Tandem amplification of a plasmid-borne tet(A) variant gene confers tigecycline resistance in Escherichia coli.

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.

poxtA amplification and mutations in 23S rRNA confer enhanced linezolid resistance in Enterococcus faecalis.

OBJECTIVES: This study aimed to explore the evolutionary patterns and resistance mechanisms of an Enterococcus faecalis strain harbouring poxtA under linezolid exposure. METHODS: A poxtA-carrying E. faecalis electrotransformant DJH702 with a linezolid minimum inhibitory concentration of 4 mg/L was exposed to increasing concentrations of linezolid (8-64 mg/L). The derived strains growing at 8, 16, 32 and 64 mg/L, designed DJH702_8, DJH702_16, DJH702_32 and DJH702_64, were obtained. The amplification and overexpression of poxtA were measured using sequencing and RT-PCR, the fitness cost by competition assays and the stability of the repeat units by serial passage. RESULTS: In all derived strains, high-level linezolid resistance develops through poxtA amplification. The relative copy numbers and transcription levels of poxtA were significantly increased. However, in the presence of higher linezolid concentrations, DJH702_32 and DJH702_64 showed reduced poxtA copy numbers and transcription levels compared with DJH702_8 and DJH702_16, but additional mutations in the 23S rRNA (G2505A). IS1216E-mediated formation of translocatable units with subsequent tandem amplification of these translocatable units supported the gain of poxtA segments. However, these amplicons were not stable and were lost frequently in the absence of a linezolid selection pressure. The amplification of the poxtA region did not result in a fitness cost, but mutations in 23S rRNA did. CONCLUSIONS: poxtA-carrying E. faecalis electrotransformants used two distinct mechanisms to resist linezolid selection pressure: at lower concentrations, strains prioritized increasing poxtA expression levels, while at higher concentrations, a combination of increased poxtA expression and mutations in 23S rRNA was observed.

Mobile Oxazolidinone Resistance Genes in Gram-Positive and Gram-Negative Bacteria.

Seven mobile oxazolidinone resistance genes, including cfr, cfr(B), cfr(C), cfr(D), cfr(E), optrA, and poxtA, have been identified to date. The cfr genes code for 23S rRNA methylases, which confer a multiresistance phenotype that includes resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A compounds. The optrA and poxtA genes code for ABC-F proteins that protect the bacterial ribosomes from the inhibitory effects of oxazolidinones. The optrA gene confers resistance to oxazolidinones and phenicols, while the poxtA gene confers elevated MICs or resistance to oxazolidinones, phenicols, and tetracycline. These oxazolidinone resistance genes are most frequently found on plasmids, but they are also located on transposons, integrative and conjugative elements (ICEs), genomic islands, and prophages. In these mobile genetic elements (MGEs), insertion sequences (IS) most often flanked the cfr, optrA, and poxtA genes and were able to generate translocatable units (TUs) that comprise the oxazolidinone resistance genes and occasionally also other genes. MGEs and TUs play an important role in the dissemination of oxazolidinone resistance genes across strain, species, and genus boundaries. Most frequently, these MGEs also harbor genes that mediate resistance not only to antimicrobial agents of other classes, but also to metals and biocides. Direct selection pressure by the use of antimicrobial agents to which the oxazolidinone resistance genes confer resistance, but also indirect selection pressure by the use of antimicrobial agents, metals, or biocides (the respective resistance genes against which are colocated on cfr-, optrA-, or poxtA-carrying MGEs) may play a role in the coselection and persistence of oxazolidinone resistance genes.

Mobilization of tet(X4) by IS1 Family Elements in Porcine Escherichia coli Isolates.

The dissemination mechanism of the high-level tigecycline resistance gene tet(X4) in porcine Escherichia coli was investigated. tet(X4) and other antimicrobial resistance genes were located on the plasmids p1919D3-1 and p1919D62-1 and flanked by two or three copies of IS1 family elements, which can form one to three translocatable units (TUs). Using a reduced transposition model, IS1A was experimentally demonstrated to mediate the transposition of tet(X4) from a suicide plasmid into the E. coli chromosome.

Recombination events that occur in a poxtA-carrying Enterococcus faecium during the conjugation process.

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.

Global distribution, dissemination and overexpression of potent multidrug efflux pump RE-CmeABC in Campylobacter jejuni.

OBJECTIVES: To investigate the global distribution, dissemination and overexpression of RE-CmeABC in Campylobacter jejuni. METHODS: WGS information for 433 RE-cmeABC-positive C. jejuni isolates (including 18 isolates sequenced in this study and 415 isolates from GenBank) was used for the generation of minimum-spanning trees with STs. WGS information for 95 representative RE-cmeABC-positive C. jejuni isolates was used for phylogenetic analysis. RT-PCR was conducted to evaluate the association between inverted repeat (IR) sequence diversity in the RE-CmeABC promoter region and RE-cmeABC gene expression. RESULTS: WGS analysis revealed the global distribution of RE-cmeABC among C. jejuni from more than 10 countries. MLST results indicated that various STs were involved in the dissemination of RE-cmeABC, with ST2109 being the most predominant ST. Phylogenetic analysis revealed the close relationship between RE-cmeABC-carrying C. jejuni isolates from poultry and humans. The IR polymorphism in the RE-CmeABC promoter region is associated with the overexpression of RE-cmeABC, which was demonstrated experimentally by RT-PCR. CONCLUSIONS: To the best of our knowledge, our analysis represents the first view of the global distribution of RE-CmeABC, which is horizontally transferable and diffused regionally in a clonal manner. The close relationship of RE-cmeABC-positive C. jejuni from poultry and humans supports the potential of these isolates for zoonotic transmission. Overexpressed RE-CmeABC in C. jejuni will increase the fitness of the corresponding bacteria and be of advantage under antimicrobial selection.

