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.

Excision and integration of unconventional circularizable structures involving the erm(B) gene in enterococci.

Traditionally, insertion sequences (ISs) play a major role in disseminating antimicrobial resistance genes (ARGs) in bacteria through transposition and translocation, forming regions that contain multiple ARGs flanked by single or multiple copies of IS. In addition, unconventional circularizable structures (UCSs), lacking recombinase genes but being surrounded by directly repeated sequences (DRs) of various sizes which do not contain transposase genes, were reported to be involved in the dissemination of ARGs. In this study, a novel UCS was identified on plasmid pE508-2 in E. faecalis E508, which carried a 24,411 bp multiresistance gene cluster, consisting of the resistance genes aphA3, lnu(B), lsa(E), spw, aac(A)-aph(D), lnu(B), dfrG, and two copies of aadE flanked by copies of erm(B). PCR assays revealed that three types of UCSs with lengths of 7235, 16,437, and 23,673 bp were formed, each of which contained the respective resistance genes and one copy of erm(B). Using erm(B)-negative and -positive strains, we demonstrated that erm(B)-carrying UCSs failed to transfer into an erm(B)-negative strain, but could integrate into an erm(B)-positive strain in a new site adjacent to a pre-existing erm(B) gene by natural transformation. Database searches revealed that erm(B)-flanked multiresistance gene regions, which might be able to form the respective UCSs, are present among various bacteria from different sources in various countries. In summary, this study experimentally demonstrated the excision and integration of UCS involving structures that include erm(B). The widespread presence of these UCSs in various Gram-positive bacteria highlights its role in the dissemination of ARGs among bacterial pathogens.

A Small Multihost Plasmid Carrying erm(T) Identified in Enterococcus faecalis.

The aim of this study was to determine the mobile genetic elements involved in the horizontal transfer of erm(T) in Enterococcus faecalis, and its transmission ability in heterologous hosts. A total of 159 erythromycin-resistant enterococci isolates were screened for the presence of macrolide resistance genes by PCR. Whole genome sequencing for erm(T)-carrying E. faecalis E165 was performed. The transmission ability in heterologous hosts was explored by conjugation, transformation, and fitness cost. The erm(T) gene was detected only in an E. faecalis isolate E165 (1/159), which was located on a 4,244-bp small plasmid, designed pE165. Using E. faecalis OG1RF as the recipient strain, pE165 is transferable. Natural transformation experiments using Streptococcus suis P1/7 and Streptococcus mutans UA159 as the recipients indicated it is transmissible, which was also observed by electrotransformation using Staphylococcus aureus RN4220 as a recipient. The erm(T)-carrying pE165 can replicate in the heterologous host including E. faecalis OG1RF, S. suis P1/7, S. mutans UA159, and S. aureus RN4220 and conferred resistance to erythromycin and clindamycin to all hosts. Although there is no disadvantage of pE165 in the recipient strains in growth curve experiments, all the pE165-carrying recipients had a fitness cost compared to the corresponding original recipients in growth competition experiments. In brief, an erm(T)-carrying plasmid was for the first time described in E. faecalis and as transmissible to heterologous hosts.

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.

Plasmid Fusion and Recombination Events That Occurred during Conjugation of poxtA-Carrying Plasmids in Enterococci.

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 IS1216E-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.

erm(T)-Mediated Macrolide-Lincosamide Resistance in Streptococcus suis.

To investigate the presence and location of erm(T) in clinical Streptococcus suis isolates and explore the transmission ability and fitness cost of erm(T)-carrying mobile genetic elements among S. suis isolates, MICs were determined by broth microdilution. The presence of erm(T) in S. suis was detected by PCR. The genetic environment of erm(T) in S. suis was explored by whole-genome sequencing (WGS) analysis. Intraspecies and interspecies transmission were examined by electrotransformation. The fitness cost associated with the carriage of an erm(T)-harboring plasmid or an integrative and conjugative element (ICE) was examined by competition experiments. Of 237 nonduplicate strains, erm(T) was detected in 2 S. suis strains (SC262-ST954 and SC117-ST1314), with its location on a 5,125-bp plasmid in S. suis SC262 and on a 64,013-bp ICESsuSC117 in S. suis SC117, respectively. Both the erm(T)-carrying plasmid pSC262 and the ICESsuSC117 were transmissible by transformation. Plasmid pSC262 can replicate and express macrolide-lincosamide resistance in heterologous hosts, including S. aureus and S. pneumoniae. Both the erm(T)-carrying plasmid and the ICE posed a fitness cost to the host S. suis isolate. To the best of our knowledge, this is the first report of the macrolide-lincosamide-streptogramin B resistance gene erm(T) in S. suis. Its location on a plasmid or an ICE will aid in its transmission. The low detection rate of erm(T) gene among the S. suis population might be due to the fitness cost of the erm(T)-carrying plasmid and ICE. IMPORTANCE Macrolide and lincosamide resistance due to the presence of erm(T) have posed a challenge for the treatment of Gram-positive pathogens. Although the low detection rate of erm(T) gene among the S. suis population due to the fitness cost of the erm(T)-carrying plasmid and ICE, the presence of erm(T) in S. suis and its potential transmission to other Gram-positive pathogens will be of important significance.

