Plasmid Conjugation Experiments Research Articles

Salmonella Typhimurium with Eight Tandem Copies of blaNDM-1 on a HI2 Plasmid.

Carbapenem-resistant Salmonella has recently aroused increasing attention. In this study, a total of four sequence type 36 Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) isolates were consecutively isolated from an 11-month-old female patient with a gastrointestinal infection, of which one was sensitive to carbapenems and three were resistant to carbapenems. Via antibiotic susceptibility testing, a carbapenemases screening test, plasmid conjugation experiments, Illumina short-reads, and PacBio HiFi sequencing, we found that all four S. Typhimurium isolates contained a blaCTX-M-14-positive IncI1 plasmid. One carbapenem-sensitive S. Typhimurium isolate then obtained an IncHI2 plasmid carrying blaNDM-1 and an IncP plasmid without any resistance genes during the disease progression. The blaNDM-1 gene was located on a new 30 kb multiple drug resistance region, which is flanked by IS26 and TnAs2, respectively. In addition, the ST_F0903R isolate contained eight tandem copies of the ISCR1 unit (ISCR1-dsbD-trpF-ble-blaNDM-1-ISAba125Δ1), but an increase in MICs to carbapenems was not observed. Our work further provided evidence of the rapid spread and amplification of blaNDM-1 through plasmid. Prompting the recognition of carbapenem-resistant Enterobacterales and the initiation of appropriate infection control measures are essential to avoid the spread of these organisms.

Involvement of Tn3 transposon in formation and transmission of hypervirulent and carbapenem-resistant Klebsiella pneumoniae

ABSTRACT Hypervirulent and carbapenem-resistant Klebsiella pneumoniae poses a severe threat to public health for its high pathogenicity, transmissibility, and drug resistance. This study aims to explore the evolutionary path and the role of Tn3 transposon in the virulence and carbapenem resistance transmission of K. pneumoniae . Bioinformatics analysis and some experimental tests such as plasmid conjugation experiments, antimicrobial susceptibility testing, and virulence-associated tests were performed to explore the virulence and drug resistance transmission. The complete genome sequencing, S1 nuclease pulsed-field gel electrophoresis, and bioinformatics analysis were used to investigate their transmission mechanism mediated by Tn3 transposons. Three hypervirulent and carbapenem-resistant K. pneumoniae isolates, which were obtained from different patients at different time points in the same ward, harbored a virulence plasmid and a carbapenem resistance plasmid. They were confirmed to have first evolved from hypervirulent isolates and then acquired a bla KPC -positive plasmid (CR-hvKp evolutionary pattern). The non-conjugative virulence plasmid pVir could be transferred to other bacterial strains via mobilization by the conjugative IncN/U-type plasmid pKPC, as well as by fusing with the conjugative pKPC plasmid (mediated by Tn3-based homologous recombination) to be self-transmissible, thus transferring drug resistance and virulence. The cointegration, pVir/KPC fusion plasmid, was further resolved into pVir and pKPC between the duplicated copies of the Tn3 transposon resolution site mediated by site-specific recombination. Therefore, Tn3 transposons can mediate hypervirulent and carbapenem-resistant K. pneumoniae strains transferring drug resistance and virulence to other bacteria. We must be vigilant to emerging transposon-mediated hypervirulent and carbapenem-resistant pathogens. IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae (CRKP) is resistant to most common antibiotics, becoming the most important and prevalent nosocomial opportunity pathogen. Besides, K. pneumoniae can also cause severe community-acquired infections, such as primary liver abscess and endophthalmitis. These pathogens are commonly referred to as hvKp. CRKP and hvKp have evolved separately, each occupying its own clonal lineage and exhibiting a variety of properties. Our study provides important insights into the evolutionary events related to the arousal of virulence and drug resistance in K. pneumoniae through plasmid transmission, mediated by Tn3 transposon. Our study also provides evidence that multiple mechanisms contribute to the successful transfer of non-conjugative virulence plasmid, and the involvement of transposons enhances the efficiency. A good knowledge of its transmission mechanisms is fundamental to finding effective strategies to combat these threatening pathogens. Transposons are widely present in bacteria, spreading resistance and virulence genes between the environment and humans. Therefore, emerging transposon-mediated hypervirulent and carbapenem-resistant pathogens should be highly valued.

Functional Portrait and Genomic Feature of Carbapenem-Resistant Pseudomonas mendocina Harboring blaNDM-1 and blaIMP-1 in China.

