Eija Hyytiä-Trees

Microbiological quality and shelf-life of vacuum-packaged 'gravad' rainbow trout stored at 3 and 8 °C

Microbiological and sensory changes of vacuum-packaged ‘gravad’ rainbow trout slices were studied during storage at 3 and 8 °C. At the time of spoilage, after 27 and 20 days of storage at 3 and 8 °C, respectively, both mesophilic viable counts (MVC) and psychrotrophic viable counts (PVC) reached 106–107cfu/g at 3 °C and 107–108 cfu/g at 8 °C. H2S-producing bacteria constituted a high proportion of the PVCs and lactic acid bacteria (LAB) counts were lower than the other determined bacterial counts. Sensory scores decreased with increasing MVC and PVC. The judges considered samples unfit for human consumption at MVC and PVC levels exceeding 106 and 107 cfu/g for samples stored at 3 and 8 °C, respectively. At respective levels of 107 and 108 cfu/g, most of the samples were deemed unfit. The main reasons for sensory rejection at both storage temperatures were the lack of the typical product odour or an ammonia off-odour and colour change to dark violet. The shelf-lives of the rainbow trout slices based on microbiological and sensory analyses were 20 days and 18 days at 3 and 8 °C, respectively.

Recent developments and future prospects in subtyping of foodborne bacterial pathogens

Infections caused by foodborne bacterial pathogens continue to be a major public health issue around the world. During the past decade, pulsed-field gel electrophoresis (PFGE) has become the gold standard for molecular subtyping and source tracking of most foodborne bacteria. Owing to problems inherent in PFGE technology, new methods have been developed focusing on DNA sequence-based subtyping. This review discusses the feasibility of using multilocus sequence typing, multiple-locus variable-number tandem repeat analysis, single nucleotide polymorphisms, microarrays, whole genome sequencing and mass spectrometry for subtyping foodborne bacterial pathogens.

Hospital-Acquired Listeriosis Outbreak Caused by Contaminated Diced Celery—Texas, 2010

BACKGROUND Listeria monocytogenes causes often-fatal infections affecting mainly immunocompromised persons. Sources of hospital-acquired listeriosis outbreaks can be difficult to identify. We investigated a listeriosis outbreak spanning 7 months and involving 5 hospitals. METHODS Outbreak-related cases were identified by pulsed-field gel electrophoresis (PFGE) and confirmed by multiple-locus variable-number tandem-repeat analysis (MLVA). We conducted patient interviews, medical records reviews, and hospital food source evaluations. Food and environmental specimens were collected at a hospital (hospital A) where 6 patients had been admitted before listeriosis onset; these specimens were tested by culture, polymerase chain reaction (PCR), and PFGE. We collected and tested food and environmental samples at the implicated processing facility. RESULTS Ten outbreak-related patients were immunocompromised by ≥1 underlying conditions or treatments; 5 died. All patients had been admitted to or visited an acute-care hospital during their possible incubation periods. The outbreak strain of L. monocytogenes was isolated from chicken salad and its diced celery ingredient at hospital A, and in 19 of >200 swabs of multiple surfaces and in 8 of 11 diced celery products at the processing plant. PCR testing detected Listeria in only 3 of 10 environmental and food samples from which it was isolated by culturing. The facility was closed, products were recalled, and the outbreak ended. CONCLUSIONS Contaminated diced celery caused a baffling, lengthy outbreak of hospital-acquired listeriosis. PCR testing often failed to detect the pathogen, suggesting its reliability should be further evaluated. Listeriosis risk should be considered in fresh produce selections for immunocompromised patients.

