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This study reported the involvement of a gene cluster from a conjugative plasmid in the biofilm formation of Escherichia coli . We used a novel EZ-Tn 5 transposon technique to generate a transposon library and used arbitrarily primed PCR to detect the insertion sites in biofilm formation-deficient mutants. To validate the function of candidate biofilm formation genes, the genes were cloned into plasmid pBluescript II SK (+) and transformed into E. coil DH5α. Biofilm production from the transformants was then assessed by phenotypic biofilm formation using Crystal Violet staining and microscopy. A total of 3,000 transposon mutants of E. coli DH5α-p253 were screened, of which 28 were found to be deficient in biofilm formation. Further characterization revealed that 24/28 mutations were detected with their insertions in chromosome, while the remaining 4 mutations were evidenced that the functional genes for biofilm formation were harbored in the plasmid. Interestingly, the plasmid sequencing showed that these four transposon mutations were all inserted into a fimbriae-associated gene cluster ( fim -cluster). This fim -cluster is a hybrid segment spanning a 7,949 bp sequence, with a terminal inverted repeat sequence and two coding regions. In summary, we performed a high-efficiency screening to a library constructed with the EZ-Tn5-based transposon approach and identified the gene clusters responsible for the biofilm production of E. coli , especially the genes harbored in the plasmid. Further studies are needed to understand the spread of this novel plasmid-mediated biofilm formation gene in clinical E. coli isolates and the clinical impacts.

We reported the complete nucleotide sequence of a tet (X4)-carrying plasmid, pSTB20-1T, from a tigecycline-resistant Escherichia coli isolate in China. Sequence analysis indicated that pSTB20-1T contains a hybrid plasmid backbone and a tet (X4)-containing multidrug resistance region, likely originated through recombination of multiple plasmids. tet (X4) was flanked by two IS CR2 , which may be responsible for tet (X4) mobilization. The occurrence and transmission of this novel hybrid plasmid may exacerbate the spread of the clinically significant tet (X4) gene., (Copyright © 2019 American Society for Microbiology.)

Tigecycline is one of the last-resort antibiotics to treat complicated infections caused by both multidrug-resistant Gram-negative and Gram-positive bacteria 1 . Tigecycline resistance has sporadically occurred in recent years, primarily due to chromosome-encoding mechanisms, such as overexpression of efflux pumps and ribosome protection 2,3 . Here, we report the emergence of the plasmid-mediated mobile tigecycline resistance mechanism Tet(X4) in Escherichia coli isolates from China, which is capable of degrading all tetracyclines, including tigecycline and the US FDA newly approved eravacycline. The tet(X4)-harbouring IncQ1 plasmid is highly transferable, and can be successfully mobilized and stabilized in recipient clinical and laboratory strains of Enterobacteriaceae bacteria. It is noteworthy that tet(X4)-positive E. coli strains, including isolates co-harbouring mcr-1, have been widely detected in pigs, chickens, soil and dust samples in China. In vivo murine models demonstrated that the presence of Tet(X4) led to tigecycline treatment failure. Consequently, the emergence of plasmid-mediated Tet(X4) challenges the clinical efficacy of the entire family of tetracycline antibiotics. Importantly, our study raises concern that the plasmid-mediated tigecycline resistance may further spread into various ecological niches and into clinical high-risk pathogens. Collective efforts are in urgent need to preserve the potency of these essential antibiotics.

Objectives: To investigate the prevalence and transmission of 16S rRNA methylase genes among Salmonella isolates from food animals in China., Methods: A total of 310 Salmonella isolates collected from food animals in seven provinces of China during 2016-17 were screened for 16S RMTase genes. The clonal relationship of the 16S RMTase-producing isolates and their plasmid contents were also characterized., Results: rmtB and armA were respectively identified in 12 and 1 Salmonella enterica serovar Indiana (Salmonella Indiana) isolates from farmed ducks. These 13 isolates concurrently expressed high-level resistance to amikacin, cefotaxime and ciprofloxacin. They were assigned to seven distinct PFGE patterns and the high similarity among 10 of the 12 rmtB-carrying isolates suggests clonal expansion. The rmtB gene was co-transferred with blaCTX-M-27-qepA and qepA in eight and two of the isolates, respectively, and was located on F2:A1:B1 plasmids with sizes of 135 and 100 kb, respectively. These 10 rmtB-bearing plasmids showed four restriction patterns with a high similarity. Four representative rmtB-bearing plasmids were fully sequenced and they exhibited remarkable similarity and possessed typical FII backbones. The primary differences were located in the region between blaTEM-1 and ycgA. Furthermore, a novel MDR region (13.5 kb) was identified that contained qepA, rmtB and blaCTX-M-27., Conclusions: This is the first report, to our knowledge, of the prevalence and complete sequences of plasmids simultaneously containing rmtB, qepA and blaCTX-M-27. These findings underscore a major public health threat posed by epidemic F2:A1:B1 plasmids bearing qepA-rmtB-blaCTX-M-27 that are circulating in XDR Salmonella Indiana clonal isolates from waterfowl husbandry.