The purpose of this research was to analyze the functional portraits and genomic features of carbapenem-resistant Pseudomonas mendocina carrying NDM-1 and IMP-1. The resistance mechanism of the strain was verified by in vivo experiments. Genomic data were aligned and analyzed in the NCBI database. Growth curve measurements were used to describe the growth characteristics of the bacteria. The virulence of P. mendocina strain was analyzed by serum killing assay and biofilm formation assay. Plasmid conjugation experiments were performed to verify the transferability of plasmids carrying drug-resistance genes. The P. mendocina strain was highly resistant to carbapenems. In addition, ST typing is unknown and has been submitted to Genebank. The strain carried two carbapenemase genes, including NDM-1 and IMP-1. Among them, blaNDM-1 was located on a 5.62832 Mb chromosome, and blaIMP-1 was located on a 172.851 Kb transferable plasmid, which was a very close relative of pIMP-NY7610 in China. The strain also had a variety of virulence genes, which were expressed in the siderophore, capsule, pilus, alginate, flagella, etc. The study suggests that the functional portrait and genomic features of carbapenem-resistant P. mendocina harboring blaNDM-1 and blaIMP-1 are unique to China. This outcome represents antibiotic resistance exhibited in the genus Pseudomonas by acquiring chromosomes and plasmid genes. The monitoring and supervision of antimicrobial usage must be strengthened since the multi-drug-resistant and moderately virulent P. mendocina will attract much attention in the near future.

Guidelines for the estimation and reporting of plasmid conjugation rates

Conjugation is a central characteristic of plasmid biology and an important mechanism of horizontal gene transfer in bacteria. However, there is little consensus on how to accurately estimate and report plasmid conjugation rates, in part due to the wide range of available methods. Given the similarity between approaches, we propose general reporting guidelines for plasmid conjugation experiments. These constitute best practices based on recent literature about plasmid conjugation and methods to measure conjugation rates. In addition to the general guidelines, we discuss common theoretical assumptions underlying existing methods to estimate conjugation rates and provide recommendations on how to avoid violating these assumptions. We hope this will aid the implementation and evaluation of conjugation rate measurements, and initiate a broader discussion regarding the practice of quantifying plasmid conjugation rates.

A clinical KPC-producing Klebsiella michiganensis strain carrying IncFII/IncFIA (HI1)/IncFIB (K) multiple replicon plasmid.

Klebsiella michiganensis is an increasingly important bacterial pathogen causing nosocomial infections in clinical patients. In this study, we described the molecular and genomic characteristics of a carbapenem-resistant K. michiganensis strain KM166 cultured from a one-month premature baby's blood sample. KM166 showed lower biofilm forming ability in optical density (OD) than K. pneumoniae NTUH-K2044 (0.271 ± 0.027 vs. 0.595 ± 0.054, p = 0.001), and the median lethal dose (0.684 lg CFU/mL) was lower than K. pneumoniae strain NTUH-K2044 (6.679 lg CFU/mL). A IncFII/IncFIA(HI1)/IncFIB(K) multiple replicon plasmid in KM166 was identified carrying three replicon types. It has low homology to Escherichia coli pMRY09-581ECO_1 and the highest homology similarity to the INcFIA/INcFII(p14)-type plasmid in K. michiganensis strain fxq plasmid pB_KPC, suggesting that this multiple replicon plasmid was unlikely to have been transmitted from E. coli and probably a transfer of repFIB replicon genes from other K. michiganensis strains into the INcFIA/INcFII(p14)-type plasmid of KM166 had occurred. Mapping of the gene environment revealed that bla KPC-2 in KM166 plasmid 3 had high identity and same Tn3-tnpR-IS481-bla KPC-2-klcA_1 genomic context structure with K. pneumoniae strain JKP55, plasmid pKPC-J5501, and bla KPC-2-carrying plasmid proved to be autonomously transferred under the help of mobile genetic elements into Escherichia coli 600 by plasmid conjugation experiment. In conclusion, we have characterized a K. michiganensis strain carrying multi-replicon IncFII/IncFIA(HI1)/IncFIB(K) plasmid and bla KPC-2-carrying IncFII(p14)/IncFIA plasmid in this study, which provided insights about the evolutionary diversity of plasmids carried by K. michiganensis.

Molecular Epidemiology and Drug Resistant Mechanism of Carbapenem-Resistant Klebsiella pneumoniae in Elderly Patients With Lower Respiratory Tract Infection.