Emergence of a Novel tet(L) Variant in Campylobacter spp. of Chicken Origin in China.

Tetracyclines are widely used in veterinary medicine and food animal production. Campylobacter members are major foodborne pathogens, and their resistance to tetracycline has been widely reported in different countries. To date, Tet(O), a ribosomal protection protein, is the only confirmed Tet resistance determinant in Campylobacter spp. Here, we reported the detection and characterization of a novel Tet resistance element in Campylobacter spp. of chicken origin. This gene is identified to be a variant of tet(L), which encodes an efflux pump for Tet resistance. The variant was detected in 14 of the 82 tetracycline-resistant Campylobacter isolates collected from chickens in Henan, China. Cloning of the tet(L) variant into tetracycline-susceptible Campylobacter jejuni NCTC 11168 confirmed its function in conferring resistance to tetracycline and doxycycline. In addition, this tet(L) variant elevated the MIC (4-fold increase) of tigecycline in the heterologous Escherichia coli host. Sequencing analysis indicated the tet(L) variant was located within a multidrug-resistance genomic island (MDRGI) containing tet(L) variant IS1216E-ORF1-fexA-Δtnp-IS1216E-tet(O)-tnpV-repA This MDRGI is inserted into conserved gene potB on the chromosome. Multilocus sequence type (MLST) analysis revealed that both clonal expansion and horizontal transfer were involved in the dissemination of the tet(L) variant. These findings reveal the emergence of a new Tet resistance determinant in Campylobacter spp., which may facilitate their adaptation to the antimicrobial selection pressure in chickens.

Studies on the role of IS1216E in the formation and dissemination of poxtA-carrying plasmids in an Enterococcus faecium clade A1 isolate.

OBJECTIVES: To analyse the role of IS1216E in the dissemination of the phenicol-oxazolidinone-tetracycline resistance gene poxtA in an Enterococcus faecium clade A1 isolate. METHODS: MICs were determined by broth microdilution. The poxtA-positive isolate was typed by MLST. The two plasmids were characterized by PCR, conjugation, S1-PFGE, Southern blot hybridization and WGS analysis. The presence of translocatable units (TUs) was examined by PCR and sequencing. RESULTS: Isolate E1077 contains the 217661 bp conjugative plasmid pE1077-217 and the 23710 bp mobilizable plasmid pE1077-23. pE1077-217 harbours erm(B), aac(A)-aph(D), aadE, spw, lsa(E), lnu(B), aphA3 and dfrG, whereas pE1077-23 carries a Tn6657-like transposon containing poxtA and fexB. pE1077-23 was apparently formed by an IS1216E-mediated composite transposon-plasmid fusion event, involving a replicative transposition process. Conjugation experiments showed that pE1077-23 is mobilizable by pE1077-217. Moreover, a novel 31742 bp plasmid, pT-E1077-31, was found in a transconjugant. WGS analysis indicated that pT-E1077-31 was formed by the integration of a Tn6657-derived, IS1216E-based translocatable unit, which carried fexB and poxtA, into a copy of pE1077-23. CONCLUSIONS: This study showed the presence of two cointegrate formation events in the formation and spread of a poxtA/fexB-carrying plasmid in E. faecium. One was the integration of a transposon into a plasmid while the other was the integration of a TU into a different site of the same type of plasmid-borne transposon from which it originated. In both events, IS1216E played a major role, suggesting that IS1216E-mediated transposition and translocation processes aid the dissemination and persistence of important antimicrobial resistance genes, such as poxtA, among enterococci.

A novel multiresistance gene cluster located on a plasmid-borne transposon in Listeria monocytogenes.

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.

Tn6674 Is a Novel Enterococcal optrA-Carrying Multiresistance Transposon of the Tn554 Family.

The novel 12,932-bp nonconjugative multiresistance transposon Tn6674 was identified in the chromosomal DNA of a porcine Enterococcus faecalis strain. Tn6674 belongs to the Tn554 family of transposons. It shares the same arrangement of the transposase genes tnpA, tnpB, and tnpC with Tn554 However, in addition to the Tn554-associated resistance genes spc and erm(A), Tn6674 harbored the resistance genes fexA and optrA Circular forms of Tn6674 were detected and suggest the functional activity of this transposon.

Analysis of a poxtA- and optrA-co-carrying conjugative multiresistance plasmid from Enterococcus faecalis.

