Characterization of a Multiresistance Plasmid Carrying the optrA and cfr Resistance Genes From an Enterococcus faecium Clinical Isolate.

Enterococcus faecium E35048, a bloodstream isolate from Italy, was the first strain where the oxazolidinone resistance gene optrA was detected outside China. The strain was also positive for the oxazolidinone resistance gene cfr. WGS analysis revealed that the two genes were linked (23.1 kb apart), being co-carried by a 41,816-bp plasmid that was named pE35048-oc. This plasmid also carried the macrolide resistance gene erm(B) and a backbone related to that of the well-known Enterococcus faecalis plasmid pRE25 (identity 96%, coverage 65%). The optrA gene context was original, optrA being part of a composite transposon, named Tn6628, which was integrated into the gene encoding for the ζ toxin protein (orf19 of pRE25). The cfr gene was flanked by two ISEnfa5 insertion sequences and the element was inserted into an lnu(E) gene. Both optrA and cfr contexts were excisable. pE35048-oc could not be transferred to enterococcal recipients by conjugation or transformation. A plasmid-cured derivative of E. faecium E35048 was obtained following growth at 42°C, and the complete loss of pE35048-oc was confirmed by WGS. pE35048-oc exhibited some similarity but also notable differences from pEF12-0805, a recently described enterococcal plasmid from human E. faecium also co-carrying optrA and cfr; conversely it was completely unrelated to other optrA- and cfr-carrying plasmids from Staphylococcus sciuri. The optrA-cfr linkage is a matter of concern since it could herald the possibility of a co-spread of the two genes, both involved in resistance to last resort agents such as the oxazolidinones.

Spread of colistin resistance gene mcr-1 in Italy: characterization of the mcr-1.2 allelic variant in a colistin-resistant blood isolate of Escherichia coli.

mcr-1.2, an allelic variant of the transferable colistin resistance gene mcr-1, was characterized in a colistin-resistant blood isolate of Escherichia coli. It was harbored by an IncX4-type plasmid (33,293 bp). Despite its low prevalence, the potentially worrying spread of the mcr-1 gene, particularly its mcr-1.2 variant, in Italy requires increasing surveillance.

Molecular Characterization of Italian Isolates of Fluoroquinolone-Resistant Streptococcus agalactiae and Relationships with Chloramphenicol Resistance.

A total number of 368 clinical isolates of Streptococcus agalactiae (group B Streptococcus, GBS) were collected in 2010-2016 from three hospitals in a region of central Italy. Fluoroquinolone (FQ)-resistant isolates were selected using levofloxacin. Levofloxacin-resistant (LR) strains (11/368, 2.99%) were characterized for several features, and their FQ resistance was analyzed phenotypically and genotypically using seven additional FQs. Their gyrA and parC quinolone resistance-determining regions were sequenced. Of the 11 LR isolates, 10 showed high-level and 1 low-level resistance. The former isolates exhibited higher minimal inhibitory concentrations also of the other FQs and all shared one amino acid substitution in ParC (Ser79Phe) and one in GyrA (Ser81Leu); only Ser79Phe in ParC was detected in the low-level LR isolate. The 11 LR strains exhibited distinctive relationships between their susceptibilities to non-FQ antibiotics and typing data. Remarkably, despite the very rare occurrence of chloramphenicol resistance in S. agalactiae, no <4 of the 11 LR isolates were chloramphenicol-resistant. Studies of GBS resistance to FQs in Europe remain scarce, notwithstanding the emergence of multidrug-resistant isolates. The incidence of LR GBS isolates is still limited in Italy, consistent with the moderate (though growing) rates reported in Europe, and much lower than the very high rates reported in East Asia. The intriguing relationships between FQ and chloramphenicol resistance deserve further investigation.

pHTß-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 pHTß-like conjugative plasmid, named pHTß17i48) were investigated by several methods, including PCR mapping and sequencing, S1-PFGE followed by Southern blotting and hybridization, and WGS. Results: Two locations of repApHTß 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 pHTß17i48 (∼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 pHTß17i48 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 pHTß17i48. The cointegrates could resolve to yield the multiresistance plasmids and a pHTß17i48 derivative carrying an IS1216 (unlike the pHTß17i48 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.

