Gianluca Morroni

Detection in Italy of two clinical Enterococcus faecium isolates carrying both the oxazolidinone and phenicol resistance gene optrA and a silent multiresistance gene cfr

Sir, In a century in which the issue of emerging antibiotic resistance is being dominated by severe concerns chiefly regarding Gram-negative organisms, the multiresistance gene cfr is probably the greatest emerging problem in Gram-positive pathogens, particularly staphylococci and enterococci. The concern over this problem is motivated not only by the fact that the resistance involves linezolid—widely used in serious infections caused by MDR Gram-positive organisms, often as a last-resort drug—but also, and critically, by the fact that the frequent location of cfr on conjugative plasmids makes the resistance transferable. Now, the report in China of a second plasmid-borne transferable gene, optrA, conferring efflux-mediated oxazolidinone (including second-generation tedizolid) and phenicol resistance in enterococcal isolates adds further to the concern. As soon as the first report, with the sequence of an Enterococcus faecalis plasmid (pE349) carrying optrA (accession no. KP399637), became available as an Advance Access article in the Journal of Antimicrobial Chemotherapy, we decided to test for the optrA gene in 81 Enterococcus isolates from blood samples, which make up the first batch of an enterococcal collection we had recently started for a study including cfr screening. Identification at the species level was performed using VITEK 2 (bioMérieux, Marcy-l’Étoile, France). The cfr and optrA genes were sought by PCR using primer pairs internal to either gene: respectively, the known pair cfr-fw and cfr-rv, yielding a 746 bp amplicon, and the specially designed pair optrA-fw and optrA-rv, which yielded a 422 bp amplicon (Table S1, available as Supplementary data at JAC Online). Two Enterococcus faecium isolates were positive for cfr and two were positive for optrA. Much to our surprise, there were in fact only two positive isolates (E20818 and E35048), since each carried both optrA and cfr. The antibiotic MICs and other features for the two isolates are reported in Table 1. Both isolates had a relatively low linezolid MIC, 4 mg/L, a value that is regarded as ‘susceptible’ according to EUCAST and ‘intermediate’ according to CLSI. Both had a tedizolid MIC of 2 mg/L; breakpoints for resistance have recently been established by EUCAST for staphylococci and b-haemolytic streptococci (.0.5 mg/L) and for viridans group streptococci (.0.25 mg/L). The two isolates were also examined for mutations in 23S ribosomal RNA (not detected) and for the phenicol exporter genes fexA and fexB (not detected). The two isolates exhibited closely related SmaI-PFGE profiles; one (E35048) was investigated for molecular traits. Sequencing demonstrated that optrA and cfr displayed high-level DNA identities (98% and 99%, respectively) to the respective reference sequences (accession numbers KP399637 and AJ57936). Three amino acid changes were detected in the protein sequence of cfr and 21 (4 of which were already reported in Chinese isolates) in the protein sequence of optrA compared with the respective reference sequences. The results of long PCR assays seeking a possible linkage between optrA and cfr were negative. The genetic contexts of both genes proved capable of undergoing excision in circular form, and were completely sequenced. The sequence of the minicircle containing optrA (3350 bp), deposited under accession no. KT892063, included a transposase gene downstream of optrA. This transposase gene exhibited 70% DNA identity and 65% amino acid identity to a chromosomal transposase from Clostridium sticklandii (accession no. FP565809). The minicircle (3405 bp) containing the cfr gene and one intact IS, ISEnfa5, was almost identical to a cfr genetic context described in Staphylococcus lentus (accession no. KF049005). Considering the low MICs of linezolid, florfenicol and chloramphenicol (Table 1), in spite of the prezsence of two resistance genes acting by different mechanisms (cfr perturbing the ribosome function and optrA providing for active efflux), RT–PCR experiments were performed to check the actual transcription of the two genes (Figure S1). We found that optrA was transcribed, whereas cfr was not. Although the exact mechanism of nontranscription is still being investigated, preliminary data indicate a 52 bp deletion in the regulatory region upstream of cfr. Interestingly, a cfr gene failing to mediate resistance to oxazolidinones and phenicols has been described in a porcine E. faecalis isolate in China. Our collection of Enterococcus blood isolates is still in progress, and the overall results of the survey will be assessed and

