Seventy-three cultures of Clostridium difficile isolated both during, and in the period immediately following, an outbreak of infection in a group of three hospitals, were characterized by one-dimensional sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) of whole-cell proteins. Each protein pattern was characterized by the presence of one or two dense bands which were highly reproducible. The protein patterns were used as the basis for a numerical analysis which divided the strains into five phenons (electrophoretic or EP types). The majority, 60 of the 73 cultures, belonged to a single phenon which included strains from both patients and the environment. We conclude that high-resolution SDS–PAGE of proteins provides an effective method for typing C. difficile and therefore for tracing the possible spread of epidemic strains in hospitals and other institutions, thereby allowing a better understanding of the epidemiology of the organism.

The genomic DNA of 39 strains of Salmonella typhi isolated from local residents and patients who had visited countries in the Asian region was analysed for restriction fragment length polymorphisms (RFLP). Pulsed-field gel electrophoretic (PFGE) analysis of Xba I- and Spe I-generated genomic restriction fragments established 22 PFGE types whereas phage typing differentiated the 39 isolates into 9 distinct phage types. This study showed that PFGE is more discriminatory than phage typing as it is capable of subtyping S. typhi strains of the same phage types. Genetic relatedness among the isolates was determined. Seven major clusters were identified at SABSof > 0–80 and the remaining 13 isolates were distributed into minor clusters which were related at SABS of less than O.80. In conclusion, PFGE analysis in conjunction with distance matrix analysis served as a useful tool for delineating common S. typhi phage types of diverse origins from different geographical localesand separated in time.

Escherichia coli O157.H7 was found in 10 of 3570 (0·28%) faecal samples from dairy cattle in 5 of 60 herds (8·3%). Several tentative associations with manure handling and feeding management practices on dairy farms were identified. Faecal/urine slurry samples, bulk milk samples, and milk filters from dairy herds were negative for E. coli 0157.H7. E. coli 0157.H7 was also isolated from 10 of 1412 (0·71 %) faecal samples from pastured beef cattle in 4 of 25 (16%) herds. The prevalence of E. coli 0157. H7 excretion in feedlot beef cattle was 2 of 600 (0·33 %). The identification of cattle management practices associated with colonization of cattle by E. coli 0157.H7 suggests the possibility that human E. coli O157.H7 exposure may be reduced by cattle management procedures.

From March to October 1993. 15 cases of haemolytic-uraemic syndrome (HUS) in children were detected in a large area of northern Italy, where only 8 cases had occurred in the previous 5 years. Analysis of stool and serum specimens obtained from 14 cases showed evidence of Verotoxin-producing Escherichia coli (VTEC) infection in 13. Serum antibodies to the E. coli O157 lipopolysaccharide (LPS) were found in 8 patients and to the O111 LPS in 2. An O86 VTEC was isolated from another patient. Fourteen children needed dialysis, and 1 died. No obvious epidemiologic link was observed among cases, most of whom lived in small townships. A case-control study did not show an association between HUS and food or exposure to cattle, but suggested an association with contact with chicken coops (OR = 6·5. 95% C.I. 1·2–34·9). However, VTEC were not isolated from stool samples obtained from the chicken coops involved. The risk factors for VTEC infection related to living in rural settlements, including the exposure to live poultry, should be considered in outbreak investigations.

The incidence of Clostridium difficile-associated diarrhoea (CDAD) was investigated retrospectively at a 690-bed teaching hospital for the period 1983–92. Our aims were to determine: (i) the distribution by age and sex of patients with CDAD, (ii) the possibility of a seasonal trend and, (iii) the influence of infection control procedures, contamination of the hospital environment and the use of third-generation cephalosporins. The laboratory diagnosis of CDAD was based on demonstration of the organism by stool culture and/or detection of specific cytotoxin in stool filtrates. C. difficile was detected in 917 patients who were being investigated for diarrhoeal illness. Yearly isolations varied from a low of 49 in 1983 to a high of 120 in 1990 (Chi square for linear trend 128·8; P < 0·005). Most patients were elderly, with 63% aged 60 years or more; the majority (59%) were female. The relationship between culture of C. difficile and detection of cytotoxin in faecal extracts was also examined. Sixty percent of a sample of 132 isolates from patients in whom faecal cytotoxin was not detected produced cytotoxin in vitro, suggesting that culture is a more sensitive indicator of infection with C. difficile than cytotoxin detection. When the total number of faecal specimens received in the laboratory was used as a denominator there was an increase in the number of incident cases of CDAD between 1983 and 1990, apart from 1986. When occupied bed days was used as the denominator a similar trend was observed with a peak in 1990. These increases correlated with an increase in the use of third-generation cephalosporins at SCGH between 1983 and 1989 (Pearson's correlation coefficient, 0·90). The introduction of Body Substance Isolation in 1989, in conjunction with other infection control procedures, appears to have halted the rise, despite a continuing use of broad-spectrum cephalosporins. In order to reduce the number of cases of CDAD, either a reduction in levels of environmental contamination or a reduction in the use of third-generation cephalosporins is required. If this can be achieved the economic consequences, in terms of an opportunity cost, will be considerable.

