An outbreak of infection due to verocytotoxin-producing Escherichia coli O157 in four families: the influence of laboratory methods on the outcome of the investigation.

Three members of family A, who had diarrhoea on 20 October, lived on a small arable farm which had 10 cattle. Manure from the animals was used to fertilize the ground for growing potatoes which were then offered for retail sale, unwashed, directly from the farm. The mother from family B bought potatoes, which were covered with manure, from family A in early November and over the subsequent 10 days she became ill with diarrhoea and her daughter and son both became ill with bloody diarrhoea. The mother from family C visited family B while the daughter from the latter family was symptomatic; the mother developed diarrhoea several days later. The mother and two sons from family D visited family B while the son from the latter family was symptomatic; the first son developed bloody diarrhoea 6 days later which progressed to development of haemolytic-uraemic syndrome. Direct culture of faecal samples onto cefixime rhamnose sorbitol MacConkey agar failed to isolate E. coli O157 from any of the symptomatic patients, and direct culture onto cefixime tellurite sorbitol MacConkey agar isolated the organism from only one patient. In contrast, a combination of isolation of E. coli O157 by immunomagnetic separation and detection of E. coli O157-specific secretory IgA, suggested E. coli O157 infection in all eight symptomatic patients, but not in any of the family members who were not ill. Two children who excreted the organism for 60 and 89 days respectively were the only two patients who did not develop a secretory IgA response. E. coli O157 was not isolated from potatoes from the farm and faecal samples from the farm animals were not available for examination. The study illustrates the need to use the most sensitive methods available during the investigation and follow up of cases of E. coli O157 infection.     

香港海鸥形菌检验技术的建立与应用

目的:建立香港海鸥形菌(Laribacter hongkongensis,LH)的社区获得性胃肠炎患者及淡水鱼类标本采样、运输、培养、分离及鉴定方法。方法:于2005年~2006年,采集社区获得性胃肠炎患者及淡水鱼类新鲜粪样,分别接种SS、XLD、CCDA、TCBS、MacConkey琼脂(MA)以及改良头孢哌酮MacConkey琼脂(CMA)等6种培养基,对典型的菌落,进行常规生化试验,凡氧化酶、触酶试验阳性以及三糖铁试验阴性的菌株,采用特异性PCR检测确诊。结果:社区获得性胃肠炎患者的LH检出率为0.53%(4/759);淡水鱼类草鱼、鲢鱼与鲈鱼的阳性率分别为9.30%、8.33%和5.88%。结论:本文建立的LH表型与基因型诊断技术适合于LH的诊断;当前LH缺乏血清学诊断,发展LH的PCR诊断方法具有现实意义。     

Studies on the isolation of Salmonella dublin.

Abattoir drain swabs, bovine faeces and a few other veterinary samples were examined for the presence of Salmonella dublin. Three selective agar media and four enrichment broths were investigated. The two most efficient plating media were deoxycholate citrate agar and brilliant green MacConkey agar. Wilson and Blair''s bismuth sulphite agar (de Loureiro''s modifications) was least successful. Selenite F broth, whether incubated at 37 or 43 degrees C., was better than the other enrichment broths used which contained a triphenyl methane dye as one of the selective ingredients.     

Influence of multiple plating from fluid media on salmonella isolation from animal feeding stuffs.

The influence of multiple plating of fluid cultures on salmonella isolation from animal feeding stuffs was examined. Four plating were made from broth culture after 24 h at 37 degrees C and four platings from selenite enrichment from 24 h at 43 degrees C. Selenite enrichment followed broth culture which was used as a pre-enrichment stage. Brilliant green MacConkey agar plates were employed for broth subculture and brilliant green MacConkey and desoxycholate citrate agars for selenite subculture. The eight brilliant green plates subcultured from broth and selenite were examined for salmonellas after incubation for 24 h at 37 degrees C. The four desoxycholate citrate agars after 24 h at 37 degrees C were used for motility enrichment. The food sample size was a single 100 g instead of 4 x 25 g cultured in an earlier study. This pooling of samples aimed at technical economy. Quadruple plating played an important part in salmonella isolation from 100 g specimens. The combination of multiple plating with motility enrichment was the most successful technique used.     

Comparison of selenite F, Muller-Kauffmann tetrathionate and Rappaport's medium for salmonella isolation from chicken giblets after pre-enrichment in buffered peptone water.

