Application of capsular sequence typing (CST) to serotype non-viable Streptococcus pneumoniae isolates from an old collection.

Serotyping of Streptococcus pneumoniae is essential for monitoring changes in the pneumococcal population and the impact of vaccines. Recently, various DNA-based methods have become available and are increasingly used because they are cheaper and easier to perform than the Quellung reaction. Our aim was to apply a DNA-based method, capsular sequence typing (CST), to a collection of non-viable lyophilized pneumococcal isolates dating from the 1980s to elucidate the serotypes circulating in Italy 30 years ago. As a preliminary evaluation of the method, CST was applied to 68 recent pneumococcal isolates representative of the most common serotypes circulating in Italy in invasive pneumococcal disease (IPD) previously serotyped by the Quellung reaction. CST was then applied to 132 lyophilized non-viable isolates. A serotype-specific polymerase chain reaction (PCR), using primers suggested by the Centers for Disease Control and Prevention (CDC), was performed when CST did not yield a univocal serotype. Considering the control isolates, CST concordance with the Quellung reaction was 95.6 %. For the non-viable lyophilized isolates, CST identified a univocal serotype for 59.4 % of the isolates. This percentage increased to 78.1 % if CST was combined with serotype-specific PCR. The most frequent serotypes in the collection of non-viable strains were: 3 (15.6 %), 14 (11.7 %), 35B (5.5 %), 19A (5.5 %), and 8 (4.7 %). CST proved to be a valid method for serotyping pneumococcal strains and provided information about pneumococcal serotypes present in Italy 30 years ago. The combination of CST with serotype-specific PCR was an effective strategy to identify pneumococcal serotypes that can be suggested also for routine laboratories.

Colistin resistance superimposed to endemic carbapenem-resistant Klebsiella pneumoniae: a rapidly evolving problem in Italy, November 2013 to April 2014.

Consecutive non-replicate clinical isolates (n=191) of carbapenem non-susceptible Enterobacteriaceae were collected from 21 hospital laboratories across Italy from November 2013 to April 2014 as part of the European Survey on Carbapenemase-producing Enterobacteriaceae (EuSCAPE) project. Klebsiella pneumonia carbapenemase-producing K. pneumoniae (KPC-KP) represented 178 (93%) isolates with 76 (43%) respectively resistant to colistin, a key drug for treating carbapenamase-producing Enterobacteriaceae. KPC-KP colistin-resistant isolates were detected in all participating laboratories. This underscores a concerning evolution of colistin resistance in a setting of high KPC-KP endemicity.

Epidemic diffusion of KPC carbapenemase-producing Klebsiella pneumoniae in Italy: results of the first countrywide survey, 15 May to 30 June 2011.

Carbapenem-resistant Enterobacteriaceae (CRE) are emerging as a public health problem in various settings. In Italy, a rapid and remarkable increase of carbapenem-non-susceptible Klebsiella pneumoniae has been reported since 2010. Here we report on the results of a countrywide cross-sectional survey, carried out from 15 May to 30 June 2011 to investigate the diffusion of CRE in Italy and to characterise the most prevalent resistance mechanisms and their dissemination patterns. CRE were reported from most (23 of 25) participating laboratories, with an overall proportion of 3.5% and 0.3% among consecutive non-duplicate clinical isolates of Enterobacteriaceae from inpatients (n=7,154) and outpatients (n=6,595), respectively. K. pneumoniae was the most frequent species (proportion of carbapenem-non-susceptible isolates: 11.9%), while a minority of CRE of other species were detected. Carbapenemase production was detected in the majority (85%) of CRE. KPC-type enzymes were by far the most common (89.5% of carbapenemase producers), followed by VIM-1 (9.2%) and OXA-48 (1.3%). KPC-producing K. pneumoniae (KPC-KP) were detected in most centres and contributed majorly to the epidemic dissemination of CRE recently observed in our country. Dissemination of KPC-KP was mostly sustained by strains of clonal complex 258 (ST-258 producing KPC-2 or KPC-3, and ST-512 producing KPC-3), while a minority belonged to ST-101.

Carbapenem non-susceptible Klebsiella pneumoniae from Micronet network hospitals, Italy, 2009 to 2012.