We report a novel fusion plasmid, pP2-3T, cointegrating sequence type 3 (ST3)-IncHI2 with an IncFII plasmid backbone mediating multidrug resistance (MDR) and virulence. Phylogenetic analysis and comparative genomics revealed that pP2-3T and other MDR ST3-IncHI2 plasmids clustered together, representing a unique IncHI2 lineage that exhibited high conservation in backbones of plasmids but possessed highly genetic plasticity in various regions by acquiring numerous antibiotic resistance genes and fusing with other plasmids. Surveillance studies should be performed to monitor multiresistance IncHI2 plasmids among Enterobacteriaceae ., (Copyright © 2018 American Society for Microbiology.)

IncX4 plasmids are associated with the dissemination of the mcr-1 genes in Enterobacteriaceae. We screened IncX4 plasmids among 2,470 isolates of Enterobacteriaceae and determined the mcr-1 positive isolates. Forty-three isolates were observed to carry IncX4 type plasmid, among which 13 were identified to carry mcr-1 gene. Three representative mcr-1-positive IncX4 plasmids were selected for high-throughput sequencing. Comparative genomics showed that the mcr-1-carrying IncX4 plasmids exhibit remarkable similarity in the backbone, and the major distinction lies in the region containing mcr-1. The major variable regions of all the IncX4 plasmids were fully characterized by PCR-RFLP. The results revealed that the mcr-1 was located on the Variable Region I of IncX4 plasmids in 11 E. coli isolates. Among them, nine E. coli strains possess an epidemic pCSZ4-like IncX4 plasmid containing mcr-1. ISApl1 was presumably involved in the transposition of the mcr-1 cassette and then was lost. Similar genetic contexts were found in different plasmids, even the E. coli chromosome, implying the acquisition of mcr-1 by a unique common mechanism.

Since initial identification in China, the widespread geographical occurrence of plasmid-mediated colistin resistance gene mcr-1 in Enterobacteriaceae has been of great concern. In this study, a total of 22 Salmonella enterica were resistant to colistin, while only five isolates which belonged to ST34 Salmonella enterica serovar Typhimurium (S. Typhimurium) were mcr-1 positive. Four of them shared nearly identical PFGE type, although they were from different host species and diverse geographical locations. All the mcr-1-positive S. Typhimurium exhibited multi-resistant phenotypes including ampicillin, streptomycin, gentamicin, florfenicol, nalidixic acid, tetracycline, trimethoprim-sulfamethox, in addition to colistin. The oqxAB and aac(6')-Ib-cr genes were present alone or in combination in four (80.0%) and five (100%) isolates, respectively. The mcr-1 gene was located on a transferable IncI2 plasmid in the four genetically related strains. In the other one strain, mcr-1 was located on an approximately 190 kb IncHI2 plasmid. In conclusion, we report five mcr-1-positive S. Typhimurium/ST34 isolates. Both clonal expansion and horizontal transmission of IncI2-type plasmids were involved in the spread of the mcr-1 gene in Salmonella enterica from food-producing animals in China. There is a great need to monitor the potential dissemination of the mcr-1 gene.

We report the complete nucleotide sequence of a plasmid, pA31-12, carrying blaCTX-M-55 and mcr-1 from a chicken Escherichia coli isolate. pA31-12 has an IncI2 replicon that displays extensive sequence similarity with pHN1122-1-borne blaCTX-M-55 and pHNSHP45-borne mcr-1 Insertion sequences ISEcp1 and ISApl1 are responsible for the mobilization of blaCTX-M-55 and mcr-1, respectively. The colocalization of mcr-1 with an extended-spectrum β-lactamase gene on a conjugative plasmid may accelerate the dissemination of both genes by coselection., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