Infection by carbapenem-resistant Klebsiella pneumoniae (CRKp) hampers the treatment of elderly patients with lower respiratory tract infection (LRTI); however, relevant data with respect to the characteristics of CRKp in elderly patients with LRTIs are limited. In the present study, K. pneumoniae isolated from elderly patients with LRTIs was collected and identified by VITEK-MS. VITEK 2 compact was used for drug sensitivity test to screen CRKps, and broth dilution method was used for drug sensitivity of tigecycline and colistin. The resistance genes, virulence genes, and serotypes of CRKps were detected via polymerase chain reaction. The homology of CRKps was analyzed via PFGE and MLST. Moreover, plasmid conjugation experiment was carried out to determine the transferability of carbapenem resistance. PCR-based replicon typing (PBRT) and S1 nuclease-PFGE were conducted for plasmid profiling. From January 2019 to August 2019, 258 elderly patients with LRTIs caused by K. pneumoniae were observed; of these, 31 (12.02%) infections were caused by CRKp strains. Majority of the patients were admitted to the intensive care unit and neurosurgery wards. Intracranial hemorrhage and pneumonia were the most common underlying diseases. Furthermore, 29 patients infected by CRKp had been exposed to various antimicrobial drugs before the positive culture. All isolates exhibited high resistance to β-lactam antibiotics. The predominant carbapenem resistance gene was blaKPC−2, and CRKps carrying blaKPC−2 were all ST11 type. Two blaNDM−5 carrying isolates were assigned to ST307 and ST1562, respectively. Conjugative assays revealed that plasmids harboring blaNDM−5 gene were self-transmissible. Plasmid analysis suggested that two blaNDM−5 were located on a ~45 kb IncX3 type plasmid. The high incidence of CRKp in elderly patients with LRTIs indicates the urgent need for further surveillance and strict infection control measures.

The Plasmid-Borne tet(A) Gene Is an Important Factor Causing Tigecycline Resistance in ST11 Carbapenem-Resistant Klebsiella pneumoniae Under Selective Pressure

The emergence and prevalence of tigecycline-resistant Klebsiella pneumoniae have seriously compromised the effectiveness of antimicrobial agents in the treatment of infections. To explore the role of the plasmid-borne tet(A) gene in tigecycline resistance in carbapenem-resistant K. pneumoniae (CRKP), a total of 63 CRKP isolates were collected from a tertiary hospital in Hangzhou, China. The minimum inhibitory concentration (MIC) of tigecycline, mutation rate of tet(A) gene, genetic surroundings of tet(A)-carrying transmissible plasmid and the contribution of tet(A) mutation to tigecycline resistance were analyzed using antimicrobial susceptibility test, whole-genome sequencing, tigecycline resistance evolution experiment, and plasmid conjugation experiment. Our results showed that 52.4% (33 isolates) of the test isolates carried the tet(A) gene; among them, 75.8% (25 isolates) exhibited a tigecycline non-susceptible phenotype (MIC = 4 mg/L). Three clonal groups (cluster I, cluster II, and cluster III) were identified in these tet(A)-bearing isolates. All 17 isolates belonged to serotype KL21 (cluster I), which differed by only 13 SNPs, suggesting a clonal spread of tet(A)-positive ST11 K. pneumoniae with serotype KL21 occurred in the sampling hospital. The induction of tigecycline resistance experiments showed that 71.4% of strains evolved tet(A) mutations and developed a high-level tigecycline resistance. Eight amino acid substitutions were identified in these mutants. The most common amino acid substitution was A370V, followed by S251A and G300E. Twelve isolates carrying tet(A) mutants succeeded in the filter mating experiment with a conjugation efficiency of 10–3–10–8. Tigecycline MICs in E. coli EC600 transconjugants with a mutated tet(A) were 2 to 8-fold higher than those in E. coli EC600 transconjugants with a wild-type tet(A). One ColRNAI/IncFII type and two IncFII type tet(A)-bearing conjugative plasmids were identified in this study, including a class 1 integron containing multiple antibiotic resistance genes, i.e., tet(A), qnrS1, blaLAP–2, catA2, sul2, and dfrA14. Our study revealed the wide-spread situation of plasmid-borne tet(A) gene in clinical CRKP, and mutation of tet(A) is a potential driven force that lead to tigecycline resistance.

Resistance of Klebsiella pneumoniae Strains Carrying bla NDM-1 Gene and the Genetic Environment of bla NDM-1.