Six hundred and eighty three samples of chicken giblets were examined for salmonellas. Three hundred and forty nine of these were neck and crop specimens and 224 were combined liver and heart samples. Two hundred and ten, in all, contained salmonellas. The technique of examination included pre-enrichment in buffered peptone water at 37 degrees C for 18 h and subculture to three enrichment media: Muller-Kauffmann tetrathionate, selenite F and Rappaport''s magnesium chloride malachite green broth. Inocula from buffered peptone water to 10 ml of tetrathionate and selenite were 1 ml in each case. The inoculum from the pre-enrichment medium to 10 ml of Rappaport was 0.005 ml. Tetrathionate and selenite were incubated at 43 degrees C for 48 h. Rappaport''s medium was incubated at 37 degrees C for 48 h. Subcultures from all three enrichment broths were made at 24 h and 48 h to brilliant green MacConkey agar. Selective agars were incubated at 37 degrees C for 24 h. The most successful technique for salmonella isolation used Rappaport''s medium, which was significantly more efficient than either tetrathionate or selenite. This finding reinforces results obtained using sewage polluted natural water as test material and it is suggested that routine examination of environment samples for salmonellas could be based on Rappaport''s medium alone. If S. typhi, S. dublin or subgenus III salmonellas were likely to be present in the sample, the technique described here would require modification.     

Comparison of antibody responses and virus shedding following administration of trivalent oral poliomyelitis vaccines prepared either in monkey or human diploid cell substrates.

Nineteen (22.9%) of 83 sera collected before vaccination from adult volunteers aged 21-64 years were without neutralizing antibody to poliomyelitis at levels of 0.15 i.u./ml for types I and II and 0.1 i.u./ml for type III. Some correlations were found between the history of previous vaccination and the presence of antibody but these were not well defined. Vaccination with a single dose of trivalent oral polio vaccine elicited fourfold or greater antibody responses to one or more poliomyelitis types in 53 (63.9%) volunteers, the percentage antibody resposnes being inversely related to the titre of antibody present before vaccination. Types I, II or III poliomyelitis virus were recovered from 76.8% of faecal samples collected 1 week after vaccination. The percentage recovery progressively declined thereafter until virus was recovered from 10.5% of samples collected 6 weeks after vaccination. Type for type, the titres and percentages of antibody responses and virus shedding in faeces were similar following trivalent oral poliomyelitis vaccines whether prepared in monkey or human diploid cell substrates. Some change in reproductive capacity temperature (r.c.t./40) marker was found in faecal isolates from volunteers vaccinated with monkey kidney and human diploid grown vaccines but no change in ''d'' marker was found.     

Evaluation of a rapid screening method for detection of antimicrobial resistance in the commensal microbiota of the gut

The assessment of antimicrobial resistance among commensal bacteria is an indicator of the spread of antimicrobial resistance. Rapid screening methods for detection of antimicrobial-resistant faecal Escherichia coli directly on MacConkey plates have been successfully adopted but suffer from lack of standardisation. The purpose of this study was to evaluate a direct plating method (DPM) for detection of antimicrobial-resistant faecal E. coli and to compare it with a conventional method. Faecal samples were collected from 71 healthy children from Peru and Bolivia. In the DPM, a faecal swab was directly plated onto a MacConkey agar plate and antimicrobial disks were applied onto the seeded plate. Raw data were obtained by direct reading of the plate and were subjected to confirmatory analysis. Good concordance between the DPM and a conventional method was observed in detecting carriage of resistant E. coli, with a higher sensitivity for the DPM. Analysis of the results allowed interpretive criteria to be defined for DPM raw data. The DPM showed good sensitivity and specificity at very low cost (ten times cheaper than the conventional method) to investigate the faecal carriage of drug-resistant E. coli. It may represent a useful tool to conduct large-scale resistance surveillance studies and to monitor resistance control programmes cost effectively, particularly in low-resource countries.     

Observations on the occurrence of Salmonella cholerae-suis and other salmonellas in two herds of feeder pigs.

Modified brilliant green agar (BGA), Muller-Kauffmann tetrathionate, Rappaport''s and selenite F broths were compared for their efficiency in isolating salmonellas from pigs and their excreta. It was concluded that BGA and Rappaport''s broth were the media of choice. Where searches were made for Salmonella cholerae-suis alone, the use of a trehalose McConkey agar provided a rapid method of differentiating S. cholerae-suis, which does not ferment trehalose, from the majority of other salmonellas, which do ferment trehalose. Casualties were collected from two farms where infection with S. cholerae-suis was endemic. The isolation rates of S. cholerae-suis from different carcase sites were compared in order to determine the relative importance of the salivary, upper respiratory and faecal routes of excretion. S. cholerae-suis was isolated from numerous carcase sites in carriers including the salivary glands, tonsils, trachea and lungs. However, isolations from the nasal passages, mouth, pharynx and gastro-intestinal tract of carriers were either infrequent or absent. When, in a further study, S. cholerae-suis was isolated from only 3/414 faeces, 1/170 nasal swabs and not at all from 170 oral swabs taken from live pigs, it was concluded that there must be more significant modes of transmission than from the salivary glands, upper respiratory or gastro-intestinal tracts. Cannibalism was considered to be a possibility. In contrast to S. cholerae-suis, other salmonellas were frequently isolated from randomly collected faeces and from the gastro-intestinal tract as well as other sites in casualties.     