Carbapenem-resistant Klebsiella pneumoniae has recently been reported as a new, multidrug-resistant nosocomial pathogen in several hospitals from various Italian regions. Through Micronet, a new Italian sentinel laboratory-based surveillance network, we studied the trend of non-susceptibility of K. pneumoniae to selected carbapenems (imipenem and/or meropenem) in 14 of the 15 hospitals participating in the network. Analysis of data from 1 January 2009 to 30 April 2012 revealed a statistically significant increasing trend (p<0.01) in the proportion of carbapenem non-susceptible K. pneumoniae isolates from clinical specimens (from 2.2 % in 2009 to 19.4% in 2012). The increase in the proportion of non-susceptibility was very large for isolates from the respiratory tract (from 5.3% in 2009 to 38.5% in 2012) and blood (from 5.4% in 2009 to 29.2% in 2012). The results demonstrate the urgent need in Italy for infection control, guidelines, antibiotic stewardship programmes and utilisation of surveillance systems, such as Micronet, which are capable of receiving data from hospitals in real time for many pathogens and types of clinical specimens.

Contribution of serotype and genetic background to biofilm formation by Streptococcus pneumoniae.

Streptococcus pneumoniae is the main cause of a variety of infections in children and the elderly ranging from otitis media to pneumonia. In recent years, biofilm formed by S. pneumoniae has begun to attract attention for a possible role in strains fitness and/or virulence. We evaluated the ability to form biofilm in a collection of clinical isolates, including antibiotic-resistant isolates whose genetic background had been previously ascertained. It appears that biofilm formation is a rather common feature among pneumococci, an observation which would fit with some types of infections caused by this microorganism (i.e. otitis, meningitis), which have often been associated with the ability to form biofilm. Antibiotic-susceptible isolates were able to form thicker biofilms compared to resistant strains, although no specific association could be observed with either serotypes or clones. This lack of association between the ability to form biofilm and any of the characters examined, while being a very common feature of pneumococci, may be suggestive of an important role for biofilm in pneumococcal ecology.

DNA microarray-based characterisation of Panton-Valentine leukocidin-positive community-acquired methicillin-resistant Staphylococcus aureus from Italy.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) isolates are widespread in many countries, with varying distribution and epidemiology. The aim of this study was to collect and characterise the CA-MRSA isolates circulating in Italy, since only some case reports have been published. Eighteen Panton-Valentine-positive CA-MRSA isolates were collected from different Italian hospitals during the period 2005-2009 from severe infections (skin and soft tissue infections, n = 10; necrotising pneumonia, n = 7; and sepsis, n = 1). Accessory gene regulator (agr) typing, staphylococcal cassette chromosome (SCC) mec typing, spa typing, multi-locus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and DNA microarray were applied to categorise isolates into clones and to compare the relevant genetic features of each clone. Six different clones were identified, the most common (7 out of 18 isolates, 38.8%) being agrI/ST8/SCCmecIV, corresponding to the USA300 clone. Six out of the seven USA300 isolates did not harbour the arginine catabolic mobile element (ACME). Four strains (22.2%) were agrIII/ST80/SCCmecIV, corresponding to the European clone. Two of the other clones, namely, agrIII/ST88/SCCmecV and agrIII/ST772/SCCmecV, corresponded to CA-MRSA clones rarely found in other countries and probably originating from Africa or the Indian subcontinent. The results of microarray hybridisations showed that the distribution of resistance genes and other virulence factors was specific to each clone. Some characteristics could be exploited as specific markers for a clone or a group of isolates, e.g. the mer operon, recovered only in ACME-negative USA300 strains. DNA microarray contributed to a more complete description of the variety of different CA-MRSA clones circulating in Italy.

Vancomycin-heteroresistant phenotype in invasive methicillin-resistant Staphylococcus aureus isolates belonging to spa type 041.

The aim of this study was to characterise invasive methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains from Italy and to investigate the presence of heteroresistant vancomycin-intermediate S. aureus (h-VISA). Eighty-two MSSA and 66 MRSA strains obtained from 19 laboratories were submitted to in vitro susceptibility testing; MRSA strains were also analysed by the macro Etest (MET) and vancomycin population analysis profiles (PAP) to detect the presence of h-VISA. Genotyping included the detection of agr locus, SCCmec typing, spa typing and multilocus sequence typing (MLST). By Etest, 66% of all isolates showed a minimum inhibitory concentration (MIC) >or=1.5 microg/ml and two MRSA strains were categorised as VISA (MIC = 3 microg/ml). Twelve MRSA strains were positive by MET; of these, 9 (14% of all MRSA) were confirmed as h-VISA by PAP. MRSA strains were assigned to 14 spa types, with t001, t008 and t041 including 77% of the isolates. The most common spa type, t041, characterised as ST228/273-MRSA-I (CC5) and comprising 24 isolates, included one VISA and eight h-VISA. This is the first description of a close association between h-VISA and t041, a spa type common in Italy and in other European countries, that highlights the importance of molecular typing to identify clones of special clinical relevance.