Multilaboratory Validation Study of Standardized Multiple-Locus Variable-Number Tandem Repeat Analysis Protocol for Shiga Toxin–Producing Escherichia coli O157: A Novel Approach to Normalize Fragment Size Data Between Capillary Electrophoresis Platforms

The PulseNet USA subtyping network recently established a standardized protocol for multiple-locus variable-number tandem repeat analysis (MLVA) to characterize Shiga toxin-producing Escherichia coli O157. To enable data comparisons from different laboratories in the same database, reproducibility and high quality of the data must be ensured. The aim of this study was to test the robustness and reproducibility of the proposed standardized protocol by subjecting it to a multilaboratory validation process and to address any discrepancies that may have arisen from the study. A set of 50 strains was tested in 10 PulseNet participating laboratories that used capillary electrophoresis instruments from two manufacturers. Six out of the 10 laboratories were able to generate correct MLVA types for 46 (92%) or more strains. The discrepancies in MLVA type assignment were caused mainly by difficulties in optimizing polymerase chain reactions that were attributed to technical inexperience of the staff and suboptimal quality of reagents and instrumentation. It was concluded that proper training of staff must be an integral part of technology transfer. The interlaboratory reproducibility of fragment sizing was excellent when the same capillary electrophoresis platform was used. However, sizing discrepancies of up to six base pairs for the same fragment were detected between the two platforms. These discrepancies were attributed to different dye and polymer chemistries employed by the manufacturers. A novel software script was developed to assign alleles based on two platform-specific (Beckman Coulter CEQ8000 and Applied Biosystems Genetic Analyzer 3130xl) look-up tables containing fragment size ranges for all alleles. The new allele assignment method was validated at the PulseNet central laboratory using a diverse set of 502 Shiga toxin-producing Escherichia coli O157 isolates. The validation confirmed that the script reliably assigned the same allele for the same fragment regardless of the platform used to size the fragment.

Safety Evaluation of Sous Vide-Processed Products with Respect to Nonproteolytic Clostridium botulinum by Use of Challenge Studies and Predictive Microbiological Models

ABSTRACT Sixteen different types of sous vide-processed products were evaluated for safety with respect to nonproteolytic group IIClostridium botulinum by using challenge tests with low (2.0-log-CFU/kg) and high (5.3-log-CFU/kg) inocula and two currently available predictive microbiological models, Food MicroModel (FMM) and Pathogen Modeling Program (PMP). After thermal processing, the products were stored at 4 and 8°C and examined for the presence of botulinal spores and neurotoxin on the sell-by date and 7 days after the sell-by date. Most of the thermal processes were found to be inadequate for eliminating spores, even in low-inoculum samples. Only 2 of the 16 products were found to be negative for botulinal spores and neurotoxin at both sampling times. Two products at the high inoculum level showed toxigenesis during storage at 8°C, one of them at the sell-by date. The predictions generated by both the FMM thermal death model and the FMM and PMP growth models were found to be inconsistent with the observed results in a majority of the challenges. The inaccurate predictions were caused by the limited number and range of the controlling factors in the models. Based on this study, it was concluded that the safety of sous vide products needs to be carefully evaluated product by product. Time-temperature combinations used in thermal treatments should be reevaluated to increase the efficiency of processing, and the use of additional antibotulinal hurdles, such as biopreservatives, should be assessed.

Inhibition of growth of nonproteolytic Clostridium botulinum type B in sous vide cooked meat products is achieved by using thermal processing but not nisin.

