Fabio D'Ambrosio

Antibiotic Susceptibility and Serotype Distribution of Streptococcus pneumoniae Causing Meningitis in Italy, 1997–1999

Because few data are available in Italy regarding antimicrobial susceptibility and serotype distribution of invasive Streptococcus pneumoniae strains, meningeal isolates collected at Italian hospitals during the years 1997-1999 were studied. The 12 most common serogroups, representing > 85% of the isolates, were 14, 23, 6, 4, 3, 9, 19, 8, 1, 12, 18, and 7 (in order of frequency). The serogroups identified in children < 5 years old were more limited in number: 80% are included in the 7-valent conjugate vaccines. Penicillin resistance was observed in 14 (9.5%) of 148 strains and increased from 5% in the first part of the study to 13% in the last part. Only 2 strains were fully penicillin resistant, and these belonged to serotype 9V. Thirty percent of the strains, mostly belonging to serogroups 14 or 6 and carrying either the ermB or the mef genes, were resistant to erythromycin.

Detection of intestinal and extra-intestinal strains of enterotoxigenic Bacteroides fragilis by the HT-29 cytotoxicity assay

Bacteroides fragilis strains with enterotoxic activity can be isolated from the faeces of newborn farm animals with diarrhoea and are called enterotoxigenic B. fragilis (ETBF). These strains can now be detected in an in-vitro cytotoxicity assay with HT-29 cells. In this study, 146 B. fragilis strains (95 faecal and 40 extra-intestinal isolates) and 64 Bacteroides isolates belonging to species other than B. fragilis were tested for their ability to produce enterotoxin. Sixteen strains of ETBF were identified; all belonged to the fragilis species and represented 11% of all B. fragilis examined. The prevalence was similar among extraintestinal and faecal strains, 11.5% and 10%, respectively. The production of enterotoxin in clinical isolates appeared to be associated with infections where tissue destruction was more prominent. Enterotoxigenicity was not associated with the presence of a plasmid and the plasmid profiles of ETBF strains that harboured plasmids were different. These results show that enterotoxin production by human isolates of B. fragilis is not uncommon and could represent a new virulence factor of B. fragilis.

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.

Genotypes of invasive pneumococcal isolates recently recovered from Italian patients.

ABSTRACT We examined 73 recent invasive pneumococcal isolates within selected areas of Italy for genotypic variability. Thirty-three genomic macrorestriction types were found, three of which represented multiple serotypes. Restriction fragment patterns of pbp2b, pbp2x, and pspA were conserved within the majority of isolates that shared macrorestriction types. Of the nine macrorestriction types found among the 22 penicillin-nonsusceptible Streptococus pneumoniae (PNSP) isolates, seven comprised isolates with allelic profiles showing five to seven allelic matches to profiles in the multilocus sequence typing database (www.mlst.net ); however, three of the seven profiles represented serotypes not previously associated with these clonal clusters. Two PNSP macrorestriction types represented new clones with unique allelic profiles. Allelic profiles obtained from isolates of 3 of the 25 macrorestriction types found among the 51 penicillin-susceptible S. pneumoniae (PSSP) isolates were closely related to previously described profiles. One PSSP isolate was a novel type 24F isolate related to the multiresistant clone France9V-3. This work reports new PNSP strains and new serotype-clone associations.

Inferring the potential success of pneumococcal vaccination in Italy: serotypes and antibiotic resistance of Streptococcus pneumoniae isolates from invasive diseases.

To evaluate the potential impact of antipneumococcal vaccination in Italy, Streptococcus pneumoniae isolates from invasive disease were collected from 65 laboratories in the years 1997-2000. Of the 503 isolates examined, 15% were from children <5 years and 34% from adults > or = 65 years. The most frequent serogroups were, in ranking order, 14, 19, 6, and 23. Overall, 93.8% of the isolates belonged to serogroups enclosed in the 23-valent polysaccharide vaccine. Among children isolates, serotypes 14, 6B, and 23F comprised 60% of the isolates; overall, 72% of the isolates belonged to serotypes included in the heptavalent conjugate vaccine. Penicillin nonsusceptible isolates (10%) belonged to a limited number of serogroups, being more common in serogroups 19 and 9 and in the nonvaccine serogroups 24 and 35. Erythromycin-resistant isolates (29%) belonged to several serogroups, more frequently to serogroups 14, 6, and 19. Both vaccines are potentially able to prevent the majority of resistant infections in the respective age groups in Italy.

Increase of pneumococcal serotype 19A in Italy is due to expansion of the piliated clone ST416/CC199

The emergence of Streptococcus pneumoniae serotype 19A, following use of the heptavalent pneumococcal conjugate vaccine (PCV7), has been favoured by multiple antibiotic resistance of this serotype and by other unknown factors. The aim of this study was to examine 19A isolates from invasive pneumococcal diseases (IPD) obtained before and after PCV7 implementation to ascertain which characteristics, including the presence of pili, might have favoured the emergence of this serotype in Italy. All S. pneumoniae isolates from IPD collected at the Italian National Institute of Health in the years 2001-2003 and 2006-2009 were serotyped. The 19A isolates were submitted to antimicrobial susceptibility testing by Etest and were genotyped by a combination of pulsed field gel electrophoresis (PFGE) and Multi Locus Sequence Typing (MLST). The presence of the pilus islets PI-1 and PI-2 was detected by PCR assays targeting a marker gene in each islet. The proportion of 19A isolates from IPD significantly increased from 4 % in 2001-2003 to 12 % in 2006-2009. This was largely due to the expansion of a clone characterized by sequence type (ST) 416, clonal complex (CC) 199, already present in Italy before PCV7 implementation. This clone included isolates susceptible to penicillin and containing PI-1 genes. Other CCs contributed to the emergence of serotype 19A: CC63 and CC193, already present in 2001-2003, and new-emerging CCs or clones such as CC230, CC320 and ST5204, that include drug-resistant and/or pilus-positive isolates. The expansion of serotype 19A in Italy might have been favoured not only by antibiotic resistance, but also by other bacterial factors such as the presence of pili.

