Emergence of High-Level Gentamicin Resistance in Streptococcus agalactiae Hypervirulent Serotype IV ST1010 (CC452) Strains by Acquisition of a Novel Integrative and Conjugative Element.

Streptococcus agalactiae (group B streptococci, GBS) is responsible for severe infections in both neonates and adults. Currently, empiric antimicrobial therapy for sepsis and meningitis is the combined use of penicillin and gentamicin due to the enhanced bactericidal activity. However, high-level gentamicin resistance (HLGR) abrogates the synergism. The rate of HLGR was investigated within a dataset of 433 GBS strains collected from cases of invasive disease in both adults and neonates as well as from pregnant carriers. GBS isolates (n = 20, 4.6%) presented with HLGR (gentamicin MIC breakpoint >1024 mg/L) that was differently diffused between strains from adults or neonates (5.2% vs. 2.8%). Notably, 70% of HLGR GBS strains (14 isolates) were serotype IV. Serotype IV HLGR-GBS isolates were susceptible to all antibiotics tested, exhibited the alpha-C/HvgA/PI-2b virulence string, and belonged to sequence type 1010 (clonal complex (CC) 452). The mobile element that harbored the HLGR aac(6')-aph(2)″ gene is a novel integrative and conjugative element (ICE) about 45 kb long, derived from GBS 515 ICE tRNALys. The clonal expansion of this HLGR hypervirulent serotype IV GBS CC452 sublineage may pose a threat to the management of infections caused by this strain type.

Invasive Group B Streptococcal Disease in Neonates and Infants, Italy, Years 2015-2019.

Invasive infections by group B streptococci (iGBS) are the leading cause of sepsis and meningitis in the first three months of life worldwide. The clinical and microbiological characteristics of neonatal and infant iGBS in Italy during the years 2015-2019 were investigated. Voluntary-based surveillance reported 191 cases (67 early-onset (EOD) and 124 late-onset disease (LOD)) and 89 bacterial isolates were received. The main clinical manifestations were sepsis (59.2%) followed by meningitis (21.5%), bacteremia (12.0%) and septic shock (6.3%). Hospitalized preterm babies accounted for one third of iGBS and constituted the most fragile population in terms of mortality (8.2%) and brain damage (16.4%). GBS serotype III was predominant in EOD (56%) and caused almost all LOD (95%). The rate of resistance to clindamycin reached 28.8%. Most of clindamycin-resistant GBS strains (76%) were serotype III-ST17 and possessed the genetic markers of the emerging multidrug resistant (MDR) CC-17 sub-clone. Our data revealed that iGBS is changing since it is increasingly reported as a healthcare-associated infection (22.6%), mainly caused by MDR-CC17. Continuous monitoring of the clinical and microbiological characteristics of iGBS remains of primary importance and it represents, at present, the most effective tool to support prevention strategies and the research on the developing GBS vaccine.

Two Overlapping Clusters of Group B Streptococcus Late-onset Disease in a Neonatal Intensive Care Unit.

OBJECTIVES: Current predominant routes of group B Streptococcus (GBS) transmission in preterm neonates admitted to neonatal intensive care unit (NICU) are poorly defined. We report 2 overlapping clusters of GBS late-onset disease (LOD) from June to September 2015 in an Italian NICU. METHODS: During the outbreak, possible sources of transmission (equipment, feeding bottles and breast pumps) were swabbed. Specimens from throat and rectum were collected on a weekly basis from all neonates admitted to NICU. Colonized or infected neonates had cohorting. Bacterial isolates were characterized by serologic and molecular typing methods. RESULTS: GBS was isolated in 2 full-term and 7 preterm neonates. Strains belonged to serotype III, with 3 different sequence types (ST17, ST182 and ST19). Full-term neonates were colonized with GBS strains unrelated to the clusters (ST182 and ST19). Two distinct ST17 strains caused 2 clusters in preterm neonates: a first cluster with 1 case of LOD and a second, larger cluster with 6 LOD in 5 neonates (one of them had recurrence). ST17 strains were isolated from vaginorectal and milk samples of 2 mothers. Two preterm neonates had no evidence of colonization for weeks, until they presented with LOD. CONCLUSIONS: Molecular analyses identified the presence of multiclonal GBS strains and 2 clusters of 7 cases of GBS-LOD. The dynamics of transmission of GBS within the NICU were complex. Breast milk was suspected to be one of the possible sources. In a research setting, the screening of GBS carrier mothers who deliver very preterm could contribute to the tracking of GBS transmission.