ObjectiveRegional dissemination is the major cause of the widespread prevalence of a plasmid-encoding NDM-1 enzyme. We investigated the drug resistance, joint efficiency, and gene environment of a Klebsiella pneumoniae strain carrying blaNDM–1 gene.Materials and MethodsCarbapenem-non-susceptible strains were analyzed using the VITEK 2 Compact. Strains carrying blaNDM–1 were identified using polymerase chain reaction and sequencing. Antimicrobial susceptibility testing and plasmid conjugation experiments were then conducted. Strains carrying blaNDM–1 were subjected to Southern blot analysis. After the gene mapping of blaNDM–1, library construction, and sequencing, plasmids were subsequently spliced and genotyped using the software Glimmer 3.0, and then analyzed using Mauve software.ResultsAmong 1735 carbapenem-non-susceptible strains, 54 strains of blaNDM–1-positive bacteria were identified, which consisted of 44 strains of K. pneumoniae, 8 strains of Acinetobacter baumannii and 2 strains of Escherichia coli. Strains carrying blaNDM–1 had a resistance rate of more than 50% in most antibiotics. Plasmid conjugation between strains carrying blaNDM–1 and E. coli strain J53 had a success rate of 50%. Southern blot analysis indicated that each strain had multiple plasmids containing blaNDM–1. Among the five plasmids containing blaNDM–1 in K. pneumoniae for sequencing, two plasmids with complete sequences were obtained. The findings were as follows: (i) The p11106 and p12 plasmids were highly similar to pNDM-BTR; (ii) the p11106 and p12 plasmids showed differences in the 20–30 kb region (orf00032–orf00043) from the other six plasmids; and (iii) blaNDM–1 was located at orf00037, while ble was found at orf00038. Two tnpA genes were located in the upstream region, and orf00052 (tnpA) in the 36 kb region was in the downstream sequence.ConclusionblaNDM–1-containing bacteria exhibit multidrug resistance, which rapidly spreads and is transferred through efficient plasmid conjugation; the multidrug resistance of these bacteria may be determined by analyzing their drug-resistant plasmids. The presence of ble and tnpA genes suggests a possible hypothesis that blaNDM–1 originates from A. baumannii, which is retained in K. pneumoniae over a long period by transposition of mobile elements.

Multispecies Outbreak of Verona Integron-Encoded Metallo-ß-Lactamase-Producing Multidrug Resistant Bacteria Driven by a Promiscuous Incompatibility Group A/C2 Plasmid.

Antibiotic resistance is often spread through bacterial populations via conjugative plasmids. However, plasmid transfer is not well recognized in clinical settings because of technical limitations, and health care-associated infections are usually caused by clonal transmission of a single pathogen. In 2015, multiple species of carbapenem-resistant Enterobacteriaceae (CRE), all producing a rare carbapenemase, were identified among patients in an intensive care unit. This observation suggested a large, previously unrecognized plasmid transmission chain and prompted our investigation. Electronic medical record reviews, infection control observations, and environmental sampling completed the epidemiologic outbreak investigation. A laboratory analysis, conducted on patient and environmental isolates, included long-read whole-genome sequencing to fully elucidate plasmid DNA structures. Bioinformatics analyses were applied to infer plasmid transmission chains and results were subsequently confirmed using plasmid conjugation experiments. We identified 14 Verona integron-encoded metallo-ß-lactamase (VIM)-producing CRE in 12 patients, and 1 additional isolate was obtained from a patient room sink drain. Whole-genome sequencing identified the horizontal transfer of blaVIM-1, a rare carbapenem resistance mechanism in the United States, via a promiscuous incompatibility group A/C2 plasmid that spread among 5 bacterial species isolated from patients and the environment. This investigation represents the largest known outbreak of VIM-producing CRE in the United States to date, which comprises numerous bacterial species and strains. We present evidence of in-hospital plasmid transmission, as well as environmental contamination. Our findings demonstrate the potential for 2 types of hospital-acquired infection outbreaks: those due to clonal expansion and those due to the spread of conjugative plasmids encoding antibiotic resistance across species.

First Reported Nosocomial Outbreak Of NDM-5-Producing Klebsiella pneumoniae In A Neonatal Unit In China.

PurposeCarbapenem-resistant Klebsiella pneumoniae (CRKP) have emerged worldwide and also being a major threat to children and neonate. In this study, we describe a nosocomial outbreak of NDM-5-producing Klebsiella pneumoniae in neonatal unit of a teaching hospital in China from September 2015 to September 2016.Patients and methodsWe collected 12 carbapenem-resistant K. pneumoniae outbreak strains from 12 newborns and characterized these isolates for their antimicrobial susceptibility, clone relationships, and multi-locus sequence types using vitek-2 compact system, pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Resistant genes were detected by using PCR and sequencing. Plasmid conjugation experiment was carried out to determine the transferability of carbapenem resistance. PCR-based replicon typing (PBRT), S1 nuclease-PFGE, and southern blotting were conducted for plasmid profiling.ResultsAll 12 K. pneumoniae isolates were resistant to carbapenems and carried blaNDM-5, blaTEM-1 and blaSHV-11. Furthermore, PFGE analysis showed that NDM-5-producing K. pneumoniae were clonally related and MLST assigned them to sequence type 337. Conjugative assays showed that plasmids harboring blaNDM-5 gene were self-transmissible. Plasmid analysis suggested that all blaNDM-5 gene located on a ~45 kb IncX3 type plasmid.ConclusionTo the best of our knowledge, this is the first report of a clone outbreak of blaNDM-5-carrying K. pneumoniae isolates from neonates. There is an urgent need for effective infection control measures to prevent blaNDM-5 variants from becoming epidemic in the neonates in the future.

