Metagenomic Shotgun Analyses Reveal Complex Patterns of Intra- and Interspecific Variation in the Intestinal Microbiomes of Codfishes.

The relative importance of host-specific selection or environmental factors in determining the composition of the intestinal microbiome in wild vertebrates remains poorly understood. Here, we used metagenomic shotgun sequencing of individual specimens to compare the levels of intra- and interspecific variation of intestinal microbiome communities in two ecotypes (NEAC and NCC) of Atlantic cod (Gadus morhua) that have distinct behavior and habitats and three Gadidae species that occupy a range of ecological niches. Interestingly, we found significantly diverged microbiomes among the two Atlantic cod ecotypes. Interspecific patterns of variation are more variable, with significantly diverged communities for most species' comparisons, apart from the comparison between coastal cod (NCC) and Norway pout (Trisopterus esmarkii), whose community compositions are not significantly diverged. The absence of consistent species-specific microbiomes suggests that external environmental factors, such as temperature, diet, or a combination thereof, comprise major drivers of the intestinal community composition of codfishes.IMPORTANCE The composition of the intestinal microbial community associated with teleost fish is influenced by a diversity of factors, ranging from internal factors (such as host-specific selection) to external factors (such as niche occupation). These factors are often difficult to separate, as differences in niche occupation (e.g., diet, temperature, or salinity) may correlate with distinct evolutionary trajectories. Here, we investigate four gadoid species with contrasting levels of evolutionary separation and niche occupation. Using metagenomic shotgun sequencing, we observed distinct microbiomes among two Atlantic cod (Gadus morhua) ecotypes (NEAC and NCC) with distinct behavior and habitats. In contrast, interspecific patterns of variation were more variable. For instance, we did not observe interspecific differentiation between the microbiomes of coastal cod (NCC) and Norway pout (Trisopterus esmarkii), whose lineages underwent evolutionary separation over 20 million years ago. The observed pattern of microbiome variation in these gadoid species is therefore most parsimoniously explained by differences in niche occupation.

Modelling microbial growth in modified-atmosphere-packed hake (Merluccius merluccius) fillets stored at different temperatures.

Market globalization and changes in purchasing habits pose a challenge to the fishery industry because of the short shelf life of fish products. In view of this scenario, it would be very helpful if tools capable of predicting the shelf-life of fish could be developed. Thus, the objective of this study was to employ a modelling approach capable of predicting the evolution of the microbiota of hake fillets packaged under a modified atmosphere (MAP) rich in CO2 (50% CO2 / 50% N2) when stored at temperatures ranging between 1 and 10 °C. Growth curves of ten microbial groups were obtained at four different temperatures and fitted with the Baranyi model. Photobacterium showed high growth rates in hake fillets (0.99 days-1 at 4 °C), similar to those of Shewanella, lactic acid bacteria, and non-specific microbial groups investigated, and significantly higher than those of Pseudomonas. Furthermore, no lag phase was observed for Photobacterium regardless of the temperature investigated. On the other hand, Enterobacteriaceae and moulds and yeasts displayed low growth fitness, and their counts increased by <1.5-2 Log10 cycles along the incubation period regardless of storage temperature. The influence of storage temperature on growth parameters (λ, µmax and Yend) was subsequently studied, and secondary models were developed for the eight most relevant microbial groups. All of the final equations developed in this study showed R2 values ≥0.90, and RMSE values ≤0.50. In addition, results obtained in this investigation strongly suggest that Photobacterium would be the main responsible microorganism for the spoilage of hake fillets stored under MAP conditions (50% CO2/50% N2) along the entire range of temperatures investigated (1-10 °C).

New icing media for quality enhancement of chilled hake (Merluccius merluccius) using a jumbo squid (Dosidicus gigas) skin extract.

BACKGROUND: An advanced strategy for chilled fish preservation, based on the inclusion in ice of an extract of jumbo squid (Dosidicus gigas) skin (JSS), is proposed. Aqueous solutions including acetic acid-ethanol extracts of JSS were tested at two different concentrations as icing media, with the effects on the quality evolution of chilled hake (Merluccius merluccius) being monitored. RESULTS: A significant inhibition (P < 0.05) of microbial activity (aerobes, psychrotrophs, Enterobacteriaceae, proteolytic bacteria; pH, trimethylamine) was obtained in hake corresponding to the icing batch including the highest JSS concentration. Additionally, fish specimens from such icing conditions showed an inhibitory effect (P < 0.05) on lipid hydrolysis development, while no effect (P > 0.05) was depicted for lipid oxidation. Sensory analysis (skin and mucus development; eyes; gills; texture; external odour; raw and cooked flesh odour; flesh taste) indicated a shelf life extension of chilled hake stored in ice including the highest JSS concentration. CONCLUSION: A profitable use of JSS, an industrial by-product during jumbo squid commercialisation, has been developed in the present work, which leads to a remarkable microbial inhibition and a significant shelf life extension of chilled hake. In agreement with previous research, ommochrome pigments (i.e. lipophilic-type compounds) would be considered responsible for this preservative effect. © 2016 Society of Chemical Industry.

Endocrine effects of real-life mixtures of persistent organic pollutants (POP) in experimental models and wild fish.