We report the complete nucleotide sequence of a plasmid carrying the multiresistance gene cfr. This plasmid was isolated from an Escherichia coli strain of swine origin in 2011. This 37,672-bp plasmid, pSD11, had an IncX4 backbone similar to those of the IncX4 plasmids obtained from the United States and Australia, in which the cfr gene was flanked by two copies of IS26 and a truncated Tn1331 was inserted., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

To study the characteristics of plasmids harboring oqxAB among bla(CTX-M)-negative Escherichia coli isolates and search for oqxAB-harboring plasmids similar to plasmids carrying oqxAB-bla(CTX-M) reported previously, conjugation experiment was performed for 115 randomly selected oqxAB-positive but bla(CTX-M)-negative E. coli isolates from diseased animals in Guangdong, China. S1 nuclease pulsed-field gel electrophoresis (PFGE) and southern blotting experiments were performed to investigate the location of oqxAB and other resistance genes. The EcoRI digestion profiles of the plasmids with oqxAB were also analyzed. The clonal relatedness of donor isolates was investigated by PFGE. In this study, 32 oqxAB transconjugants were successfully obtained and most transconjugants showed multidrug resistances. Eleven replicon combination types were found in these transconjugants. floR and oqxAB were found on the same plasmids in all nine transconjugants resistant to florfenicol. The sequences between floR and oqxAB were identical in most transconjugants and the two genes were both linked with tnp in insertion sequences. Nine F18:A-:B1 plasmids with only oqxAB shared identical EcoRI digestion profiles and the profiles were also identical with that of a plasmid carrying oqxAB-bla(CTX-M) found previously. Co-transfer of plasmids carrying oqxAB and fosA3, respectively, was also observed in one isolate. This study demonstrates the dissemination of oqxAB among bla(CTX-M)-negative E. coli isolates was mainly mediated by identical F18:A-:B1 plasmids. A novel arrangement of regions between floR and oqxAB might play an important role in the dissemination of floR-oqxAB. This is the first description of the genetic environment of the relationship between oqxAB and floR in E. coli.

Background: The association of PMQR and ESBLs in negative-bacteria isolates has been of great concern. The present study was performed to investigate the prevalence of co-transferability of oqxAB and bla CTX-M genes among the 696 Escherichia coli (E. coli) isolates from food-producing animals in South China, and to characterize these plasmids., Methods: The ESBL-encoding genes (bla(CTX-M), bla(TEM) and bla(SHV)), and PMQR (qnrA, qnrB, qnrS, qnrC, qnrD, aac(6')-Ib-cr, qepA, and oqxAB) of these 696 isolates were determined by PCR and sequenced directionally. Conjugation, S1 nuclease pulsed-field gel electrophoresis (PFGE) and Southern blotting experiments were performed to investigate the co-transferability and location of oqxAB and bla(CTX-M). The EcoRI digestion profiles of the plasmids with oqxAB-bla(CTX-M) were also analyzed. The clonal relatedness was investigated by PFGE., Results: Of the 696 isolates, 429 harbored at least one PMQR gene, with oqxAB (328) being the most common type; 191 carried bla(CTX-M), with bla(CTX-M-14) the most common. We observed a significant higher prevalence of bla(CTX-M) among the oqxAB-positive isolates (38.7%) than that (17.4%) in the oqxAB-negative isolates. Co-transferability of oqxAB and bla(CTX-M) was found in 18 of the 127 isolates carrying oqxAB-bla(CTX-M). These two genes were located on the same plasmid in all the 18 isolates, with floR being on these plasmids in 13 isolates. The co-dissemination of these genes was mainly mediated by F33:A-: B- and HI2 plasmids with highly similar EcoRI digestion profiles. Diverse PFGE patterns indicated the high prevalence of oqxAB was not caused by clonal dissemination., Conclusion: bla(CTX-M) was highly prevalent among the oqxAB-positive isolates. The co-dissemination of oqxAB-bla(CTX-M) genes in E. coli isolates from food-producing animals is mediated mainly by similar F33:A-: B- and HI2 plasmids. This is the first report of the co-existence of oqxAB, bla(CTX-M), and floR on the same plasmids in E. coli.

qnrD, unlike other qnr genes, is mainly located on small nonconjugative plasmids. We investigated the presence of qnrD among 1,373 Enterobacteriaceae isolates in China. Twelve qnrD-positive strains were detected, and all were nonsusceptible to fluoroquinolones. The complete sequence of plasmids showed that the qnrD determinants were located on two plasmids with a respective size of ~4.2 and 2.7 k-bp. Interestingly, the identification of qnrD in this study revealed the highest prevalence of Proteeae among Enterobacteriaceae identified.

