidemiology and distribution of blaKPC-2 and blaNDM-1 genes in K. pneumoniae based on 1579 global genomes and carbapenem-resistant K. pneumoniae strains from hospital were analyzed using bioinformatics. The possible core structure carrying blaKPC-2 or blaNDM-1 genes was investigated among global data.ResultsA total of 19 carbapenem-resistant K. pneumoniae were isolated in a tertiary hospital. All isolates had a multi-resistant pattern and eight kinds of resistance genes. The phylogenetic analysis showed all isolates in the hospital were dominated by two lineages composed of ST11 and ST25, respectively. ST11 and ST25 were the major ST type carrying blaKPC-2 and blaNDM-1 genes, respectively. Among 1579 global genomes data, 147 known ST types (1195 genomes) have been identified, while ST258 (23.6%) and ST11 (22.1%) were the globally prevalent clones among the known ST types. Genetic environment analysis showed that the ISKpn7-dnaA/ISKpn27 -blaKPC-2-ISkpn6 and blaNDM-1-ble-trpf-nagA may be the core structure in the horizontal transfer of blaKPC-2 and blaNDM-1, respectively. In addition, DNA transferase (hin) may be involved in the horizontal transfer or the expression of blaNDM-1.ConclusionThere was clonal transmission of carbapenem-resistant K. pneumoniae in the tertiary hospital in China. The prevalence and distribution of blaKPC-2 and blaNDM-1 varied by countries and were driven by different transposons carrying the core structure. This study shed light on the genetic environment of blaKPC-2 and blaNDM-1 and offered basic information about the mechanism of carbapenem-resistant K. pneumoniae dissemination.

A Novel Transposon, Tn6518, Mediated Transfer of mcr-3 Variant in ESBL-Producing Aeromonas veronii.

PurposeThe aim of this study was to determine the prevalence and transmission mechanism of mcr-3 in Aeromonas spp. isolated from chicken cloaca.Materials and MethodsA. veronii w55 was isolated from chicken in 2008. PCR assay was used to detect mcr genes and putative circular intermediate. Susceptibility testing was identified by the microdilution method. WGS was performed to obtain the whole sequence. S1-PFGE and DNA southern hybridization were used to study the location of mcr-3.6.ResultsPCR-based analysis indicated that 1 out of 55 Aeromonas spp. isolates was mcr-3-positive. Whole-genome sequencing revealed that the strain A. veronii w55 belonged to novel sequence type ST514 and had two adjacent chromosomally located mcr variants, mcr-3.6 and mcr-3-like. The mcr-3.6 and mcr-3-like genes showed 93.67% and 82.84% nucleotide sequence identity, respectively, to original mcr-3 from E. coli. A. veronii w55 also exhibited resistance to extended-spectrum β-lactams and was positive for blaPER-3, and this is the first time to report blaPER-3 in A. veronii. Genetic environment analysis revealed that the segment of mcr-3.6-mcr-3-like-dgkA was flanked by five insertion sequence elements originated from Aeromonas species, and the structure of ISAs2-ISAhy2-ISAs20-mcr-3.6-mcr-3-like-dgkA-ISAs2 was designated as a novel transposon Tn6518, in which an 8405-bp circular intermediate carrying two mcr-3 variants can be looped out.ConclusionThis result suggested the mcr-3 variant genes could be disseminated between various Aeromonas species via transposon-mediated transmission.

Characterization of a blaNDM-1-carrying IncHI5 plasmid from Enterobacter cloacae complex of food-producing animal origin

To characterize an NDM-1-encoding multiresistance IncHI5 plasmid from Enterobacter cloacae complex of chicken origin. Carbapenemase genes were detected by PCR and Sanger sequencing. The MICs for the E. cloacae complex isolate and its transformant were determined by the agar dilution and broth microdilution methods. Conjugation and electrotransformation were performed to assess the horizontal transferability of the carbapenemase plasmid. Plasmid DNA was isolated from the transformant and fully sequenced using Illumina HiSeq and PacBio platforms. Plasmid stability was investigated by sequential passages on antibiotic-free medium. A circular intermediate was detected by inverse PCR and Sanger sequencing. Plasmid pNDM-1-EC12 carried a conserved IncHI5 backbone and exhibited an MDR phenotype. All antimicrobial resistance genes were clustered in a single MDR region. Genetic environment analysis revealed that the blaNDM-1 gene was in a novel complex integron, In469. Based on sequence analysis, the blaNDM-1-carrying region was thought to be inserted by homologous recombination. Inverse PCR indicated that an ISCR1-mediated circular intermediate can be formed. Plasmid pNDM-1-EC12 was stably maintained both in the parental strain and the transformant without selective pressure. Comprehensive analysis of IncHI5-type plasmids suggested that they may become another key vehicle for rapid transmission of carbapenemase genes. To the best of our knowledge, this is the first report of a fully sequenced IncHI5 plasmid recovered from an E. cloacae complex strain of food-producing animal origin. Co-occurrence of blaNDM-1 with genes encoding resistance to other antimicrobial agents on the same IncHI5 plasmid may result in the co-selection of blaNDM-1 and facilitates its persistence and rapid dissemination.

