A model system for the study of phage conversion of Salmonella enterica serotype Enteritidis is reported. Temperate phages 1,2,3 and 6 from the phage typing scheme were used to convert several individually recognized phage types into others. Phage type 4 was converted to PT8, PT6a to PT4, PT6a to PT7, PT13 to PT13a and PT15 to PT11; some new phage lysis patterns were also detected.

This model was used to examine the relationships between phage types within a previously denned clonal lineage, SECLIII, to establish whether or not Enteritidis like Salmonella enterica serotype Typhi and Salmonella enterica serotype Paratyphi B possessed type determining phages. We were able to convert PT1 to PT20, and PT15 to PT11.

This study evaluated the epidemiology of Pseudomonas aeruginosa keratitis in contact lens (CL) wearers; the relationships between CL storage case contamination and CL hygiene practice and between CL hygiene and the development of keratitis. Sixteen CL wearers with keratitis were compared with 44 asymptomatic controls. Lens hygiene practice was assessed and CL care materials, domestic water sites and endogenous sites were evaluated microbiologically. Poor CL hygiene was not associated with Ps. aeruginosa keratitis. There was an association between keratitis and bacterial contamination of the CL and storage case (P < 0·0005). Lens and storage case contamination were not significantly associated with poor hygiene. No domestic or endogenous source for Ps. aeruginosa was found. Causative organisms may be derived from other sources, but CLs and CL storage cases provide a favourable environment for Ps. aeruginosa colonization. Changing the CL care environment to one less favourable for Ps. aeruginosa may help to eliminate this problem.

Foot-and-mouth disease (FMD) was the first animal disease to be attributed to a virus, and the second virus to be discovered [1]. It is a positive-sense, singlestranded RNA picornavirus and the sole member of the genus Aphthovirus. Each infectious virus particle contains a single strand of RNA approximately 8-5 kb long. This is translated into a single polypeptide which is then cleaved into the structural and non-structural virus proteins.

Using pulsed-field gel electrophoresis (PFGE) and 16S rRNA (rrn) analysis (ribotyping). the in viro derivation of strains of Salmonella enteritidis PTs 9a and 7 from a strain of S. enteritidis PT 4 has been demonstrated. All strains were isolated from a single patient over a 6-week period. Further studies have demonstrated that in terms of pulsed-field profile and ribotype, the genotypes of the patient-derived strains differed from those of the reference strains of the respective phage types. It is concluded that when used in combination, these methods can provide evidence of phylogenetic relationships in apparently unrelated S. enteritidis phage types isolated during pathogenesis of disease.

Consumption of milk contaminated with Campylobacter jejuni has been described as a cause of human enteritis. Although faecal contamination of milk with the organism has frequently been described, direct milk excretion of Campylobacter jejuni into milk has rarely been linked with cases of human infection. We describe the investigations undertaken following the isolation of Campylobacter jejuni from samples of unpasteurized milk prior to retail. Results of epidemiological investigations including typing of Campylobacter jejuni isolates using pyrolysis mass spectrometry, Penner and Lior serotyping, biotyping, phage typing and restriction fragment length polymorphism analysis provided convincing evidence implicating direct milk excretion of Campylobacter jejuni by one asymptomatic dairy cow as the source of the milk contamination and the cause of local cases of human enteritis.

Molecular variation within and between plasmids of Salmonella enterica serotype Dublin was analysed. Such variation has been demonstrated in the serotype-specific plasmids (SSP's) of Typhimurium and Enteritidis. The two aims of this study were to determine the plasmid diversity in a host-adapted serotype and also the incidence of molecular variation in the SSP among strains of Dublin using restriction endonuclease fragmentation pattern (REFP) analysis with Pst1, Sma1 and EcoRV. Sixty-five strains were examined from seven countries. Plasmid profile and REFP analysis showed that none of the strains was plasmid-free. Seventy-seveń percent of the strains possessed the 72 kb SSP either alone or in combination with another plasmid; 23 % harboured plasmids which were molecular variants of the SSP. Four of the variants were more closely related to each other than to the reference SSP and were harboured by Dublin isolated from both the USA and Europe. A further three were shown to be cointegrate plasmids and were similarly distributed. Thirty-two percent of strains possessed the SSP alone. None of the UK strains was resistant to any of the antimicrobial agents tested whereas 74 % of the remaining strains were resistant to between one and five antimicrobial agents. This study corroborates previous findings concerning the high degree of stability of the SSP and confirmed the clonal nature of Dublin. Co-resident plasmids provided evidence of sub-clones within localized geographical areas.