Neonatal Group B Streptococcus Infections: Prevention Strategies, Clinical and Microbiologic Characteristics in 7 Years of Surveillance.

BACKGROUND: The characteristics of group B streptococcus (GBS) neonatal disease in a period of 7 years are reported. METHODS: The estimation of the neonatal GBS disease risk and prevention strategies adopted at delivery in absence of national guidelines was evaluated by the analysis of 3501 questionnaires. Notification of 194 neonatal GBS infections was recorded. In addition, 115 strains from neonatal early-onset disease (EOD) and late-onset disease, respectively, plus 320 strains from pregnant women were analyzed by molecular typing methods and for antibiotic resistance. RESULTS: Preterm deliveries, precipitous labor and GBS negatively screened mothers were the prominent causes for an inadequate or lack of intrapartum antibiotic prophylaxis and EOD occurrence. The superimposable serotype distribution of GBS strains from EOD and from antenatal screening confirmed the vertical transmission from mother to neonate as the cause of disease. On the contrary, late-onset disease was almost exclusively caused by the internationally diffused clonal complex 17. Erythromycin resistance was detected in 17% of strains. Resistance to clindamycin was 15.3 %. CONCLUSIONS: The administration of intrapartum antibiotic prophylaxis to negatively GBS screened women in presence of risk factors was a deviation from the recommendations issued by the Centers for Disease Control and Prevention, and it should deserve further consideration. Routine surveillance and molecular typing of circulating clones are essential for the effective management of the neonatal GBS disease.

A fatal case of streptococcal toxic shock syndrome caused by Streptococcus suis carrying tet (40) and tet (O/W/32/O), Italy.

We report the first human fatal case of streptococcal toxic shock syndrome (STSS) caused by Streptococcus suis serotype 2 carrying the tetracycline efflux tet (40) gene and the tetracycline ribosomal protection tet (O/W/32/O) gene. The patient was splenectomized. The case was characterized by multi-organ dysfunction and disseminated intravascular coagulopathy, in accordance with the clinical parameters of STSS. More investigations are needed to improve the epidemiology and the pathogenesis of S. suis in human infection.

Tetanus in Italy 2001-2010: a continuing threat in older adults.

Despite being a completely preventable disease, tetanus cases continue to occur in Italy and notification and hospitalization rates have been reported to be higher with respect to European and other industrialized countries. We examined statutory notification, hospitalization, mortality and seroprevalence data to describe tetanus epidemiology in Italy from 2001 to 2010. A total of 594 tetanus cases were notified, with an average annual incidence of 1.0/1,000,000 population. Most cases were unvaccinated or incompletely vaccinated. Eighty percent of cases occurred in subjects aged >64 years and a higher proportion of females with respect to males were reported in this age group. The annual number of hospital admissions was 1.4-1.7 times greater than the number of notifications in the same year. The mean annual number of reported deaths was 21. Seroprevalence data show progressively higher susceptibility levels with increasing age. Over 50% of persons aged 45-64 years and over two thirds of subjects ≥65 years had tetanus antibody levels <0.01 IU/ml. Results show that tetanus is a continuing problem in Italy and, as in other countries, most cases occur in older adults, especially elderly women. The observed differences in notification and hospitalization rates suggest underreporting by physicians. In recent years, Italy has accounted for most cases reported annually in the European Union (EU) but different case definitions are used. In Italy, a confirmed case is one that meets the clinical case definition while the EU case definition classifies confirmed cases as those with laboratory confirmation of disease. The incidence of clinical tetanus in Italy is ten-fold higher than in other industrialized countries, like Australia and Canada, likely due to higher susceptibility levels in Italy. In view of the low prevalence of tetanus antibodies in adults ≥45 years, strategies to improve vaccine uptake in this population group need to be implemented.

Characteristics of neonatal GBS disease during a multicentre study (2007-2010) and in the year 2012.

INTRODUCTION: The characteristics of Group B Streptococcal (GBS) early onset (EOD) and late onset (LOD) neonatal infections in Italy were analyzed. Two periods were considered, a first 3-years period (2007-2010), when notification of GBS infections was enforced under the auspices of the Italian Ministry of Health, and a second 1 year period (2012) when reporting on neonatal GBS disease continued on voluntary basis. METHODS: A standardized form was used to collect data on cases of neonatal GBS disease. They included both maternal and neonatal data. RESULTS AND DISCUSSION: The two surveys underlined that preterm deliveries, precipitous labor and negatively GBS screened mothers are common causes of EOD occurrence, possibly explained by inadequate, or lack of, intrapartum antibiotic prophylaxis. Nevertheless, measures for reducing prevention failures and EOD incidence by an higher adherence to prevention strategies, as the Centre for Disease Control recommendations, are still possible and should be encouraged.