Genomic Analysis Of A KPC-2-Producing Klebsiella Pneumoniae ST11 Outbreak From A Teaching Hospital In Shandong Province, China.

PurposeKlebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae bacteria causes nosocomial infections worldwide. However, KPC-producing K. pneumoniae outbreak has never been reported in Shandong Province, China. The purpose of our study was to elucidate the epidemiological and drug resistance mechanisms of KPC-producing K. pneumoniae strains collected from a large teaching hospital in Shandong during the outbreak. Moreover, we attempted to characterize the genetic environment and phylogenetic analysis of blaKPC-2 in outbreak isolates.MethodsWe monitored a 64-day outbreak of infection in a general hospital in Shandong Province, and the bacteria causing the infection were all ST11-type K. pneumoniae. The genotype correlation of KPC-producing K. pneumoniae isolates was assessed by whole-genome sequencing (WGS) phylogenetic analysis. Subsequent studies included antibiotic susceptibility testing, multilocus sequence typing (MLST) and S1-pulsed-field gel electrophoresis (S1-PFGE), Southern blot hybridization.ResultsFrom February 1, 2018 to April 5, 2018, 14 KPC-producing K. pneumoniae isolates from different wards were collected. All 14 isolates were resistant to carbapenems and carried the extended-spectrum β-lactamase (ESBL) gene as well as fosA, and sul genes. Whole-genome analysis showed that all 14 the outbreak isolates were all ST11 type. The blaKPC-2 carrying plasmids were all belong to IncFIIK2 type, and the size ranged from 94 kb to 368 kb.ConclusionAs far as we know, this report first describes the genomics characterization of KPC-2-producing K. pneumoniae outbreak isolates from Shandong Province, China. In our study, these isolates appeared to be cloned, and ST11 K. pneumoniae was the major clone caused the outbreak. Therefore, routine surveillance of such strains in this region is urgently warranted.

Prevalence and molecular epidemiology characteristics of carbapenem-resistant Escherichia coli in Heilongjiang Province, China.

ObjectiveThis retrospective study was conducted to determine the prevalence and molecular epidemiology characteristics of carbapenem-resistant Escherichia coli (CRE).MethodsA total of 593 Escherichia coli (E. coli) isolates were recovered from pigs and urban river from 2009 to 2014 in Heilongjiang Province of China. Forty CRE including 22 strains isolated from fecal samples of pigs and 18 strains isolated from water samples were selected. PCR detection of resistance determinants, multi-locus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and phylogenetic groups were performed to characterize CRE isolates. Conjugation experiments, plasmid stability testing, PCR-based replicon typing (PBRT), and PCR mapping were conducted to analyze blaNDM-carrying plasmids. In vitro time–growth studies and competition experiments were carried out to assess the fitness impact of NDM carriage.ResultsFive NDM-1-positive E. coli isolates were identified from water samples. Genetic environment analysis revealed that a cluster of genes (ISAba125-blaNDM-1-bleMBL-ΔtrpF) was detected in all of the NDM-1-positive isolates. Conjugation assays showed that blaNDM-1 could be successfully transferred to E. coli J53 from 5 donor strains at frequencies of 4.6×10−5 to 2.6×10−2. The plasmids from all transconjugants belonged to different plasmid replicon types including IncA/C (n=2), IncFII (n=1) and IncX3 (n=2). In vitro time–growth studies revealed that blaNDM-1 did not have a significant impact on cell proliferation. Meanwhile, competition experiments showed that the acquisition of blaNDM-1 can place an energy burden on the bacterial host and incur fitness cost. However, plasmid stability testing showed that blaNDM-1-carrying plasmid remained stable in the hosts after seven passages without antimicrobial selection.ConclusionThe study revealed the early molecular epidemiology and dissemination characteristics of CRE. In addition, the overall antimicrobial resistance in E. coli recovered from water samples is higher than the strains isolated from fecal samples of pigs. Furthermore, we isolated and identified five NDM-1-producing E. coli strains from water samples.