Molecular surveillance and population structure analysis of methicillin-susceptible and methicillin-resistant Staphylococcus aureus in high-risk wards.

In this study we report the results of analysis of 253 isolates of Staphylococcus aureus (132 methicillin [meticillin]-resistant S. aureus [MRSA] isolates and 121 methicillin-susceptible S. aureus [MSSA] isolates) from 209 patients admitted to 18 high-risk wards of six hospitals located in Florence, Italy, over an 8-month period during which a program of epidemiological surveillance of hospital-acquired infections was conducted. The majority (69%) of the 87 reported S. aureus infections were caused by MRSA. No outbreak events have been reported. All the isolates were typed by amplified fragment length polymorphism (AFLP), and AFLP profiles were analyzed in order to define similarity groups. The discriminatory power of AFLP is very high with MSSA (Simpson index of diversity [D], 95.9%), whereas its resolution capability with MRSA (D, 44.7%) is hampered by the well-known high clonality of these populations (the main MRSA group accounted for 74% of the MRSA isolates). Combining AFLP, improved by visual inspection of polymorphisms, with multiplex PCR greatly increases MRSA resolution (D, 85.5%), resolving the MRSA population to a level that is one of the highest reported in the literature. Widespread and sporadic clones of MSSA and MRSA were identified, and their diffusion in the different hospitals and wards over the surveillance period was studied. The understanding of MSSA and MRSA population structures should be the starting point for the design of a more rational surveillance program for S. aureus species, maximizing benefits and reducing the cost of infection control strategies.

Characterization of Listeria monocytogenes isolates from human listeriosis cases in Italy.

The objective of this study was to characterize by serotyping, pulsed-field gel electrophoresis (PFGE), and PCR amplification of virulence genes and markers of epidemic clones I, II, and III (ECI, ECII, and ECIII) 54 human isolates from apparently sporadic cases of infection occurring in the Lombardy region and in the province of Florence, Tuscany, Italy, in the years 1996 to 2007. Listeria monocytogenes isolates were provided by the clinical microbiology laboratories of the Lombardy region and the "Careggi" Hospital of Florence, Tuscany, Italy. Serotyping, PFGE after digestion with the AscI and ApaI enzymes, and PCR amplification for the inlA, inlC, and inlJ genes and ECI, ECII, and ECIII markers were performed according to procedures described previously. Twenty-five (46.3%) L. monocytogenes isolates were assigned to serotype 1/2a, 23 (42.6%) to serotype 4b, and 6 (11.1%) to serotype 1/2b. Thirty-one AscI pulsotypes were recognized among the 54 human isolates. Eleven molecular subtype clusters, of which eight included indistinguishable pulsotypes and three included closely related pulsotypes, were shared by two to seven isolates. Fifteen isolates exhibited unique AscI pulsotypes. Three groups of clustered isolates and two apparently sporadic isolates generated EC amplicons. All strains tested positive for the inlA, inlC, and inlJ genes. Based on the results of serotyping and molecular typing, there were 11 occasions when L. monocytogenes strains with the same subtype were isolated from more than one listeriosis case. A total of 39 out of 54 isolates (72.2%) were attributed to molecular subtype clusters. The results of the study suggest that routine subtyping of L. monocytogenes strains from human listeriosis cases could allow more-timely detection of outbreaks possibly caused by food-borne isolates from a common source and could lead to control of ongoing food exposure, thus preventing the occurrence of more cases.

Molecular typing reveals frequent clustering among human isolates of Listeria monocytogenes in Italy.

In Italy, the annual incidence of reported cases of listeriosis amounts in recent years (2004 to 2006) to 0.8 case per million inhabitants. Our study is a subtyping analysis by serotyping, ribotyping, and pulsed-field gel electrophoresis analysis of 44 human isolates from apparently sporadic cases of infection in the Lombardy region and in the Province of Florence, Italy, in the years 1996 to 2007. Based on the results of the different subtyping methods, 10 occasions were detected when strains of L. monocytogenes with the same subtype were isolated from more than one listeriosis case. A total of 28 (66.7%) of 44 isolates were attributed to molecular subtype clusters. Our data support the use of sensitive molecular approaches to identify and trace L. monocytogenes isolates responsible for foodborne outbreaks of human listeriosis.

Use of the DiversiLab semiautomated repetitive-sequence-based polymerase chain reaction for epidemiologic analysis on Acinetobacter baumannii isolates in different Italian hospitals.

Acinetobacter baumannii is typically a nosocomial pathogen. Epidemiologic tools that can rapidly trace the spread of hospital-associated infections due to this microorganism are essential. Currently, amplified fragment length polymorphism and pulsed-field gel electrophoresis using ApaI, a macrorestriction enzyme, are the molecular techniques most widely used to type this microorganism. Unfortunately, they are technically demanding, requiring also well-trained personnel, and are time consuming. The aims of this study are 1) to evaluate the usefulness of the semiautomated repetitive-sequence-based polymerase chain reaction (rep-PCR) for typing A. baumannii, comparing this method with another semiautomated technique, such as ribotyping, and 2) to acquire information about the incidence, the clinical significance, and the susceptibility patterns of this microorganism in 13 different Italian hospitals in a 4-week period (total study population, >14000 beds). Twenty-eight A. baumannii were isolated in 7 different hospitals; 21 strains were analyzed with molecular methods. Automated ribotyping distinguished 6 different clusters of isolates, whereas rep-PCR appeared to be more discriminating, allowing us to distinguish 8 different clusters. Our study confirms the good discriminatory power of the semiautomated rep-PCR. Although expensive, this method is simple, fast, and reproducible, and in our opinion, it could be used in a hierarchic approach as a 1st-line typing tool if results of analysis are required in a short period or if a large number of isolates have to be analyzed.

