S1 Nuclease Pulsed-field Gel Electrophoresis Research Articles

Complete Genome Sequences of Two Novel KPC-2-Producing IncU Multidrug-Resistant Plasmids From International High-Risk Clones of Escherichia coli in China.

The rapidly increasing prevalence of Klebsiella pneumoniae carbapenemase 2 (KPC-2)-producing bacteria has become a serious challenge to public health. Currently, the blaKPC–2 gene is mainly disseminated through plasmids of different sizes and replicon types. However, the plasmids carrying the blaKPC–2 gene have not been fully characterized. In this study, we report the complete genome sequences of two novel blaKPC–2-harboring incompatibility group U (IncU) plasmids, pEC2341-KPC and pEC2547-KPC, from international high-risk clones of Escherichia coli isolated from Zhejiang, China. Two KPC-2-producing E. coli isolates (EC2341 and EC2547) were collected from clinical samples. Whole-genome sequencing (WGS) analysis indicated that EC2341 and EC2547 belonged to the ST410 and ST131 clones, respectively. S1-nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern blot and conjugation experiments confirmed the presence of the blaKPC–2 gene on the pEC2341-KPC plasmid and that this was a conjugative plasmid, while the blaKPC–2 gene on the pEC2547-KPC plasmid was a non-conjugative plasmid. In addition, plasmid analysis further revealed that the two blaKPC–2-harboring plasmids have a close evolutionary relationship. To the best of our knowledge, this is the first report of E. coli strains carrying the blaKPC–2 gene on IncU plasmids. The emergence of the IncU-type blaKPC–2-positive plasmid highlights further dissemination of blaKPC–2 in Enterobacteriaceae. Therefore, effective measures should be taken immediately to prevent the spread of these blaKPC–2–positive plasmids.

First Report of Coexistence of bla SFO-1 and bla NDM-1 β-Lactamase Genes as Well as Colistin Resistance Gene mcr-9 in a Transferrable Plasmid of a Clinical Isolate of Enterobacter hormaechei.

Many antimicrobial resistance genes usually located on transferable plasmids are responsible for multiple antimicrobial resistance among multidrug-resistant (MDR) Gram-negative bacteria. The aim of this study is to characterize a carbapenemase-producing Enterobacter hormaechei 1575 isolate from the blood sample in a tertiary hospital in Wuhan, Hubei Province, China. Antimicrobial susceptibility test showed that 1575 was an MDR isolate. The whole genome sequencing (WGS) and comparative genomics were used to deeply analyze the molecular information of the 1575 and to explore the location and structure of antibiotic resistance genes. The three key resistance genes (blaSFO–1, blaNDM–1, and mcr-9) were verified by PCR, and the amplicons were subsequently sequenced. Moreover, the conjugation assay was also performed to determine the transferability of those resistance genes. Plasmid files were determined by the S1 nuclease pulsed-field gel electrophoresis (S1-PFGE). WGS revealed that p1575-1 plasmid was a conjugative plasmid that possessed the rare coexistence of blaSFO–1, blaNDM–1, and mcr-9 genes and complete conjugative systems. And p1575-1 belonged to the plasmid incompatibility group IncHI2 and multilocus sequence typing ST102. Meanwhile, the pMLST type of p1575-1 was IncHI2-ST1. Conjugation assay proved that the MDR p1575-1 plasmid could be transferred to other recipients. S1-PFGE confirmed the location of plasmid with molecular weight of 342,447 bp. All these three resistant genes were flanked by various mobile elements, indicating that the blaSFO–1, blaNDM–1, and mcr-9 could be transferred not only by the p1575-1 plasmid but also by these mobile elements. Taken together, we report for the first time the coexistence of blaSFO–1, blaNDM–1, and mcr-9 on a transferable plasmid in a MDR clinical isolate E. hormaechei, which indicates the possibility of horizontal transfer of antibiotic resistance genes.