Co-carrying of KPC-2, NDM-5, CTX-M-3 and CTX-M-65 in three plasmids with serotype O89: H10 Escherichia coli strain belonging to the ST2 clone in China

Carbapenem-resistant Enterobacteriaceae strains as a new serious threat for the public health have been increasingly reported worldwide. In this study, one multi-resistant Escherichia coli strain ZSH6 which co-carried blaKPC-2, blaNDM-5 and blaCTX-M, was isolated from human blood sample. By using plasmid conjugation experiments, ZSH6 was found to harbor three plasmids carrying the blaNDM-5 gene, the blaKPC-2 and blaCTX-M gene, respectively. Whole-genome sequencing of ZSH6 yielded 122 scaffolds of chromosomal DNA and three circular plasmids including pZSH6-blaKPC-2 (46,319 bp), pZSH6-blaNDM-5 (46,161bp) and pZSH6-blaCTX-M (184,723). The isolate was classified to Sequence Type 2 and to the O89: H10 serotype. The results of genome analyses revealed that ZSH6 carried three virulence factors (capU, gad and iss) and twenty resistance genes [blaKPC-2blaNDM-5, blaCTX-M-3, blaCTX-M-65, blaTEM-1, floR, tet(A), tet(B), dfrA17, aadA5, sul1, mdf(A), mph(A), erm(B), aph(3′)-Ia, aph(3′)-Ib, aph(4)-Ia, aph(6)-Id, aac(3)-Iva, aac(3)-IId]. Therefore, the co-existence of such a large number of resistance genes in multiple plasmids making ZSH6 highly resistant to almost all kinds of commonly used antibiotics, and brings a serious challenge for resistance control and clinical treatment of infections caused by this bacterium.

High Prevalence of bla NDM Variants Among Carbapenem-Resistant Escherichia coli in Northern Jiangsu Province, China.

The continuous emergence of carbapenem-resistant Escherichia coli (CRECO) presents a great challenge to public health. New Delhi metallo-lactamase (NDM) variants are widely disseminated in China, so the research on the prevalence and transmission of diverse blaNDM variants is urgently needed. In the present study, 54 CRECO isolates were collected from 1,185 Escherichia coli isolates in five hospitals in Northern Jiangsu Province, China from September 2015 to August 2016. Antimicrobial susceptibility tests, PCR detection of resistance determinants, multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed to characterize these strains. Plasmid conjugation experiments were carried out to determine the transferability of resistant genes from selected isolates. PCR-based replicon typing (PBRT), S1 nuclease-PFGE, and Southern blotting were conducted for plasmid profiling. Carbapenemase genes were detectable in all CRECO isolates, among which thirty-one CRECO isolates were found to carry blaNDM−5 (54.7%), while, blaNDM−1, blaNDM−7, blaNDM−4, blaNDM−9, and blaKPC−2 were identified in 14, five, two, one, and one isolates, respectively. MLST results revealed 15 different STs and four new STs were first reported to be linked with NDM-producing isolates. PFGE typing showed that no more than two isolates with the same ST appeared to the same band pattern except three ST410 isolates. Twenty-six selected NDM-producing isolates were successfully transferred to E. coli J53 by conjugation experiments. Notably, 50.0% (13/26) of blaNDM variants were found to be carried by ~55 kb IncX3 plasmid. Our study reported a high prevalence of blaNDM variants, especially blaNDM−5, in Northern Jiangsu province, China. Diverse blaNDM variants were mainly carried by ~55 kb IncX3 plasmids, suggesting that the fast evolution and high transferability of this kind of plasmid promote the high prevalence of blaNDM variants. Therefore, large-scale surveillance and effective infection control measures are also urgently needed to prevent diverse blaNDM variants from becoming epidemic in the future.

CTX-M-55-type ESBL-producing Salmonella enterica are emerging among retail meats in Phnom Penh, Cambodia.