Reinterpreting a community outbreak of Salmonella enterica serotype Enteritidis in the light of molecular typing.

BACKGROUND: In November 2005, a large outbreak due to Salmonella enterica serotype Enteritidis (S. Enteritidis) was observed within children who had eaten their meals at 53 school cafeterias in Florence and the surrounding area. A total of 154 isolates of S. Enteritidis were recovered from human cases between November 2005 and January 2006. All strains were assigned phage type 8 (PT8) and a common XbaI pulsotype. This paper reports the findings of a molecular epidemiological investigation performed on 124 strains of S. Enteritidis isolated in the years 2005 and 2006 in Florence and the surrounding area, including the epidemic isolates. METHODS: One hundred twenty-four human isolates of S. Enteritidis identified in the period January 2005 - December 2006 were submitted to molecular typing by single enzyme - amplified fragment length polymorphism (SE-AFLP). RESULTS: Molecular subtyping by SE-AFLP yielded five different profiles. In the pre-epidemic phase, type A included 78.4% of isolates, whereas only three (8.1%) belonged to type C. All isolates, but one, of the epidemic phase were indistinguishable and attributed to type C. In the post-epidemic period, a polymorphic pattern of SE-AFLP types was again recognized but type C accounted for 73.3% of the isolates during the first six months of 2006, whereas during the remaining six months type A regained the first place, including 52.0% of the isolates. CONCLUSION: The epidemic event was attributed to the emergence and clonal expansion of a strain of S. Enteritidis PT8-SE-AFLP type C. Circulation of the epidemic clone was much more extensive than the surveillance and traditional laboratory data demonstrated.

A trial of preventing early- and late-onset Group B streptococcal sepsis with combined intrapartum chemoprophylaxis and universal neonatal screening.

BACKGROUND: Prevention of early-onset Group B Streptococcal (GBS) infection has been attempted by employing universal maternal screening for GBS, intrapartum chemoprophylaxis, and a single dose of penicillin given to neonates in the first hour of life. This strategy, however, does not seem to prevent the occurrence of late-onset neonatal group B streptococcal disease. STUDY DESIGN: We assessed early and late-onset GBS disease with the use of a before-after study designed to evaluate the implementation of intrapartum antimicrobial prophylaxis. Moreover, universal neonatal screening for colonization of GBS was carried out with swabs of the external ear canal. Newborns with GBS colonization received a preventive treatment with oral amoxicillin for 10 days. RESULTS: Early-onset GBS infection decreased from 0.5 per thousand at baseline period to 0.19 per thousand at the study period. The incidence of late-onset GBS disease decreased from 1:1348 (0.74 per thousand) to 1:20,710 (0.048 per thousand). The overall cost for universal neonatal screening paid for by the Italian Health System in the study period was 31,065 US dollars with an antibiotic prophylaxis cost of 2,399 US dollars. CONCLUSIONS: A combined strategy based on GBS culture screening and assessment of risk factors for perinatal GBS disease can significantly reduce the rate of both early and late-onset GBS infections.

Pseudomonas aeruginosa sepsis in stem cell transplantation patients.

We report the epidemiological investigation of an outbreak of Pseudomonas aeruginosa infection in 6 patients who shared, during different periods, the same 2 rooms of a bone marrow transplantation unit. Phenotypic and molecular analysis of isolates from patients and from the environment strongly suggested a single, environmental source of infection.

Staphylococcus aureus epidemic in a neonatal nursery: a strategy of infection control.

The risk of nosocomial infection due to Staphylococcus aureus in fullterm newborns is higher under hospital conditions where there are overcrowded nurseries and inadequate infection control techniques. We report on an outbreak of skin infection in a Maternity Nursery (May 21, 2000) and the measures undertaken to bring the epidemic under control. These measures included: separating neonates already present in the nursery on August 23, 2000 from ones newly arriving by creating two different cohorts, one of neonates born before this date and one of neonates born later; restricting healthcare workers caring for S. aureus- infected infants from working with non-infected infants; disallowing carrier healthcare workers from caring for patients; introducing contact and droplet precautions (including the routine use of gowns, gloves, and mask); ensuring appropriate disinfection of potential sources of contamination. A representative number of isolates were typed by genomic DNA restriction length polymorphism analysis by means of pulsed-field gel electrophoresis (PFGE). Among the 227 cases of skin lesions, microbiological laboratory analyses confirmed that 175 were staphylococcal infections. The outbreak showed a gradual reduction in magnitude when the overcrowding of the Nursery was reduced by separating the newborns into the two different Nurseries (two cohorts). The genotyping of the strains by PFGE confirmed the nurse-to-newborn transmission of S. aureus. The measures adopted for controlling the S. aureus outbreak can, in retrospect, be assessed to have been very effective.