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Objectives: To elucidate the mechanism of tigecycline resistance in Escherichia coli that is mediated by the tet(A) variant gene., Methods: E. coli strain 573 carried a plasmid-borne tet(A) variant gene, tentatively designated tet(A)TIG, that conferred decreased tigecycline susceptibility (MIC 0.5 mg/L). When exposed to increasing concentrations of tigecycline (0.25-8 mg/L), mutants growing at 2, 4 and 8 mg/L were obtained and sequenced. Copies of plasmid and tet(A)TIG relative to the chromosomal DNA in the mutants were determined by WGS and quantitative PCR (qPCR). Expression of tet(A)TIG in the mutants was evaluated by RT-qPCR. The tet(A)TIG-carrying plasmids were visualized by S1-PFGE and Southern blot hybridization. PCR served for the detection of a tet(A)TIG-carrying unconventional circularizable structure (UCS)., Results: Tigecycline resistance with maximum MICs of 16 mg/L was seen in E. coli mutants selected in the presence of tigecycline. Compared with the parental strain, the relative copy number and transcription level of tet(A)TIG in the mutants increased significantly in the presence of 2, 4 and 8 mg/L tigecycline, respectively. With increasing tigecycline selection pressure, the tet(A)TIG-carrying plasmids in the mutants increased in size, correlating with the number of tandem amplificates of a ΔTnAs1-flanked UCS harbouring tet(A)TIG. These tandem amplificates were not stable in the absence of tigecycline., Conclusions: Tigecycline resistance is due to the tandem amplification of a ΔTnAs1-flanked tet(A)TIG-carrying plasmid-borne segment in E. coli. The gain/loss of the tandem amplificates in the presence/absence of tigecycline represents an economic way for the bacteria to survive in the presence of tigecycline., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Linezolid plays a crucial role in the treatment of infections caused by multiresistant Gram-positive bacteria. The poxtA gene not only confers oxazolidinone and phenicol resistance but also decreases susceptibility to tetracycline. In this study, we investigated structural changes in mobilizable poxtA -carrying plasmids in enterococci which occurred during conjugation experiments using S1-PFGE (pulsed-field gel electrophoresis), Southern blot hybridization, and whole-genome sequencing (WGS) analysis. Two poxtA -carrying strains were identified in Enterococcus faecalis E006 and Enterococcus lactis E843, respectively. E. faecalis E006 contains the 121,520-bp conjugative plasmid pE006-121 and the 19,832-bp mobilizable poxtA -carrying plasmid pE006-19, while E. lactis E843 contains the 171,930-bp conjugative plasmid pE843-171 and the 27,847-bp mobilizable poxtA -carrying plasmid pE843-27. Moreover, both poxtA -carrying plasmids were mobilized by their respective conjugative plasmid in enterococci by plasmid fusion; one was generated by homologous recombination in E. faecalis through an identical 864-bp homologous region in the plasmids of the parental strain, while another was generated by an IS 1216E -mediated plasmid integration in E. lactis , involving a replicative transposition. IMPORTANCE Until now, all the poxtA genes described in enterococci, including E. faecalis, E. faecium, and E. hirae, are plasmid-borne, suggesting that plasmids play an important role in the dissemination of the poxtA gene among enterococci. This study showed that the mobilizable poxtA -carrying plasmid could transfer with the help of conjugative plasmid in enterococci via plasmid fusion, with one generated by homologous recombination in E. faecalis, and another by replicative transposition in E. lactis . During both the fusion events, the poxtA -carrying plasmids changed from nonconjugative to conjugative, leading to the generation and enhanced dissemination of the larger phenicol-oxazolidinone-tetracycline resistance-encoding plasmids in enterococci.