The safety of refrigerated processed foods of extended durability (REPFEDs) with respect to nonproteolytic Clostridium botulinum is under continuous evaluation. In the present study, mild (P7.0(85.0) values 0 to 2 min [P, pasteurization value; z-value 7.0 degrees C; reference temperature 85.0 degrees C]) and increased (P7.0(85.0) values 67 to 515 min) heat treatments were evaluated in relation to survival of nonproteolytic C. botulinum type B spores in sous vide processed ground beef and pork cubes. The use of two concentrations of nisin in inhibition of growth and toxin production by nonproteolytic C. botulinum in the same products was also evaluated. A total of 96 samples were heat processed and analyzed for C. botulinum by BoNT/B gene-specific polmerase chain reaction and for botulinum toxin by a mouse bioassay after storage of 14 to 28 days at 4 and 8 degrees C. Predictably, after mild processing all samples of both products showed botulinal growth, and one ground beef sample became toxic at 8 degrees C. The increased heat processing, equivalent to 67 min at 85 degrees C. resulted in growth but not toxin production of C. botulinum in one ground beef sample in 21 days at 8 degrees C: in the pork cube samples no growth was detected. The increased heating of both products resulted in higher sensory quality than the milder heat treatment. Nisin did not inhibit the growth of nonproteolytic C. botulinum in either product; growth was detected in both products at 4 and 8 degrees C, and ground beef became toxic with all nisin levels within 21 to 28 days at 8 degrees C. Aerobic and lactic acid bacterial counts were reduced by the addition of nisin at 4 degrees C. The study demonstrates that the mild processing temperatures commonly employed in sous vide technology do not eliminate nonproteolytic C. botulinum type B spores. The intensity of each heat treatment needs to be carefully evaluated individually for each product to ensure product safety in relation to nonproteolytic C. botulinum.

Characterization of Salmonella enterica serovar Enteritidis isolates recovered from blood and stool specimens in Thailand.

BackgroundBacteremia due to Salmonella spp. is a life-threatening condition and is commonly associated with immune compromise. A 2009 observational study estimated risk factors for the ten most common non-typhoidal Salmonella (NTS) serovars isolated from Thai patients between 2002–2007. In this study, 60.8% of Salmonella enterica serovar Enteritidis isolates (n = 1517) were recovered from blood specimens and infection with Salmonella serovar Enteritidis was a statistically significant risk factor for bacteremia when compared to other NTS serovars. Based on this information, we characterized a subset of isolates collected in 2008 to determine if specific clones were recovered from blood or stool specimens at a higher rate. Twenty blood isolates and 20 stool isolates were selected for antimicrobial resistance testing (MIC), phage typing, PFGE, and MLVA.ResultEight antibiogrammes, seven MLVA types, 14 XbaI/BlnI PFGE pattern combinations, and 11 phage types were observed indicating considerable diversity among the 40 isolates characterized. Composite analysis based on PFGE and MLVA data revealed 22 genotypes. Seven of the genotypes containing two or more isolates were from both stool and blood specimens originating from various months and zones. Additionally, those genotypes were all further discriminated by phage type and/or antibiogramme. Ninety percent of the isolates were ciprofloxacin resistant.ConclusionsThe increased percentage of bloodstream infections as described in the 2009 observational study could not be attributed to a single clone. Future efforts should focus on assessing the immune status of bacteriaemic patients and identifying prevention and control measures, including attribution studies characterizing non-clinical (animal, food, and environmental) isolates.

Outbreak of Shiga-toxigenic Escherichia coli O157:H7 infections associated with rodeo attendance, Utah and Idaho, 2009.

OBJECTIVES In summer 2009, the Utah Department of Health investigated an outbreak of Shiga-toxigenic Escherichia coli (STEC) O157:H7 (O157) illness associated with attendance at multiple rodeos. MATERIALS AND METHODS Patients were interviewed regarding exposures during the week before illness onset. A ground beef traceback investigation was performed. Ground beef samples from patient homes and a grocery store were tested for STEC O157. Rodeo managers were interviewed regarding food vendors present and cattle used at the rodeos. Environmental samples were collected from rodeo grounds. Two-enzyme pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA) were performed on isolates. RESULTS Fourteen patients with primary STEC O157 illness were reported in this outbreak. Isolates from all patients were indistinguishable by PFGE. Isolates from nine patients had identical MLVA patterns (main outbreak strain), and five had minor differences. Thirteen (93%) patients reported ground beef consumption during the week before illness onset. Results of the ground beef traceback investigation and ground beef sampling were negative. Of 12 primary patients asked specifically about rodeo attendance, all reported having attended a rodeo during the week before illness onset; four rodeos were mentioned. All four rodeos had used bulls from the same cattle supplier. An isolate of STEC O157 identified from a dirt sample collected from the bullpens of one of the attended rodeos was indistinguishable by PFGE and MLVA from the main outbreak strain. DISCUSSION Recommendations were provided to rodeo management to keep livestock and manure separate from rodeo attendees. This is the first reported STEC O157 outbreak associated with attendance at multiple rodeos. Public health officials should be aware of the potential for rodeo-associated STEC illness.

