A Shigella sonnei outbreak traced to imported basil--the importance of good typing tools and produce traceability systems, Norway, 2011.

On 9 October 2011, the University Hospital of North Norway alerted the Norwegian Institute of Public Health (NIPH) about an increase in Shigella sonnei infections in Tromsø. The isolates had an identical 'multilocus variable-number tandem repeat analysis' (MLVA) profile. Most cases had consumed food provided by delicatessen X. On 14 October, new S. sonnei cases with the same MLVA-profile were reported from Sarpsborg, south-eastern Norway. An outbreak investigation was started to identify the source and prevent further cases. All laboratory-confirmed cases from both clusters were attempted to be interviewed. In addition, a cohort study was performed among the attendees of a banquet in Tromsø where food from delicatessen X had been served and where some people had reported being ill. A trace-back investigation was initiated. In total, 46 cases were confirmed (Tromsø= 42; Sarpsborg= 4). Having eaten basil pesto sauce or fish soup at the banquet in Tromsø were independent risk factors for disease. Basil pesto was the only common food item that had been consumed by confirmed cases occurring in Tromsø and Sarpsborg. The basil had been imported and delivered to both municipalities by the same supplier. No basil from the specific batch was left on the Norwegian market when it was identified as the likely source. As a result of the multidisciplinary investigation, which helped to identify the source, the Norwegian Food Safety Authority, together with NIPH, planned to develop recommendations for food providers on how to handle fresh plant produce prior to consumption.

Potentially human-pathogenic Escherichia coli O26 in Norwegian sheep flocks.

A national survey of Escherichia coli O26 in Norwegian sheep flocks was conducted, using fecal samples to determine the prevalence. In total, 491 flocks were tested, and E. coli O26 was detected in 17.9% of the flocks. One hundred forty-two E. coli O26 isolates were examined for flagellar antigens (H typing) and four virulence genes, including stx and eae, to identify possible Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC). Most isolates (129 out of 142) were identified as E. coli O26:H11. They possessed eae and may have potential as human pathogens, although only a small fraction were identified as STEC O26:H11, giving a prevalence in sheep flocks of only 0.8%. Correspondingly, the sheep flock prevalence of atypical EPEC (aEPEC) O26:H11 was surprisingly high (15.9%). The genetic relationship between the E. coli O26:H11 isolates was investigated by pulsed-field gel electrophoresis (PFGE) and multilocus variable number tandem repeat analysis (MLVA), identifying 63 distinct PFGE profiles and 22 MLVA profiles. Although the MLVA protocol was less discriminatory than PFGE and a few cases of disagreement were observed, comparison by partition mapping showed an overall good accordance between the two methods. A close relationship between a few isolates of aEPEC O26:H11 and STEC O26:H11 was identified, but all the E. coli O26:H11 isolates should be considered potentially pathogenic to humans. The present study consisted of a representative sampling of sheep flocks from all parts of Norway. This is the first large survey of sheep flocks focusing on E. coli O26 in general, including results of STEC, aEPEC, and nonpathogenic isolates.

Outbreak of Shigella sonnei infection in Norway linked to consumption of fresh basil, October 2011.

We report a Shigella sonnei outbreak of 46 cases that occurred in Norway during October 2011. Two municipalities were involved. A large cluster (42 cases)was concentrated in north Norway, while a smallcluster (4 cases) occurred in the south-east region.Epidemiological evidence and trace back investigations have linked the outbreak to the consumption of imported fresh basil. The product has been withdrawn from the market. No further cases have been reported since 25 October.

The effect of lactic acid sprays on the keeping qualities of meat during storage.

Spraying the meat surface of skinned cow heads with 1% v/v lactic acid resulted in significant reduction in total viable counts of bacteria at storage temperatures of 4, 15 and 20 degrees C. The number of coliform bacteria was also reduced at all three temperatures but the reductions were not statistically significant on most occasions. However, after five and two days at 15 degrees C and 20 degrees C, respectively, when the initial effect of acid appeared to be lost, the number of coliforms on sprayed heads exceeded those on unsprayed heads. The shelf lives of all sprayed heads were observed to have been extended for about three days at 4 degrees C and one day for both 15 degrees C and 20 degrees C.

