Maria Del Grosso

Decrease of vancomycin-resistant enterococci in poultry meat after avoparcin ban.

In Italy, 18 months after the ban of avoparcin, the percentage of poultry meat samples containing vanA gene-positive vancomycin-resistant enterococci fell from 14.6% to 8%.

Tn2009, a Tn916-like element containing mef(E) in Streptococcus pneumoniae

ABSTRACT The association between the macrolide efflux gene mef(E) and the tet(M) gene was studied in two clinical strains of Streptococcus pneumoniae that belonged to serotypes 19F and 6A, respectively, and that were resistant to both tetracycline and erythromycin. The mef(E)-carrying element mega (macrolide efflux genetic assembly; 5,511 bp) was found to be inserted into a Tn916-like genetic element present in the chromosomes of the two pneumococcal strains. In both strains, mega was integrated at the same site, an open reading frame identical to orf6 of Tn916. The new composite element, Tn2009, was about 23.5 kb and, with the exception of the tet(M)-coding sequence, appeared to be identical in both strains. By sequencing of the junction fragments of Tn2009 at the site of insertion into the chromosome, it was possible to show that (i) the insertion site was identical in the two clinical strains and (ii) the integration of Tn2009 caused a 9.5 kb-deletion in the pneumococcal chromosome. It was not possible to detect the conjugal transfer of Tn2009 to a recipient pneumococcal strain; however, transfer of the whole element by transformation was shown to occur. It is possible to hypothesize that Tn2009 relies on transformation for its spread among clinical strains of S. pneumoniae.

Clonal Spread of a Vancomycin-Resistant Enterococcus faecium Strain among Bloodstream-Infecting Isolates in Italy

ABSTRACT Recent data indicated that the rate of vancomycin resistance in bloodstream-infecting enterococcal isolates in Italy is one of the highest in Europe. The aims of this study were to characterize bloodstream-infecting vancomycin-resistant enterococci (VRE) obtained from various Italian hospitals and to establish whether the isolates were clonally related. During the years 2001 to 2003, a total of 39 VRE isolates were obtained from 19 hospital laboratories in various areas of Italy. Species identification and resistance genotypes of the isolates were obtained by multiplex PCR. Further characterization included antibiotic susceptibility testing, pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA, detection of virulence genes (esp and hyl), and multilocus sequence typing (MLST) of selected isolates. VRE were identified as 31 Enterococcus faecium (VREfm) isolates and 8 E. faecalis isolates. All but one isolate carried the vanA gene; one VREfm isolate carried the vanB gene. Analysis of the PFGE profiles showed that 28 VREfm isolates shared a similar electrophoretic profile, designed type 1, and were clonally related. All type 1 isolates were resistant to ampicillin, streptomycin, gentamicin, and rifampin and were positive for the esp gene. MLST identified an allelic profile (ST78) comprising purK allele 1, belonging to the C1 clonal lineage, characteristic of human infection and hospital outbreak isolates. The vanB-carrying VREfm isolate, of PFGE type 2, was shown to be a single-locus variant of ST78. Our data indicate that the recent increase in the number of bloodstream infections caused by VRE in Italy is due to the spread of a hospital-adapted, multidrug-resistant VREfm clone belonging to an internationally disseminated lineage.

The mef(E)-Carrying Genetic Element (mega) of Streptococcus pneumoniae: Insertion Sites and Association with Other Genetic Elements

ABSTRACT The structure of the macrolide efflux genetic assembly (mega) element, its genomic locations, and its association with other resistance determinants and genetic elements were investigated in 16 Streptococcus pneumoniae isolates carrying mef(E), of which 1 isolate also carried tet(M) and 4 isolates also carried tet(M) and erm(B). All isolates carried a mega element of similar size and structure that included the operon mef(E)-msr(D) encoding the efflux transport system. Among tetracycline-susceptible isolates, six different integration sites were identified, five of which were recognized inside open reading frames present in the R6 genome. In the five isolates also carrying tet(M), mega was inserted in different genetic contexts. In one isolate, it was part of previously described Tn916-like element Tn2009. In another isolate, mega was inserted in a transposon similar to Tn2009 that also included an erm(B) element. This new composite transposon was designated Tn2010. Neither Tn2009 nor Tn2010 could be transferred by conjugation to pneumococcal or enterococcal recipients. In the three isolates in which mega was not physically linked with tet(M), this gene was associated with erm(B) in transposon Tn3872, a Tn916-like element. Homologies between the chromosomal insertions of these composite transposons and sequences of multidrug-resistant pneumococcal genomes in the databases indicate the presence of preferential sites for the integration of composite Tn916-like elements carrying multiple resistance determinants in S. pneumoniae.