Salmonella enterica can lead to intestinal diarrhea, and the emergence and spread of cephalosporin-resistant Salmonella have brought great challenges to clinical treatment. Therefore, this study investigated the prevalence and transmission of bla CTX-M genes among S. Typhimurium from diarrhoeal outpatients in Guangdong, China, from 2010 to 2017. A total of 221 bla CTX-M-positive isolates were recovered from 1,263 S. Typhimurium isolates from the facal samples of diarrhoea patients in 45 general hospitals from 11 cities. The most popular CTX-M gene was bla CTX-M-55 (39.6%, 72/182) in the CTX-M-1 group, followed by bla CTX-M-14 (22.5%, 41/182) and bla CTX-M-65 (19.2%, 35/182) in the CTX-M-9 group. The isolates that carried bla CTX-M-9G had significantly higher resistance rates to multiple antibacterials compared with bla CTX-M-1G (p < 0.01). Meanwhile, PFGE analysis not only showed the clonal transmission of bla CTX-M-55/14/65-positve isolates of diarrhoeal outpatients' origins from different hospitals in Guangdong province, but also the characteristic of bla CTX-M-55/14/65-positve isolates' bacterial persistence. Multilocus sequence typing (MLST) analysis indicated that these S. Typhimurium isolates possessed ST34 and ST19. Furthermore, genomic Beast phylogenomic analysis provided the evidence of a close relationship of bla CTX-M-positive S. Typhimurium isolates between the outpatients and pork. Most bla CTX-M-55/14/65 genes were transmitted by non-typeable or IncI1/IncFII/IncHI2 plasmids with the size of ranging from ~80 to ~280 kb. Moreover, whole-genome sequencing (WGS) analysis further revealed that bla CTX-M-55/14/65 coexisted with other 25 types of ARGs, of which 11 ARGs were highly prevalent with the detection rates >50%, and it first reported the emergence of bla TEM-141 in S. Typhimurium. This study underscores the importance of surveillance for bla CTX-M-positive microbes in diarrhea patients. [ABSTRACT FROM AUTHOR]

Objectives: To determine the transmission and molecular characteristics of blaNDM-producing Escherichia coli between companion animals and their healthcare providers at veterinary clinics in Guangzhou, China.Methods: A total of 359 samples from companion animals and their healthcare providers were collected at 14 veterinary clinics in Guangzhou, China. Genomic characteristics and clonal relationships for blaNDM-positive E. coli and complete plasmid sequences were characterized based on WGS data from combined Illumina and MinION platform reads.Results: Forty-five blaNDM-positive bacteria were recovered from companion animals (n = 43) and their healthcare providers (n = 2) at 10 veterinary clinics. Overall, E. coli (73.3%, 33/45) and Klebsiella pneumoniae (13.3%, 6/45) were the most prevalent species among the seven species of blaNDM-positive bacteria. Four blaNDM variants (blaNDM-1, blaNDM-4, blaNDM-5 and blaNDM-7) were identified in 45 blaNDM-positive bacteria and blaNDM-5 was the most prevalent (77.8%, 35/45). WGS indicated that the most prevalent STs were ST405 (8/33), ST453 (6/33), ST457 (6/33) and ST410 (5/33) among the 33 blaNDM-positive E. coli isolates. Phylogenomics and PFGE analysis revealed that clonal spread of blaNDM-positive ST453 E. coli isolates between companion animals and their healthcare providers was evident. In addition, two novel IncFIB plasmids carrying blaNDM-4 (pF765_FIB and pG908_FIB) were found in this study and indicated that IS26 may promote the horizontal transmission of blaNDM between different plasmid types.Conclusions: In this study we conducted a large-scale investigation on the prevalence of blaNDM-positive E. coli isolates from companion animals and their healthcare providers and revealed the clonal spread of blaNDM-positive E. coli isolates between these two groups. [ABSTRACT FROM AUTHOR]