Four hundred and thirty-four isolates of Salmonella enterica serotype Enteritidis were studied. They were grouped into five subsets defined by either the collection criteria or the parameter which formed the basis for subsequent analysis. Seventy-seven per cent harboured the serotype-specific plasmid (SSP). In 55% of the isolates this was the sole plasmid. Molecular variation in the SSP was detected in 17 (5%) of the isolates on the basis of restriction enzyme fragmentation pattern (REFP) analysis using Pst I and Sma I. The SSP variants were further characterized using additional restriction enzymes chosen to optimize the information content and analysed using a coefficient of similarity.

A variant SSP designated pOG690 showed greater resemblance to the SSP of Salmonella enterica serotype Typhimurium than Enteritidis; 89% and 68% respectively for Pst I and 79% and 55% respectively for Sma I. In respect of the Pst I data pOG690 shared at least 55 kb of DNA with the Typhimurium SSP and 37 kb with the SSP of Enteritidis. This variant was associated with poultry (duck, goose, chicken) and all isolates belonged to phage type 9b. Other variants were associated with phage types 4, 6, 6a, 9a, 11, 15 and 24. The epidemiological implications of these results are discussed.

In response to the Latin American cholera epidemic, El Salvador began a prevention programme in April 1991. The first case was confirmed in August, and 700 cases were reported within 3 months. A matched case-control study was conducted in rural La Libertad Department in November 1991. Illness was associated with eating cold cooked or raw seafood (odds ratio [OR] = 7·0; 95% confidence limits [CL] = 1·4, 35·0) and with drinking water outside the home (OR = 8·8; 95% CL = 1·7, 44·6). Assertion of knowledge about how to prevent cholera (OR = 0·2; 95% CL = 0·1, 0·8) and eating rice (OR = 0·2; 95% CL = 0·1, 0·8) were protective. More controls than patients regularly used soap (OR = 0·3; 95% CL = 0·1, 1·0). This study demonstrated three important points for cholera prevention: (1) seafood should be eaten cooked and hot; (2) populations at risk should be taught to treat household drinking water and to avoid drinking water outside the home unless it is known to be treated; and (3) education about hygiene can be an important tool in preventing cholera.

Broiler flocks on two Dutch poultry farms were screened weekly for the presence of Campylobacter in fresh caecal droppings during eight consecutive production cycles. Hatchery and fresh litter samples were taken at the start of each new cycle. Water, feed, insects, and faeces of domestic animals, present on the farms were also included in the sampling. Penner seretyping of isolates was used to identify epidemiological factors that contribute to Campylobacter colonization in the broiler flocks. Generally, broiler flocks became colonized with Campylobacter at about 3–4 weeks of age with isolation percentages of 100%, and stayed colonized up to slaughter. A similar pattern of serotypes was found within the various broiler houses on one farm during one production cycle. New flocks generally showed also a new pattern of serotypes. Most serotypes isolated from the laying hens, pigs, sheep and cattle were different from those isolated from the broilers at the same time. Campylobacter serotypes from darkling beetles inside the broiler houses were identical to the ones isolated from the broilers. No Campylobacter was isolated from any of the hatchery, water, feed or fresh litter samples. Conclusive evidence of transmission routes was not found, but results certainly point towards horizontal transmission from the environment. Horizontal transmission from one broiler flock to the next one via a persistent contamination within the broiler house, as well as vertical transmission from breeder flocks via the hatchery to progeny, did not seem to be very likely.

In May 1992, 81 bacteriologically confirmed cases of typhoid fever (TF) were identified in all districts of Tabuk City in northwestern Saudi Arabia. Attack rates (AR) in residential districts ranged from 0·9–10·3 per 10000. Confirmed cases included 9 workers in the city's referral hospital, King Khalid Hospital (AR 140/10000), 2 in families of medical staff, 57 in the community (AR 4·4/10000) and 13 in a local military cantonment (AR 0·8/10000). The outbreak began with the onset of TF in the three areas within 5 days, continued for 7 weeks, and ended 2 weeks after chlorination began. Among water sources, the odds ratio (OR) was highest (2·6; 95% confidence interval [CI] 1·25–5·39) for water purchased from reverse osmosis (RO) plants, especially RO plants supplied by one well (ASUW) (OR = 7·05; 95% CI 2.51–20·7). The aquifer for ASUW lay partially beneath a depression where city sewage collected. Unchlorinated water samples from ASUW 1 month after the outbreak ended yielded coliforms. ASUW probably became contaminated with Salmonella typhi when KKH demand overtaxed the aquifer and drew in surface water. Membranes in RO plants using this unchlorinated well water could then become fouled with S. typhi. RO plants, which are common throughout Saudi Arabia, need close monitoring. WTater for RO must be prechlorinated to prevent microbiologic fouling of the membranes.