Detection of Salmonella from raw food samples using Dynabeads anti-Salmonella and a conventional reference method.

A Dynal core method has been established using Dynabeads anti-Salmonella to detect Salmonella from all categories of food samples. The protocol consists of the standard pre-enrichment of samples in buffered peptone water followed by immunomagnetic separation and subsequent selective enrichment of the bead-bacteria complexes in Rappaport-Vassiliadis Soya Peptone broth before plating onto Salmonella selective media. This modified IMS cultural method is intended to replace or augment traditional cultural methods used for Salmonella detection due to its specificity and increased sensitivity. The optional direct plating of bead-bacteria complexes onto solid media using a swab-streak technique is suitable for processed foods or samples known to have a history of very low resident flora. In an evaluation using 100 naturally contaminated samples, this IMS core method detected 39 of the 44 positive samples detected by all the methods combined, compared to 31 detected by the conventional ISO 6579 reference method. Furthermore, in ten different food matrices inoculated with low levels (1-5 cells/25 g) of twenty Salmonella serovariants, frozen for one month before being examined, the IMS core method, showed a 90% concordance with the ISO method and isolated two more Salmonella positive samples than the conventional ISO method.

Immunomagnetic separation of Salmonella from foods and their detection using immunomagnetic particle (IMP)-ELISA.

An immunomagnetic particle based ELISA (IMP-ELISA) for the detection of Salmonella from foods has been developed using Dynabeads anti-Salmonella (Dynal, Oslo, Norway). Appropriate sample preparation protocols to allow rapid detection of Salmonella serovariants in processed (powdered egg products) and non-processed (raw chicken) samples have been established. Pre-enriched broths of heat processed samples likely to harbour only low levels of competitive enteric flora, were boiled and used directly for IMP-ELISA. For non-heat processed or raw samples likely to contain higher numbers of such competing organisms, live Salmonella cells were first isolated by immunomagnetic separation (IMS) from standard pre-enrichment broths, and then post-selectively enriched for a short time in M-broth followed by boiling before IMP-ELISA. The total assay time including sample preparation was under 26 h for both types of procedure, with a lower detection limit of 10(5) Salmonella cells/ml of sample. In an evaluation of naturally contaminated poultry samples, all 45 of 48 samples previously shown to contain salmonellae in a comparison of ISO, IMS-Plating, Salmonella-Tek ELISA (Organon Teknika, Inc. Durham, NC) and a modification of the latter based on IMS, were identified as positive. None of the other methods gave positives for all 45.

IMS: a new selective enrichment technique for detection of Salmonella in foods.

In a study designed to evaluate the performance of Dynabeads Anti-Salmonella, immunomagnetic separation (IMS) followed by plating (IMS-Plating) proved far superior to the conventional ISO Salmonella methodology and the Modified Semi-solid Rappaport-Vassiliadis (MSRV) method. Salmonella species were isolated and detected from 135 out of the 180 diverse poultry samples by IMS analysis as against 98 by the conventional method and 33 by the MSRV technique. All results were confirmed biochemically and serologically. It appeared from the data generated that some of the salmonellae isolated using IMS were greatly inhibited in Rappaport-Vassiliadis enrichment broth at 42 degrees C and to a lesser extent in selenite cystine broth at 37 degrees C. A moderately selective plating medium like XLD proved to be better in isolating these possible sensitive wild-type strains of salmonellae than the more selective BGA.

Identification of thermotolerant Campylobacter species from poultry using an enzyme-labelled oligonucleotide DNA probe.

A commercially available enzyme-labelled DNA probe for human Campylobacter strains has been tested and also found to hybridize with DNA from C. jejuni and C. coli isolates from poultry. DNA from 11 enteric, non-campylobacter organisms, included in the test as negative controls, failed to hybridize with the probe indicating that the probe might be used for identification of campylobacter from poultry too.

Use of ferrous sulphate and immunomagnetic separation to recover Salmonella enteritidis from raw eggs.