This study aimed to determine the prevalence and transmission characteristics of New Delhi metallo β-lactamase (NDM)-producing Escherichia coli from ducks in Guangdong, China. In this study, a total of 28 NDM-producing E. coli isolates were recovered from 88 unduplicated diseased duck samples (31.8%) from veterinary clinics in Guangzhou, Foshan, Qingyuan, and Huizhou. Two variants, bla NDM−1 and bla NDM−5, were detected and the latter was present in 89.6% of the isolates (25/28). Multilocus sequence typing (MLST) analysis indicated that these E. coli isolates possessed six distinct STs, and ST156 was the most prevalent followed by ST648, ST746, ST354, ST10, and ST162. In addition, phylogenomic analysis found that two of the isolates that were recovered from a single sample possessed different genomes, and the bla NDM-carrying IncX3 plasmids may be horizontal transfer between E. coli isolates in the intestinal tracts of ducks. Whole-genome sequencing (WGS) analysis further revealed that bla NDM co-existed with other 25 types of antimicrobial resistance genes (ARGs), of which 16 ARGs were highly prevalent with detection rates >50%, and a high incidence of coproducing bla NDM and mcr-1 E. coli isolates (22/88, 25.0%) was detected in ducks. This study underscores the importance of surveillance for bla NDM-harboring microbes in ducks. [ABSTRACT FROM AUTHOR]

Objectives: To clarify the transmission mechanism of the blaCTX-M-64 gene between Escherichia coli and Salmonella isolates from food animals.Methods: A total of 329 E. coli and 60 Salmonella isolates collected from food animals in 2016 were screened for the presence of blaCTX-M-64 genes. The blaCTX-M-64-positive isolates were typed and plasmid and chromosome DNA was sequenced to determine the genetic context of blaCTX-M-64 and the plasmid types present.Results: The blaCTX-M-64 gene was identified in only three E. coli isolates but was the predominant gene in the Salmonella isolates (n = 9). These 12 CTX-M-64-positive isolates were all resistant to ampicillin, cefotaxime, ceftiofur, ceftriaxone, ceftazidime and florfenicol and 9 were resistant to ciprofloxacin. The blaCTX-M-64 gene was located on transferable IncI2 plasmids and an IncHI2 plasmid in three E. coli and one Salmonella isolate, respectively. The remaining eight Salmonella isolates contained blaCTX-M-64 integrated into the chromosome. Different genetic contexts of blaCTX-M-64 genes were found among the 12 isolates: ISEcp1-blaCTX-M-64-orf477-A/C on IncI2 plasmids of 3 E. coli isolates; ΔISEcp1-blaCTX-M-64-orf477-A/C in the chromosome of 1 Salmonella isolate; and ISEcp1-blaCTX-M-64-orf477 on the IncHI2 plasmid and chromosome of 8 Salmonella isolates.Conclusions: To the best of our knowledge, this is the first report of chromosomally encoded CTX-M-64 in Salmonella isolates. ISEcp1-mediated transposition is likely to be responsible for the spread of blaCTX-M-64 between different plasmids and chromosomes in Enterobacteriaceae especially E. coli and Salmonella. [ABSTRACT FROM AUTHOR]

Objectives: The emergence and spread of plasmid-encoded tet(X3/X4) genes that confer high-level tigecycline and eravacycline resistance in Escherichia coli and Acinetobacter spp. pose serious threats to human and animal health. We developed a rapid and robust assay to detect Tet(X3/X4) in Gram-negative bacteria based on eravacycline degradation by the presence of the Tet(X) enzyme in the test strain.Methods: This tetracycline inactivation method (TIM) is based on the degradation of eravacycline by the Tet(X3/X4)-producing strain, which results in reduced eravacycline activity against an acid-producing thermophile Bacillus stearothermophilus indicator strain. For Tet(X)-negative strains, eravacycline retains its antimicrobial activity. Coupled with a pH-sensitive dye (bromocresol purple), the reduced colorimetric inhibition zone can be measured to determine the production of Tet(X3/X4). One hundred and eighteen isolates, including 30 tet(X4)-positive E. coli, 30 tet(X3)-positive Acinetobacter spp. and 58 tet(X)-negative E. coli and Acinetobacter spp., were examined to evaluate the performance of this TIM.Results: The sensitivity and specificity for E. coli carrying tet(X4) was 96.7% and 100%, respectively, and for Acinetobacter spp. carrying tet(X3) both were 100%. The TIM assay can be completed within 6.5 h.Conclusions: The TIM is a simple, rapid and cost-effective method for the detection of plasmid-mediated high-level tigecycline resistance in E. coli and Acinetobacter spp. [ABSTRACT FROM AUTHOR]