The effect of antibiotic therapy on the faecal excretion of Salmonella typhimurium by experimentally infected chickens.

Chickens in groups of 40 were infected orally with a nalidixic acid-resistant mutant of Salmonella typhimurium and then fed continuously on diets containing ampicillin, chloramphenicol, furazolidone, neomycin, oxytetracycline, polymixin, spectinomycin, streptomycin or a mixture of trimethoprim and sulphadiazine. The amount of S. typhimurium excreted in their faeces was estimated at intervals by culture on brilliant green agar containing sodium nalidixate, both direct and after enrichment in selenite broth; the amount of Escherichia coli excreted was estimated by culture on MacConkey agar. The feeding of diets containing 500 mg./kg. of ampicillin, furazolidone, neomycin, polymixin, spectinomycin or streptomycin or 100 mg./kg. of trimethoprim and 500 mg./kg. of sulphadiazine for 46 days reduced to a varying degree the amount of S. typhimurium and E. coli excreted, the greatest reduction in S. typhimurium being brought about by the last treatment. The effect was less obvious when the concentration of the antibiotics in the food was decreased fivefold. An important reason for the very limited effect of some of the antibiotics was the emergence of antibiotic-resistant populations of S. typhimurium and E. coli. High concentrations of antibiotic-resistant organisms also arose in the faeces of the chickens fed diets containing tetracyclines and chloramphenicol, treatments which had no apparent effect on the amount of S. typhimurium and E. coli excreted. Much of the antibiotic resistance encountered was determined by R factors, a particular R factor usually being found in the E. coli populations of individual chickens before it was found in their S. typhimurium populations. No S. typhimurium or E. coli were isolated that possessed R factors determining resistance to polymixin, furazolidone or trimethoprim. No S. typhimurium or E. coli were isolated that were polymixin-resistant and no S. typhimurium that were furazolidone-resistant. The few trimethoprim-resistant S. typhimurium isolated were thymine-dependent. The feeding of diets containing the higher concentrations of trimethoprim/sulphadiazine, neomycin, furazolidone or ampicillin for 9 days reduced the amount of S. typhimurium excreted. After the withdrawal of these diets, the amount of S. typhimurium excreted increased to the numbers found in chickens given ordinary diets throughout; the chickens that had been given trimethoprim/sulphadiazine or furazolidone did not remain faecal excreters of S. typhimurium longer than the chickens that had been given ordinary diets. Similar results were obtained with trimethoprim/sulphadiazine when the start of the 9-day treatment period was delayed for an extra 9 days or when it was extended to 18 days.     

Rapid detection of intestinal carriage of Klebsiella pneumoniae producing KPC carbapenemase during an outbreak

Two different approaches are described for rapid detection of intestinal carriage of Klebsiella pneumoniae producing KPC-type carbapenemase (KPC-KP), based on PCR amplification of DNA extracts from rectal swabs (K-PCR), and on direct plating of rectal swabs on to MacConkey agar with a meropenem disc and a meropenem plus 3-aminophenylboronic acid disc (direct KPC screening test, DKST). K-PCR and DKST were tested with a total of 101 samples from 65 patients, during an outbreak. Although less sensitive, DKST could detect high-level carriage, which appears to be common among infected and colonised patients, while being very cheap and easy to perform, and requiring only basic facilities.     