Contaminated eggs or foods containing eggs have been a source of food borne salmonellosis, with a significant proportion of these outbreaks being attributed to Salmonella enteritidis. Since the level of contamination in individual eggs or a pool of such eggs may be low, enrichment to increase cell numbers can take several days. Pre-enrichment of raw blended eggs which have been supplemented with ferrous sulphate at a concentration of 35 mg/l, for 6 h at 37 degrees C, significantly enhanced the growth of Salmonella. Using Dynabeads Anti-Salmonella (Dynal, Oslo), a new commercial product for selective enrichment of Salmonella from food samples, we have defined a protocol based on immunomagnetic separation, specially for raw eggs. The application of this protocol enables definitive detection of Salmonella enteritidis from eggs within 30 h.

Detection of Clostridium perfringens type A enterotoxin in faecal and food samples using immunomagnetic separation (IMS)-ELISA.

A simple, rapid and sensitive immunoassay, based on immunomagnetic particles (Dynabeads M-280) was developed for detection and quantitation of Clostridium perfringens type A enterotoxin from faecal and food extracts. The assay had a detection limit of 2.5 ng/ml enterotoxin in homogenates of faeces and inoculated meat extracts. The specificity was confirmed by both crossed immunoelectrophoresis and Western immunoblotting techniques, using a purified enterotoxin as standard.

The effect of lactic acid sprays on Campylobacter jejuni inoculated onto poultry carcasses.

Spraying poultry carcasses with 1% lactic acid 10 min after inoculation with Campylobacter jejuni, resulted in a significant reduction in the number of the bacteria after 4 h at 4 degrees C. Some of the inoculated cells, however, survived for at least 144 h. Spraying 10 min after inoculation with 2% lactic acid, totally eliminated all inoculated C. jejuni within 24 h. On the other hand, spraying 24 h after inoculation, with either 1% or 2% lactic acid did not eliminate all the bacteria. Inoculated C. jejuni on poultry carcasses not sprayed with lactic acid, survived at 4 degrees C throughout the sampling period (up to 144 h) and showed little tendency to decrease in number even when the carcasses started to deteriorate. Resident campylobacters on poultry carcasses were significantly reduced by the lactic acid treatment. Frozen and thawed chickens appeared to show a graying of the skins immediately after spraying with lactic acid, slightly stronger with 2% lactic acid, but the colour reverted to normal after 24 h. We were not able to observe any colour change on the fresh broiler chickens after lactic acid treatment. Our results indicated that lactic acid had a significant bactericidal effect on C. jejuni on both naturally and artificially contaminated poultry carcasses. This effect, however, became manifest only several hours after acid treatment.

Enumeration of thermotolerant Campylobacter spp. from poultry carcasses at the end of the slaughter-line.

AIM: To enumerate Campylobacter on poultry carcasses at the end of the slaughter-line, and investigate the extent to which Campylobacter from a positive flock were transmitted to other flocks during slaughter. METHODS AND RESULTS: The presence (in caeca) and the level (from carcasses) of Campylobacter were determined. The isolates were fingerprinted by amplified fragment length polymorphism (AFLP). A total of three of 13 broiler flocks and three of four-layer flocks harboured caecal Campylobacter. Carcasses from the caeca-positive broiler flocks were Campylobacter positive with numbers ranging from 2.6 x 10(4) to 2.6 x 10(6) CFU per carcass. Two caeca-negative broiler flocks, slaughtered directly after the positive broiler flocks, had the first carcasses contaminated with Campylobacter, with numbers below 2 x 10(4) CFU per carcass of the same AFLP haplotypes as the preceding flock. Campylobacter was detected on carcasses from only one of the caeca-positive layer flocks in numbers below 2 x 10(4) CFU per carcass. No Campylobacter was detected on carcasses from a flock succeeding the positive-layer flocks. CONCLUSION: Carcasses from Campylobacter-positive broiler flocks were heavily contaminated with Campylobacter, and transmitted low levels of Campylobacter to carcasses from negative flocks, slaughtered directly after. Campylobacter-positive layer flocks had low numbers of Campylobacter on the carcasses. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate limited cross-contamination of Campylobacter between flocks at the slaughterhouse, reducing the advantage of logistic slaughter.

Genetic variability of Campylobacter jejuni isolated from fresh and frozen broiler carcasses.