Objectives: To investigate the presence and transferability of the poxtA gene and identify the genetic context of poxtA in two enterococcal plasmids from swine., Methods: MICs were determined by broth microdilution. A total of 114 porcine enterococci with florfenicol MICs of ≥16 mg/L were screened for the presence of the poxtA gene by PCR. Transferability of poxtA was investigated by conjugation and transformation. The poxtA-carrying plasmids were completely sequenced using the Illumina Miseq and PacBio RSII platform. The presence of circular intermediates was examined by inverse PCR., Results: The poxtA gene was present in 57.9% (66/114) of the florfenicol-resistant porcine enterococci. Two poxtA-carrying plasmids, pE035 and pE076, were identified. The conjugative 121524 bp plasmid pE035 carried poxtA and optrA along with the resistance genes erm(A), erm(B), aac(A)-aph(D), lnu(G), fexB, dfrG and bcrABDR. Three mobile elements, comprising a mobile dfrG locus, a mobile bcrABDR locus and an unconventional circularizable structure containing aac(A)-aph(D), were located on this plasmid and all proved to be active by inverse PCR. The non-conjugative 19832 bp plasmid pE076 only carried poxtA and fexB. After transfer, both the transconjugant and the transformant displayed elevated MICs of the respective antimicrobial agents., Conclusions: To the best of our knowledge, this is the first report of the co-location of the oxazolidinone resistance genes poxtA and optrA on a conjugative multiresistance plasmid from a porcine enterococcal strain. In addition, the presence of three mobile elements in such a plasmid will aid in the persistence and dissemination of poxtA and optrA among enterococci., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Eight rmtB-carrying avian Escherichia coli strains from a farm in China were characterised in our previous study, but little is known about the backbones and entire multiresistance regions (MRRs) of these plasmids. Here, three rmtB-carrying IncI1 ST136 plasmids were analysed by whole-plasmid sequencing and were compared. These plasmids were composed of an 83 470-bp IncI1 backbone carrying genes responsible for plasmid replication, transfer, maintenance and stability functions, as well as a 17 330-bp MRR for pEC006 and pEC007, and a 34 626-bp MRR for pEC008. Plasmid pEC006 was not transferable, thus truncation of the traI gene may explain the inability to conjugate. pEC008 harboured the bla TEM-1 , rmtB, aacC2, tetA, floR and strAB genes as well as a class 1 integron cassette array (|dfrA12|orfF|aadA2|), which were interspersed with different mobile elements, including Tn2, Tn1721, Tn1722, Tn5393, ISCfr1, IS5057, ISCR1 and ISCR2, and three copies of IS26. The MRR of pEC008 may have resulted from transposition of Tn1722 into the plasmid backbone. Acquisition and rearrangement of MRRs demonstrated the accumulation of different resistance determinants., (Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

To evaluate the temporal change in the plasmid-mediated quinolone resistance (PMQR) determinants from 2001 to 2007 in chicken, a total of 532 chicken Escherichia coli isolates were screened for PMQR determinants by polymerase chain reaction and sequencing. The prevalence of qnr genes, aa(6')-Ib-cr, and qepA were 9.8%, 11.7%, and 0.75%, respectively. Among the qnr determinants, qnrA-, qnrB-, and qnrS-type genes were detected in 4 (0.75%), 21 (3.9%), and 27 (5.1%) of the examined isolates, respectively. None of the isolates carried qnrC gene. Ciprofloxacin resistance increased over time (p < 0.01), and a clear trend of increase in the prevalence of qnr and aac(6')-Ib-cr genes among the isolates was shown from 2001 to 2007 (p < 0.01). Pulsed-field gel analysis showed that the PMQR-positive isolates were not clonally related and genetically diverse. Quinolone resistance was transferred by conjugation from qnrB-, qnrS-, and aac(6')-Ib-cr-positive isolates to recipient E. coli. The qnrB and aac(6')-Ib-cr alleles were located on the plasmids with the size of 49 and 50 kb, respectively. However, the qnrS alleles were located on different plasmids with sizes from 57.4 to 88.6 kb, indicating diverse genetic backgrounds. The increasing frequency of ciprofloxacin resistance in E. coli was associated with increasing prevalence of qnr genes and aac(6')-Ib-cr (r(s) = 0.964, p = 0.00045). This survey showed that PMQR determinants were highly prevalent in chicken E. coli isolates in China with a trend of increase from 2001 to 2007. Horizontal transfer and widespread use of quinolone antimicrobials may have contributed to the spread of PMQR determinants in the poultry production system. The widespread dissemination of PMQR could potentially fuel the rapid development of fluoroquinolones resistance.