Objectives: The emergence of carbapenemase-positive Enterobacteriaceae poses a serious threat to public health worldwide. Here we conducted a molecular surveillance study on carbapenem-resistant Enterobacteriaceae (CRE) colonization among migratory birds at Qinghai Lake in China.Methods: A total of 420 samples from migratory birds and their surrounding environment were collected at three sites along the Qinghai Lake bird island. Carbapenem-non-susceptible isolates were identified by 16S rDNA sequencing and MALDI-TOF MS. Carbapenemase producers were determined by Carba NP testing. Antimicrobial susceptibility testing, transfer ability and PFGE were also performed, and 46 isolates from different pulsotypes were analysed by WGS.Results: Three hundred and fifty isolates were carbapenemase producers based on Carba NP testing, while 233 Klebsiella spp. and 2 Escherichia coli isolates were NDM-5-carriers. PFGE was performed and showed that the isolates were grouped into five pulsotypes; among these, type A was predominant (86.7%, n = 202) and belonged to a novel Klebsiella lineage, ST1697. WGS analysis indicated that ST1697 strains may be a hybrid of the recombination of Klebsiella quasipneumoniae subsp. similipneumoniae and Klebsiella pneumoniae genomes.Conclusions: This high frequency of carbapenemase producers in migratory birds is unexpected. These results provide new insight into the spread of antibiotic resistance, and highlight that continued vigilance for MDR carbapenemase-producing Enterobacteriaceae in migratory birds is urgently needed. [ABSTRACT FROM AUTHOR]

Objectives To investigate the prevalence and transmission of 16S rRNA methylase genes among Salmonella isolates from food animals in China. Methods A total of 310 Salmonella isolates collected from food animals in seven provinces of China during 2016–17 were screened for 16S RMTase genes. The clonal relationship of the 16S RMTase-producing isolates and their plasmid contents were also characterized. Results rmtB and armA were respectively identified in 12 and 1 Salmonella enterica serovar Indiana (Salmonella Indiana) isolates from farmed ducks. These 13 isolates concurrently expressed high-level resistance to amikacin, cefotaxime and ciprofloxacin. They were assigned to seven distinct PFGE patterns and the high similarity among 10 of the 12 rmtB -carrying isolates suggests clonal expansion. The rmtB gene was co-transferred with bla CTX-M-27- qepA and qepA in eight and two of the isolates, respectively, and was located on F2:A1:B1 plasmids with sizes of 135 and 100 kb, respectively. These 10 rmtB- bearing plasmids showed four restriction patterns with a high similarity. Four representative rmtB -bearing plasmids were fully sequenced and they exhibited remarkable similarity and possessed typical FII backbones. The primary differences were located in the region between bla TEM-1 and ycgA. Furthermore, a novel MDR region (13.5 kb) was identified that contained qepA, rmtB and bla CTX-M-27. Conclusions This is the first report, to our knowledge, of the prevalence and complete sequences of plasmids simultaneously containing rmtB, qepA and bla CTX-M-27. These findings underscore a major public health threat posed by epidemic F2:A1:B1 plasmids bearing qepA - rmtB - bla CTX-M-27 that are circulating in XDR Salmonella Indiana clonal isolates from waterfowl husbandry. [ABSTRACT FROM AUTHOR]

Abstract The cfr gene associated with linezolid resistance has attracted wide attention. However, little is known about its prevalence and mode of transmission in Enterococcus faecalis. In this study, we investigate the prevalence and genetic environment of the cfr gene in 91 E. faecalis isolates collected from swine faecal swabs in 30 farms in Guangdong Province, China in 2012. A relatively high prevalence of cfr was identified in E. faecalis isolates (11/91, 12.1%) by PCR. All the cfr- positive E. faecalis strains had a multidrug-resistance phenotype including erythrocin, tetracycline, gentamicin, kanamicin and ciprofloxacin, except vancomycin and linezolid. Molecular typing indicated that ST475 and ST16 were the most common types in cfr- positive E. faecalis strains. In addition, we demonstrated that all the cfr genes were located on plasmids by S1 -PFGE and Southern blotting. A 12 kb cfr- positive plasmid (pE30) was identified in most (9/11) E. faecalis strains, but it couldn't mediate resistance to linezolid in the transconjugant. Sequence analysis showed that the pE30 was a pCPPF5-like plasmid and the region surrounding the cfr gene was the same as a cfr -carrying IS Enfa5 -composite element in Streptococcus plasmid pStrcfr with 4 bp direct repeat (GTAT) on both sides. In conclusion, the cfr gene which had no linezolid resistance phenotype was present in multidrug-resistance E. faecalis strains, and the clonal spread of ST475 and ST16 strains and the horizontal transfer of the pCPPF5-like plasmids have contributed to the dissemination of cfr. Highlights • A relatively high prevalence of cfr was found in E. faecalis isolates from swine in China. • A prevent cfr -carrying pCPPF5-like plasmids was found and it contributed to 'non-linezolid resistance' phenotype in E. faecalis isolates. [ABSTRACT FROM AUTHOR]