Characterization of novel conjugative multiresistance plasmids carrying cfr from linezolid-resistant Staphylococcus epidermidis clinical isolates from Italy

OBJECTIVES The objective of this study was to investigate the genetic environment of the cfr gene from two linezolid-resistant clinical isolates of Staphylococcus epidermidis from Italy. METHODS The two strains (SP1 and SP2) were phenotypically and genotypically characterized. Transferability of cfr was assessed by electrotransformation and conjugation. The genetic contexts of cfr were investigated by PCR mapping, sequencing and comparative sequence analyses. RESULTS SP1 and SP2 belonged to ST23 and ST83, respectively. In both strains, the cfr gene was located on a plasmid, which could be transferred to Staphylococcus aureus by transformation and conjugation. In isolate SP1, linezolid resistance mediated by mutations in 23S rRNA and the L3 ribosomal protein was also detected. pSP01, the cfr-carrying plasmid from strain SP1, had a larger number of additional resistance genes and was sequenced (76 991 bp). It disclosed a distinctive mosaic structure, with four cargo regions interpolated into a backbone 95% identical to that of S. aureus plasmid pPR9. Besides cfr, resistance genes distributed in the cargo regions included blaZ, lsa(B), msr(A) and aad, and a gene cluster for resistance to heavy metals. A closely related cfr plasmid (pSP01.1, ∼ 49 kb), differing from pSP01 by the lack of a large cargo region with some resistance genes, was detected in strain SP2. CONCLUSIONS The conjugative multiresistance plasmid pSP01 is the first cfr-carrying plasmid to be sequenced in Italy. This is the first time cfr has been found: (i) in association with blaZ, msr(A) and heavy metal resistance genes; and (ii) in an S. epidermidis strain (SP2) belonging to ST83.

Tn5253 family integrative and conjugative elements carrying mef(I) and catQ determinants in Streptococcus pneumoniae and Streptococcus pyogenes.

ABSTRACT The linkage between the macrolide efflux gene mef(I) and the chloramphenicol inactivation gene catQ was first described in Streptococcus pneumoniae (strain Spn529), where the two genes are located in a module designated IQ element. Subsequently, two different defective IQ elements were detected in Streptococcus pyogenes (strains Spy029 and Spy005). The genetic elements carrying the three IQ elements were characterized, and all were found to be Tn5253 family integrative and conjugative elements (ICEs). The ICE from S. pneumoniae (ICESpn529IQ) was sequenced, whereas the ICEs from S. pyogenes (ICESpy029IQ and ICESpy005IQ, the first Tn5253-like ICEs reported in this species) were characterized by PCR mapping, partial sequencing, and restriction analysis. ICESpn529IQ and ICESpy029IQ were found to share the intSp23FST81 integrase gene and an identical Tn916 fragment, whereas ICESpy005IQ has int5252 and lacks Tn916. All three ICEs were found to lack the linearized pC194 plasmid that is usually associated with Tn5253-like ICEs, and all displayed a single copy of a toxin-antitoxin operon that is typically contained in the direct repeats flanking the excisable pC194 region when this region is present. Two different insertion sites of the IQ elements were detected, one in ICESpn529IQ and ICESpy029IQ, and another in ICESpy005IQ. The chromosomal integration of the three ICEs was site specific, depending on the integrase (intSp23FST81 or int5252). Only ICESpy005IQ was excised in circular form and transferred by conjugation. By transformation, mef(I) and catQ were cotransferred at a high frequency from S. pyogenes Spy005 and at very low frequencies from S. pneumoniae Spn529 and S. pyogenes Spy029.

Transduction of the Streptococcus pyogenes bacteriophage Φm46.1, carrying resistance genes mef (A) and tet (O), to other Streptococcus species

