An international trial of quantitative PCR for monitoring Legionella in artificial water systems.

AIMS: To perform an international trial to derive alert and action levels for the use of quantitative PCR (qPCR) in the monitoring of Legionella to determine the effectiveness of control measures against legionellae. METHODS AND RESULTS: Laboratories (7) participated from six countries. Legionellae were determined by culture and qPCR methods with comparable detection limits. Systems were monitored over ≥10 weeks. For cooling towers (232 samples), there was a significant difference between the log mean difference between qPCR (GU l(-1) ) and culture (CFU l(-1) ) for Legionella pneumophila (0·71) and for Legionella spp. (2·03). In hot and cold water (506 samples), the differences were less, 0·62 for Leg. pneumophila and 1·05 for Legionella spp. Results for individual systems depended on the nature of the system and its treatment. In cooling towers, Legionella spp. GU l(-1) always exceeded CFU l(-1) , and usually Legionella spp. were detected by qPCR when absent by culture. The pattern of results by qPCR for Leg. pneumophila followed the culture trend. In hot and cold water, culture and qPCR gave similar results, particularly for Leg. pneumophila. There were some marked exceptions with temperatures ≥50°C, or in the presence of supplementary biocides. Action and alert levels for qPCR were derived that gave results comparable to the application of the European Guidelines based on culture. Algorithms are proposed for the use of qPCR for routine monitoring. CONCLUSIONS: Action and alert levels for qPCR can be adjusted to ensure public health is protected with the benefit that remedial actions can be validated earlier with only a small increase in the frequency of action being required. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms it is possible to derive guidelines on the use of qPCR for monitoring the control of legionellae with consequent improvement to response and public health protection.

Assessment of the microbiological safety of edible roasted nut kernels on retail sale in England, with a focus on Salmonella.

There is little published information on the prevalence of Salmonella in edible nut kernels. A study in early 2008 of edible roasted nut kernels on retail sale in England was undertaken to assess the microbiological safety of this product. A total of 727 nut kernel samples of different varieties were examined. Overall, Salmonella and Escherichia coli were detected from 0.2 and 0.4% of edible roasted nut kernels. Of the nut varieties examined, Salmonella Havana was detected from 1 (4.0%) sample of pistachio nuts, indicating a risk to health. The United Kingdom Food Standards Agency was immediately informed, and full investigations were undertaken. Further examination established the contamination to be associated with the pistachio kernels and not the partly opened shells. Salmonella was not detected in other varieties tested (almonds, Brazils, cashews, hazelnuts, macadamia, peanuts, pecans, pine nuts, and walnuts). E. coli was found at low levels (range of 3.6 to 4/g) in walnuts (1.4%), almonds (1.2%), and Brazils (0.5%). The presence of Salmonella is unacceptable in edible nut kernels. Prevention of microbial contamination in these products lies in the application of good agricultural, manufacturing, and storage practices together with a hazard analysis and critical control points system that encompass all stages of production, processing, and distribution.

Large outbreak of salmonella phage type 1 infection with high infection rate and severe illness associated with fast food premises.

OBJECTIVES: To describe the epidemiology of an outbreak of Salmonella enteritidis phage type 1 (PT1) infection associated with a fast food premises, and to identify the causative factors leading to an acute outbreak with high attack rate and severe illness including hospital admission. STUDY DESIGN: Integrated descriptive study of epidemiology, food and environmental microbiology, and professional environmental health assessment, supplemented by a case-case analytical study. METHODS: Cases were identified through multiple sources and were interviewed to identify food items consumed. Descriptive epidemiology of all cases and a case-case analytical study of risk factors for severe illness were undertaken. Microbiological investigation included analysis and typing of pathogens from stools, blood and environmental surfaces. Professional environmental heath assessment of the premises was undertaken. RESULTS: S. enteritidis PT1 was recovered from two-thirds of faecal samples. Three cases had dual infection with enterotoxin-producing Clostridium perfringens. S. enteritidis PT1 was isolated from 14 of 40 food samples examined and C. perfringens was isolated from eight food samples. Environmental health inspection of the premises revealed multiple deficiencies, including deficits in food preparation and hygiene consistent with multiple cross-contamination, and time-temperature abuse of sauces widely used across menu items. Severe cases were associated with consumption of chips and salad. CONCLUSIONS: Outbreaks from fast food premises have been infrequently described. This outbreak demonstrates the potential for fast food premises, with multiple deficiencies in food preparation and hygiene, to produce large, intense community outbreaks with high attack rates and severe illness, highly confined in space and time.