Several typing systems have been described for Campylobacter jejuni and C. coli, to assess the complex epidemiology of these important enteric pathogens. In the present study two typing methods, slide agglutination according to the Lior scheme, and the demonstration of restriction-fragment length polymorphisms (RFLP) of flagellar genes, have been used in parallel on a set of 194 strains. This set comprised 118 sero-reference strains of C. jejuni and C. coli of the Lior scheme, as well as 76 clinical isolates. All isolates were serotyped and subjected to PCR for amplification of flagellar genes, and the PCR product was restricted with Alu I. Flagellar genes could be amplified in 152 strains. Among 85 seroreference strains, 74 different RFLP patterns were observed, and among 67 clinical isolates, there were 36 patterns. There was only limited correlation between flagellar RFLP and the Lior serogroup, and the variability of patterns in serogroups HL2 and HL4 were as marked as the variability between serogroups. Flagellar gene RFLP patterns are shown to be stable, highly discriminatory epidemiologic markers.

Extensive DNA sequence diversity was noted in Helicobacter pylori flagellin genes flaA and flaB. PCR amplified sequences from 49 isolates were digested with AluI. HindIII. MboI or MspI, the resultant patterns were compared between the different isolates and these used to differentiate the isolates from each other. Evidence that the extensive diversity that was found in these genes is the result of reassortment of sequences between strains in the bacterial population is presented, such that a comparatively small number of individual sequence mutations can recombine together in random combinations to form a greater number of distinct alleles. Geographical differences in the predominant patterns in the flaA alleles were also observed and could reflect regional differences either in the human host population or in the bacterial population. In view of the genetic complexity of this species, molecular typing schemes designed to identify related strains may falsely associate strains if the methods do not characterize sufficient genetic sites to exclude chance associations of genetic markers in strains which are actually not closely related to each other.

Thirty-six encoded isolates of Escherichia coli, 32 of which were of serotype O157. were examined by pyrolysis mass spectrometry (PyMS). Thirty-one of the serotype O157 isolates possessed the flagellar antigen H7 and produced Verocytotoxin (VT). the other isolate serotyped as H45 and was non-toxigenic. Eighteen of the VT-producing E. coli (VTEC) isolates were from sporadic disease in residents of the Northern Region. Standard principal component (PC) and canonical variate (CV) analysis of the data distinguished only the four non-O157 isolates from the remainder which were indistinguishable by this approach. A similarity matrix based on differences between individual CV means distinguished a further ten isolates. The matrix correctly clustered 2 pairs of isolates from siblings and 4 isolates from an affected family. A further 5 clusters of 3 or more isolates and 6 pairs of isolates were defined. These groupings proved to be homogenous for toxin phenotype but occasionally entrained isolates of dissimilar phage type. However, in general, PyMS-derived clustering of apparently sporadic isolates accorded with geographical locations as determined by postcode. PyMS, which is a quick and high volume capacity phenotypic technique. may be a useful addition to existing methods in the investigation of the epidemiology of sporadic VTEC disease.

A collection of 64 clinical and environmental Vibrio cholerae non-O1 strains isolated in Asia and Peru were characterized by molecular methods and antibiotic susceptibility testing. All strains were resistant to at least 1 and 80% were resistant to two or more antibiotics. Several strains showed multiple antibiotic resistance (≥ three antibiotics). Plasmids most often of low molecular weight were found in 21/64 (33%) strains. The presence of plasmids did not correlate with antibiotic resistance or influence ribotype patterns. In colony hybridization studies 63/64 (98%) V. cholerae non-O1 strains were cholera toxin negative, whereas only strains recovered from patients were heat-stable enterotoxin positive. Forty-seven Bgl I ribotypes were observed. No correlation was shown between ribotype and toxin gene status. Ribotype similarity was compared by cluster analysis and two main groups of 13 and 34 ribotypes was found. Ribotyping is apparently a useful epidemiological tool in investigations of V. cholerae non-O1 infections.

We studied 7 patients with nosocomial Legionnaires' disease to determine the relationship between isolates of Legionella pneumophila recovered from potable water and those recovered from patients. Potable water was cultured from all rooms in which patients had stayed prior to the diagnosis of Legionnaires' disease. The 38 isolates of L. pneumophila (31 environmental, 7 patient) were resolved into 9 distinct patterns by pulse-field gel electrophoresis (PFGE), 3 by plasmid content and 2 each with monoclonal antibodies and conventional agarose gel electrophoresis of small fragments of DNA.