Φm46.1 – Streptococcus pyogenes bacteriophage carrying mef(A) and tet(O), respectively, encoding resistance to macrolides (M phenotype) and tetracycline – is widespread in S. pyogenes but has not been reported outside this species. Φm46.1 is transferable in vitro among S. pyogenes isolates, but no information is available about its transferability to other Streptococcus species. We thus investigated Φm46.1 for its ability to be transduced in vitro to recipients of different Streptococcus species. Transductants were obtained from recipients of Streptococcus agalactiae, Streptococcus gordonii, and Streptococcus suis. Retransfer was always achieved, and from S. suis to S. pyogenes occurred at a much greater frequency than in the opposite direction. In transductants Φm46.1 retained its functional properties, such as inducibility with mitomycin C, presence both as a prophage and as a free circular form, and transferability. The transductants shared the same Φm46.1 chromosomal integration site as the donor, at the 3′ end of a conserved RNA uracil methyltransferase (rum) gene, which is an integration hotspot for a variety of genetic elements. No transfer occurred to recipients of Streptococcus pneumoniae, Streptococcus oralis, and Streptococcus salivarius, even though rum-like genes were also detected in the sequenced genomes of these species. A largely overlapping 18-bp critical sequence, where the site-specific recombination process presumably takes place, was identified in the rum genes of all recipients, including those of the species yielding no transductants. Growth assays to evaluate the fitness cost of Φm46.1 acquisition disclosed a negligible impact on S. pyogenes, S. agalactiae, and S. gordonii transductants and a noticeable fitness advantage in S. suis. The S. suis transductant also displayed marked overexpression of the autolysin-encoding gene atl.

Characterization of poxtA, a novel phenicol-oxazolidinone-tetracycline resistance gene from an MRSA of clinical origin.

Objectives To characterize a novel phenicol-oxazolidinone-tetracycline resistance gene, named poxtA, identified in a previously described MRSA strain that was highly resistant to linezolid and also carried the cfr gene. Methods The poxtA gene was identified by bioinformatic analysis of the whole genome sequence of Staphylococcus aureus AOUC-0915. The poxtA gene was cloned in a shuttle plasmid vector and expressed in Escherichia coli, S. aureus and Enterococcus faecalis to investigate the protein function. Comparative sequence analyses at the protein and genetic levels were carried out using standard procedures. Results The poxtA gene encodes a protein that is 32% identical to OptrA and exhibits structural features typical of the F lineage of the ATP-binding cassette (ABC) protein superfamily that cause antibiotic resistance by ribosomal protection. Expression of poxtA in E. coli, S. aureus and E. faecalis was able to decrease susceptibility to phenicols, oxazolidinones and tetracyclines. A database search identified the presence of poxtA in E. faecalis, Enterococcus faecium and Pediococcus acidilactici strains, mostly of animal origin, and revealed the presence of poxtA homologues in the genomes of some Clostridiales. Analysis of the genetic context revealed that poxtA was located in a composite transposon-like structure containing two IS1216 elements. Conclusions A novel resistance gene, named poxtA, encoding a protein of the antibiotic resistance (ARE) ABC-F lineage, was identified in the genome of an MRSA of clinical origin. PoxtA can confer decreased susceptibility to phenicols, oxazolidinones and tetracyclines and is associated with a putative mobile element that could contribute to its horizontal dissemination.

Comparison of the Antimicrobial Activities of Four Honeys From Three Countries (New Zealand, Cuba, and Kenya)

Skin and chronic wound infections are an increasing and urgent health problem worldwide. Their management is difficult and the development of antibiotic resistance by both planktonic and biofilm-associated bacteria necessitates the use of alternative treatments. The purpose of this study was to compare the antimicrobial activity of four honeys from different floral and geographical origins: Melipona beecheii honey (Cuba) and three Apis mellifera honeys [Manuka honey (New Zealand), A. mellifera honey (Cuba), and African honey (Kenya)]. The physicochemical parameters were within the ranges reported for these honeys and M. beecheii honey stood out due to its acidic character. An agar incorporation technique was used to determine the minimum active dilution of each honey against 52 clinical isolates (34 Gram-positive, 17 Gram-negative, and 1 Candida albicans). The antibiofilm activity of honeys was tested by assessing their ability to inhibit biofilm formation and to disrupt preformed biofilms. Overall, M. beecheii honey had the highest antimicrobial and antibiofilm activity, although a marked disruption in preformed biofilms was shared by all tested honeys. Structural changes induced by M. beecheii honey on Staphylococcus aureus and Pseudomonas aeruginosa cells were observed by transmission electron microscopy suggesting that this honey has a potent antimicrobial action and may be an excellent candidate for the development of topical preparations for the treatment of infected wounds.