A new mosaic integrative and conjugative element from Streptococcus agalactiae carrying resistance genes for chloramphenicol (catQ) and macrolides [mef(I) and erm(TR)].

OBJECTIVES: To investigate the genetic basis of catQ-mediated chloramphenicol resistance in Streptococcus agalactiae. METHODS: Two clinical strains of catQ-positive chloramphenicol-resistant S. agalactiae (Sag236 and Sag403) were recently isolated, typed (MLST, PFGE pulsotypes, capsular types) and their antibiotic resistances investigated by phenotypic and genotypic approaches. Several molecular methods (PCR mapping, restriction assays, Southern blotting, sequencing and sequence analysis, conjugal transfer assays) were used to determine the genetic context of catQ and characterize a genetic element detected in the isolates. RESULTS: Sag236 and Sag403 shared the same ST (ST19), but exhibited a different capsular type (III and V, respectively) and pulsotype. Both harboured the macrolide resistance genes mef(I) and erm(TR) and the tetracycline resistance gene tet(M). Accordingly, they were resistant to chloramphenicol, erythromycin and tetracycline. catQ and mef(I) were associated in an IQ module that was indistinguishable in Sag236 and Sag403. In mating assays, chloramphenicol and erythromycin resistance proved transferable, at low frequency, only from Sag236. Transconjugants carried not only catQ and mef(I), but also erm(TR), suggesting a linkage of the three resistance genes in a mobile element, which, though seemingly non-mobile, was also detected in Sag403. The new element (designated ICESag236, ∼110 kb) results from recombination of two integrative and conjugative elements (ICEs) originally described in different streptococcal species: S. agalactiae ICESagTR7, carrying erm(TR); and Streptococcus pneumoniae ICESpn529IQ, carrying the prototype IQ module. CONCLUSIONS: These findings strengthen the notion that widespread streptococcal ICEs may form mosaics that enhance their diversity and spread, broaden their host range and carry new cargo genes.

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.

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.

Macrolide resistance gene erm(TR) and erm(TR)-carrying genetic elements in Streptococcus agalactiae: characterization of ICESagTR7, a new composite element containing IMESp2907.

OBJECTIVES: The objective of this study was to investigate macrolide-resistant Streptococcus agalactiae isolates harbouring erm(TR), an erm(A) gene subclass, with emphasis on their erm(TR)-carrying genetic elements. Four erm(TR)-carrying elements have been described to date: three closely related (ICE10750-RD.2, Tn1806 and ICESp1108) in Streptococcus pyogenes, Streptococcus pneumoniae and S. pyogenes, respectively; and one completely different (IMESp2907, embedded in ICESp2906 to form ICESp2905) in S. pyogenes. METHODS: Seventeen macrolide-resistant erm(TR)-positive S. agalactiae isolates were phenotypically and genotypically characterized. Their erm(TR)-carrying elements were explored by analysing the distinctive recombination genes of known erm(TR)-carrying integrative and conjugative elements (ICEs) and by PCR mapping. The new genetic context and organization of IMESp2907 in S. agalactiae were explored using several experimental procedures and in silico analyses. RESULTS: Five isolates harboured ICE10750-RD.2/Tn1806, five isolates harboured ICESp1108 and five isolates bore unknown erm(TR)-carrying elements. The remaining two isolates, exhibiting identical serotypes and pulsotypes, harboured IMESp2907 in a new genetic environment, which was further investigated in one of the two isolates, SagTR7. IMESp2907 was circularizable in S. agalactiae, as described in S. pyogenes. The new IMESp2907 junctions were identified based on its site-specific integration; the att sites were almost identical to those in S. pyogenes. In strain SagTR7, erm(TR)-carrying IMESp2907 was embedded in an erm(TR)-less internal element related to ICE10750-RD.2/Tn1806, which, in turn, was embedded in an ICESde3396-like element. The resulting whole ICE, ICESagTR7 (∼129 kb), was integrated into the chromosome downstream of the rplL gene, and was excisable in circular form and transferable by conjugation. CONCLUSIONS: This is the first study exploring erm(TR)-carrying genetic elements in S. agalactiae.

