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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.

This paper presents the results of an Italian multicentric study conducted in the period April 1999-April 2000 in 64 clinical microbiology laboratories, aimed at collecting antimicrobial resistance data using a standard European protocol (EARSS Project, European Antimicrobial Surveillance System). A total of 1701 strains of Staphylococcus aureus were isolated from blood and 367 strains of Streptococcus pneumoniae were isolated from blood (72.7%) or from CSF (27.3%). Methicillin resistance in S. aureus was found to be 42.1%; it was significantly higher in adults (RR = 6.6, CI 95% 2.5-17.0), in hospitals of Centre and North of Italy (respectively RR = 1.45, CI 95% 1.1-2.0; RR = 1.6, CI 95% 1.1-2.2), and in intensive care and surgery units (respectively RR = 1.8, IC 95% 1.6-2.0 and RR = 1.7, CI 95% 1.4-1.9). Penicillin resistance in S. pneumoniae was found to be 12.1%; it was higher in South Italy (RR = 3.5, CI 95% 1.9-6.6, in meningitis compared to sepsis (RR = 2.5, CI 95% 1.4-2.5) and in intensive care units compared to other departments (RR = 2.2, CI 95% 1.0-4.8). The level of erythromycin resistance in S. pneumoniae was of 28.2%, reaching 56.4% in penicillin resistant strains. These results suggest that a continued surveillance and effective control measures are required.

Antimicrobial resistance represents a critical problem in human medicine today, from both clinical and economic points of view. In 1999, the Council of the European Union included antibiotic resistance among the public health priorities in the Community, and a resolution entitled 'A strategy against the microbial threat' was issued (1). According to this document, the establishment or strengthening of a surveillance system for the collection of reliable and comparable data on a national and international scale is one of the main interventions needed to control and prevent antibiotic resistance. Surveillance systems should also integrate data about the use and prescription of antibiotics (2).

The precursor of the Bacteroides fragilis metalloprotease enterotoxin was cloned and expressed in Escherichia coli, which was not able to process the precursor into the biologically active enterotoxin. Mouse antiserum elicited to the recombinant precursor reacted with the purified enterotoxin and with a crude enterotoxin preparation from an enterotoxigenic strain. The antiserum neutralized the cytotoxic activity of the enterotoxin in HT-29 cells.

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.

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.

The 36 kDa antigen represents the major EDTA-extracted protein of Clostridium difficile strains belonging to electrophoretic group 2. Antibodies to this antigen are found in sera of patients with C. difficile-associated diarrhoea. The 36 kDa antigen was extracted from C. difficile C253 by EDTA and purified by gel filtration (Sephacryl S300) and ion exchange chromatography (DEAE-Trisacryl M). The molecular weight of the purified protein was 36 kDa as determined by SDS-PAGE, also in non-reducing conditions. By gel filtration, the molecular size appeared to be 72 kDa and was not modified by the presence of EDTA or SDS in the column buffer. Since IEF showed a single isoelectric form of pI 4.6, the protein could be a homodimeric molecule. Immunofluorescence demonstrated that the 36 kDa antigen was located on the surface of the C. difficile cell. Monospecific antiserum raised in rabbits reacted positively with the 36 kDa protein of most group 2 C. difficile strains isolated from antibiotic-associated diarrhoea (AAD) outbreaks in Italy and other European countries.

30 strains of Cl. difficile isolated from faeces of patients with pseudomembranous colitis (PMC), antibiotic associated diarrhoea (AAC) and other intestinal disorders and from faeces of asymptomatic carriers were studied for production of toxins. Tissue culture assay was used for the detection of cytotoxin (toxin B) and ileal loop test for enterotoxin (toxin A). All Cl. difficile isolates from patients with PMC and AAC were found to produce cytotoxin, whereas enterotoxin was demonstrated only in approximately 70% of strains.

The "in vitro" susceptibility of 48 strains of "Clostridium difficile" to 12 antimicrobial agents was determined by agar dilution method. All isolates were susceptible to ampicillin, metronidazole, piperacillin, vancomycin and N-formimidoylthienamycin, resistant or intermediate to the new cephalosporins: cefoxitin, cefotaxime and moxalactam. For clindamycin, the MIC distribution appeared to be bimodal with 41.7% of the strains susceptible to 8 micrograms/ml and 58.3% resistant to 128 micrograms/ml; 90% of these strains were also resistant to erythromycin, tetracycline and chloramphenicol. The percentage of resistant strains in the isolates from patients with pseudomembranous colitis is significantly higher than in the isolates from patients with antibiotic-associated colitis.

Two hospital outbreaks of Clostridium difficile diarrhoea in two general surgery units of different hospitals are described. Moreover, the results of a study on the circulation of C. difficile in a neurosurgery unit following two cases of colitis are reported. C. difficile strains isolated from patients and environment have been typed using antimicrobial susceptibility testing and electrophoretic profiles of EDTA-extracted proteins. The majority of strains isolated in each hospital shared the same protein profile and the same pattern of antibiotic susceptibility. This provides strong evidence of cross-infection and hospital acquisition of C. difficile.

The human normal intestinal flora prevents the colonization of exogenous bacteria, maintaining a constant microecology: this property is called "colonization resistance". In leukemia patients antibiotics used for prevention and/or therapy of infectious episodes can alter the intestinal microecology, so that the gut can represent the trigger zone for generalized septicemia. Moreover cytotoxic drugs used in these patients can favour intestinal disturbances. In our study we evaluated the in vitro activity of three commonly used antineoplastic drugs (Daunorubicin, Cytosine arabinoside, Methotrexate) against aerobic and anaerobic intestinal bacteria and Clostridium difficile that is the aetiological agent of pseudomembranous colitis. Daunorubicin proved to be the most active inhibiting, in concentration ranging from 16 to 128 micrograms/ml, 50% of Bacteroides strains and 90% of Clostridium difficile and Enterococci strains tested. Methotrexate showed activity only against some Bacteroides strains, while Cytosine arabinoside had no activity at all. We conclude that in these patients the use of these drugs may represent another factor of risk altering the intestinal flora and so lowering the colonization resistance.

Intestinal microflora changes in ten adult and healthy subjects after storage of specimen with different modalities, temperatures and times were evaluated quantitatively and qualitatively. Aerobic components (Enterobacteriaceae, Enterococcus, Staphylococcus and Bacillus spp.) did not show considerable variations of concentration in all maintenance conditions. Anaerobic components showed different capacity of survival for every researched genus: Clostridia and Lactobacilli, even kept frozen for one month in glycerol broth at -70 degrees C, maintained a stable viable count; Bacteroides, Fusobacteria, Veillonellae and Peptostreptococci were found stable after one week, while after one month showed a loss of concentration superior to 4 Log; Eubacteria were found remarkably reduced after seven days and completely lost after one month.

In this preliminary study we have examined 12 healthy men aged between 20-60 years, by three specimens each. All the isolated could be assigned to genera by biochemical tests and gas-liquid chromatography. The results showed a 10(8)/g concentration of aerobe bacteria in faeces and a 10(10)/g concentration of anaerobe non sporing bacteria. The results of the count of Clostridia showed a variability among the examined individuals. The predominant groups of aerobes in the faeces were found to be E. coli and S. faecalis spp. and the predominant group of non sporing anaerobes were Bacteroides and Bificobacteria. These results were similar to the data obtained from foreign AA., showing the predominance of anaerobes in the faeces and particularly of Bacteroides.