A multiplex PCR assay for the direct identification of the capsular type (Ia to IX) of Streptococcus agalactiae.

A multiplex PCR assay for the identification of serotypes Ia to IX of Streptococcus agalactiae was developed. By using a single PCR reaction containing a mix of 19 primers the assay identified each serotype by the analysis of the unique two or three bands pattern on agarose gel.

Molecular epidemiology and distribution of serotypes, surface proteins, and antibiotic resistance among group B streptococci in Italy.

Group B streptococci (GBS) comprising three different sets of isolates (31 invasive, 36 noninvasive, and 24 colonizing isolates) were collected in Italy during the years 2002 to 2005. Clonal groups were established by pulsed-field gel electrophoresis (PFGE), and selected isolates were studied by multilocus sequence typing (MLST). GBS isolates were also characterized by classical and molecular techniques for serotyping and protein gene and antibiotic resistance profiling. Some serotypes were significantly associated with a particular isolate population: serotype Ia more frequently corresponded to invasive strains than other strains, serotype V was more frequently encountered among noninvasive strains, and nontypeable strains were more common among isolates from carriers. Four major clonal groups accounted for 52.7% of all isolates: PFGE type 1/clonal complex 1 (CC1) comprised mainly serotype V isolates carrying the alp3 gene, PFGE type 2/CC23 encompassed serotype Ia isolates with the alp1 or alpha gene, PFGE type 3/CC17 comprised serotype III isolates carrying the rib gene, and PFGE type 4/CC19 consisted mainly of serotype II isolates possessing the rib gene. The same serotypes were shared by isolates of different clonal groups, and conversely, isolates belonging to the same clonal groups were found to be of different serotypes, presumably due to capsular switching by the horizontal transfer of capsular genes. Erythromycin resistance (prevalence, 16.5%; 15 resistant isolates of 91) was restricted to strains isolated from patients with noninvasive infections and carriers, while tetracycline resistance was evenly distributed (prevalence, 68.1%; 62 resistant isolates of 91). Most erythromycin-resistant GBS strains were of serotype V, were erm(B) positive, and belonged to the PFGE type 1/CC1 group, suggesting that macrolide resistance may have arisen both by clonal dissemination and by the horizontal transfer of resistance genes.

emm Types, virulence factors, and antibiotic resistance of invasive Streptococcus pyogenes isolates from Italy: What has changed in 11 years?

To investigate the epidemiology and characteristics of invasive group A streptococcal (GAS) disease over 11 years in Italy, this study compared the emm types and the superantigen toxin genes speA and speC as well as the erythromycin, clindamycin, and tetracycline susceptibilities of 207 invasive GAS strains collected during two national enhanced surveillance periods (1994 to 1996 and 2003 to 2005) and the time between each set of surveillance periods. The present study demonstrated that emm1 strains were consistently responsible for about 20% of invasive GAS infections, while variations in the frequencies of the other types were noted, although the causes of most cases of invasive infections were restricted to emm1, emm3, emm4, emm6, emm12, and emm18. During the 1994 to 1996 surveillance period, an emm89 epidemic clone spread across the northern part of Italy. A restricted macrolide resistance phenotype-type distribution of the bacteriophage-encoded speA toxin as well as of macrolide resistance genes was noted over time. Indeed, the recent acquisition of macrolide resistance in previously susceptible emm types was observed.

Therapeutic failures of antibiotics used to treat macrolide-susceptible Streptococcus pyogenes infections may be due to biofilm formation.