Recurrent skin infection associated with nasal carriage of Panton-Valentine leukocidin-positive methicillin-susceptible Staphylococcus aureus closely related to the EMRSA-15 clone.

We report the case of a soldier with recurrent skin infection associated with nasal carriage of a Panton-Valentine leukocidin (PVL)-producing methicillin-susceptible Staphylococcus aureus (MSSA), closely related to the EMRSA-15 clone. MSSA isolates causing infection not requiring hospitalization usually go unnoticed; however, their typing may be useful to understand the global distribution of successful staphylococcal lineages related to epidemic clones. PVL-positive MSSA strains might serve as reservoirs from which virulent methicillin-resistant strains may evolve and spread.

Epidemiology of Staphylococcus aureus in Italy: First nationwide survey, 2012.

A 3-month epidemiological study to determine the prevalence and antibiotic resistance of Staphylococcus aureus nosocomial infections was performed in 52 centres throughout Italy in 2012. A total of 21,873 pathogens were analysed. The prevalence of S. aureus among all nosocomial pathogens isolated in that period was 11.6% (n=2541), whilst the prevalence of methicillin-resistant S. aureus (MRSA) among the S. aureus was 35.8% (n=910). All tested antimicrobials demonstrated ≥92.2% susceptibility against methicillin-susceptible S. aureus, with the exception of clindamycin (89.7%) and erythromycin (84.2%). Among MRSA, percentages of resistance ranged from 12.6% to >39% for tetracycline, rifampicin, clindamycin and gentamicin; higher percentages were found for erythromycin (65.4%) and fluoroquinolones (72.3-85.8%). Overall, the glycopeptide minimum inhibitory concentration (MIC) distribution showed that 58.3% of strains possessed MICs of 1-2mg/L and few strains were linezolid- or daptomycin-resistant. Molecular characterisation was performed on 102 MRSA selected from Northern, Central and Southern regions. Five major clones were found: Italian/ST228-I (t001-t023-t041-t1686-t3217), 33.3%; USA500/ST8-IV (t008), 17.6%; E-MRSA15/ST22-IVh (t020-t025-t032-t223), 16.7%; USA100/ST5-II (t002-t653-t1349-t2164-t3217-t388), 14.7%; and Brazilian/ST239/241-III (t030-t037), 3.9%. Five PVL-positive CA-MRSA isolates, belonging to USA300 and minor clones, were also identified. In conclusion, this first nationwide surveillance study showed that in Italy, S. aureus infections accounted for 11.6% of all nosocomial infections; MRSA accounted for approximately one-third of the S. aureus isolates and these were multidrug-resistant organisms. Five major MRSA epidemic clones were observed and were inter-regionally distributed, with ST228-SCCmecI becoming predominant.

Antibiotic Resistance Related to Biofilm Formation in Klebsiella pneumoniae.

The Gram-negative opportunistic pathogen, Klebsiella pneumoniae, is responsible for causing a spectrum of community-acquired and nosocomial infections and typically infects patients with indwelling medical devices, especially urinary catheters, on which this microorganism is able to grow as a biofilm. The increasingly frequent acquisition of antibiotic resistance by K. pneumoniae strains has given rise to a global spread of this multidrug-resistant pathogen, mostly at the hospital level. This scenario is exacerbated when it is noted that intrinsic resistance to antimicrobial agents dramatically increases when K. pneumoniae strains grow as a biofilm. This review will summarize the findings about the antibiotic resistance related to biofilm formation in K. pneumoniae.

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

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 intSp 23FST81 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 (intSp 23FST81 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.

Genetic determinants and elements associated with antibiotic resistance in viridans group streptococci.