Emergence of blaNDM-9-bearing tigecycline-resistant Klebsiella aerogenes of chicken origin

The aim of this study was to characterise a tigecycline-resistant blaNDM-9-bearing Klebsiella aerogenes strain (HNHF1) of chicken origin. Strain HNHF1 was characterised by phenotypic antimicrobial susceptibility testing, PCR, conjugation assays, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), whole-genome sequencing and bioinformatics analysis. The blaNDM-9 gene was located on an IncHI2 plasmid (pHNHF1_NDM-9) carrying various antimicrobial resistance genes. Moreover, the genetic context ΔISAba125-blaNDM-9-bleMBL-trpF is similar to other blaNDM-bearing genetic contexts. TA cloning experiments showed that tet(A) variants may play a partial role in high-level tigecycline resistance in HNHF1. This is the first report of a tigecycline-resistant blaNDM-9-bearing IncHI2 plasmid in a K. aerogenes ST4 isolate of animal origin, which poses a great threat to public health. Further comprehensive surveillance is needed.

Emergence of transferable ceftazidime–avibactam resistance in KPC-producing Klebsiella pneumoniae due to a novel CMY AmpC β-lactamase in China

ObjectivesTo evaluate the molecular mechanisms of ceftazidime/avibactam (CAZ/AVI) resistance in six Klebsiella pneumoniae strains that co-produce K. pneumoniae carbapenemase (KPC)-2 and a novel variant of CMY cephalosporinase in a Chinese hospital. MethodsAntimicrobial susceptibility was determined by broth microdilution. Whole-genome sequencing (WGS) was performed to investigate potential resistance determinants. Plasmid conjugation, electroporation, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) hybridization and cloning experiment were carried out to investigate the resistance plasmids and genes. ResultsA high level of CAZ/AVI resistance was observed in six KPC-Kp strains (MIC 128 mg/L). Five strains were isolated in 2015 and one in 2016, before the approval of CAZ/AVI in China. Sequence analysis indicated that all the strains belonged to sequence type (ST) 11 and uniformly carried a novel CMY AmpC β-lactamase gene, designated blaCMY-172. When compared with CMY-2, CMY-172 has a deletion of three consecutive amino acids (K290, V291 and A292) in the R2-loop region and a non-synonymous amino acid substitution at position 346 (N346I). The blaCMY-172-bearing plasmid, pKPCZA02_4, was 93.3 Kb, IncI1-I type, and conjugative; blaCMY-172 was located in an IS1294-mediated transposon. Plasmid conjugation and DNA fragment cloning proved that blaCMY-172 was responsible for CAZ/AVI resistance. ConclusionsOur study identified conjugative plasmid-mediated blaCMY-172 as a new mechanism for CAZ/AVI resistance in clinical KPC-Kp strains. Careful monitoring of CAZ/AVI susceptibility is imperative for preventing the spread of the resistance gene.

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.

Genomic characterization of conjugative plasmids carrying the mcr-1 gene in foodborne and clinical strains of Salmonella and Escherichia coli