Objectives: To investigate transferability of the poxtA-carrying plasmids in Enterococcus faecium and the mechanism of recombination that occurs during the conjugation process.Methods: MICs were determined by broth microdilution. Transferability of the poxtA-carrying plasmids in E. faecium was investigated by conjugation. The mechanism of recombination that occurred during the conjugation process was explored by S1-PFGE and WGS.Results: E. faecium strain Fac90 carries two plasmids, designated pFac90-154 and pFac90-54, respectively. Six transconjugants with different characteristics were obtained. In transconjugant T90-1, a plasmid-chromosome fusion event led to the integration of plasmid pFac90-154 from the donor E. faecium strain Fac90 into the chromosomal DNA of the recipient strain Enterococcus faecalis JH2-2. In transconjugants T90-2, -3 and -4, losses or additions of different-sized plasmid segments most likely occurred due to IS1216-mediated recombination. In transconjugants T90-5 and -6, two large plasmids with sizes of 101 656 and 149 526 bp were formed by plasmid fusion.Conclusions: To the best of our knowledge, this is the first report showing the integration of pFac90-154 from E. faecium Fac90 into the chromosomal DNA of recipient E. faecalis JH2-2 via homologous recombination. Besides, we showed that five new plasmid types were formed by genetic rearrangements. These recombination events resulted simultaneously in the formation of various types of mosaic plasmids with multiple resistance genes and/or conjugation characteristics, which might promote the transmission of diverse plasmids encoding resistance genes among enterococci. Thus, these data significantly expand our knowledge regarding conjugative events, establishing a dual role of conjugation in both dissemination of resistance genes and plasmid evolution. [ABSTRACT FROM AUTHOR]

Objectives: To identify the genetic context and the transferability of the multiresistance gene lsa(E) in Listeria monocytogenes.Methods: MICs were determined by broth microdilution. Transferability of lsa(E) was investigated by conjugation, electrotransformation and natural transformation. The lsa(E)-carrying plasmid was sequenced using the Illumina MiSeq and PacBio RSII platforms. The presence of translocatable units (TUs) was examined by PCR.Results: The 85 555 bp non-conjugative multiresistance plasmid pNH1 from L. monocytogenes harboured nine antimicrobial resistance genes including a multiresistance gene cluster, consisting of the genes aphA3, erm(B), aadE, spw, lsa(E) and lnu(B), and in addition the genes dfrG, tet(S) and catA8 were also located on plasmid pNH1 The multiresistance gene cluster, and each of the genes tet(S), catA8 and cadA were flanked by IS1216 elements. PCR identified four types of TUs, consisting of either the multiresistance gene cluster and one copy of IS1216, the catA8 gene and one copy of IS1216, or both, but also the tet(S) gene and one copy of IS1216, respectively. Natural transformation into Streptococcus mutans UA159 yielded transformants that harboured a novel 13 208 bp transposon, designated Tn6659. This transposon consisted of the multiresistance gene cluster bounded by IS1216 copies. All transformants displayed elevated MICs of the respective antimicrobial agents. At the integration site in the transformants, 8 bp direct target duplications (5'-ATTCAAAC-3') were found immediately up- and downstream of Tn6659.Conclusions: To the best of our knowledge, this is the first report of this novel multiresistance gene cluster and the gene catA8, flanked by IS1216 elements located on a plasmid of L. monocytogenes. Moreover, a novel functionally active multiresistance transposon was identified. [ABSTRACT FROM AUTHOR]

Background: The acquired optrA gene, which encodes a ribosomal protection protein of the ABC-F family, can confer cross-resistance to linezolid and florfenicol, posing a serious therapeutic challenge to both human and veterinary medicine. Purpose: The objective of this study was to investigate the two Enterococcus faecalis (E. faecalis) plasmids for their fine structure, their transferability and the presence of mobile antimicrobial resistance loci. Methods: To elucidate their fine structure, the two plasmids were completely sequenced and the sequences analysed. Besides conjugation experiments, inverse PCR assays were conducted to see whether minicircles are produced from the mobile antimicrobial resistance loci. Results: Two pheromone-responsive conjugative optrA-carrying plasmids from E. faecalis, pE211 and pE508 were identified, which can transfer with frequencies of 2.6 ×10−2 and 3.7 ×10−2 (transconjugant per donor), respectively. In both plasmids, optrA was located on the novel mobile optrA locus with different sizes (12,834 bp in pE211 and 7,561 bp in pE508, respectively), flanked by two copies of IS1216 genes in the same orientation. Inverse PCR revealed that circular forms can be generated, consisting of optrA and one copy of IS1216, indicating they are all active. The 77,562 bp plasmid pE211 also carried Tn558 and a mobile bcrABDR locus, and the 84,468 bp plasmid pE508 also harbored the genes fexA, tet(L), tet(O/W/32/O) and a mobile aac(A)-aph(D) locus. Conclusion: The presence of mobile genetic elements in these plasmids renders them flexible and these elements will aid to the persistence and dissemination of these plasmids among enterococci and potentially also other gram-positive bacteria. [ABSTRACT FROM AUTHOR]