Infection with verocytotoxin-producing Escherichia coli O157 during a visit to an inner city open farm

Two cases of Escherichia coli O157 infection occurred in children after visiting an inner city open farm. Subsequently faecal samples collected from animal pens and samples of composted mixed animal manure and vegetable waste were examined for E. coli O157 by enrichment culture, immunomagnetic separation and culture of magnetic beads to cefixime tellurite sorbitol MacConkey agar. Strains of E. coli O157 were characterized by hybridization with DNA probes for VT1, VT2 and eaeA, plasmid profile analysis, phage typing and pulsed field gel electrophoresis (PFGE). Verocytotoxin-producing E. coli O157 strains were isolated from faecal samples from a cow, a horse, 3 breeds of pigs, 2 breeds of sheep and 2 breeds of goats and from 2 samples of compost which had been processed for 3 months. All strains were phage type 21, hybridized with probes for VT2 and eaeA but not with one for VT1, harboured 92 and 2 kb plasmids and gave indistinguishable banding patterns with PFGE. Although only two culture-confirmed cases of infection had been identified, the farm had over 100,000 visitors per year and so it was closed as a precaution both to allow a thorough investigation and to prevent further cases. The investigation identified many factors which may have contributed to transmission of E. coli O157 infection. Most of these were readily resolved by appropriate corrective measures and as there were no further cases associated with the farm during the ensuing 4 weeks it then re-opened. These cases highlight the risk, especially to young children, of acquiring zoonotic infections during visits to open farms and emphasize the need for adequate guidance and supervision before and during such visits.     

Isolations of subgenus III salmonellas (arizonas) in Cardiff, 1959-1971

Isolations of subgenus III salmonellas (arizonas) made in the regional Public Health Laboratory, Cardiff, between 1959 and 1971 are reviewed. The techniques of isolation are listed for the various materials examined. The necessity of using bismuth sulphite agar as a plating medium is stressed, as some strains might appear on brilliant green MacConkey agar as rapid lactose fermenters and be missed. The serotypes isolated in Cardiff are discussed with reference to isolations by other authors. The culture of a subgenus III salmonella from pig faeces is described. It is thought that this is the first record of such an isolation from a pig in the United Kingdom.     

从冻鸡中分离出山梨醇发酵阳性大肠杆菌O157的报告

大肠杆菌(E．coli)O157属于大肠杆菌的一个血清型，是引起出血性肠炎的主要病原体。山梨醇不发酵是目前进行E．coliO157筛选时的重要依据。本实验室用免疫法从冻鸡腿中分离出三株山梨醇发酵阳性的E．coli O157，提示选用免疫法代替山梨醇发酵特征来进行E．coli O157检测的必要性。     

Immunomagnetic separation as a sensitive method for isolating Escherichia coli O157 from food samples.

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 O157, 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 O157 of different combinations of phage type, H antigen and toxin genotype were all recovered from initial inocula of two organisms/g by IMS. Non-specific 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.     

Prevalence of enteric bacteria that inhibit growth of enterohaemorrhagic Escherichia coli O157 in humans

Enterohaemorrhagic Escherichia coli O157 (O157) is infectious to humans, particularly children, at very low doses and causes not only haemorrhagic colitis but also other serious symptoms. To investigate an association between intestinal bacterial flora and resistance to such infections, we screened faecal samples for the presence of enteric bacteria that are able to suppress the growth of O157. Samples from 303 individuals, 35 children (aged < or =6 years) and 268 adults (aged 20-59 years), were examined. Colonies with different appearances on sorbitol MacConkey agar medium were screened for the production of bacteriocins inhibitory for O157 in an overlay agar plate assay. O157-inhibiting strains were isolated from 52 individuals. The prevalence of these bacteria tended to rise with age, and was significantly higher among 40- to 59-year-old adults (23/101, 22.8%) than among children (3/35, 8.6%; P<0.05). To test the hypothesis that these bacteriocin-producing strains contribute to resistance against O157 in human adults, we examined faecal samples of 25 healthy O157 carriers. Inhibitory bacteria were more prevalent among the latter (9/25, 36.0%) than among age-matched subjects who did not carry O157 (49/268, 18.3%). It appears, therefore, that inhibitory bacteria in the human gut may play a role in inhibiting propagation of O157 and/or suppressing expression of virulence factors by this pathogen.     

婴幼儿配方米粉中肠杆菌科细菌检测

目的对婴幼儿配方米粉中肠杆菌科细菌污染情况进行调查,为今后制定相应标准及技术壁垒提供科学依据。方法选择3种基础肠道分离培养基组合应用,对几种常见的肠杆菌科细菌在3种基础肠道分离培养基上进行生物学特性反应差异比较,生化鉴定。结果应用伊红美蓝、麦康凯、山梨醇麦康凯3种基础肠道分离培养基组合,观察其不同形态特征及生物学特性反应,可初步判别及肠杆菌科中不同的属以及同属不同种。对市场抽检的52件婴幼儿配方米粉,检出肠杆菌科细菌27株,阳性率51.92%。其中肺炎克雷伯氏菌9株,阪崎肠杆菌4株,阴沟肠杆菌5株,聚团肠杆菌5株。大肠埃希菌4株,检出率分别为17.30%、7.69%、9.61%、9.61%、7.69%。结论加强对婴幼儿谷类食品中的细菌污染的控制很重要。     