To date, the genomic characterization of foodborne bacteria carrying the mobile colistin resistance gene mcr-1 and transferability of mcr-1-carrying plasmids between wild-type bacteria have not been studied in detail. We retrospectively investigated the prevalence of mcr-1-positive Salmonella and Escherichia coli isolated from retail food products, food supply chain, and clinical samples across China in the last decade (2006–2017). The horizontal transfer of mcr-1-bearing plasmids and their complete nucleotide sequence were characterized using conjugation, S1-nuclease pulsed-field gel electrophoresis, and Southern hybridization, coupled with Illumina and Nanopore sequencing. Twelve (0.67%) mcr-1-positive strains were identified from 1789 non-duplicated isolates, including 1283 Salmonella and 506 E. coli. Eleven (21.7‰) mcr-1-positive E. coli strains were identified from the isolates collected during 2006–2017, which was significantly more prevalent than mcr-1-positive Salmonella in this period (0.78‰, 1/1283; P < 0.05). All donors (nine mcr-1-positive isolates, designated as D1–9) were multidrug resistant with diverse antimicrobial susceptibility profiles and were resistant to polymyxin B. mcr-1-carrying plasmids in donors were successfully transferred to recipients at frequencies of 4.6 × 10−2 to 6.25 × 10−4 per recipient cell. A ~33 kb IncX4-type plasmid in D1, which carried mcr-1 with the insertion sequence IS26 upstream, was effectively transferred to recipient R1. D2, D4, and their corresponding transconjugants harbored a ~60 kb IncI2-type plasmid with the insertion sequence ISApl1 upstream of mcr-1. A ~60 kb conjugative mcr-1-bearing plasmid derived from D5 also belonged to type IncI2 and contained the mcr-1 gene cassette ISEcp1-blaCTX-M-55-mcr-1-PAP2. In conclusion, mcr-1 was commonly detected in type IncI2, IncX4, and IncHI2 plasmids and mcr-1 encoding colistin resistance was successfully transferred between foodborne and clinical strains of E. coli and Salmonella isolated in China. mcr-1 gene cassettes including IS26-mcr-1-PAP2, ISAPl1-mcr-1-PAP2, and ISEcp1-blaCTX-M-55-mcr-1-PAP2 can be transferred horizontally between wild-type bacteria via conjugation. The new mcr-1 gene cassette discovered in this study provided evidence for co-transfer of mcr-1 and other antibiotic resistance genes. This finding suggests that antibiotics, other than colistin, should be used in combination for the treatment and prevention of diseases caused by foodborne pathogens. The co-transfer of mcr-1 and other antibiotic resistance genes will also enhance antibiotic resistance in foodborne pathogens.

Heterogeneity and Diversity of mcr-8 Genetic Context in Chicken-Associated Klebsiella pneumoniae.

Increasing mobile colistin resistance, mediated by the mcr gene family, in Enterobacteriaceae has become a global concern. Among the 10 reported mcr genes, mcr-8 was first identified in Klebsiella pneumoniae, which could cause severe infections with high mortality. Information about the prevalence and genetic context of mcr-8 is still lacking. In this study, we found that mcr-8 was present in 9.83% of K. pneumoniae isolates of chicken origin. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting showed that the mcr-8 gene was located on a plasmid in all of the isolates. The genetic context of the plasmids exhibited considerable diversity from the whole-genome sequence through Illumina and MinION long-read sequencing. Mutations in two-component systems may function synergistically with mcr-8, resulting in extremely high resistance to colistin. In addition to colistin resistance, these plasmids also contained genes conferring resistance to beta-lactams, tetracycline, aminoglycosides, sulfonamides, macrolides, chloramphenicol, and florfenicol. Therefore, these findings indicate that the genetic context of mcr-8 is heterogeneous and diverse and that mcr-8 and certain chromosomal mechanisms jointly contribute to high-level colistin resistance in K. pneumoniae strains, which provides new insights into the resistance mechanisms of K. pneumoniae.

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.

Investigation of tigecycline resistant Escherichia coli from raw meat reveals potential transmission among food-producing animals

To investigate the characteristics of tet(X4)-carrying E. coli from retail raw meat, three hundred and eleven retail raw meat samples including chicken, pork, duck, and beef were collected for screening for tigecycline resistant isolates in Sichuan Province, China. We detected 25 (8.0%, 95%CI: 5.3%–11.6%) tet(X4) positive E. coli isolates from 311 retail meat with the highest prevalence (13.4%, 95%CI: 7.3%–21.8%) observed in pork. All the tet(X)-carrying isolates were analyzed by antimicrobial susceptibility testing, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), conjugation experiments, and whole genome sequencing (WGS) to study the phylogenetic relationship and genomic characteristics. Conjugation and S1-PFGE profiling showed 6 isolates (24.0%) successfully transferred the resistant phenotype with frequencies from 3.63 × 10−5 to 7.68 × 10−2. Simultaneously, tet(X4), floR, and the characteristic structure of adjacent abh gene and cassette flanked by ISCR2 were identified in all 25 isolates, and a structure of abh-tet(X4)-ISCR2-virD2-floR was aligned as an important genetic pattern around tet(X4). Two dominant sequence types (STs) ST1638 (n = 4) and ST48 (n = 4) were identified by in silico MLST. Single nucleotide polymorphisms analysis revealed two potential clone-like transmission clusters which showed high concordance with the local spatio-temporal epidemiology, suggesting that clonal transmission events were very likely. The presence of tet(X4) in multiple different clone complexes (CCs) and the identification of two clone-like transmission indicates its potentially extensive and successful spread among different food-producing animals.