Salmonella enterica is a leading cause of human gastroenteritis. S. enterica strains that produce ESBLs (ESBL-Salm) remain rare in Europe and North America, but less is known about their prevalence among animal-derived foods in countries with weaker food safety practices and unregulated veterinary antibiotic use. To examine the prevalence and characteristics of ESBL-Salm from retail meats in Phnom Penh, Cambodia. We tested fish, pork and chicken from two markets for ESBL- and carbapenemase-producing Salmonella from September-December 2016, using cefotaxime- and ertapenem-supplemented media, respectively. ESBL-Salm were sequenced and their genomes characterized. We performed plasmid conjugation experiments to assess the co-transferability of ESBL-encoding genes and MDR phenotypes. Twenty-six of 150 fish and meat samples (17%) were positive for ESBL-Salm, including 10/60 fish (17%), 15/60 pork (25%) and 1/30 chicken (3%). Carbapenemase-producing Salmonella strains were not detected. Pork-origin ESBL-Salm were primarily serotypes Rissen (10/15) or a monophasic variant of Typhimurium 4,5,12:i:- (3/15), whereas Saintpaul (3/10) and Newport (4/10) were more common among fish. Most ESBL enzymes were encoded by blaCTX-M-55 genes (24/26) harboured on conjugative IncA/C2 (n = 14) or IncHI2 (n = 10) plasmids. Resistance to up to six additional drug classes was co-transferred by each plasmid type. ESBL-Salm were resistant to almost every antibiotic recommended for severe salmonellosis treatment. CTX-M-55-type S. enterica are highly prevalent among pork and fish from Phnom Penh markets and their spread appears to be mediated by MDR IncA/C2 and IncHI2 plasmids. Food safety must be improved and veterinary antibiotic use should be regulated to protect public health.

Use of Colistin and Other Critical Antimicrobials on Pig and Chicken Farms in Southern Vietnam and Its Association with Resistance in Commensal Escherichia coli Bacteria.

ABSTRACTAntimicrobial resistance (AMR) is a global health problem, and emerging semi-intensive farming systems in Southeast Asia are major contributors to the AMR burden. We accessed 12 pig and chicken farms at key stages of production in Tien Giang Province, Vietnam, to measure antimicrobial usage and to investigate the prevalence of AMR to five critical antimicrobials (β-lactams, third-generation cephalosporins, quinolones, aminoglycosides, and polymyxins) and their corresponding molecular mechanisms among 180 Escherichia coli isolates. Overall, 94.7 mg (interquartile range [IQR], 65.3 to 151.1) and 563.6 mg (IQR, 398.9 to 943.6) of antimicrobials was used to produce 1 kg (live weight) of chicken and pig, respectively. A median of 3 (out of 8) critical antimicrobials were used on pig farms. E. coli isolates exhibited a high prevalence of resistance to ampicillin (97.8% and 94.4% for chickens and pigs, respectively), ciprofloxacin (73.3% and 21.1%), gentamicin (42.2% and 35.6%), and colistin (22.2% and 24.4%). The prevalence of a recently discovered colistin resistance gene, mcr-1, was 19 to 22% and had strong agreement with phenotypic colistin resistance. We conducted plasmid conjugation experiments with 37 mcr-1 gene-positive E. coli isolates and successfully observed transfer of the gene in 54.0% of isolates through a plasmid of approximately 63 kb, consistent with one recently identified in China. We found no significant correlation between total use of antimicrobials at the farm level and AMR. These data provide additional insight into the role of mcr-1 in colistin resistance on farms and outline the dynamics of phenotypic and genotypic AMR in semi-intensive farming systems in Vietnam.IMPORTANCE Our study provides accurate baseline information on levels of antimicrobial use, as well as on the dynamics of phenotypic and genotypic resistance for antimicrobials of critical importance among E. coli over the different stages of production in emerging pig and poultry production systems in Vietnam. E. coli isolates showed a high prevalence of resistance (>20%) to critically important antimicrobials, such as colistin, ciprofloxacin, and gentamicin. The underlying genetic mechanisms identified for colistin (the mcr-1 gene) and quinolone (gyrA gene mutations) are likely to play a major role in AMR to those compounds. Conjugation experiments led to the identification of a 63-kb plasmid, similar to one recently identified in China, as the potential carrier of the mcr-1 gene. These results should encourage greater restrictions of such antimicrobials in Southeast Asian farming systems.

The study of genotype and plasmid transfer of carbapenem-resistant Enterobacteriaceae carrying blaNDM-1 with blaIMP-4 or blaKPC-2