The Macrolide Resistance Genes erm(B) and mef(E) Are Carried by Tn2010 in Dual-Gene Streptococcus pneumoniae Isolates Belonging to Clonal Complex CC271

ABSTRACT The genetic elements carrying macrolide resistance genes in Streptococcus pneumoniae isolates belonging to CC271 were investigated. The international clone Taiwan19F-14 was found to carry Tn2009, a Tn916-like transposon containing tet(M) and mef(E). The dual erm(B) mef(E) isolates carried Tn2010, which is similar to Tn2009 with the addition of a putative new transposon, the erm(B) genetic element.

Pneumococcal carriage in young children one year after introduction of the 13-valent conjugate vaccine in Italy.

Background In mid 2010, the 7-valent pneumococcal conjugate vaccine (PCV7) was replaced by the 13-valent conjugate vaccine (PCV13) for childhood immunization in Italy. Our objective in this study was to obtain a snapshot of pneumococcal carriage frequency, colonizing serotypes, and antibiotic resistance in healthy children in two Italian cities one year after PCV13 was introduced. Methods Nasopharyngeal swabs were obtained from 571 children aged 0-5 years from November 2011-April 2012. Pneumococcal isolates were serotyped and tested for antimicrobial susceptibility. Penicillin and/or erythromycin non-susceptible isolates were analyzed by Multi Locus Sequence Typing (MLST). Results Among the children examined, 81.2% had received at least one dose of PCV7 or PCV13 and 74.9% had completed the recommended vaccination schedule for their age. Among the latter, 57.3% of children had received PCV7, 27.1% PCV13, and 15.6% a combination of the two vaccines. The overall carriage rate was 32.9%, with children aged 6-35 months the most prone to pneumococcal colonization (6-23 months OR: 3.75; 95% CI: 2.19-6.43 and 24-35 months OR: 3.15, 95%CI: 2.36-4.22). A total of 184 pneumococcal isolates were serotyped and divided into PCV7 (5.4%), PCV13 (18.0%), and non-PCV13 (82.0%) serotypes. Serotypes 6C, 24F, and 19A were the most prevalent (10.3%, 8.6%, and 8.1%, respectively). The proportion of penicillin non-susceptible (MIC >0.6 mg/L) isolates was 30.9%, while 42.3% were erythromycin resistant. Non-PCV13 serotypes accounted for 75.4% and 70.8% of the penicillin and erythromycin non-susceptible isolates, respectively. Conclusions Our results revealed low rates of PCV7 and PCV13 serotypes in Italian children, potentially due to the effects of vaccination. As the use of PCV13 continues, its potential impact on vaccine serotypes such as 19A and cross-reactive serotypes such as 6C will be assessed, with this study providing a baseline for further analysis of surveillance isolates.

Antibiotic-Resistant Invasive Pneumococcal Clones in Italy

ABSTRACT A total of 105 multiple-antibiotic-resistant invasive pneumococcal isolates recovered in Italy from 2001 to 2003 were genetically characterized. Of these, 40 were penicillin-nonsusceptible (PNSSP) and 65 were penicillin-susceptible (PSSP) Streptococcus pneumoniae strains. Among the PNSSP isolates, 8 and 11 different restriction profiles were obtained for the pbp2b and pbp2x genes, respectively. Clonal groups were established on the basis of analysis of both pulsed-field gel electrophoresis (PFGE) types and multilocus sequence typing (MLST). Several international clones, such as Spain23F-1/ST81, Spain6B-2/ST90, Spain9V-3/ST156, and Sweden15A-25/ST263, were identified among the PNSSP isolates. Other, smaller clones, such as the minor Spanish 19F clone/ST88 and Denmark14-32/ST230, were also found. Among the PSSP isolates, clones related to England14-9/ST9, Greece6B-22/ST273, and Portugal19F-21/ST177 were found. In addition, two large clones comprised nonvaccine serotypes. One, comprising serotype 3 isolates, corresponded to the clone Netherlands3-31/ST180; the other, comprising serotype 15B/C isolates, ST474, was not related to any previously described clone. Two small clusters related to the newly described clones Greece21-30/ST193 and Netherlands15B-37/ST199 included isolates with unrelated PFGE profiles. An unusual finding was the inability to obtain the MLST allelic profile for an isolate of serotype 19A, belonging to the Sweden15A-25/ST263 clone, due to a large deletion of the xpt gene. Capsular switching was observed among both PNSSP and PSSP isolates and involved also serotypes not included in the 7-valent pneumococcal conjugate vaccine (PCV7), such as serotypes 15B/C and 19A. Since antibiotic-resistant nonvaccine serotype clones are present in Italy, continuous monitoring of pneumococcal epidemiology should be carried out in the PCV7 era.