A pseudo-outbreak of salmonellosis.

during July 1991, a single laboratory reported an increased number of an unusual salmonella isolate. An outbreak control team was convened. A case was defined as an individual with diarrhoea from whose faecal sample Salmonella hadar was isolated after 1 July 1991. By 30 July, 90 isolates had been identified and 57 persons interviewed including 39 primary cases. Interviews failed to identify any common features among the cases. A review of the laboratory procedures revealed that the selenite enrichment medium was inoculated using the spoon from the stool collection kit after it was used to emulsify the faecal sample with saline for microscopy. Salmonella hadar was isolated from this saline. Once this practice was stopped, no further isolates of S. hadar were made. This pseudo-outbreak is a powerful reminder to verify the existence of an outbreak, especially when epidemiological data are inconsistent.     

Pseudo-outbreak of Pseudomonas aeruginosa and Serratia marcescens related to bronchoscopes.

OBJECTIVE: To investigate an apparent outbreak involving simultaneous isolation of Pseudomonas aeruginosa and Serratia marcescens from bronchoalveolar lavage (BAL) samples. DESIGN: Retrospective and prospective cohort studies using chart review, environmental sampling, and ribotyping of all available isolates. Cleaning and disinfection procedures for the bronchoscopes were also evaluated. SETTING: A 380-bed private hospital in S?o Paulo, Brazil PATIENTS: Forty-one patients who underwent bronchoscopic procedures between December 1994 and October 1996 and from whom P. aeruginosa and S. marcescens were concomitantly isolated. Bronchoscopes and related items were microbiologically assessed. RESULTS: P. aeruginosa and S. marcescens were simultaneously isolated from BAL samples 12.6% of the time (41 of 324) during the epidemic period versus 1.8% of the time (1 of 54) in the pre-epidemic period (P = .035). Ribotyping revealed two strains of P. aeruginosa and one of S. marcescens that were isolated from BAL samples of patients with no signs of respiratory tract infection, suggesting a pseudo-outbreak. Evaluation of bronchoscope disinfection revealed that inappropriate methods were being used. Implementation of simple control measures resulted in a significant decrease in simultaneous isolation of these species. CONCLUSION: Prevention of pseudo-outbreaks requires meticulous use of preventive measures for infection-prone medical procedures.     

Detection of campylobacter in gastroenteritis: comparison of direct PCR assay of faecal samples with selective culture

The prevalence of campylobacter gastroenteritis has been estimated by bacterial isolation using selective culture. However, there is evidence that certain species and strains are not recovered on selective agars. We have therefore compared direct PCR assays of faecal samples with campylobacter culture, and explored the potential of PCR for simultaneous detection and identification to the species level. Two hundred unselected faecal samples from cases of acute gastroenteritis were cultured on modified charcoal cefoperazone deoxycholate agar and subjected to DNA extraction and PCR assay. Culture on CCDA indicated that 16 of the 200 samples contained 'Campylobacter spp.'. By contrast, PCR assays detected campylobacters in 19 of the 200 samples, including 15 of the culture-positive samples, and further identified them as: C. jejuni (16), C. coli (2) and C. hyointestinalis (1). These results show that PCR offers a different perspective on the incidence and identity of campylobacters in human gastroenteritis.     

Rubella antibody measured by radial haemolysis. Characteristics and performance of a simple screening method for use in diagnostic laboratories.

A simple method for preparing radial haemolysis gels for rubella antibody screening is described. In use it gave clear zones of haemolysis when a standard serum was tested at dilutions down to 5.6 i.u./ml rubella antibody. In five laboratories 8404 sera were screened by the method and the results were read by comparing zones of haemolysis with that of a standard serum diluted to contain 15 i.u/ml antibody. A zone greater than or equal to 15 i.u./ml, indicating immunity, was given by 7433 (88.4%) of the sera. No zone indicating susceptibility was seen with 748 (8.9%) sera. Small zones, less than 15 i.u./ml standard, were given by 189 (2.2%) sera, and in only 34 cases (0.4%) did non-specific haemolysis interfere with the test readings. Further testing of the radial haemolysis interfere with the test readings. Further testing of the radial haemolysis negative and low positive sera by the haemagglutination inhibition test gave rise to some discrepant results which are discussed.