Objective To analyze the genotype and plasmid transfer of Enterobacteriaceae carring blaNDM-1 with blaIMP-4 or blaKPC-2. Methods From April 2012 to October 2014, a total of 33 non-repeatitive carbapenem-resistant Enterobacteriaceae (including Imipenem-resistant, meropenem-resistant or Ertapenem-resistant) were isolated from 5 hospitals in Wenzhou and Hangzhou. Identification and antimicrobial susceptibility test were performed using Vitek2 Compact automatic microbiology analyzer. Phenotypes of carbapenemase were screened using modified Hodge test and ethylenediamine tetraacetic acid-disk synergy test. Extended spectrum β lactamase test was determined by the double disk combination test which was recommended by Clinical and Laboratory Standards Institute. AmpC activity was tested by a three-dimensional Cefoxitin method. Drug resistant genes including blaNDM-1 and linkage of ISAba125-NDM were detected by polymerase chain reaction (PCR). The purified PCR products were cloned and sequenced. Plasmid conjugation experiment and elimination method were carried out to test partial bacterial strain and K. pneumoniae carrying blaNDM-1 with blaIMP-4 or blaKPC-2. Results Of the 33 non-repeatitive carbapenem-resistant Enterobacteriaceae, 28 were strains of K. pneumoniae, 1 strain of K. oxytoca, 2 strains of Escherichia coli, 1 strain of K. planticola and 1 strain of E. cloacae. Thirteen strains were isolated from Hospital of Sir Run Run Shaw of Zhejiang University, thirteen from Wenzhou Hospital of Traditional Chinese Medicine, one from Wenzhou People's Hospital, three from the First Affiliated Hospital of Wenzhou Medical University and three from Wenzhou Hospital of Integrated Traditional Chinese and Western Medicine. Thirty-one strains were confirmed as carbapenemase-producing with 24 of blaKPC-2, 3 of blaNDM-1, 1 of blaNDM-5 and 3 of blaIMP-4. Among them, one strain carried blaNDM-1 with blaIMP-4 and one strain carried blaNDM-1with blaKPC-2, respectively. The plasmid transfer and conjugation experiment was performed between strains carrying blaNDM-1 and Escherichia coli EC600 or K. pneumoniae ATCC13833 and genes of blaNDM-1 and ISAba125-NDM were obtained. Conclusions blaKPC-2 gene is the popular carbapenemase genotype. blaNDM-1 or blaNDM-5 may be correlated with linkage gene of ISAba125-NDM. Coexistence of blaNDM-1 carrying blaIMP-4 or blaKPC-2 is detected in the same strain, respectively. Enough importance should be attached to the strains, because most of them are multiple drug resistance with related genes located in the plasmid which is easily spread between strains. Key words: Carbapenemase; Enterobacteriaceae; NDM-1; Genotype

Function of the CRISPR-Cas System of the Human Pathogen Clostridium difficile.

ABSTRACTClostridium difficile is the cause of most frequently occurring nosocomial diarrhea worldwide. As an enteropathogen, C. difficile must be exposed to multiple exogenous genetic elements in bacteriophage-rich gut communities. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems allow bacteria to adapt to foreign genetic invaders. Our recent data revealed active expression and processing of CRISPR RNAs from multiple type I-B CRISPR arrays in C. difficile reference strain 630. Here, we demonstrate active expression of CRISPR arrays in strain R20291, an epidemic C. difficile strain. Through genome sequencing and host range analysis of several new C. difficile phages and plasmid conjugation experiments, we provide evidence of defensive function of the CRISPR-Cas system in both C. difficile strains. We further demonstrate that C. difficile Cas proteins are capable of interference in a heterologous host, Escherichia coli. These data set the stage for mechanistic and physiological analyses of CRISPR-Cas-mediated interactions of important global human pathogen with its genetic parasites.

Analysis of the mechanism of drug resistance of VIM-2-type metallo-β-lactamase-producing Acineto- bacter baumannii isolated from burn patients and its homology