Microbiological study of fresh herbs from retail premises uncovers an international outbreak of salmonellosis.

This Local Authorities Co-ordinators of Regulatory Services/Health Protection Agency study was prompted by the increasing concern regarding the microbiological safety of ready-to-eat salad vegetable products, particularly fresh herbs. During May to October 2007, 3760 ready-to-eat fresh herbs, of different varieties, were sampled across the UK to assess their microbiological safety in relation to salmonella contamination and levels of Escherichia coli. Sixty (1.6%) herb samples were found to be of unsatisfactory quality according to Regulation (EC) No. 2073/2005 on the microbiological criteria of foodstuffs, i.e. contaminated with Salmonella spp. and/or containing E. coli at >10(3) cfu/g. When criteria in the PHLS Microbiological Guidelines for some ready-to-eat foods (2000) were used, 117 (3.9%) of herb samples were of unsatisfactory quality due to the presence of salmonella and/or E. coli at > or = 10(2) cfu/g. Eighteen (0.5%) samples of six different herb types were contaminated with Salmonella spp.: identified as serotypes Senftenberg (8), Agona (2), Anatum (1), Durban (1), Javiana (1), Mgulani (1), Montevideo (1), Unnamed (I 16:g, t: z42) (1), Virchow (1) and mixed Newport & Virchow (1). In each case the retailer and the UK Food Standards Agency were immediately informed and remedial action taken. Samples contaminated with S. Senftenberg were specifically associated with basil grown in Israel. Thirty-two human cases of S. Senftenberg infection were subsequently identified throughout England and Wales and a further 19 in Scotland, Denmark, The Netherlands and the USA. The strain of S. Senftenberg identified from the basil and that from cases had an indistinguishable molecular profile, suggesting a likely connection between consumption of basil and human infection. The presence of Salmonella spp. is unacceptable in ready-to-foods such as fresh herbs. This study highlights the necessity of applying good agricultural and hygiene practices pre-, during and post-harvest, at processing, retail and use. These practices help to prevent cross-contamination and/or bacterial growth occurring in these products. Best practice is to store and display such products at, or below, 8 degrees C as this inhibits bacterial growth.

A national outbreak of verotoxin-producing Escherichia coli O157 associated with consumption of lemon-and-coriander chicken wraps from a supermarket chain.

A national outbreak of verotoxin-producing Escherichia coli O157 infection affected five English regions and Wales. Twelve cases were associated with lemon-and-coriander chicken wrap from a single supermarket chain consumed over a 5-day period. An outbreak investigation aimed to identify the source of infection. Descriptive epidemiology and phenotypic and genotypic tests on human isolates indicated a point-source outbreak; a case-control study showed a very strong association between consumption of lemon-and-coriander chicken wrap from the single supermarket chain and being a case (OR 46.40, 95% CI 5.39-infinity, P=0.0002). Testing of raw ingredients, products and faecal samples from staff in the food production unit did not yield any positive results. The outbreak was probably caused by one contaminated batch of an ingredient in the chicken wrap. Even when current best practice is in place, ready-to-eat foods can still be a risk for widespread infection.

Survey of Salmonella contamination of raw shell eggs used in food service premises in the United Kingdom, 2005 through 2006.

This survey was launched after an unusual number of Salmonella Enteritidis outbreaks associated with the use of eggs in food service premises in England and Wales. Between November 2005 and December 2006, 9,528 eggs (1,588 pooled samples of 6 eggs) were collected from 1,567 food service premises in the United Kingdom, most of which (89%) were produced in the United Kingdom. Salmonella was isolated from 6 (0.38%) pools of eggs. Of these, 5 (0.31%) were Salmonella Enteritidis, which were further characterized to phage types (PTs): PT 4 (0.19%), PT 8 (0.06%), and PT 12 (0.06%). Salmonella Mbandaka was also isolated (0.06%). Salmonella was detected from five and one of pooled eggs samples that were produced in the United Kingdom and Germany, respectively; these were from different producers. The study showed evidence of poor egg storage and handling practices in food service premises, in that 55% did not store eggs under refrigerated conditions; 20.7% of eggs had expired "best before" dates or were in use after 3 weeks of lay, indicating poor stock rotation; and 37.1% pooled eggs not intended for immediate service. Eggs are a commonly consumed food that may occasionally be contaminated with Salmonella at different rates, according to their country of origin. The food service sector needs to be aware of this continuing hazard, receive appropriate food safety and hygiene training on storage and usage of raw shell eggs, adopt appropriate control measures, and follow advice provided by national food agencies in order to reduce the risk of infection.