No evidence of colonization with community-acquired methicillin-resistant Staphylococcus aureus in HIV-1-infected men who have sex with men.

To assess the prevalence of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) colonization in HIV-1-infected men who have sex with men (MSM), a cross-sectional study was conducted on 104 persons attending a large STI/HIV unit in Rome, Italy in the period June 2007-June 2008. Swabs obtained from both anterior nares and S. aureus isolates were characterized by phenotypic and genotypic methods. A total of 24 individuals (23.1%) were colonized with S. aureus but none carried MRSA. No statistically significant association between colonization with S. aureus and behavioural, clinical, virological or immunological characteristics was identified. This study indicates a lack of circulation of CA-MRSA in HIV-1-infected MSM in Italy and underscores large epidemiological differences between the USA and a European country, so that only locally conducted epidemiological studies can provide insight into the local circulation of CA-MRSA in general and selected populations.

Characterization of macrolide efflux pump mef subclasses detected in clinical isolates of Streptococcus pyogenes isolated between 1999 and 2005.

The macrolide efflux mechanism of resistance, mef, was characterized in community-acquired respiratory tract infections with Streptococcus pyogenes. Fifty-four (4.6%) M phenotype isolates were screen tested as negative for mef(A). Of these 54 isolates, 5 (0.4%), 27 (2.3%), and 1 (0.1%) were considered to be mef(I) positive, a novel mosaic variant of mef, or a novel subclass of mef, respectively. This study shows (i) the definitive presence of mef(E) in S. pyogenes and its global distribution, (ii) the presence of a mosaic variant of mef composed of mef(A) and mef(E), (iii) the previously undescribed presence of mef(I) in S. pyogenes, and (iv) the presence of a novel subclass of mef in S. pyogenes.

What is MRSA?

For decades methicillin-resistant Staphylococcus aureus (MRSA) has been considered the prototype of multi-resistant nosocomial pathogens, causing infections in high-risk patients. Changes in the healthcare system, coupled with the evolution of this versatile microorganism, have transformed MRSA into a cause of community-onset infections, in both patients who have contact with the healthcare system and patients without such a risk factor. New lineages of MRSA, defined as community acquired (CA)-MRSA, have emerged that have a propensity to cause infections in young individuals without risk factors. CA-MRSA primarily causes skin infections and, rarely, necrotising pneumonia. In the USA, these strains belong to a single widespread clone, designated USA300, while in Europe they belong to a variety of clones. Most strains carry genes for the Panton-Valentine leukocidin, whose role in diseases is under debate. In subjects living in the community who have contact with the healthcare system, MRSA strains of the nosocomial type are a frequent cause of infection and of pneumonia in particular. The detection of a large MRSA reservoir in pigs and the finding that professionally exposed individuals are colonised, has further shown that it is necessary to closely follow the epidemiology of MRSA if we want to combat it effectively.

Population structure of invasive Streptococcus pneumoniae isolates in Italy prior to the implementation of the 7-valent conjugate vaccine (1999-2003).

A total of 773 pneumococcal isolates were collected from a nationwide surveillance of invasive pneumococcal diseases during 1999-2003 prior to the implementation of the 7-valent conjugate vaccine (PCV7) in Italy. The isolates included vaccine serotypes (VS, 393 isolates), vaccine-related serotypes (VRS, 93), and nonvaccine serotypes (NVS, 279). The ten most prevalent serotypes were: 14 (16.4%), 3 (8.4%), 23F (8%), 19F (7.4%), 4 (5.9%), 7F (5.8%), 9V (5.3%), 6B (4.9%), 19A (4.7%), and 1 (3.7%). VRS or NVS isolates showed a lower rate of penicillin or drug resistance than VS. Representative isolates of the major VS, VRS, and NVS were genotyped by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The isolates examined were found to belong to 18 international clones and to eight newly described clones. VS isolates sharing clonal groups with VRS or NVS were also detected. Evidence of a past history of capsular switching events was observed in five clones.

Colonization and infection due to carbapenemase-producing Enterobacteriaceae in liver and lung transplant recipients and donor-derived transmission: a prospective cohort study conducted in Italy.