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.

A prophage and two ICESa2603-family integrative and conjugative elements (ICEs) carrying optrA in Streptococcus suis.

OBJECTIVES: To investigate the presence and transfer of the oxazolidinone/phenicol resistance gene optrA and identify the genetic elements involved in the horizontal transfer of the optrA gene in Streptococcus suis. METHODS: A total of 237 S. suis isolates were screened for the presence of the optrA gene by PCR. Whole-genome DNA of three optrA-positive strains was completely sequenced using the Illumina MiSeq and Pacbio RSII platforms. MICs were determined by broth microdilution. Transferability of the optrA gene in S. suis was investigated by conjugation. The presence of circular intermediates was examined by inverse PCR. RESULTS: The optrA gene was present in 11.8% (28/237) of the S. suis strains. In three strains, the optrA gene was flanked by two copies of IS1216 elements in the same orientation, located either on a prophage or on ICESa2603-family integrative and conjugative elements (ICEs), including one tandem ICE. In one isolate, the optrA-carrying ICE transferred with a frequency of 2.1 × 10-8. After the transfer, the transconjugant displayed elevated MICs of the respective antimicrobial agents. Inverse PCRs revealed that circular intermediates of different sizes were formed in the three optrA-carrying strains, containing one copy of the IS1216E element and the optrA gene alone or in combination with other resistance genes. CONCLUSIONS: A prophage and two ICESa2603-family ICEs (including one tandem ICE) associated with the optrA gene were identified in S. suis. The association of the optrA gene with the IS1216E elements and its location on either a prophage or ICEs will aid its horizontal transfer.

Novel lnu(G) gene conferring resistance to lincomycin by nucleotidylation, located on Tn6260 from Enterococcus faecalis E531.

Objectives: To identify a novel putative lincosamide resistance gene determinant in a swine Enterococcus faecalis E531 exhibiting a lincosamide resistance/macrolide susceptibility (L R M S ) phenotype and to determine its location and genetic environment. Methods: The whole genomic DNA of E. faecalis E531, which tested negative for the known lincosamide nucleotidyltransferase genes, was sequenced. A putative lincosamide resistance gene determinant was cloned into an Escherichia coli - E. faecalis shuttle vector (pAM401) and transformed into E. faecalis JH2-2. The MICs were determined by the microbroth dilution method. Inactivity of lincomycin was examined by UPLC-MS/MS. Inverse PCR and primer walking were used to explore the genetic environment based on the assembled sequence. Results: A novel resistance gene, designated lnu (G), which encodes a putative lincosamide nucleotidyltransferase, was found in E. faecalis E531. The deduced Lnu(G) amino acid sequence displayed 76.0% identity to Lnu(B) in Enterococcus faecium . Both E. faecalis E531 and E. faecalis JH2-2 harbouring pAM401- lnu (G) showed a 4-fold increase in the MICs of lincomycin, compared with E. faecalis JH2-2 or E. faecalis JH2-2 harbouring empty vector pAM401 only. UPLC-MS/MS demonstrated that the Lnu(G) enzyme catalysed adenylylation of lincomycin. The genetic environment analysis revealed that the lnu (G) gene was embedded into a novel putative transposon, designated Tn 6260 , which was active. Conclusions: A novel lincosamide nucleotidyltransferase gene lnu (G) was identified in E. faecalis . The location of the lnu (G) gene on a mobile element Tn 6260 makes it easy to disseminate.

Functional analysis of a bacitracin resistance determinant located on ICECp1, a novel Tn916-like element from a conjugative plasmid in Clostridium perfringens.

Bacitracins are mixtures of structurally related cyclic polypeptides with antibiotic properties. They act by interfering with the biosynthesis of the bacterial cell wall. In this study, we analyzed an avian necrotic enteritis strain of Clostridium perfringens that was resistant to bacitracin and produced NetB toxin. We identified a bacitracin resistance locus that resembled a bacitracin resistance determinant from Enterococcus faecalis. It contained the structural genes bcrABD and a putative regulatory gene, bcrR. Mutagenesis studies provided evidence that both bcrA and bcrB are essential for bacitracin resistance, and that evidence was supported by the results of experiments in which the introduction of both the bcrA and bcrB genes into a bacitracin-susceptible C. perfringens strain was required to confer bacitracin resistance. The wild-type strain was shown to contain at least three large, putatively conjugative plasmids, and the bcrRABD locus was localized to an 89.7-kb plasmid, pJIR4150. This plasmid was experimentally shown to be conjugative and was sequenced. The sequence revealed that it also carries a tpeL toxin gene and is related to the pCW3 family of conjugative antibiotic resistance and toxin plasmids from C. perfringens. The bcr genes were located on a genetic element, ICECp1, which is related to the Tn916 family of integrative conjugative elements (ICEs). ICECp1 appears to be the first Tn916-like element shown to confer bacitracin resistance. In summary, we identified in a toxin-producing C. perfringens strain a novel mobile bacitracin resistance element which was experimentally shown to be essential for bacitracin resistance and is carried by a putative ICE located on a conjugative plasmid.