To study the drug resistance of Acinetobacter baumannii (AB) producing VIM-2-type metallo-β-lactamase (MBL) isolated from burn patients of our ward against carbapenem antibiotics and its homology. A total of 400 strains of AB (identified) were isolated from sputum, urine, blood, pus, and wound drainage. of burn patients hospitalized in our ward from September 2011 to March 2014. Drug resistance of the 400 strains of AB to 15 antibiotics, including compound sulfamothoxazole, aztreonam, etc. , was tested using the automatic microorganism identifying and drug sensitivity analyzer. Among the carbapenems-resistant AB isolates, modified Hodge test was applied to screen carbapenemase-producing strains. The carbapenemase genes of the carbapenemase-producing strains, and the mobile genetic elements class I-integron (Intl1) gene and conserved sequence (CS) of carbapenemase-producing strains carrying blaVIM-2 gene were determined with PCR and DNA sequencing. For carbapenemase-producing strains carrying blaVIM-2 gene, synergism test with imipenem-ethylene diamine tetraacetic acid (EDTA) and enhancement test with imipenem-EDTA and ceftazidime-EDTA were used to verify the MBL-producing status. Drug resistance of the VIM-2-type MBL-producing AB strains was analyzed. For VIM-2-type MBL-producing AB strains, plasmid conjugation experiment was used to explore the transfer of plasmid; outer membrane protein (OMP) CarO gene was detected by PCR. For VIM-2-type MBL-producing AB strains carrying CarO gene, the protein content of CarO was analyzed with sodium dodecyl sulfate polyacrylamide gel electro- phoresis. The repetitive consensus sequence of Enterobacteriaceae genome PCR (ERIC-PCR) was carried out for gene typing of VIM-2-type MBL-producing AB strains to analyze their homology. (1) The resistant rates of the 400 strains of AB against levofloxacin and compound sulfamethoxazole were low. A total of 381 carbapenems-resistant AB strains were screened, including 240 carbepenemase-producing strains. (2) Out of the 240 carbepenemase-producing strains, 18 strains were found to harbor the blaVIM-2 gene, accounting for 7.5%; 133 strains carried the blaTEM-1 gene, accounting for 55.42%; 195 strains carried the blaOXA23 gene, accounting for 81.25%; 188 strains carried the bla(armA) gene, accounting for 78.33%. (3) Eighteen carbepenemase-producing strains which carried the bla(VIM-2) gene were found to carry the Intl1 gene, showing the Intl1-VIM linkage. Simultaneously, Intl1 variable area CS showed diversity. (4) Eighteen carbepenemase-producing strains which carried the blaVIM-2 gene were verified to produce MBL. The resistant rates of the 18 strains of AB against compound sulfamethoxazole were the lowest, followed by levofloxacin and cefoperazone/sulbactam, and those against the other antibiotics were above 60.00%. (5) Through multiple joint tests, plasmid conjugation experiment positive transfer strain was not found in 18 VIM-2-type MBL-producing AB strains. (6) Nine out of the 18 VIM-2-type MBL-producing AB strains were found to carry CarO gene. The OMP CarO of VIM-2-type MBL-producing AB strains carrying CarO gene was lost or lowered in the protein content. (7) The 18 VIM-2-type MBL-producing AB strains were classified into 6 genotypes by the ERIC-PCR. There were respectively 6, 4, 3, and 1 stain (s) in genotypes A, B, C, and F, and there were 2 strains in genotypes D and E respectively. The resistance mechanism of AB against carbapenems is mainly mediated by blaTEM-1, blaOXA-23, and bla(arma); meanwhile, VIM-2-type MBL-producing and lack or change in OMP CarO are attributable to carbapenems resistance of clinically isolated AB from burn wards, and the Intl1 gene may take a part in blaVIM-2 gene transmission.

Identification and characterization of the first Escherichia coli strain carrying NDM-1 gene in China.

New Delhi metallo-β-lactamase-1 (NDM-1), an acquired class B carbapenemase, is a significant clinical threat due to its extended hydrolysis of β-lactams including carbapenems. In this study, we identified the first confirmed clinical isolate of Escherichia coli BJ01 harboring bla NDM-1 in China. The isolate is highly resistant to all tested antimicrobials except polymyxin. bla NDM-1, bla CTX-M-57, and bla TEM-1 were identified in the isolate. bla NDM-1 was transferable to E. coli EC600 and DH5α in both plasmid conjugation experiments and plasmid transformation tests. BJ01 was identified as a new sequence type, ST224, by multilocus sequence typing. Analysis of genetic environment shows complex transposon-like structures surrounding the bla NDM-1 gene. Genetic analysis revealed that the region flanking bla NDM-1 was very similar to previously identified NDM-positive Acinetobacter spp. isolated in China. The findings of this study raise attention to the emergence and spread of NDM-1-carrying Enterobacteriaceae in China.

Gene Clusters Located on Two Large Plasmids Determine Spore Crystal Association (SCA) in Bacillus thuringiensis Subsp. finitimus Strain YBT-020

Crystals in Bacillus thuringiensis are usually formed in the mother cell compartment during sporulation and are separated from the spores after mother cell lysis. In a few strains, crystals are produced inside the exosporium and are associated with the spores after sporulation. This special phenotype, named ‘spore crystal association’ (SCA), typically occurs in B. thuringiensis subsp. finitimus. Our aim was to identify genes determining the SCA phenotype in B. thuringiensis subsp. finitimus strain YBT-020. Plasmid conjugation experiments indicated that the SCA phenotype in this strain was tightly linked with two large plasmids (pBMB26 and pBMB28). A shuttle bacterial artificial chromosome (BAC) library of strain YBT-020 was constructed. Six fragments from BAC clones were screened from this library and discovered to cover the full length of pBMB26; four others were found to cover pBMB28. Using fragment complementation testing, two fragments, each of approximately 35 kb and located on pBMB26 and pBMB28, were observed to recover the SCA phenotype in an acrystalliferous mutant, B. thuringiensis strain BMB171. Furthermore, deletion analysis indicated that the crystal protein gene cry26Aa from pBMB26, along with five genes from pBMB28, were indispensable to the SCA phenotype. Gene disruption and frame-shift mutation analyses revealed that two of the five genes from pBMB28, which showed low similarity to crystal proteins, determined the location of crystals inside the exosporium. Gene disruption revealed that the three remaining genes, similar to spore germination genes, contributed to the stability of the SCA phenotype in strain YBT-020. Our results thus identified the genes determining the SCA phenotype in B. thuringiensis subsp. finitimus.