OBJECTIVES: A prospective cohort study was conducted in Italy in order to describe the microbiologic aspects of colonization/infection by carbapenemase-producing Enterobacteriaceae (CPE) in donors and recipients of lung and liver transplants and the possible CPE transmission from donors to recipients. METHODS: Between 15 January 2014 and 14 January 2015, all recipients of solid organ transplants (SOT) at ten lung and eight liver transplantation centres and the corresponding donors were enrolled. Screening cultures to detect CPE were performed in donors, and screening and clinical cultures in recipients with a 28-day microbiologic follow-up after receipt of SOT. Detection of carbapenemase genes by PCR, genotyping by multilocus sequence typing, and pulsed-field gel electrophoresis and whole-genome sequencing were performed. RESULTS: Of 588 screened donors, 3.4% were colonized with CPE. Of the liver first transplant recipients (n = 521), 2.5% were colonized before receipt of SOT and 5% acquired CPE during follow-up. CPE colonization was higher in lung first transplant recipients (n = 111, 2.7% before SOT and 14.4% after SOT). CPE infections occurred in 1.9% and 5.3% of liver or lung recipients, respectively. CPE isolates were mostly Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae belonging to CG258. Three events of donor-recipient CPE transmission, confirmed by whole-genome sequencing and/or pulsed-field gel electrophoresis, occurred in lung recipients: two involving K. pneumoniae sequence type 512 and one Verona integron-encoded metallo-ß-lactamase (VIM)-producing Enterobacter aerogenes. CONCLUSIONS: This study showed a low risk of donor-recipient CPE transmission, indicating that donor CPE colonization does not necessarily represent a contraindication for donation unless colonization regards the organ to be transplanted. Donor and recipient screening remains essential to prevent CPE transmission and cross-infection in transplantation centres.

Colonization by multidrug-resistant organisms in long-term care facilities in Italy: a point-prevalence study.

OBJECTIVES: To determine prevalence and risk factors for colonization by multidrug-resistant organisms (MDROs) in long-term care facility (LTCF) residents in Italy. Genotypes of MDRO isolates were investigated. METHODS: A point-prevalence study was conducted at 12 LTCFs located in four Italian cities (2 February to 14 March 2015). Rectal swabs, faeces and nasal/auxiliary swabs were cultured for extended-spectrum ß-lactamase (ESBL)- and/or carbapenemase-producing Enterobacteriaceae, Clostridium difficile and methicillin-resistant Staphylococcus aureus (MRSA) respectively. Antimicrobial susceptibility testing, detection of ESBL and/or carbapenemase genes and molecular typing of MDROs were performed. Risk factors for colonization were determined by univariate and multivariate analysis. RESULTS: A total of 489 LTCF residents aged ≥65 years were enrolled. The prevalence of colonization by ESBL-producing Enterobacteriaceae, MRSA and C. difficile was 57.3% (279/487), 17.2% (84/487) and 5.1% (21/409) respectively. Carriage rate of carbapenemase-producing Enterobacteriaceae was 1% (5/487). Being bedridden was a common independent risk factor for colonization by all MDROs, although risk factors specific for each MDRO were identified. ESBL-producing Escherichia coli carriage was associated with the sequence type (ST) 131-H30 subclone, but other minor STs predominated in individual LTCF or in LTCFs located in the same city, suggesting a role for intrafacility or local transmission. Similarly, MRSA from LTCF residents belonged to the same spa types/ST clones (t008/ST8 and t032/ST22) commonly found in Italian acute-care hospitals, but infrequent spa types were recovered in individual LTCFs. The prevalent C. difficile PCR ribotypes were 356/607 and 018, both common in Italian acute-care hospitals. CONCLUSIONS: MDRO colonization is common among residents in Italian LTCFs.

Bacterial isolates from severe infections and their antibiotic susceptibility patterns in Italy: a nationwide study in the hospital setting.