Canonical Single Nucleotide Polymorphisms (SNPs) for High-Resolution Subtyping of Shiga-Toxin Producing Escherichia coli (STEC) O157:H7.

The objective of this study was to develop a canonical, parsimoniously-informative SNP panel for subtyping Shiga-toxin producing Escherichia coli (STEC) O157:H7 that would be consistent with epidemiological, PFGE, and MLVA clustering of human specimens. Our group had previously identified 906 putative discriminatory SNPs, which were pared down to 391 SNPs based on their prevalence in a test set. The 391 SNPs were screened using a high-throughput form of TaqMan PCR against a set of clinical isolates that represent the most diverse collection of O157:H7 isolates from outbreaks and sporadic cases examined to date. Another 30 SNPs identified by others were also screened using the same method. Two additional targets were tested using standard TaqMan PCR endpoint analysis. These 423 SNPs were reduced to a 32 SNP panel with the almost the same discriminatory value. While the panel partitioned our diverse set of isolates in a manner that was consistent with epidemiological data and PFGE and MLVA phylogenies, it resulted in fewer subtypes than either existing method and insufficient epidemiological resolution in 10 of 47 clusters. Therefore, another round of SNP discovery was undertaken using comparative genomic resequencing of pooled DNA from the 10 clusters with insufficient resolution. This process identified 4,040 potential SNPs and suggested one of the ten clusters was incorrectly grouped. After its removal, there were 2,878 SNPs, of which only 63 were previously identified and 438 occurred across multiple clusters. Among highly clonal bacteria like STEC O157:H7, linkage disequilibrium greatly limits the number of parsimoniously informative SNPs. Therefore, it is perhaps unsurprising that our panel accounted for the potential discriminatory value of numerous other SNPs reported in the literature. We concluded published O157:H7 SNPs are insufficient for effective epidemiological subtyping. However, the 438 multi-cluster SNPs we identified may provide the additional information required.

Emergence and clonal dissemination of Salmonella enterica serovar Enteritidis causing salmonellosis in Mauritius

INTRODUCTION For decades, Salmonella enterica serovar Enteritidis has been among the most prevalent serovars reported worldwide. However, it was rarely encountered in Mauritius until 2007; since then the number of non-typhoidal Salmonella serogroup O:9 (including serovar Enteritidis) increased. A study was conducted to investigate the genetic relatedness between S. Enteritidis isolates recovered in Mauritius from food and clinical specimens (stool, blood, and exudate). METHODOLOGY Forty-seven isolates of S. Enteritidis obtained in 2009 from human stools, blood cultures and exudates, and from food specimens were characterized by antimicrobial susceptibility testing and Multiple-Locus Variable-number tandem repeat Analysis (MLVA). RESULTS With the exception of a single isolate which demonstrated intermediate susceptibility to streptomycin, all isolates were pansusceptible to the 14 antimicrobials tested. Thirty seven out of the 47 isolates (78.7%) exhibited an indistinguishable MLVA profile which included isolates from ready-to-eat food products, chicken, and human clinical isolates from stool, blood and exudate. CONCLUSIONS The presence of highly related strains in both humans and raw chicken, and the failure to isolate the serovar from other foods, suggests that poultry is the main reservoir of S. Enteritidis in Mauritius and that the majority of human cases are associated with chicken consumption which originated from one major producer. Stool isolates were indistinguishable or closely related to blood and exudate isolates, indicating that, besides gastroenteritis, the same strain caused invasive infections. Control of S.Enteritidis by poultry breeders would lower the financial burden associated with morbidity in humans caused by this organism in Mauritius.