Background: CMY-2 is the most prevalent pAmpC β-lactamase, but the chromosomal blaCMY-2 gene transfer via horizontal transmission has been seldom reported. This study aimed to describe an ISEcp1-mediated transposition of a chromosomal blaCMY-2 gene from Escherichia coli into a small endogenous ColE1-like plasmid, resulting in elevated resistance to extended-spectrum cephalosporins. Methods: Three ESCs-resistant ST641 E. coli strains EC6413, EC4103 and EC5106 harbored the blaCMY-2 gene. S1- PFGE, I-ceu I-PFGE, Southern blotting and electroporation experiments were performed to investigate the location and transferability of blaCMY-2. The genetic context and gene expression of blaCMY-2 in the original isolates and the corresponding electroporants were explored by PCR mapping, primer walking strategy and RT-qPCR. Results: The blaCMY-2-containing region (ISEcp1-blaCMY-2-∆blc-∆yggR-∆tnp1-orf7-orf8-orf9-∆tnp2-∆hsdR) was transposed into endogenous ColE1-like plasmid pSC137 in the process of electroporation at very low frequencies (10–8–10–9). The transpositions resulted in novel larger blaCMY-2-harboring ColE1-like plasmids with size of 14,845 bp, enabling increase in MICs of 2 to 8-fold for cefotaxime, ceftiofur, and ceftazidime in recipient strains over their respective original counterparts. Transcriptional level analysis revealed that the increased blaCMY-2 expression was correlated with elevated MIC values of cephalosporins. The blaCMY-2 transposition unit was identical to that in a clinical isolate E. coli TN44889 from France isolated in 2004. Conclusions: Our results firstly demonstrated that ISEcp1 mediated a transposition of chromosome-borne blaCMY-2 into an endogenous ColE1-like plasmid by electroporation. Amplification of the blaCMY-2 gene facilitates the strain adaptation to a changed environment with an elevated antibiotic pressure. [ABSTRACT FROM AUTHOR]

Two colistin-resistant Escherichia coli strains (FS13Z2S and FS3Z6C) possessing chromosomally encoded mcr-1 isolated from swine were characterised. Whole-genome sequencing revealed that in strain FS13Z2S mcr-1 occurred in triplicate in the chromosome with another copy encoded on a pHNSHP45-2-like IncHI2 plasmid, whereas in strain FS3Z6C only one copy mcr-1 was inserted in the chromosome. It seems likely that the triplication of chromosomal copies of mcr-1 in FS13Z2S is due to intramolecular transposition events via a composite transposon containing an mcr-1 cassette bracketed by two copies of insertion sequence IS Apl1 , and the pap2 gene at the insertion site was truncated by an IS 1294 -like element. In plasmid pFS13Z2S and the chromosome of strain FS3Z6C, only a single copy of IS Apl1 was present upstream of the mcr-1 cassette. The two strains exhibited similar colistin minimum inhibitory concentrations (MICs) and featured phosphoethanolamine addition to lipid A, without regard to the copy number of mcr-1 . The mcr-1 -harbouring plasmid was unstable in wild-type strain FS13Z2S and was quickly lost after 7 days of passage on colistin-free Luria–Bertani broth containing 0.5% SDS, but the mcr-1 copies on the chromosome persisted. These results reveal that the single copy of mcr-1 could result in modification of lipopolysaccharide (LPS) and cause colistin resistance in E. coli . Acquisition of multiple copies of mcr-1 , especially on the chromosome, would facilitate stable persistence of colistin resistance in the host strain. [ABSTRACT FROM AUTHOR]