The most frequent agents of severe bacterial infections and their antibiotic susceptibility patterns were determined in patients admitted to 45 Italian hospitals over the years 2002-2003. The most common diagnoses were: sepsis (33.8%), pneumonia (9.4%), intravascular catheter-associated infections (9.3%) and ventilator-associated pneumonia (8.1%). Overall, 5115 bacterial isolates were identified from 4228 patients. Three bacterial species, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, accounted for more than 50% of the isolates. Other prevalent bacterial isolates were Staphylococcus epidermidis and Enterococcus faecalis, while Acinetobacter baumanii ranked third among all Intensive Care Unit (ICU) isolates. 7% of S. aureus had intermediate resistance to vancomycin. Although E. faecalis displayed no vancomycin resistance, 34% of vancomycin-resistant isolates were found among Enterococcus faecium, one of the highest rates found to date, emphasizing the difference between these two enterococcal species. All the Gram-positive pathogens were susceptible to linezolid, with the exception of approximately 2% of the enterococcal isolates that were intermediate with a minimum inhibitory concentration (MIC)=4 microg/ml. Almost 10% of Escherichia coli, 14% of Klebsiella pneumoniae, 22% of Serratia marcescens and 50% of Enterobacter cloacae were non-susceptible to cefotaxime. Amikacin was the most active antibiotic against P. aeruginosa that showed lack of susceptibility to ceftazidime, gentamicin, piperacillin and ciprofloxacin ranging from 20 to 35%. Finally, Acinetobacter baumanii showed a high level of resistance to all the antibiotics tested including imipenem (58%). The results obtained in this study, the first of its kind in Italy, offer indications for guiding empirical therapy and implementing specific interventions to fight antibiotic-resistant bacterial infections and their transmission in the hospital setting in Italy.

[Antibiotic-resistance in Italy: activity of the first year of the surveillance project AR-ISS]. / Antibiotico-resistenza in Italia: un anno di attività del progetto di sorveglianza AR-ISS.

The antibiotic resistance surveillance project AR-ISS, started in 2001, is based on a network of 62 sentinel microbiological laboratories throughout the country. The laboratories collect and transmit data to the Istituto Superiore di Sanità on the antibiotic susceptibility of bloodstream isolates of 7 species: Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis/faecium, Klebsiella pneumoniae/oxytoca ed Escherichia coli. They also send selected bacterial strains for further characterization. Results of the first year of surveillance are presented and are compared with data from the previous study EARSS-Italia and from other European countries. Oxacillin resistance in S. aureus appears to be stable, however, it remains one of the highest in Europe (41,5%). No strain with intermediate susceptibility or resistance to vancomycin has been isolated. In S. pneumoniae, the level of penicillin resistance is moderate (10,8%), but macrolide resistance has increased greatly (37,6% versus 28,6% of the previous study), following a tendency common to several European countries. Unexpectedly, vancomycin resistance in E. faecium was found to be 18%, the highest in Europe. Presumptive ESBL production in Gram-negative organisms can be estimated at 20% in Klebsiella and 1% in E. coli. Ampicillin and ciprofloxacin resistance in E. coli (respectively 50% and 18%) are among the highest in Europe. In conclusion, the rate of antibiotic resistance in the species studied is worrisome and requires continuing monitoring. Although some activities of AR-ISS need improvements, the surveillance has the potentiality to produce relevant and representative data about antibiotic resistance in Italy that can be used for comparison at the European level.

Evaluation of gas-liquid chromatography for the rapid diagnosis of Clostridium difficile associated disease.

Direct gas-liquid chromatography of faecal specimens with isocaproic acid as a marker was used for the rapid diagnosis of Clostridium difficile associated diarrhoeal diseases. Ninety stools were examined and results were compared with conventional culture on selective medium and cytotoxin assay in tissue culture. Using a combined analysis of isocaproic acid and butyric acid peak heights we defined three categories: positive, negative, and indeterminate. When the indeterminate group was excluded, the positive and negative predictive values of gas-liquid chromatography analysis were 86.9% and 85% respectively compared with culture and 71.4% and 95% respectively compared with cytotoxin assay.

Comparison of antisera in the fluorescent antibody test for detection of Bacteroides spp in clinical specimens.

Twenty-two known strains of the Bacteroides fragilis group of organisms and 67 clinical specimens from a variety of sites were examined by fluorescent antibody test (IFA) using two different antisera fro the rapid detection of B fragilis group of organisms. A previously reported Barts' pooled antisera was compared with a commercially produced Fluoretec kit antisera and the findings were related to routine anaerobic culture and gas liquid chromatography for short chain fatty acids. The Barts' antisera was more sensitive (88%) but less specific (88%) than the kit (sensitivity 50%, specificity 98%). This indicates that Barts' antisera picks up more positive cultures than the kit. The predictive value of a positive test was 82% for Barts' antisera and 93% for the kit. There were higher numbers of false-negatives with the kit (13/26) than with the Barts' (3/26). The predictive value of a negative test was 92% for Barts' antisera and 75% for the kit, indicating that a negative IFA test with Barts' antisera is a reliable index of the absence of the B fragilis group of organisms from clinical specimens. The implications for the use of this test in a routine laboratory are discussed.

Antimicrobial susceptibility of invasive Streptococcus pneumoniae in Italy by agar dilution method and E test.