The microbiological quality of potable water on board ships docking in the UK and the Channel Islands: an association of Port Health Authorities and Health Protection Agency Study.

Providing safe potable water onboard vessels presents particular challenges and contamination can occur directly from source waters as well as during loading, storage and distribution. Between May and October 2005, 950 potable water samples were collected from 342 ships docking at ports. Comparison with Guidelines found 9% of samples contained coliforms, Escherichia coli or enterococci and 2.8% had faecal indicators (E. coli or enterococci). Action levels of aerobic colony count (ACC) bacteria were detected in 20% (22 degrees C) and 21.5% (37 degrees C) of samples. ACC results from one-off sampling are not informative as this does not enable port health authorities to monitor ACC trends. They should be removed as a routine criterion for remedial action and vessels should adopt the WHO Water Safety Plan approach, whilst continuing to monitor water quality with public health-based indicators (e.g. chlorine residual, coliforms, E. coli and enterococci). Logistic regression analyses identified practices associated with water quality. Practices protective against coliforms, E. coli or enterococci in potable supplies were: good hose hygiene, processing water onboard, maintaining free chlorine residual at >or=0.2 mg/L. This emphasizes the importance of good hygiene during potable water loading and maintaining adequate disinfection of supplies onboard.

Public health and economic costs of investigating a suspected outbreak of Legionnaires' disease.

This paper provides one of the first assessments of the burden of both the public health investigation and the economic costs associated with an apparent outbreak of Legionnaires' disease (LD) in South East London. In addition to epidemiological, microbiological and environmental investigations, we collected data on the staff time and resources committed by the 11 main organizations responsible for managing the outbreak. Of the overall estimated costs of 455,856 pounds, only 14% (64,264 pounds) was spent on investigation and control of the outbreak compared with 86% (391,592 pounds) spent on the hospital treatment of the patients. The time and money spent on public health services in this investigation appear to represent good value for money considering the potential costs of a major outbreak, including the high case-fatality rate in LD generally and the high health-care costs. Further research is needed to determine optimum strategies for the cost-effective use of health system resources in investigations of LD. Whether the threshold for investigation of cases should be based on observed incidence rates or the cost-effectiveness of investigations, or both, should be debated further.

Survey of Salmonella contamination of non-United Kingdom-produced raw shell eggs on retail sale in the northwest of England and London, 2005 to 2006.

This survey was prompted by a change in the epidemiology of Salmonella Enteritidis infections in England and Wales and elsewhere in Europe and, to our knowledge, is the first survey to provide information on Salmonella contamination of non-United Kingdom eggs on retail sale. Based on 10,464 non-United Kingdom eggs (1744 pooled samples of six eggs) purchased between March 2005 and July 2006, the total weighted prevalence estimate for all Salmonella detected in non-United Kingdom eggs was 3.3%. Of the eggs sampled, most were produced in Spain (66.3%), France (20.0%), or The Netherlands (7.4%). Salmonella was detected from 4.4 and 0.3% of eggs produced in Spain and France, respectively, with weighted prevalence estimates. Eight different Salmonella serotypes were recovered from non-United Kingdom eggs, of which Salmonella Enteritidis predominated, with an estimated prevalence of 2.6%. Salmonella Enteritidis was obtained only from Spanish eggs. Nine different phage types of Salmonella Enteritidis were identified, with phage type 1 found to be the predominant phage type. Most of the Salmonella Enteritidis isolates obtained from Spanish eggs in the survey were resistant to nalidixic acid with concomitant decreased susceptibility to ciprofloxacin (0.125 to 1.0 mg/liter) or ampicillin (8.0 mg/liter). Salmonella Enteritidis phage type 1 until now had not been detected in eggs examined as part of previous United Kingdom egg surveys but has been detected in eggs of Spanish origin examined during recent national outbreaks of Salmonella Enteritidis non-phage type 4 infections in England and Wales. Eggs are a commonly consumed food that may occasionally be contaminated with Salmonella. The rates of contamination may be linked to the origin of the eggs. Consumers and caterers need to be aware of this continuing hazard, adopt appropriate control measures, and follow advice provided by national food agencies in order to reduce the risk of infection.

Public health investigations of Salmonella Enteritidis in catering raw shell eggs, 2002-2004.