Novel Tet(L) Efflux Pump Variants Conferring Resistance to Tigecycline and Eravacycline in Staphylococcus Spp.

Tigecycline is regarded as one of the few important last-resort antibiotics to treat complicated skin and intra-abdominal infections. Members of the genus Staphylococcus are zoonotic pathogens and pose a serious threat to public health. Tigecycline resistance in this species appears to be a rare phenomenon, and the mechanisms underlying tigecycline resistance have not been fully elucidated. Here, we report two novel variants of the tet(L) gene in Staphylococcus spp. from swine in China, designed as tet(L)F58L and tet(L)A117V. The tet(L)F58L was located within a 18,720 bp chromosomal multidrug resistance gene cluster flanked by two copies of IS257 in Staphylococcus cohnii 11-B-312, while the tet(L)A117V was located on a 6,292 bp plasmid in S. haemolyticus 11-B-93, which could be transferred to S. aureus by electrotransformation. Cloning of each of the two tet(L) variants into S. aureus RN4220 showed 16- or 8-fold increases in the minimal inhibition concentrations (MICs), which can fully confer the resistance to tigecycline (MICs from 0.125 to 2 mg/liter) and eravacycline (MICs from 0.125 to 1 or 2 mg/liter), but no increase in the MICs of omadacycline, compared with the MICs of the recipient strain S. aureus RN4220. In the in vivo murine sepsis and in the murine pneumonia models, an increase in CFU of S. aureus 29213_pT93 carrying the tet(L)A117V was seen despite tigecycline treatment. This observation suggests that the tet(L)A117V and its associated gene product compromise the efficacy of tigecycline treatment in vivo and may lead to clinical treatment failure. Our finding, that novel Tet(L) efflux pump variants which confer tigecycline and eravacycline resistance have been identified in Staphylococcus spp., requires urgent attention. IMPORTANCE Tigecycline and eravacycline are both important last-resort broad spectrum antimicrobial agents. The presence of novel Tet(L) efflux pump variants conferring the resistance to tigecycline and eravacycline in Staphylococcus spp. and its potential transmission to S. aureus will compromise the efficacy of tigecycline and eravacycline treatment for S. aureus associated infection in vivo and may lead to clinical treatment failure.

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.

Analysis of two pheromone-responsive conjugative multiresistance plasmids carrying the novel mobile optrA locus from Enterococcus faecalis.

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.

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.

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.

Distribution of optrA and cfr in florfenicol-resistant Staphylococcus sciuri of pig origin.

A novel transferable oxazolidinone-phenicol resistance gene, optrA, which confers resistance to linezolid, the next-generation oxazolidinone tedizolid, and also to chloramphenicol and florfenicol, has been identified in enterococcal and staphylococcal species. Here, we investigated the epidemiology of optrA in florfenicol-resistant Staphylococcus spp. isolates of pig origin, and characterized the genetic context of oxazolidinone resistance genes in 20 optrA-positive florfenicol- and methicillin-resistant S. sciuri isolates, 11 (55%) of which also harbored the multi-resistance gene cfr. Pulsed-field gel electrophoresis and direct repeat unit (dru) typing of the 20 optrA-positive S. sciuri isolates revealed 17 patterns and four distinct dru types, respectively. Nine and six different arrangements of the optrA and cfr gene regions, respectively, were identified among the isolates. The arrangements optrA-araC-Tn558 or optrA-ΔTn558 were present in all optrA-positive isolates, and in three of them, ISEnfa5 and cfr were located immediately downstream of optrA. The cfr-carrying segment in eight isolates was similar to the corresponding region of the staphylococcal plasmid pWo28-3, in which the coexistence of cfr and optrA was identified for the first time.