BlaOXA -731 , a new chromosome-encoded blaOXA -48 -like variant in Shewanella sp. from the aquatic environment in Myanmar.

Shewanella sp., the progenitors of blaOXA-48 -like genes are increasingly reported with the possession of different blaOXA-48 -like variants. This study aims to characterize blaOXA-731 , a new variant of a blaOXA-48 -like gene identified in Shewanella sp. isolated from the aquatic environment in Myanmar. Phylogenetic analysis of the blaOXA-731 sequence with other blaOXA-48 -like variants showed that it has the highest nucleotide identity of 86.09% with blaOXA-48 . However, the active site motifs in OXA-731 were 100% identical to that in OXA-48. Whole-genome sequencing analysis showed that blaOXA-731 is not surrounded by any mobile genetic elements. The genetic context of blaOXA-731 was found as similar to other blaOXA-48 -like genes previously identified in Shewanella sp. S1 nuclease pulsed-field gel electrophoresis followed by Southern blotting confirmed the location of blaOXA-731 in the chromosome of the Shewanella genome. Cloning and expression studies showed that OXA-731 has β-lactamase activity similar to OXA-48 and OXA-181, but it has no significant carbapenemase activity. Our results showed the significance of blaOXA-48 -like-carrying Shewanella sp. in the spreading of blaOXA-48 -like genes in the community.

Prevalence of Aminoglycoside Resistance Genes and Molecular Characterization of a Novel Gene, aac(3)-IIg, among Clinical Isolates of the Enterobacter cloacae Complex from a Chinese Teaching Hospital.

Members of the Enterobacter cloacae complex are important opportunistic human pathogens capable of causing a wide variety of infections. During recent decades, aminoglycoside-resistant E. cloacae complex isolates have increasingly been reported and have become a major concern. Here, we employed high-throughput sequencing in combination with specific PCR assays to investigate the prevalence of aminoglycoside resistance genes among 170 isolates of the E. cloacae complex collected from a teaching hospital in Wenzhou, China. A total of 12 known genes [aphA-1, strA, strB, aac(6')-IIc, aadA2, aac(3)-IId, aadB, aadA1, rmtB, armA, aadA5, and aac(6')-Ie-aph(2'')-Ia] and 1 novel gene [aac(3)-IIg] were identified, with aphA-1 (71.18%), strA (55.29%), and strB (52.35%) being the most prevalent, and aac(3)-IIg was detected with a positive rate of 21.76% (37/170). The aac(3)-IIg gene was 810 bp in length and encoded a protein that shared 72 to 78% identities with previously known AAC(3)-II aminoglycoside 3-N-acetyltransferases. The MICs of gentamicin and tobramycin were 512 μg/ml and 64 μg/ml, respectively, when aac(3)-IIg was cloned into Escherichia coli DH5α. All aac(3)-IIg-positive isolates exerted broad aminoglycoside resistance profiles, mediated by the coexistence of multiple resistance genes. Moreover, aminoglycoside resistance and resistance genes were found to be transferable in most strains (24/37). Nevertheless, pulsed-field gel electrophoresis (PFGE) and dendrogram analysis showed clonal diversity among these isolates. S1 nuclease PFGE, Southern hybridization, and whole-genome sequencing indicated that aac(3)-IIg was located on transferable as well as nontransferable plasmids of various sizes. The analysis of the genetic environment suggested that aac(3)-IIg is embedded within a class 1 integron, with IS26 playing an important role in its mobility.