AIMS: In response to a dramatic change in the epidemiology of Salmonella Enteritidis in England and Wales thought to be associated with raw shell eggs, the Health Protection Agency initiated public health investigations to establish the incidence of Salmonella contamination and origin of eggs used by catering premises implicated in outbreaks of Salm. Enteritidis. METHODS AND RESULTS: Between October 2002 and November 2004, 16 971 eggs were sampled and Salmonella were recovered from 3.4%. Salmonella was isolated from 5.5% and 6.3% of Spanish and eggs of unknown origin, respectively, used in catering premises linked to outbreaks, a level significantly higher than that (1.1%) found in nonLion Quality UK eggs sampled. The small sample of UK Lion Quality eggs tested (reflecting their lack of use in premises visited) did not contain Salmonella. Several phage types of Salm. Enteritidis other than phage type 4 (PT 4) were identified with nonUK eggs. CONCLUSIONS: Eggs from Spain were implicated as a major source of infection. Eggs were contaminated more frequently with Salmonella when shells were dirty and/or cracked, and stored at above 8 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of Spanish eggs by the catering sector has been identified as a consistent significant factor in many of the outbreaks caused by Salm. Enteritidis nonPT4 in England and Wales during 2002-2004. Advice to caterers and hospitals that raw shell eggs should not be used in food that will either not be cooked or only lightly cooked should be reinforced.

Legionnaires' disease outbreak associated with a cruise liner, August 2003: epidemiological and microbiological findings.

Eight cases of Legionnaires' disease were identified among the 215 German passengers after a cruise to the Nordic Sea in August 2003. An unmatched case-control study was conducted to identify risk factors and the source of infection. In total, eight passengers fulfilled the case definition, one of these died. Forty-two passengers served as controls. The attack rate was 4%. The mean age was 60 years for cases and 62 years for controls. Prolonged exposure to the spa pool seemed to be a risk factor of infection (OR 4.85, P=0.09). Legionella pneumophila serogroup 1, monoclonal antibody (mAb) subgroup 'Knoxville' was isolated from clinical and environmental samples. DNA sequence-based typing revealed that these isolates were indistinguishable from each other. The investigation showed the importance of an interdisciplinary approach of microbiology and epidemiology as not all sites on the ship that tested positive for L. pneumophila actually posed a relevant risk for the passengers.

Culture of Helicobacter pylori from domestic water samples--the impact of strain variation on growth on solid and in liquid media.

Helicobacter pylori is an important global human pathogen and there is growing evidence from PCR assays that contaminated drinking water might be a possible source of infection in some circumstances. There are no validated protocols for direct isolation but various culture media have been developed for possible environmental sampling. Our aim here was to investigate how inter-strain variation might affect the interpretation of results with such media. Two laboratory adapted reference strains and four recent clinical isolates were tested on four solid media and in ten liquid media. Considerable variation was found between strains in their ability to recover on the different media after stress exposure (suspension in sterile tap water). Generally, clinical isolates were less robust than the laboratory-adapted strains and, overall, the former required longer recovery times. Our findings highlighted the importance of using a range of isolates for evaluations, as examination of laboratory-adapted strains alone did not provide an accurate representation of the utility of media that may be used to recover H. pylori from water.

Detection of Helicobacter pylori by PCR but not culture in water and biofilm samples from drinking water distribution systems in England.

AIMS: To investigate treated water distribution systems in England as a source of Helicobacter pylori. METHODS AND RESULTS: Water and biofilms were obtained from 11 domestic and seven educational properties and from hydrants, reservoirs and water meters supplied by three water utilities. Samples were cultured on nonselective and antibiotic containing media combined with immunomagnetic separation concentration. Viable helicobacters were not detected in any of the 151 samples but Helicobacter-specific PCR assays detected DNA in 26% of samples from domestic properties, schools and hydrants with the highest frequency in biofilms (42%). Direct sequencing of six selected amplicons confirmed >95% sequence homology to H. pylori. CONCLUSIONS: While viable helicobacters were not isolated, evidence was obtained for the presence of Helicobacter DNA, including that of H. pylori. Biofilms on surfaces within water distribution systems may act either as sites for the passive accumulation of helicobacters or as potentially important reservoirs of infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings strengthen evidence that H. pylori may be transmitted through drinking water. However, there is currently no evidence that viable cells can survive the disinfection levels used in UK mains supplies and the health risk from this source remains unclear.