Few data on antimicrobial susceptibility of invasive Streptococcus pneumoniae isolated in Italy are available. Ninety-two invasive isolates from all over the country collected from January 1997 to April 1998 were tested for sensitivity to penicillin, erythromycin, ceftriaxone, chloramphenicol, tetracycline, and trimethoprim/sulfamethoxazole by the agar dilution method. Five (5.4%) strains were resistant to penicillin (one highly, four intermediately resistant), 8 (8.7%) to chloramphenicol, 27 (29.3%) to erythromycin, 17 (18.5%) to tetracycline (16 highly, one intermediately), and 21 (22.8%) to trimethoprim/sulfamethoxazole (14 highly, 7 intermediately). All strains were susceptible to ceftriaxone, although the penicillin-resistant strain had the highest minimal inhibitory concentration. (MIC) value (0.5 microg/ml); three penicillin-resistant strains were also resistant to erythromycin. Eight strains were multi-drug resistant, being also resistant to at least three antibiotics. The commercially available E test was compared with the standard agar dilution method for the determination of MIC of penicillin, erythromycin, ceftriaxone, chloramphenicol, and trimethoprim/sulfamethoxazole. E test established the same susceptibility categories for 100% of the strains tested for penicillin and ceftriaxone, 99% for chloramphenicol, 97% for erythromycin, and 74% for trimethoprim/sulfamethoxazole. According to our results, E test was simple to perform, easy to interpret, and a valid method for susceptibility testing of S. pneumoniae. Our study shows that in Italy the rate of penicillin resistance in invasive isolates of S. pneumoniae is one of the lowest in Europe (5.4%), while the rate of erythromycin is very high (29.3%) and is reaching the highest rates of other Southern European countries.

Susceptibility and genetic relatedness of invasive Haemophilus influenzae type b in Italy.

Haemophilus influenzae type b (Hib) still causes a large portion of meningitis in children less than 5 year old in Italy because vaccination against this agent has not been fully implemented in the country. We have studied 78 Hib strains and 4 nontypable H. influenzae (NTHi) isolated from the cerebrospinal fluid of subjects with meningitis for susceptibility to ampicillin, chloramphenicol, and ceftriaxone. The macrorestriction profiles of chromosomal DNA obtained by pulsed-field gel electrophoresis (PFGE) following digestion with SmaI and ApaI were also determined. All strains except one were equally susceptible to the antibiotics tested. One Hib strain, the only beta-lactamase producer, showed an intermediate susceptibility to ampicillin (MIC = 2 microg/ml), while maintaining full susceptibility to chloramphenicol and ceftriaxone. The analysis of the PFGE patterns showed that most of the Hib isolates, including the beta-lactamase-positive Hib strain, belonged to the same clone or to closely related subclones. For three PCR-confirmed NTHi isolates, we obtained completely different PFGE profiles. In conclusion, resistance to ampicillin still appears to be a rare finding in Hib strains causing meningitis in Italy; moreover, PFGE showed that the population structure of invasive Hib is essentially clonal.

Detection and characterization of vancomycin-resistant enterococci in farm animals and raw meat products in Italy.

The emergence of vancomycin-resistant enterococci (VRE) in Europe has been ascribed to the long-time use of the glycopeptide antibiotic avoparcin as feed additive in food animals, until its ban in 1997 in EU. The pres- ence of VRE in food of animal origin is believed to represent a potential risk for the consumer. We studied the fecal carriage of VRE in broiler chickens and slaughter pigs in Italy before the avoparcin ban and eval- uated the impact of avoparcin withdrawal on the presence of VRE in raw meat products. Broilers and pigs were both found to be frequently colonized by VRE, as 36% and 24.6% of the flocks or the herds, respec- tively, were positive. Molecular typing of VRE strains by PFGE showed that animals housed in different pens within the same farm were colonized by clonally related strains. After the avoparcin ban, a decrease in the rate of VRE contamination in meat products was observed. Such a decrease was statistically significant in poultry (from 18.8% to 9.6%) but not in pork products (from 9.7% to 6.9%). The majority of VRE from all sources carried the vanA resistance gene and included Enterococcus faecium, E. faecalis, E. hirae, E. durans, and E. gallinarum. None of the strains carried the vanB gene, whereas constitutively resistant vanC-positive strains were frequently found. Our results show that avoparcin withdrawal has been successful in reducing VRE contamination in meat products. However, this measure needs to be complemented by a prudent use of glycopeptide antibiotics in human medicine.