Serotype and clonal evolution of penicillin-non susceptible invasive Streptococcus pneumoniae in the PCV7 era in Italy

ABSTRACT The percentage of invasive penicillin-nonsusceptible pneumococci (PNSSP) isolated in Italy in the seven-valent pneumococcal conjugate vaccine (PCV7) era moderately increased in comparison to the pre-PCV7 era. Increase of nonvaccine serotypes was observed among PNSSP. The most frequent PNSSP clones were the same as those identified in the pre-PCV7 era, although they were present in different proportions. Clonal expansion, emergence of new clones, and acquisition of penicillin resistance by established clones contributed to the maintenance of penicillin resistance.

Phenotypic and Genotypic Characterization of Two Penicillin-Susceptible Serotype 6B Streptococcus pneumoniae Clones Circulating in Italy

ABSTRACT Twenty-nine penicillin-susceptible serotype 6B strains isolated from patients with invasive diseases and from healthy carriers were examined by different genotyping methods. Ten groups were identified on the basis of the pulsed-field gel electrophoresis (PFGE) profiles, and two of these contained multiple isolates and were analyzed further. PFGE group 1 comprised 12 isolates, the majority of which had a multiresistant phenotype (resistance to erythromycin, clindamycin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole), corresponding to that of a clone previously described in the Mediterranean area and related to penicillin-resistant clone Spain6B-2. The pbp2b, pbp2x, dhf, and pspA genes of the isolates had identical restriction profiles; and the partial sequence of pspA was identical to that of clone Spain6B-2. In all isolates the resistance determinants erm(B) and tet(M) were inserted in a Tn1545-like element; 11 isolates carried cat as part of the integrated plasmid pC194. Multilocus sequence typing (MLST) performed with two isolates confirmed that their profiles corresponded to that of the Mediterranean clone. PFGE group 2 comprised nine strains, of which the majority showed no antibiotic resistance. Their pspA profiles were different, and the partial sequences obtained for two representative isolates indicated the presence of PspA proteins of different clades. The MLST profile of one strain was identical to that of a serotype 6B strain from the United Kingdom, while two other isolates were novel one-allele variants. This clone appears to be related (five of seven identical alleles) to two other internationally disseminated clones, Hungary19A-6 and Poland23F-16, both of which are penicillin resistant. The presence of antibiotic-susceptible isolates of this clone suggests that traits other than antibiotic resistance can make a clone successful.

Genetic Diversity of Invasive Strains of Haemophilus influenzae Type b before and after Introduction of the Conjugate Vaccine in Italy

We determined the genotypes of 95 invasive Haemophilus influenzae type b (Hib) strains collected before and after introduction of widespread Hib vaccination in Italy. No substantial change in genetic diversity was highlighted by pulsed-field gel electrophoresis. However, an upward temporal trend in proportion of strains possessing multiple copies of the capsulation b locus was detected (P = .03).

Characterization of Streptococcus pneumoniae clones from paediatric patients with cystic fibrosis.

The role of Streptococcus pneumoniae in cystic fibrosis (CF) is poorly understood. The pneumococcal population has changed over time after the introduction of the heptavalent conjugate vaccine (PCV7) and, more recently, the 13-valent conjugate vaccine (PCV13). Although serotypes and clones causing invasive pneumococcal disease or colonizing healthy children have been extensively analysed, little is known so far on the serotypes and clones of pneumococci in CF patients. The aim of this work was to investigate serotypes, antibiotic susceptibilities, genotypes and biofilm production of CF pneumococcal isolates. Overall, 44 S. pneumoniae strains collected from 32 paediatric CF patients from January 2010 to May 2012 in a large Italian CF Centre were tested for antimicrobial susceptibility testing by Etest, serotyped by the Quellung reaction and genotyped by a combination of different molecular typing methods, including pbp gene restriction profiling, pspA restriction profiling and sequencing, PFGE and multilocus sequence typing. Biofilm production by pneumococcal strains was also assessed. Penicillin non-susceptibility was 16 %. High resistance rates (>56 %) were observed for erythromycin, clindamycin and tetracycline. The most frequent serotype recovered was serotype 3 (31.8 %). The coverage of PCV7 and PCV13 was 6.8 and 47.7 %, respectively. More than 80 % of CF strains belonged to Pneumococcal Molecular Epidemiology Network (PMEN) reference clones, the most common being Netherlands(3)-ST180 (28.2 %), and Greece(21)-30/ST193 (15.4 %). All strains produced biofilm in vitro, although with large variability in biofilm formation efficiency. No correlation was found between biofilm levels and serotype, clone or antibiotic resistance. The high isolation rate of antibiotic-resistant serotype 3 pneumococci from CF patients suggests that PCV13 could increase protection from pneumococcal colonization and infection.