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.

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.

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.

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.

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.

AimsMultidrug-resistant Klebsiella pneumoniae has become a relevant healthcare-associated pathogen. Capsule, type 1 and 3 fimbriae (mrkA gene), type 2 quorum-sensing system (luxS), synthesis of D-galactan I (wbbM), LPS transport (wzm) and poly-beta-1,6-N-acetyl-D-glucosamine (pgaA) seem involved in K. pneumoniae biofilm. Nonenzymatic antibiotic resistance is related to nonexpression or mutation of porins (OmpK35 and OmpK36), and efflux pump (acrB) overexpression. The aim of this study was to analyse some virulence factors of K. pneumoniae isolates, and to evaluate possible correlations between their antibiotic resistance profile and ability to form biofilm.Methods and ResultsQuantitative biofilm production assay, congo red agar test and string test were performed on 120 isolates clustered in 56 extensively drug-resistant (XDR), 40 MDR and 24 susceptible (S) strains. Nine representative strains were analysed by real-time RT-PCR for the expression of antibiotic resistance (OmpK35, OmpK36, acrB) and biofilm production genes (mrkA, luxS, pga, wbbM, wzm) during planktonic and sessile growth. XDR isolates showed a higher ability to form biofilm (9107%) and to produce polysaccharides (7857%) when compared to MDR and S strains. In biofilm-growing XDR strains, seven of eight genes were upregulated, with the only exception of OmpK36.ConclusionsXDR strains exhibited phenotypic and genotypic features supporting a significant growth as biofilm.Significance and Impact of the StudyThis study produces new findings that highlight a positive correlation between antibiotic resistance profile and biofilm-forming ability in XDR K. pneumoniae strains. These new evidences might contribute to the progress in selection of therapeutic treatments of infections caused by K. pneumoniae resistant also to the last line of defence' antibiotics, that is, carbapenems.

Objectives: To analyse the recombination events associated with conjugal mobilization of two multiresistance plasmids, pRUM(17i48) 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. faeciumdonor (also carrying a pHT beta-like conjugative plasmid, named pHT beta(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 repA(pHT beta) were detected in both transconjugants, one on a similar to 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 beta(17i48) (similar to 50kb) 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 pHTb17i48 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 beta(17i48). The cointegrates could resolve to yield themultiresistance plasmids and a pHT beta(17i48) derivative carrying an IS1216 (unlike the pHT beta(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.

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

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 (76991 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, similar to 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.

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.

Linezolid, the first oxazolidinone to be introduced in clinical practice, has broad activity against important multi-drug-resistant Gram-positive pathogens, including meticillin-resistant staphylococci and vancomycin-resistant enterococci. Linezolid inhibits protein synthesis by binding to the peptidyl transferase centre of the bacterial ribosome. Although resistance may be mediated by transferable genes such as cfr (and, crucially for enterococci, optrA), mutations in ribosomal proteins L3 and L4 and in the central loop of domain V of 23S rDNA1 constitute the main mechanism associated with linezolid resistance among clinically isolated staphylococci.

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.

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.

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 1-2 mg/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. (C) 2015 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

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.

Фm46.1 — a 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. Фm46.1 is transferable in vitro among S. pyogenes isolates, but no information is available about its transferability to other Streptococcus species. We thus investigated Фm46.1 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 Фm46.1 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 Фm46.1 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 Фm46.1 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.

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 Tn 5253 family integrative and conjugative elements (ICEs). The ICE from S. pneumoniae (ICE Spn 529IQ) was sequenced, whereas the ICEs from S. pyogenes (ICE Spy 029IQ and ICE Spy 005IQ, the first Tn 5253 -like ICEs reported in this species) were characterized by PCR mapping, partial sequencing, and restriction analysis. ICE Spn 529IQ and ICE Spy 029IQ were found to share the int Sp 23FST81 integrase gene and an identical Tn 916 fragment, whereas ICE Spy 005IQ has int 5252 and lacks Tn 916 . All three ICEs were found to lack the linearized pC194 plasmid that is usually associated with Tn 5253 -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 ICE Spn 529IQ and ICE Spy 029IQ, and another in ICE Spy 005IQ. The chromosomal integration of the three ICEs was site specific, depending on the integrase ( int Sp 23FST81 or int 5252 ). Only ICE Spy 005IQ 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.

The term “typing” is generally used with two meanings: a methods to establish the correct taxonomic collocation of a genus/specie/biotype, b methods for discriminating different bacterial isolates of the same species in order to establish the genetic relationship among the microorganisms involved in a possible outbreak. In this paper we focus our attention on the second aspect, that represents a relevant epidemiological tools in infection prevention and control. Typing systems are traditionally based on two steps workup: the first is the study of phenotypes such as serotype, biotype, phage-type, or antimicrobial susceptibilities of the isolates and this can be easily performed in every microbiology laboratory; the second, examines the relatedness of isolates at a molecular level. Over the years many molecular methods have been developed and efficiently applied in several hospital settings. The large panorama of methods put the microbiologists in trouble to operate the proper choice. Thus, in the present paper, we have reviewed old as well new molecular typing methods in order to provide a useful guide that can represent an overview on molecular methods and particularly of their specific pro and cons.