AIMS: The aim of this study was to determine the genetic variability of Campylobacter jejuni isolates from poultry before and after freezing treatment in order to identify genotypes that would survive the treatment. METHODS AND RESULTS: C. jejuni was isolated from both fresh and frozen halves of the same carcass after freezing for 2 or more than 20 days at -20 degrees C. From 36 carcasses, representing five unrelated flocks in Norway, a total of 209 isolates were included in the study. Thirty-two of the isolates were recovered with a qualitative method while the remaining 177 were isolated using a quantitative method. Isolates were genotyped with fluorescent amplified fragment length polymorphism using MfeI and BglII restriction enzymes. Nine different genotypes were identified, however, one genotype was shown to be dominant in three different flocks. This genotype and the dominant genotype of another flock were found among isolates from fresh and frozen broiler halves. They were also shown to be identical to genotypes frequently identified among strains isolated from humans, cattle and poultry flocks in previous years. CONCLUSIONS: Freezing treatment or isolation method appeared not to select for a particular genotype. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the present study indicate that the freezing tolerance of strains is not genotype dependent.

Isolation of Shiga toxin-producing Escherichia coli O103 from sheep using automated immunomagnetic separation (AIMS) and AIMS-ELISA: sheep as the source of a clinical E. coli O103 case?

AIMS: To investigate whether a sheep flock was the original reservoir of a Shiga toxin-producing Escherichia coli (STEC) O103 strain causing a clinical human case and to compare the two diagnostic methods automated immunomagnetic separation (AIMS) and AIMS-ELISA. METHODS AND RESULTS: AIMS detected Escherichia coli O103 in 36.5% of the samples and AIMS-ELISA detected E. coli O103 in 52.1% of the samples. Polymerase chain reaction detected stx1 and eae in three of 109 E. coli O103 isolates. Pulsed field gel electrophoresis showed that the sheep and human STEC O103 were characterized by distinctly different profiles. CONCLUSIONS: The sheep flock was shown to carry STEC O103, although an association between the sheep flock and the clinical human case could neither be proven nor eliminated. Substantial agreement was found between AIMS and AIMS-ELISA, but AIMS-ELISA was less time consuming and resulted in a higher recovery of E. coli O103. SIGNIFICANCE AND IMPACT OF THE STUDY: The study shows that sheep may be carriers of STEC that are associated with human disease and that the methods described can be used to increase the sensitivity of STEC detection.

Magnetic separation techniques in diagnostic microbiology.

The principles of magnetic separation aided by antibodies or other specific binding molecules have been used for isolation of specific viable whole organisms, antigens, or nucleic acids. Whereas growth on selective media may be helpful in isolation of a certain bacterial species, immunomagnetic separation (IMS) technology can isolate strains possessing specific and characteristic surface antigens. Further separation, cultivation, and identification of the isolate can be performed by traditional biochemical, immunologic, or molecular methods. PCR can be used for amplification and identification of genes of diagnostic importance for a target organism. The combination of IMS and PCR reduces the assay time to several hours while increasing both specificity and sensitivity. Use of streptavidin-coated magnetic beads for separation of amplified DNA fragments, containing both biotin and a signal molecule, has allowed for the conversion of the traditional PCR into an easy-to-read microtiter plate format. The bead-bound PCR amplicons can also easily be sequenced in an automated DNA sequencer. The latter technique makes it possible to obtain sequence data of 300 to 600 bases from 20 to 30 strains, starting with clinical samples, within 12 to 24 h. Sequence data can be used for both diagnostic and epidemiologic purposes. IMS has been demonstrated to be a useful method in diagnostic microbiology. Most recent publications describe IMS as a method for enhancing the specificity and sensitivity of other detection systems, such as PCR, and providing considerable savings in time compared with traditional diagnostic systems. The relevance to clinical diagnosis has, however, not yet been fully established for all of these new test principles. In the case of PCR, for example, the presence of specific DNA in a food sample does not demonstrate the presence of a live organism capable of inducing a disease. However, all tests offering increased sensitivity and specificity of detection, combined with reduced time of analysis, have to be seriously evaluated.