Commentary: Nationwide Surveillance of Novel Oxazolidinone Resistance Gene optrA in Enterococcus Isolates in China from 2004 to 2014.

Citation: Morroni G, Brenciani A, Simoni S, Vignaroli C, Mingoia M and Giovanetti E (2017) Commentary: Nationwide Surveillance of Novel Oxazolidinone Resistance Gene optrA in Enterococcus Isolates in China from 2004 to 2014. Front. Microbiol. 8:1631. doi: 10.3389/fmicb.2017.01631 Commentary: Nationwide Surveillance of Novel Oxazolidinone Resistance Gene optrA in Enterococcus Isolates in China from 2004 to 2014

Stability of the cargo regions of the cfr-carrying, multiresistance plasmid pSP01 from Staphylococcus epidermidis

This study investigated the stability or instability - i.e. the ability or inability to undergo excision in circular form - of the four cargo regions (cr1 to cr4) of the novel cfr-carrying, multiresistance plasmid pSP01, arboured by a clinical Staphylococcus epidermidis isolate. Only cr4 proved unstable. The stability of cr1 and cr2 was substantially expected. Insertion sequences (ISs) played an important role in the stability of cr3 (the cfr gene context) and in the instability of cr4. Whereas the stability of cfr genetic contexts is associated with the presence of a single IS copy (istAS-istBS in cr3), their instability is associated with two identical, flanking ISs with the same orientation. cr4 is bracketed between two identical IS257 elements, and appears to behave as a composite transposon. Its instability is of interest because of the existence of a closely related cfr plasmid from S. epidermidis (pSP01.1) that differs from pSP01 only by the lack of cr4. An integration/recombination mechanism is suggested to explain how cr4 may have moved to pSP01.1 to form pSP01.

ICESp1116, the Genetic Element Responsible for erm(B)-Mediated, Inducible Erythromycin Resistance in Streptococcus pyogenes, Belongs to the TnGBS Family of Integrative and Conjugative Elements

While most integrative and conjugative elements (ICEs) encode Tyr/Ser recombinases, two ICE families have recently been described ([1][1]) that rely on DDE transposases ([2][2]) for circularization and integration into the chromosome. Such ICE families, described in Streptococcus agalactiae (Tn GBS

pHTβ-promoted mobilization of non-conjugative resistance plasmids from Enterococcus faecium to Enterococcus faecalis

Objectives: To analyse the recombination events associated with conjugal mobilization of two multiresistance plasmids, pRUM17i48 and pLAG (formerly named pDO1‐like), from Enterococcus faecium 17i48 to Enterococcus faecalis JH2–2. Methods: The plasmids from two E. faecalis transconjugants (JH‐4T, tetracycline resistant, and JH‐8E, erythromycin resistant) and from the E. faecium donor (also carrying a pHT&bgr;‐like conjugative plasmid, named pHT&bgr;17i48) were investigated by several methods, including PCR mapping and sequencing, S1‐PFGE followed by Southern blotting and hybridization, and WGS. Results: Two locations of repApHT&bgr; were detected in both transconjugants, one on a ˜50 kb plasmid (as in the donor) and the other on plasmids of larger sizes. In JH‐4T, WGS disclosed an 88.6 kb plasmid resulting from the recombination of pHT&bgr;17i48 (˜50 kb) and a new plasmid, named pLAG (35.3 kb), carrying the tet(M), tet(L), lsa(E), lnu(B), spw and aadE resistance genes. In JH‐8E, a 75 kb plasmid resulting from the recombination of pHT&bgr;17i48 and pRUM17i48 was observed. In both cases, the cointegrates were apparently derived from replicative transposition of an IS1216 present in each of the multiresistance plasmids into pHT&bgr;17i48. The cointegrates could resolve to yield the multiresistance plasmids and a pHT&bgr;17i48 derivative carrying an IS1216 (unlike the pHT&bgr;17i48 of the donor). Conclusions: Our results completed the characterization of the multiresistance plasmids carried by the E. faecium 17i48, confirming the role of pHT plasmids in the mobilization of non‐conjugative antibiotic resistance elements among enterococci. Results also revealed that mobilization to E. faecalis was associated with the generation of cointegrate plasmids promoted by IS1216‐mediated transposition.