Salmonella enteritidis PT4 was recovered from fingers following the breaking of intact shell eggs artificially contaminated in the contents with the bacterium. Kitchen utensils used to mix egg dishes were salmonella-positive, sometimes after washing. Following the preparation of batter or the mixing of eggs, S. enteritidis was recovered from work surfaces over 40 cm from the mixing bowl. The bacterium survived well in thin, dry films of either batter or egg and, from an initial level of one cell per cm2, could be recovered from formica work surfaces 24 h after contamination.

A family outbreak of Salmonella enteritidis PT4 infection is described in which home-made ice cream was identified as the vehicle of infection. The ice cream contained approximately 105S. enteritidis PT4 organisms per gm and was probably contaminated by an infected shell egg containing between 105−108 organisms. The continued relevance of the Chief Medical Officer's warning on the use of raw shell eggs is highlighted.

Home-made ice cream using the same recipe as ice cream that had been incriminated as the cause of the family outbreak of S. enteritidis PT4 infection was used to study the growth of the organism that might have occurred in the 3–4 h it took to prepare the product. When the inoculum was in the stationary phase, as it would be from shell or other cross contamination, there was a lag phase of 3 h before growth occurred at room temperature. Even when actively multiplying organisms were introduced, as may be found in an infected egg, there was less than 3 log10 increase in the salmonella count in 4 h at room temperature. It was, therefore, given the high S. enteritidis count, unlikely that the ice cream was cross-contaminated.

By contrast, raspberry sorbet at pH 3·73 proved to be lethal to a large inoculum of S. enteritidis and may be a relatively safe raw egg containing product.

Eighty-nine Salmonella enteritidis phage type 25/17 strains isolated from a localized outbreak in the German state Nordrhein-Westfalen (outbreak NWI) could not be further differentiated by biochemotyping and plasmid pattern analysis. They were submitted to a complex typing system consisting of modern physico-chemical analytical procedures. In lipopolysaccharide pattern analysis the strains proved to be homogeneous. In multilocus enzyme electrophoresis, outer membrane and whole cell protein pattern (WCPP) analysis, and Fourier transform infrared (FT–IR) spectroscopy (increasing extent of differentiation in the given order) strains deviating from each basal pattern were found. The extent of correspondence in these deviations was satisfactory.

Forty-six strains of the same sero- and phage type, however, obtained from different outbreaks, were additionally typed. The results obtained with them indicate that the data of the first group were not restricted to strains from outbreak NWI, but of general validity.

It was found that both WCPP and FT–IR represent valuable methods for the sub-grouping of bacteria.

Flagellin gene sequence polymorphisms were used to discriminate amongst 77 strains of Campylobacter jejuni from sporadic and outbreak-associated human enteric infections, and from chickens, sheep and calves. The results were assessed in relation to Lior biotyping and serotyping (Penner somatic antigens). Eight DNA PCR-RFLP patterns (genotypes) were identified by analysis of HinfI fragment length polymorphisms in flagellin gene (flaA) polymerase chain reaction (PCR) products. One genotype (F-l) was a feature of 55% of strains. Strains within the genotypes were heterogeneous with respect to somatic antigens with 12 serogroups represented amongst the C. jejuni isolates of flaA type F-l. Serogroups Pen 1. 2 and 23 were the commonest (45%) amongst the 20 different serogroups represented. Several unique clusters of isolates with diverse biotypes were defined, and one cluster (F-7/Pen 23) contained epidemiologically implicated outbreak strains as well as sheep and calf isolates. We conclude that HinfI flaA typing is reproducible and offers high typability, and its combination with serogrouping provides a novel approach to characterizing isolates of C. jejuni with improved discrimination.

The physiological behaviour of clinical Aeromonas spp. isolates was compared following culture in a conventional broth and human pooled ileostomy fluid (PIF). Protein expression was markedly affected by the growth medium, with an overall reduction in whole cell proteins in bacteria grown in ileostomy fluid. In addition, novel outer membrane proteins were produced in PIF but not in broth. The majority of A. hydrophila and A. sobria isolates produced toxin in both broth and PIF. whereas no cytotoxin positive A. caviae were found. Toxin titres were at least two doubling dilutions higher in 40% and 21% of A. hydrophila and A. sobria isolates, respectively, following culture in brain heart infusion broth compared with PIF. Bacterial adherence to Vero and A-549 cells was significantly more common in A. hydrophila (53%) and A. sobria (64%) than in A. caviae (15%) (P < 0·01). We observed increased adherence by 6 Aeromonas strains previously classified as adherence-positive, but not by 6 non-adherers, in PIF compared with brain heart infusion broth. The influence of growth medium on the expression of potential virulence determinants by Aeromonas spp. provides a rationale for the use of human ileostomy fluid in future in vitro studies, in order to simulate the nutrient conditions found in vivo.