Characterization of a fosA3 Carrying IncC-IncN Plasmid From a Multidrug-Resistant ST17 Salmonella Indiana Isolate.

The aim of this study was to investigate the characteristics of a fosA3 carrying IncC–IncN plasmid from a multidrug-resistant Salmonella isolate HNK130. HNK130 was isolated from a chicken and identified as ST17 Salmonella enterica serovar Indiana and exhibited resistance to 13 antibiotics including the cephalosporins and fosfomycin. S1 nuclease pulsed-field gel electrophoresis and Southern blot assays revealed that HNK130 harbored only one ∼180-kb plasmid carrying fosA3 and blaCTX–M–14, which was not transferable via conjugation. We further examined 107 Escherichia coli electro-transformants and identified 3 different plasmid variants, pT-HNK130-1 (69), pT-HNK130-2 (15), and pT-HNK130-3 (23), in which pT-HNK130-1 seemed to be the same as the plasmid harbored in HNK130. We completely sequenced an example of each of these variants, and all three variants were IncC–IncN multi-incompatible plasmid and showed a mosaic structure. The fosA3 gene was present in all three and bounded by IS26 elements in the same orientation (IS26-322bp-fosA3-1758bp-IS26) that could form a minicircle containing fosA3. The blaCTX–M–14 gene was located within an IS15DI-ΔIS15DI-iroN-IS903B-blaCTX–M–14-ΔISEcp1-IS26 structure separated from the fosA3 gene in pT-HNK130-1, but was adjacent to fosA3 in pT-HNK130-3 in an inverted orientation. Linear comparison of the three variants showed that pT-HNK130-2 and pT-HNK130-3 resulted from the sequence deletion and inversion of pT-HNK130-1. Stability tests demonstrated that pT-HNK130-1 and pT-HNK130-3 could be stably maintained in the transformants without antibiotic selection but pT-HNK130-2 was unstable. This is the first description of an IncC–IncN hybrid plasmid from an ST17 S. Indiana strain and indicates that this plasmid may further facilitate dissemination of fosfomycin and cephalosporin resistance in Salmonella.

Co-existence of the oxazolidinone resistance genes cfr and optrA on two transferable multi-resistance plasmids in one Enterococcus faecalis isolate from swine

ObjectivesTo identify and characterize oxazolidinone resistance genes cfr and optrA in enterococcal isolates. MethodsTwo hundred and ninety-three enterococcal isolates were screened for the presence of cfr and optrA by polymerase chain reaction. The transferability of cfr and optrA was examined by conjugation. S1 nuclease pulsed-field gel electrophoresis and Southern blotting were used to identify the location of cfr and optrA. One Enterococcus faecalis isolate carrying both cfr and optrA was sequenced in full. Resultscfr and optrA were detected in 16 (5.5%) and 170 (58.0%) enterococcal isolates, respectively. Sixteen enterococcal isolates (E. faecalis n=13, Enterococcus avium n=2, Enterococcus mundtii n=1) carried both cfr and optrA. The cfr-carrying fragment between res and theta in plasmid p4 showed 98.9% identity to the corresponding region of plasmid pEF120805 from vancomycin-resistant Enterococcus faecium. The optrA-carrying segment between tnpB and optrA in plasmid p1 showed >99.9% identity to the corresponding region of genomic DNA from E. faecalis A101. Plasmid p4 and plasmid p1 were simultaneously conjugated to E. faecalis JH2-2. ConclusionsOne hundred and seventy optrA-positive enterococci were identified in 293 enterococcal isolates from swine and the farm environment. The co-existence of cfr and optrA in E. avium and E. mundtii has been identified previously. cfr and optrA were identified on two new conjugative plasmids from one E. faecalis isolate. The optrA-carrying segment (IS1216E-optrA-IS1216E) was reported initially. Among different types of enterococcal plasmids, ISEnfa5 and IS1216E elements may play a vital role in the dissemination of cfr and optrA, respectively.