To study the characteristics of plasmids harboring oqxAB among blaCTX-M-negative Escherichia coli isolates and search for oqxAB-harboring plasmids similar to plasmids carrying oqxAB- blaCTX-M reported previously, conjugation experiment was performed for 115 randomly selected oqxAB-positive but blaCTX-M-negative E. coli isolates from diseased animals in Guangdong, China. S1 nuclease pulsed-field gel electrophoresis (PFGE) and southern blotting experiments were performed to investigate the location of oqxAB and other resistance genes. The EcoRI digestion profiles of the plasmids with oqxAB were also analyzed. The clonal relatedness of donor isolates was investigated by PFGE. In this study, 32 oqxAB transconjugants were successfully obtained and most transconjugants showed multidrug resistances. Eleven replicon combination types were found in these transconjugants. floR and oqxAB were found on the same plasmids in all nine transconjugants resistant to florfenicol. The sequences between floR and oqxAB were identical in most transconjugants and the two genes were both linked with tnp in insertion sequences. Nine F18:A-:B1 plasmids with only oqxAB shared identical EcoRI digestion profiles and the profiles were also identical with that of a plasmid carrying oqxAB- blaCTX-M found previously. Co-transfer of plasmids carrying oqxAB and fosA3, respectively, was also observed in one isolate. This study demonstrates the dissemination of oqxAB among blaCTX-M-negative E. coli isolates was mainly mediated by identical F18:A-:B1 plasmids. A novel arrangement of regions between floR and oqxAB might play an important role in the dissemination of floR- oqxAB. This is the first description of the genetic environment of the relationship between oqxAB and floR in E. coli. [ABSTRACT FROM AUTHOR]

Horizontal transfer of plasmid-encoded multidrug-resistant determinants is a major health problem and has attracted much public attention. We studied the dissemination of the efflux pump gene oqxAB located on transferable plasmid pHXY0908 between Salmonella Typhimurium and Escherichia coli in the gut of chickens. After an inoculation with Salmonella Typhimurium harboring oqxAB -bearing plasmid pHXY0908, chickens were treated with enrofloxacin and florfenicol. Inoculated, but non-treated chickens were included as a control group. Our results revealed that commensal E. coli isolates from the gut of chickens acquired the oqxAB -bearing plasmid in both treated and non-treated groups. Additionally, in the florfenicol treatment group, the average isolation rate of oqxAB -positive E. coli was significantly higher than that in the non-treated group. PFGE analysis showed that oqxAB -positive E. coli strains belonged to different patterns with one predominating. Moreover, multilocus sequence typing analysis revealed that E. coli ST533 was closely associated with the spread of oqxAB gene. qPCR analysis indicated that antibiotic administration provided selective advantages for sustaining a significantly high level of oqxAB gene from the DNA extracted from the feces. There was also a fluctuation in the intestinal microbiota with antibiotic therapy. In conclusion, the present study indicates that the oqxAB gene could be readily spread within the intestinal microflora. This could be enhanced by administrated with clinical doses of florfenicol and enrofloxacin, resulting in the enlargement of resistance gene reservoirs. In addition, ST533 E. coli isolates were found to contribute to transfer of the oqxAB gene. [ABSTRACT FROM AUTHOR]

• We reported 10 (3.0 %) tet (X5)- and tet (X6)-positive MDR A. baumannii isolates from sick animals. • Both the tet (X5)- and tet (X6)-positive A. baumannii strains could inactivate tetracyclines by hydroxylation. • The plasmid-borne tet (X5) and chromosomal tet (X6) genes shared homologous genetic structures with those of clinical strains. • A clonal spread of ST355 A. baumannii strains as well as IS CR2 -mediated transposition for tet (X) genes was discovered. The recent emergence of plasmid-mediated tigecycline resistance gene tet (X) has challenged the clinical effectiveness of tigecycline as a last-resort treatment option. During 2017–2018, 336 fecal samples from sick ducks, pigs, chickens and geese in Guangdong, China, were screened for tet (X)-positive Acinetobacter baumannii strains. Their activities on tetracyclines were determined by microbiological degradation and mass spectrometry, followed by susceptibility testing, sequence typing, gene transfer, molecular location and genomic DNA sequencing analyses. A total of 10 tet (X)-positive A. baumannii strains were isolated from ducks and chickens, including eight plasmid-borne tet (X5)-positive and two chromosomal tet (X6)-positive isolates. All of them exhibited good degradation activities on tetracyclines by hydroxylation at C11a and were multidrug-resistant to tigecycline, tetracycline, florfenicol, ciprofloxacin and trimethoprim/sulfamethoxazole. Genetically, they belonged to two sequence types (ST355, n = 8; ST1980, n = 2) that were consistent with their pulsotypes, revealing a clonal spread of ST355 A. baumannii. An IS CR2 - or IS 26 -mediated tet (X) transposition structure, homologous to those of clinical A. baumannii strains, was also identified and IS CR2 could transfer tet (X5) into the recipient Acinetobacter baylyi ADP1 at a frequency of (1.8 ± 0.3)×10−6. Therefore, more efforts are needed to evaluate the clinical impact of these tigecycline resistance genes. [ABSTRACT FROM AUTHOR]