A comprehensive analysis of the epidemiology of salmonellosis in a major hospital in Hong Kong from 1982–93 is reported. The trend of salmonella isolations over the past 12 years and changes in the occurrence of individual serotypes are delineated. A total of 5328 isolates were analyzed. Groups B (Salmonella typhimurium and S. derby) and E (S. anatum) were the commonest serogroups isolated from the intestinal tract in all age groups. A significant increase in the isolation of group D salmonellae has been observed since 1989. This is accounted for by a substantial rise in S. enteritidis isolation as seen in Western countries, despite a concomitant decrease of S. typhi. The extraintestinal isolation index (EII) is proposed as an index of the virulence potential of individual serotypes and serogroups. Group D salmonella was found to be the most invasive serogroup. While group D was the predominant serogroup isolated from extraintestinal sites in patients older than 1 year, group B serotypes (especially S. typhimurium) were more frequently seen in infants younger than 12 months.

Minced beef samples inoculated with Escherichia coli O157 were cultured in buffered peptone water supplemented with vancomycin, cefsulodin and cefixime (BPW-VCC) and subcultured to cefixime tellurite sorbitol MacConkey (CT-SMAC) agar both directly and after immunomagnetic separation (IMS) of the organism with magnetic beads coated with an antibody against E. coli O157 (Dynabeads anti-E. coli 0157, Dynal, Oslo). E. coli O157 was recovered from initial inocula of 200 organisms/g by direct subculture and 2 organisms/g by IMS. Twelve strains of E. coli 0157 of different combinations of phage type, H antigen and toxin genotype were all recovered from initial inocula of two organisms/g by IMS. Nonspecific binding of other organisms to the magnetic beads could be reduced by washing of the beads in PBS with Tween-20 0·002–0.005% E. coli O157 was not bound by magnetic coated with an unrelated antibody.

During investigation of a dairy herd that was possibly linked to a small outbreak of infection with E. coli O157, the organism was isolated from 2 of 279 forestream milk samples from individual cattle; both isolates were made only by the IMS technique. IMS is rapid, technically simple, and a specific method for isolation of E. coli O157 and will be useful in epidemiological studies.

Utilizing the Office of Population Censuses and Surveys (OPCS) Omnibus Survey, it was possible to measure the frequency with which a stool culture was obtained following episodes of diarrhoea in adults. Interviewing over 8000 adults, over a 4-month period between October 1992 and January 1993, 633 persons (7·9%) reported one episode of diarrhoea in the previous month, and 5·4 % of these individuals with diarrhoea reported that a stool had been requested for examination. No significant regional differences were observed with the sample size available. The estimate of the rate of diarrhoea in adults was just under one episode per person per year.

The study was conducted to isolate and characterize campylobacter, enterotoxigenic Escherichia coli-labile toxin (ETEC-LT), shigella and salmonella in flies and water.

The material for the study, flies (n = 300) and water samples (n = 148), was collected from different localities in and around Lahore, Pakistan. Cultivation of the samples was performed on conventional standard media. Membrane filtration technique was used for water prior to culture. Determination of ETEC-LT was done by GM1 ELISA.

Results of our study showed that flies and water were reservoirs for all the four pathogens, campylobacter, ETEC-LT, shigella and salmonella. Flies from the village were carrying fewer enteropathogens, while water from the village was found to be more contaminated as compared to the city. Campylobacter and ETEC-LT were the most frequently isolated pathogens in both flies and water.

Thus the incidence of diarrhoeal disease in children of developing countries may be decreased by providing plenty of safe drinking water, improving excreta disposal, toilet facilities and giving education in personal hygiene.

A high incidence of resistance to trimethoprim has been shown in the normal faecal flora in a population in south India. The dihydrofolate reductase (dhfr) genes mediating transferable resistance to trimethoprim have been identified. Unusually, in this study, the dhfr V was shown to be the predominant resistance gene (dhfr V 50% of tranaconjugants, dhfr Ia 30%), the dhfr Ib was also detected being distinguished from the dhfr V by an oligo-probe. However, when non-transferable resistance was considered, the dhfr Ia was the most prevalent of the dhfrs identified. All those plasmids harbouring the dhfr Ia were shown to possess Tn7. All the plasmids that probed positive for the dhfr V and the dhfr Ib were shown to be associated with the integrase of the Tn21-like transposons, but 8 of the dhfr V genes were not associated with the Tn21 resolvase. The dhfr IV was shown to be present in all seven plasmids that produced low level trimethoprim-resistance. The dhfr V. first characterized in Sri Lanka, would seem to have a local distribution in this region of Asia but is distinguishable from the dhfr Ib only by the use of an oligo-probe.