Plasmids and genes contributing to high-level quinolone resistance in Escherichia coli

Introduction: The importance of plasmid-mediated quinolone resistance (PMQR) in Enterobacterales and its high incidence has been emphasised many times. However, a clinical strain carrying more than two PMQR genes is rare. This study sequenced plasmid transconjugants from a donor strain carrying four different PMQR genes to establish their genetic locations.Methods: An Escherichia coli clinical strain displayed remarkable quinolone resistance with a ciprofloxacin MIC of 1024 mg/L carrying four PMQR genes: qnrA1, qepA1, aac(6′)1b-cr and oqxAB. When outcrossed to Escherichia coli J53 AziR, different PMQR genes were transferred and the resulting strains 7C and 8C were chosen for further characterisation. Plasmids were extracted and sequenced by the Illumina and Oxford Nanopore Technologies platforms. S1 nuclease-PFGE was used to estimate the number and size of plasmids.Results: The parental strain had three plasmid bands, as determined by PFGE. Transconjugant 8C obtained three plasmids: pMG336 (162 647 bp, F18:A-:B1:C4) carrying oqxAB; pMG335 carrying qepA1 (73 874 bp, F2:A-:B-); and pMG334 (59 724 bp, IncN (ST5)) with qnrA1 and aac(6′)1b-cr. Interestingly, strain 7C obtained plasmid pMG333 (134 435 bp), which was not present in the parental strain but was an IncN-IncF cointegrate of plasmids pMG334 and pMG335 linked via insertion sequence IS26.Conclusion: This study described the complete nucleotide sequence of three plasmids carrying four PMQR genes in a single strain and the plasmid profile obtained after outcrosses. In addition, it described a cointegrate of two plasmids formed with flanking insertion sequences.

Emerging coexistence of three PMQR genes on a multiple resistance plasmid with a new surrounding genetic structure of qnrS2 in E. coli in China

BackgroundQuinolones are commonly used for treatment of infections by bacteria of the Enterobacteriaceae family. However, the rising resistance to quinolones worldwide poses a major clinical and public health risk. This study aimed to characterise a novel multiple resistance plasmid carrying three plasmid-mediated quinolone resistance genes in Escherichia coli clinical stain RJ749.MethodsMICs of ceftriaxone, cefepime, ceftazidime, ciprofloxacin, and levofloxacin for RJ749 and transconjugant c749 were determined by the Etest method. Conjugation was performed using sodium azide-resistant E. coli J53 strain as a recipient. The quinolone resistance-determining regions of gyrA, gyrB, parC, and parE were PCR-amplified.ResultsRJ749 was highly resistant to quinolones, while c749 showed low-level resistance. S1-nuclease pulsed-field gel electrophoresis revealed that RJ749 and c749 both harboured a plasmid. PCR presented chromosomal mutation sites of the quinolone resistance-determining region, which mediated quinolone resistance. The c749 genome comprised a single plasmid, pRJ749, with a multiple resistance region, including three plasmid-mediated quinolone resistance (PMQR) genes (aac (6′)-Ib-cr, qnrS2, and oqxAB) and ten acquired resistance genes. One of the genes, qnrS2, was shown for the first time to be flanked by two IS26s. Three IS26-mediated circular molecules carrying the PMQR genes were detected.ConclusionsWe revealed the coexistence of three PMQR genes on a multiple resistance plasmid and a new surrounding genetic structure of qnrS2 flanked by IS26 elements. IS26 plays an important role in horizontal spread of quinolone resistance.

Emergence of NDM-1- and CTX-M-3-Producing Raoultella ornithinolytica in Human Gut Microbiota.