Presence of the optrA Gene in Methicillin-Resistant Staphylococcus sciuri of Porcine Origin.

A total of 57 methicillin-resistant Staphylococcus aureus (MRSA) isolates and 475 methicillin-resistant coagulase-negative staphylococci (MRCoNS) collected from pigs in the Guangdong province of China in 2014 were investigated for the presence of the novel oxazolidinone-phenicol resistance gene optrA The optrA gene was detected in 6.9% (n = 33) of the MRCoNS, all of which were Staphylococcus sciuri isolates, but in none of the MRSA isolates. Five optrA-carrying methicillin-resistant (MR) S. sciuri isolates also harbored the multiresistance gene cfr Pulsed-field gel electrophoresis (PFGE) and dru typing of the 33 optrA-carrying MR S. sciuri isolates revealed 25 patterns and 5 sequence types, respectively. S1 nuclease PFGE and Southern blotting confirmed that optrA was located in the chromosomal DNAs of 29 isolates, including 1 cfr-positive isolate. The remaining four isolates harbored a ∼35-kb pWo28-3-like plasmid on which optrA and cfr were located together with other resistance genes, as confirmed by sequence analysis. Six different types of genetic environments (types I to VI) of the chromosome-borne optrA genes were identified; these types had the optrA gene and its transcriptional regulator araC in common. Tn558 was found to be associated with araC-optrA in types II to VI. The optrA gene in types II and III was found in close proximity to the ccr gene complex of the respective staphylococcal cassette chromosome mec element (SCCmec). Since oxazolidinones are last-resort antimicrobial agents for the control of serious infections caused by methicillin-resistant staphylococci in humans, the location of the optrA gene close to the ccr complex is an alarming observation.

Co-location of the oxazolidinone resistance genes optrA and cfr on a multiresistance plasmid from Staphylococcus sciuri.

OBJECTIVES: To identify and characterize the oxazolidinone/phenicol resistance gene optrA in Staphylococcus isolates. METHODS: Fifty porcine staphylococci with florfenicol MICs of ≥16 mg/L were screened by PCR for the presence of the optrA gene. Transferability of optrA was examined by transformation and conjugation. Functionality of this gene was confirmed by cloning and expression in a susceptible Staphylococcus host. The optrA-carrying plasmid was completely sequenced and analysed. RESULTS: A single Staphylococcus sciuri was optrA positive. This isolate carried the optrA gene on the 60 563 bp multiresistance plasmid pWo28-3, which also harboured the resistance genes, cfr, fexA, aadD, ble and aacA-aphD. Plasmid pWo28-3 is composed of three regions (A, B and C). Region A, which harboured all resistance genes except optrA, showed ≥99.8% nucleotide sequence identity to the corresponding region of plasmids pJP1 and pJP1-like from Jeotgalicoccus pinnipedialis and Staphylococcus lentus, respectively. The optrA gene located in region B differed from the optrA gene of the Enterococcus faecalis plasmid pE349 by four nucleotide substitutions, which also resulted in amino acid substitutions. This optrA variant also conferred resistance to oxazolidinones and phenicols in staphylococci. The 28 genes in region C represent the plasmid backbone and were apparently acquired from staphylococci, enterococci and nosocomiicocci. CONCLUSIONS: This is the first report of the optrA gene in staphylococci and of the coexistence of optrA and cfr on the same plasmid. Dissemination of this plasmid will substantially limit the efficacy of oxazolidinones. Surveillance of optrA in staphylococci of both human and animal origin is urgently warranted.

Identification of multiresistance gene cfr in methicillin-resistant Staphylococcus aureus from pigs: plasmid location and integration into a staphylococcal cassette chromosome mec complex.

The multiresistance gene cfr was found in 8/231 porcine methicillin-resistant Staphylococcus aureus isolates. They were characterized by multilocus sequence typing, spa typing, dru typing, and staphylococcal cassette chromosome mec (SCCmec) typing as ST627-t002-dt12w-IVb, ST6-t304-dt12w-IVb, ST9-t899-dt12w-IVb, ST9-t899-dt12ae-IVb, or ST63-t899-dt12v-IVb. Different cfr gene regions were detected on plasmids of ca. 35 kb in seven isolates. For the first time, an ISEnfa4-cfr-IS256 fragment was found to be inserted upstream of the ccr genes in a chromosomal SCCmec IVb element of the remaining isolate.