MecA, a structural gene located on the chromosome of Staphylococcus aureus, characterizes methicillin-resistant S. aureus (MRSA), and femA and femB(fem) genes encode proteins which influence the level of methicillin resistance of S. aureus. In order to examine effectiveness of detecting mecA and fem genes in identification of MRSA, the presence of these genes in 237 clinically isolated strains of staphylococci was investigated by polymerase chain reaction (PCR). An amplified mecA DNA fragment of 533 base pairs (bp) was detected in 100% of oxacillin-resistant S. aureus, in 16·7 % of oxacillin-sensitive S. aureus, in 81·5% of S. epidermidis, and in 58·3% of other coagulase-negative staphylococci (CNS). While the PCR product of femA (509 bp) or femB (651 bp) was obtained from almost all the S. aureus strains except for five oxacillin-resistant strains (2·5%), neither of these genes were detected in CNS. Therefore, the detection of femA and femB together with mecA by PCR was considered to be a more reliable indicator to identify MRSA by differentiating it from mecA-positive CNS than single detection of mecA.

Discriminatory typing methods are invaluable in the investigation of outbreaks of infectious diseases. Single primers were used to generate randomly amplified polymorphic DNA (RAPD) profiles from Klebsiella pneumoniae isolates of various serotype and K. pneumoniae isolates from cases of sepsis at a Malaysian hospital and two English hospitals. RAPD profiles of acceptable reproducibility, a maximum of three minor band variations, were produced using a rapid DNA extraction method. RAPD typing of K. pneumoniae was shown to be as discriminatory as restriction fragment length polymorphism analysis using pulsed field gel electrophoresis yet quicker and less costly. The findings suggest that RAPD typing may be a useful tool for the epidemiological typing of K. pneumoniae.

Faecal samples collected from three populations of healthy adult volunteers (290 pigfarmers, 316 abattoir workers, 160 (sub)urban residents) living in the south of The Netherlands were analysed for the prevalence and degree of antibiotic resistance of Escherichia coli.

Significant differences in prevalence of resistance to amoxicillin, neomycin, oxytetracycline, sulfamethoxazole and trimethoprim were observed. The pig-farmers showed the highest percentages of resistance and the (sub)urban residents the lowest. In contrast no significant differences in high degrees of resistance were observed, except for neomycin.

Although both pigfarmers and abattoir workers have regular contact with pigs differences in prevalences of resistance were observed. However, because abattoir workers with intensive and less intensive pig(carcass) contact did not show significant differences, this is probably not the only important source of resistant E. coli in pigfarmers.

The high antibiotic use by pigfarmers (5%) and abattoir workers (8%) than by (sub)urban residents (0%) did not result in significantly different resistance percentages.

Twenty-two reference isolates and 30 local isolates of group A Streptococci were classified into 36 electrophoretic types (ET) on the basis of allozyme variation at 27 enzyme loci. Local isolates were characterized by a high frequency of M-non typable strains. M-type and ET were more closely associated in local isolates from an endemically-infected population; nevertheless, amongst the local isolates there were also strains of the same ET type with different M-types. A possible explanation is that genetic exchange between strains may introduce different M-types into strains of defined ET when these are exposed to strong selection in the presence of heavy loads of infection.

In contrast to the reported clustering of strains associated with toxic shock-like syndrome into two closely related ET clones, we found no relationship of ET phenotype to acute poststreptococcal glomerulonephritis or rheumatic fever.

To know the types of meningococcal strains in Spain, we serotyped and subtyped 743 Neisseria meningitidis isolates recovered between 1990 and 1992 from patients. A great number of serogroup B, serogroup C and non-groupable meningoccoci reacted with the serotyping reagents while many serogroup C and non-groupable isolates did not react with the serosubtyping reagents (78·2% and 54·8 % respectively); only 8·9 % of serogroup B meningococci were non-subtypeable (NST). Distribution of serotypes was similar in serogroup C and in non-groupable strains. Isolates showed great variability in antigenic phenotypes (71 in serogroup B, 20 in serogroup C and 25 in non-groupable meningococci). The most frequent antigenic combinations were 4:P1. 15 (39·8%) in serogroup B, 2b:NST (55·8%) in serogroup C and 2b:NST (35·6%) in non-groupable meningococci.