OBJECTIVES: To investigate the distribution of erythromycin, tetracycline and chloramphenicol resistance mechanisms and determinants and the relevant genetic environments and elements in viridans group streptococci (VGS). METHODS: A total of 263 VGS collected from routine throat swabs in 2010-12 and identified to the species level were studied. Antibiotic resistance determinants and the relevant genetic contexts and elements were determined using amplification and sequencing assays and restriction analysis. RESULTS: The investigation provided original information on the distribution of resistance mechanisms, determinants and genetic elements in VGS. Erythromycin-resistant isolates totalled 148 (56.3%; 37 belonging to the cMLS phenotype and 111 belonging to the M phenotype); there were 72 (27.4%) and 7 (2.7%) tetracycline- and chloramphenicol-resistant isolates, respectively. A number of variants of known genetic contexts and elements carrying determinants of resistance to these antibiotics were detected, including the mega element, Φ10394.4, Tn2009, Tn2010, the IQ element, Tn917, Tn3872, Tn6002, Tn916, Tn5801, a tet(O) fragment from ICE2096-RD.2 and ICESp23FST81. CONCLUSIONS: These findings shed new light on the distribution of antibiotic resistance mechanisms and determinants and their genetic environments in VGS, for which very few such data are currently available. The high frequency and broad variety of such elements supports the notion that VGS may be important reservoirs of resistance genes for the more pathogenic streptococci. The high rates of macrolide resistance confirm the persistence of a marked prevalence of resistant VGS in Europe, where macrolide resistance is, conversely, declining among the major streptococcal pathogens.

Genetic basis of the association of resistance genes mef(I) (macrolides) and catQ (chloramphenicol) in streptococci.

In streptococci mef(I) and catQ, two relatively uncommon macrolide and chloramphenicol resistance genes, respectively, are typically linked in a genetic module designated IQ module. Though variable, the module consistently encompasses, and is sometimes reduced to, a conserved ∼5.8-kb mef(I)-catQ fragment. The prototype IQ module was described in Streptococcus pneumoniae. IQ-like modules have subsequently been detected in Streptococcus pyogenes and in different species of viridans group streptococci, where mef(E) may be found instead of mef(I). Three genetic elements, one carrying the prototype IQ module from S. pneumoniae and two carrying different, defective IQ modules from S. pyogenes, have recently been characterized. All are integrative and conjugative elements (ICEs) belonging to the Tn5253 family, and have been designated ICESpn529IQ, ICESpy029IQ and ICESpy005IQ, respectively. ICESpy029IQ and ICESpy005IQ were the first Tn5253 family ICEs to be described in S. pyogenes. A wealth of new information has been obtained by comparing their genetic organization, chromosomal integration, and transferability. The origin of the IQ module is unknown. The mechanism by which it spreads in streptococci is discussed.

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.

Pitfalls encountered while investigating genetic elements by PCR.

The unprecedented wealth of databases that have become available in the era of next-generation sequencing has considerably increased our knowledge of bacterial genetic elements (GEs). At the same time, the advent of single-cell based approaches has brought realization that unsuspected heterogeneity may occur in the bacterial population from a single colony. The increasing use of PCR-based techniques to study new GEs requires careful consideration of the possible different PCR targets associated with different subpopulations if incorrect or incomplete interpretations are to be avoided. In this commentary, confining ourselves to our direct experience, we illustrate some examples of PCR pitfalls that may be encountered while investigating GEs.

Characterization of Tn5801.Sag, a variant of Staphylococcus aureus Tn916 family transposon Tn5801 that is widespread in clinical isolates of Streptococcus agalactiae.

Tn5801, originally detected in Staphylococcus aureus Mu50, is a Tn916 family element in which a unique int gene (int5801) replaces the int and xis genes in Tn916 (int916 and xis916). Among 62 tet(M)-positive tetracycline-resistant Streptococcus agalactiae isolates, 43 harbored Tn916, whereas 19 harbored a Tn5801-like element (Tn5801.Sag, ∼20.6 kb). Tn5801.Sag was characterized (PCR mapping, partial sequencing, and chromosomal integration) and compared to other Tn5801-like elements. Similar to Tn5801 from S. aureus Mu50, tested in parallel, Tn5801.Sag was unable to undergo circularization and conjugal transfer.