Raoultella ornithinolytica is an opportunistic pathogen of the Enterobacteriaceae family and has been implicated in nosocomial infections in recent years. The aim of this study was to characterize a carbapenemase-producing R. ornithinolytica isolate and three extended-spectrum β-lactamase (ESBL)-producing R. ornithinolytica isolates from stool samples of adults in a rural area of Shandong Province, China. The species were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rDNA sequence analysis. Antimicrobial susceptibility test showed that all four isolates were multidrug-resistant (MDR). The whole genome sequence (WGS) of these isolates was determined using an Illumina HiSeq platform, which revealed MDR-related genes. The S1 nuclease-pulsed-field gel electrophoresis (S1-PFGE) was used to characterize the plasmids carried by the R. ornithinolytica isolates. The blaNDM-1 and blaCTX-M-3 genes were probed using Southern blotting, which confirmed the location of both genes on the same plasmid with molecular weight of 336.5–398.4 kb. The transferability of blaNDM-1 and blaCTX-M was also confirmed by conjugation assays. Finally, BLAST analysis of both genes showed that mobile genetic elements were associated with the spread of drug resistance genes. Taken together, we report the presence of conjugative blaNDM-1 and blaCTX-M plasmids in R. ornithinolytica isolates from healthy humans, which indicate the possibility of inter-species transfer of drug resistance genes. To the best of our knowledge, this is the first study to isolate and characterize carbapenemase-producing R. ornithinolytica and ESBL-producing R. ornithinolytica isolates from healthy human hosts.

Emergence and Comparative Genomics Analysis of Extended-Spectrum-β-Lactamase-Producing Escherichia coli Carrying mcr-1 in Fennec Fox Imported from Sudan to China.

The aim of this study was to investigate the occurrence and genomic characteristics of extended-spectrum-β-lactamase-producing Escherichia coli (ESBL-EC) in fennec fox imported from Sudan to China. We screened 88 fecal samples from fennec fox for ESBL-EC, using cefotaxime- and meropenem-supplemented selective medium. Antimicrobial susceptibility testing was performed by the agar dilution method except for colistin and tigecycline; for colistin and tigecycline, testing was conducted by the broth microdilution method. ESBL-EC bacteria were sequenced, and their genomes were characterized. Plasmid conjugation, S1 nuclease pulsed-field gel electrophoresis (PFGE), and Southern blotting were performed for a MCR-1-producing isolate. The genetic environment of mcr-1 and ESBL genes was also investigated. A total of 29 ESBL-EC bacteria were isolated from 88 fennec fox (32.9%), while no carbapenemase producers were found. The most prevalent genotypes were the blaCTX-M-55 and blaCTX-M-14 genes, followed by blaCTX-M-15 and blaCTX-M-64 We detected nine sequence types among 29 ESBL-EC. Furthermore, the mcr-1 gene was detected in isolate EcFF273. Conjugation analysis confirmed that the mcr-1 gene was transferable. S1 PFGE, Southern blotting, and whole-genome sequencing revealed that mcr-1 and blaCTX-M-64 were both located on a 65-kb IncI2 plasmid. This study reports for the first time the occurrence of ESBL-EC in fennec fox. The high prevalence of ESBL producers and the occurrence of MCR-1 producer in fennec fox imported into China from Sudan are unexpected. In addition, it clearly demonstrated that commensal E. coli strains can be reservoirs of blaCTX-M and mcr-1, potentially contributing to the dissemination and transfer of such genes to pathogenic bacteria among fennec fox. Our results support the implication of fennec fox as a biological vector for ESBL-producing members of the Enterobacteriaceae family.IMPORTANCE The extended-spectrum-β-lactamase (ESBL)-producing members of the Enterobacteriaceae family are a global concern for both animal and human health. There is some information indicating a high prevalence of ESBL producers in food animals. Moreover, there have been an increasing number of reports on ESBL-producing strains resistant to the last-resort antibiotic colistin with the global dissemination of the plasmid-mediated mcr-1 gene, which is believed to have originated in animal breeding. However, little is known regarding the burden of ESBL-producing Enterobacteriaceae on wild animals. No data were available on the prevalence of antimicrobial resistance (AMR) among wild animals imported into China. This is the first study to investigate the microbiological and genomics surveillance investigation of ESBL colonization among fennec fox (Vulpes zerda) imported from Sudan to China, and we uncovered a high prevalence of ESBL-EC. Furthermore, the underlying mechanism of colistin resistance in an isolate that harbored mcr-1 was also investigated. Results of characterization and analysis of 29 ESBL-producing E. coli may have important implications on our understanding of the transmission dynamics of these bacteria. We emphasize the importance of improved multisectoral surveillance for colistin-resistant E. coli in this region.