Complete genome sequence of a serotype 11A, ST62 Streptococcus pneumoniae invasive isolate

BackgroundStreptococcus pneumoniae is an important human pathogen representing a major cause of morbidity and mortality worldwide. We sequenced the genome of a serotype 11A, ST62 S. pneumoniae invasive isolate (AP200), that was erythromycin-resistant due to the presence of the erm(TR) determinant, and carried out analysis of the genome organization and comparison with other pneumococcal genomes.ResultsThe genome sequence of S. pneumoniae AP200 is 2,130,580 base pair in length. The genome carries 2216 coding sequences (CDS), 56 tRNA, and 12 rRNA genes. Of the CDSs, 72.9% have a predicted biological known function. AP200 contains the pilus islet 2 and, although its phenotype corresponds to serotype 11A, it contains an 11D capsular locus. Chromosomal rearrangements resulting from a large inversion across the replication axis, and horizontal gene transfer events were observed. The chromosomal inversion is likely implicated in the rebalance of the chromosomal architecture affected by the insertions of two large exogenous elements, the erm(TR)-carrying Tn1806 and a functional prophage designated ϕSpn_200. Tn1806 is 52,457 bp in size and comprises 49 ORFs. Comparative analysis of Tn1806 revealed the presence of a similar genetic element or part of it in related species such as Streptococcus pyogenes and also in the anaerobic species Finegoldia magna, Anaerococcus prevotii and Clostridium difficile. The genome of ϕSpn_200 is 35,989 bp in size and is organized in 47 ORFs grouped into five functional modules. Prophages similar to ϕSpn_200 were found in pneumococci and in other streptococcal species, showing a high degree of exchange of functional modules. ϕSpn_200 viral particles have morphologic characteristics typical of the Siphoviridae family and are capable of infecting a pneumococcal recipient strain.ConclusionsThe sequence of S. pneumoniae AP200 chromosome revealed a dynamic genome, characterized by chromosomal rearrangements and horizontal gene transfers. The overall diversity of AP200 is driven mainly by the presence of the exogenous elements Tn1806 and ϕSpn_200 that show large gene exchanges with other genetic elements of different bacterial species. These genetic elements likely provide AP200 with additional genes, such as those conferring antibiotic-resistance, promoting its adaptation to the environment.

Genetic Resistance Elements Carrying mef Subclasses Other than mef(A) in Streptococcus pyogenes

ABSTRACT In Streptococcus pyogenes, efflux-mediated erythromycin resistance is associated with the mef gene, represented mostly by mef(A), although a small portion of strains carry different mef subclasses. We characterized the composite genetic elements, including mef subclasses other than mef(A), associated with other resistance genes in S. pyogenes isolates. Determination of the genetic elements was performed by PCR mapping. The strains carrying mosaic mef(A/E), in which the 5′ region was identical to mef(A) and the 3′ region was identical to mef(E), also carried tet(O). The two genes were found enclosed in an element similar to S. pyogenes prophage Φm46.1, designated the Φm46.1-like element. In S. pyogenes strains carrying mef(E) and tet(M), mef(E) was included in a typical mega element, and in some strains, it was physically associated with tet(M) in the composite element Tn2009. S. pyogenes strains carrying mef(I) also carried catQ; the two genes were linked in a fragment representing a portion of the 5216IQ complex of Streptococcus pneumoniae, designated the defective IQ element. In the only isolate carrying a novel mef gene, this was associated with catQ and tet(M) in a genetic element similar to the 5216IQ complex of S. pneumoniae (5216IQ-like complex), suggesting that the novel mef is in fact a variant of mef(I). This study demonstrates that the composite elements containing mef are shared between S. pyogenes and S. pneumoniae and suggests that it is important to distinguish the mef subclass on the basis of the genetic element containing it.

New Genetic Element Carrying the Erythromycin Resistance Determinant erm(TR) in Streptococcus pneumoniae

ABSTRACT erm(A) subclass erm(TR), a common macrolide resistance determinant in Streptococcus pyogenes but quite rare in Streptococcus pneumoniae, was found in a clinical S. pneumoniae isolate (AP200) from Italy. In this isolate, erm(TR) was found included in a genetic element approximately 56 kb in size that did not appear to be conjugative but could be transferred by transformation. An erm(TR)-containing DNA fragment of approximately 10 kb was sequenced and 12 open reading frames (ORFs) were identified. Upstream of erm(TR), a regulatory protein of the TetR family and the two components of an efflux pump of the ABC type were found. Downstream of erm(TR), there were ORFs homologous to a spectinomycin phosphotransferase, transposases, and a relaxase. Since the genomic sequence of S. pyogenes MGAS10750 carrying erm(TR) became available, comparison between the erm(TR)-containing genetic elements in AP200 and in MGAS10750 was performed. The region flanking erm(TR) in MGAS10750 showed identity with AP200 for 10 ORFs out of 12. PCR mapping using primers designed on the sequence of MGAS10750 confirmed that AP200 carries a genetic element similar to that of MGAS10750. In AP200 the genetic element was inserted inside an ORF homologous to spr0790 of S. pneumoniae R6, coding for a type I restriction modification system. Homologies between the insertion sites in AP200 and MGAS10750 consisted of eight conserved nucleotides, of which three were duplicated, likely representing target site duplication. The structure of the erm(TR)-carrying genetic element shows characteristics of a transposon/prophage remnant chimera. In AP200 this genetic element was designated Tn1806.