Streptococcus pyogenes infections often fail to respond to antibiotic therapy, leading to persistent throat carriage and recurrent infections. Such failures cannot always be explained by the occurrence of antibiotic resistance determinants, and it has been suggested that S. pyogenes may enter epithelial cells to escape antibiotic treatment. We investigated 289 S. pyogenes strains isolated from different clinical sources to evaluate their ability to form biofilm as an alternative method to escape antibiotic treatment and host defenses. Up to 90% of S. pyogenes isolates, from both invasive and noninvasive infections, were able to form biofilm. Specific emm types, such as emm6, appeared to be more likely to produce biofilm, although variations within strains belonging to the same type might suggest biofilm formation to be a trait of individual strains rather than a general attribute of a serotype. Interestingly, erythromycin-susceptible isolates formed a significantly thicker biofilm than resistant isolates (P < 0.05). Among resistant strains, those carrying the erm class determinants formed a less organized biofilm than the mef(A)-positive strains. Also, prtF1 appeared to be negatively associated with the ability to form biofilm (P < 0.01). Preliminary data on a selection of strains indicated that biofilm-forming isolates entered epithelial cells with significantly lower efficiency than biofilm-negative strains. We suggest that prtF1-negative macrolide-susceptible or mef(A)-carrying isolates, which are poorly equipped to enter cells, may use biofilm to escape antimicrobial treatments and survive within the host. In this view, biofilm formation by S. pyogenes could be responsible for unexplained treatment failures and recurrences due to susceptible microorganisms.

Association of group A streptococcal emm types with virulence traits and macrolide-resistance genes is independent of the source of isolation.

Streptococcus pyogenes (group A streptococci; GAS) recovered from paediatric pharyngitis (101 isolates) and asymptomatic children (79 isolates) in the same geographical area and period, as well as isolates collected during an enhanced national surveillance programme for GAS invasive diseases (79 isolates), were screened for the incidence of the streptococcal pyrogenic exotoxin (spe) genes speA and speC, as well as the macrolide-resistance genes erm(B), erm(A) subclass erm(TR) and mef(A), and typed by emm sequencing. The speA gene was detected with comparable incidence among throat isolates (13.9 % of asymptomatic children and 16.8 % of pharyngitis isolates) and in 25 % of invasive cases; in contrast, speC incidence was, surprisingly, higher in paediatric populations (55.4 % in pharyngitis isolates and 65.8 % in asymptomatic children) than in invasive isolates (30 %; P < 0.0001). Macrolide resistance was detected in 26.6, 38.0 and 37.6 % of strains belonging to invasive, asymptomatic and pharyngitis populations, respectively. The different incidences of exotoxin and antibiotic-resistance genes among populations did not appear to have an intrinsic clinical significance, but may reflect the propensity of these traits to be associated with certain emm types independent of the source from which the strains were isolated. Further investigations with larger emm-type populations are warranted to confirm this.

Molecular epidemiology and characteristics of Corynebacterium diphtheriae and Corynebacterium ulcerans strains isolated in Italy during the 1990s.

Five cases of diphtheria were reported in Italy between January 1990 and June 2001. Three cases were confirmed microbiologically by the isolation of toxigenic Corynebacterium diphtheriae (two cases) and Corynebacterium ulcerans (one case). Over the same period, 11 cases of non-toxigenic C. diphtheriae infection were reported to the Italian Public Health Institute, from which the causative organism was isolated from a skin infection in one case and from the throat in the other ten. Seven of the throat isolates were associated with fever, severe pharyngitis and tonsillitis and were all biotype gravis. Because there are no standardized breakpoints, the antimicrobial sensitivities of C. diphtheriae were determined in accordance with the National Committee for Clinical Laboratory Standards guidelines for Streptococcus spp. other than Streptococcus pneumoniae. MICs for penicillin ranged between 0.125 and 0.250 mg l(-1) and 7 out of 11 strains had a minimal bactericidal concentration (MBC)/MIC ratio >or= 32. All strains were sensitive to clindamycin (MIC

Virulence properties of type VII Streptococcus agalactiae (group B streptococci) and immunochemical analysis of capsular type polysaccharide.

Strains of a new polysaccharide type of group B streptococci (GBS), type VII, have been isolated from human carriers and invasive infections. Some of these strains bear the protein antigen c or R, as do other GBS serotypes. The capsular type polysaccharide is sialylated and this residue is involved in the immunodeterminant structure. All type VII strains examined were virulent in CD-1 mice; the LD50 after intraperitoneal (i.p.) challenge was 4.57 (SD 0.12) x10(7) cfu for the reference strain and 5.49 (SD 1.5) x10(7) cfu for clinical isolates. A particular feature of this serotype was the ability to induce septic arthritis not only when injected intravenously (i.v.), but also when injected i.p. Rabbit antiserum against the capsular type VII polysaccharide exhibited opsonic activity in a phagocytosis assay and protective activity against infection.