Using PFGE it was determined that 4 of the 7 patients were infected with L. pneumophila identical to an isolate recovered from the potable water supply in one of the rooms each had occupied prior to the diagnosis of Legionnaires' disease. Patients had resided in a mean of 3·57 rooms before a diagnosis of nosocomial Legionnaires' disease. We conclude that in the setting of contaminated potable water and nosocomial Legionnaires' disease water from all the rooms which the patient has occupied prior to this diagnosis should be cultured. PFGE of large DNA fragments discriminated best among the isolates of L. pneumophila.

A summer outbreak of severe gastroenteritis followed by haemolytic uraemic syndrome (HUS) and thrombotic thrombocytopenic purpura in a nursery school and kindergarten is described. Sandwiches prepared with green butter made with contaminated parsley were the likely vehicle of infection. The parsley originated from an organic garden in which manure of pig origin was used instead of artificial fertilizers, Cornally identical Verotoxinogenic Citrobacter freundii were found as causative agents of HUS and gastroenteritis and were also detected on the parsley.

The distribution and virulence of Vibrio cholerae serogroups other than O1 and O139 in India before, during and after the advent of O139 serogroup was investigated. A total of 68 strains belonging to 31 different ‘O’ serogroups were identified during the study period. With the exception of O53, there was no spatial or temporal clustering of any particular non-O1 non-O139 serogroup at any given place. Two of the 68 strains examined produced cholera toxin (CT) which could only be partially absorbed with anti-CT immunoglobulin G. Tissue culture assay revealed that some of the non-O1 non-O139 strains produced factors which evoked either a cell rounding or cell elongation response depending upon the medium used. This study indicates that serogroups other than O1 and O139 should also be continuously monitored.

Pilot studies to test methods to determine the incidence, agents, risk factors and socioeconomic costs of infectious intestinal disease (IID) in England were carried out as recommended by the Committee on the Microbiological Safety of Food (the Richmond Committee) by eight general practices. There were case control and enumeration studies of patients presenting to general practice with IID, a population-based prospective cohort study, and a survey of socioeconomic costs of cases of IID. Information on risk factors was obtained by questionnaire (self-administered compared with interview) and a stool sample was requested on all cases and controls. Response rates in the GP case control study were 75% for case questionnaires and 74% for stools; for controls the figures were 70% and 68% respectively. The acceptance rate into the cohort study was 49%; this was significantly higher where phone contact was made. The rate was similar if recruitment was by individual or household. Follow-up of the cohort by negative reporting was complete for up to 6 months. Direct postage by subject was required to obtain fresh stool specimens. Estimates were obtained of presentation rates of IID and the distribution of risk factors which were used to plan the main study. The pilot study demonstrated that it is possible to undertake a national study based in general practice to determine the incidence of IID in the population and presenting to GPs and its agents, risk factors and costs.

A 2-year case-control study was conducted to describe the epidemiology of Haemophilus influenzae type b (Hib) and investigate Hib disease risk factors in the Republic of Ireland. Between October 1991 and September 1993, 149 laboratory confirmed incident cases were matched with community controls. Annual Hib disease incidence was 25·4 per 100000 children under 5 years, with peak incidence (65·8 per 100000) in the 6–11 months age-group. Meningitis was the predominant clinical condition. Twenty-four (16·1%) isolates were resistant to ampicillin. Crèche or day-care attendance and the presence of chronic illness emerged as risk factors for Hib disease. Empirical first line treatment for suspected Hib infection warrants alternatives to ampicillin such as cefotaxime. Completed immunization with Hib conjugate vaccine by 6 months of age is required for maximum disease prevention. Until all children are receiving Hib vaccine on schedule, those who are crèche or day-care attendees and those with chronic illness should be prioritized for timely immunization.

Following an episode of water contamination with sewage in a rural Irish town, a community-wide survey of gastrointestinal-associated illness and health service utilization was conducted. Random sampling of households yielded residents who were surveyed using a self-administered questionnaire. Of 560 respondents from 167 (84%) households, equal proportions lived in areas known to have been exposed and unexposed to the contaminated water, although 65% of subjects reported using contaminated water. Sixty-one percent of subjects met the case definition. The most common symptoms among cases were abdominal cramps (80%), diarhoea (75%), appetite loss (69%), nausea (68%) and tiredness (66%). Mean duration of illness was 7·4 days. Only 22 % of cases attended their general practitioner. Drinking unboiled water from the exposed area was strongly associated with being a case. A substantial degree of community illness associated with exposure to contaminated water was observed. The episode ranks as one of the largest reported water-borne outbreaks causing gastrointestinal illness in recent times.