Characterization of Klebsiella pneumoniae ST11 Isolates and Their Interactions with Lytic Phages

The bacterial pathogen Klebsiella pneumoniae causes urinary tract infections in immunocompromised patients. Generally, the overuse of antibiotics contributes to the potential development and the spread of antibiotic resistance. In fact, certain strains of K. pneumoniae are becoming increasingly resistant to antibiotics, making infection by these strains more difficult to treat. The use of bacteriophages to control pathogens may offer a non-antibiotic-based approach to treat multidrug-resistant (MDR) infections. However, a detailed understanding of phage–host interactions is crucial in order to explore the potential success of phage-therapy for treatment. In this study, we investigated the molecular epidemiology of nine carbapenemase-producing K. pneumoniae isolates from a local hospital in Shanghai, China. All strain isolates belong to sequence type 11 (ST11) and harbor the blaKPC-2 gene. The S1-PFGE (S1 nuclease pulsed field gel electrophoresis) pattern of the isolates did not show any relationship to the multilocus sequence typing (MLST) profiles. In addition, we characterized phage 117 and phage 31 and assessed the potential application of phage therapy in treating K. pneumoniae infections in vitro. The results of morphological and genomic analyses suggested that both phages are affiliated to the T7 virus genus of the Podoviridae family. We also explored phage–host interactions during growth in both planktonic cells and biofilms. The phages’ heterogeneous lytic capacities against K. pneumoniae strains were demonstrated experimentally. Subsequent culture and urine experiments with phage 117 and host Kp36 initially demonstrated a strong lytic activity of the phages. However, rapid regrowth was observed following the initial lysis which suggests that phage resistant mutants were selected in the host populations. Additionally, a phage cocktail (117 + 31) was prepared and investigated for antimicrobial activity. In Luria Broth (LB) cultures, we observed that the cocktail showed significantly higher antimicrobial activity than phage 117 alone, but this was not observed in urine samples. Together, the results demonstrate the potential therapeutic value of phages in treating K. pneumoniae urinary tract infections.

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.

Analysis of combined resistance to oxazolidinones and phenicols among bacteria from dogs fed with raw meat/vegetables and the respective food items

The gene optrA is the first gene that confers resistance to the oxazolidinone tedizolid, a last resort antimicrobial agent in human medicine. In this study we investigated the presence of optrA and the multi-resistance genes poxtA and cfr in enterococci and staphylococci from (i) pet animals known to be fed raw meat and vegetables and (ii) the respective food items. We examined 341 bacterial isolates from cats and dogs, 195 bacterial isolates from supermarket food items and only one E. faecium collected from industrial food in Beijing during 2016. Thirty-five (6.5%) of the 537 isolates, including 31/376 (8.2%) enterococci and 4/161 (2.5%) staphylococci, were positive for optrA, while all isolates were negative for poxtA and cfr. S1-nuclease pulsed-field gel electrophoresis (PFGE) and Southern blotting confirmed that optrA was located in the chromosomal DNA of 19 isolates and on a plasmid in the remaining 16 isolates. Whole genome sequencing revealed several different genetic environments of optrA in plasmid- or chromosome-borne optrA genes. PFGE, multilocus sequence typing (MLST) and/or SNP analysis demonstrated that the optrA-carrying Staphylococcus and Enterococcus isolates were genetically heterogeneous. However, in single cases, groups of related isolates were identified which might suggest a transfer of closely related optrA-positive E. faecalis isolates between food items and dogs.