A series of studies have assessed the occurrence, levels, and potential adverse effects of persistent organic pollutants (POP) in fish from Lake Mjøsa. In this lake, high levels of various POP were detected in biota. Fish from the nearby Lake Losna contain background levels of POP and served as reference (controls) in these studies. Significantly higher prevalence of mycobacteriosis and pathological changes were documented in burbot (Lota lota) from Mjøsa compared to burbot from Losna. Further, transcriptional profiling identified changes in gene expression in burbot from Mjøsa compared to burbot from Losna associated with drug metabolism enzymes and oxidative stress. POP extracted from burbot liver oil from the two lakes was used to expose zebrafish (Danio rerio) during two consecutive generations. During both generations, POP mixtures from both lakes increased the rate of mortality, induced earlier onset of puberty, and skewed sex ratio toward males. However, opposite effects on weight gain were found in exposure groups compared to controls during the two generations. Exposure to POP from both lakes was associated with suppression of ovarian follicle development. Analyses of genome-wide transcription profiling identified functional networks of genes associated with weight homeostasis, steroid hormone functions, and insulin signaling. In human cell studies using adrenocortical H295R and primary porcine theca and granulosa cells, exposure to lake extracts from both populations modulated steroid hormone production with significant difference from controls. The results suggest that POP from both lakes may possess the potential to induce endocrine disruption and may adversely affect health in wild fish.

Quality and shelf-life prediction for retail fresh hake (Merluccius merluccius).

Fish quality has a direct impact on market price and its accurate assessment and prediction are of main importance to set prices, increase competitiveness, resolve conflicts of interest and prevent food wastage due to conservative product shelf-life estimations. In this work we present a general methodology to derive predictive models of fish freshness under different storage conditions. The approach makes use of the theory of optimal experimental design, to maximize data information and in this way reduce the number of experiments. The resulting growth model for specific spoilage microorganisms in hake (Merluccius merluccius) is sufficiently informative to estimate quality sensory indexes under time-varying temperature profiles. In addition it incorporates quantitative information of the uncertainty induced by fish variability. The model has been employed to test the effect of factors such as fishing gear or evisceration, on fish spoilage and therefore fish quality. Results show no significant differences in terms of microbial growth between hake fished by long-line or bottom-set nets, within the implicit uncertainty of the model. Similar conclusions can be drawn for gutted and un-gutted hake along the experiment horizon. In addition, whenever there is the possibility to carry out the necessary experiments, this approach is sufficiently general to be used in other fish species and under different stress variables.

Molecular analysis of skin bacterial assemblages from codfish and pollock after dry-salted fish production.

Dry-salted codfish and pollock are commercially important food products with a relatively long shelf life. To date, bacterial assemblages present in these products that are relevant for food safety have been monitored using only classical molecular and/or cultivation methods. The present study employed a rapid and accurate identification method involving PCR with denaturing gradient gel electrophoresis and pyrosequencing to characterize the bacterial assemblages in the skin of three closely related fishes: Gadus morhua, Gadus macrocephalus, and Theragra chalcogramma. This methodology can be crucial for timely identification of food spoilage, hazard analysis, and monitoring of critical control points during food production. Although all specimens were processed in the same factory, there were significant compositional differences in their skin bacterial communities. In general, the bacterial community was dominated by gram-negative species of the Gammaproteobacteria. Pyrosequencing yielded 90, 69, and 245 operational taxonomic units associated with G. morhua, G. macrocephalus, and T. chalcogramma, respectively. The most dominant operational taxonomic units were assigned in order to Pseudomonas sp., Serratia marcescens, Salinisphaera sp., and Psychrobacter pulmonis. Spoilage and pathogenic bacterial groups were detected in all the studied salted gadoid samples.

Photobacterium piscicola sp. nov., isolated from marine fish and spoiled packed cod.

Five isolates from marine fish (W3(T), WM, W1S, S2 and S3) and three isolates misclassified as Photobacterium phosphoreum, originating from spoiled modified atmosphere packed stored cod (NCIMB 13482 and NCIMB 13483) and the intestine of skate (NCIMB 192), were subjected to a polyphasic taxonomic study. Phylogenetic analysis of 16S rRNA gene sequences showed that the isolates were members of the genus Photobacterium. Sequence analysis using the gapA, gyrB, pyrH, recA and rpoA loci showed that these isolates formed a distinct branch in the genus Photobacterium, and were most closely related to Photobacterium aquimaris, Photobacterium kishitanii, Photobacterium phosphoreum and Photobacterium iliopiscarium. The luxA gene was present in isolates W3(T), WM, W1S, S2 and S3 but not in NCIMB 13482, NCIMB 13483 and NCIMB 192. AFLP and (GTG)5-PCR fingerprinting indicated that the eight isolates represented at least five distinct genotypes. DNA-DNA hybridizations revealed 89% relatedness between isolate W3(T) and NCIMB 192, and values below 70% with the type strains of the phylogenetically closest species, P. iliopiscarium LMG 19543(T), P. kishitanii LMG 23890(T), P. aquimaris LMG 26951(T) and P. phosphoreum LMG4233(T). The strains of this new taxon could also be distinguished from the latter species by phenotypic characteristics. Therefore, we propose to classify this new taxon as Photobacterium piscicola sp. nov., with W3(T) (=NCCB 100098(T)=LMG 27681(T)) as the type strain.

High prevalence of infections and pathological changes in burbot (Lota lota) from a polluted lake (Lake Mjøsa, Norway).

The aim of the present study was to investigate whether exposure to high levels of persistent organic pollutants (POPs) affected a fish population in Lake Mjøsa. Lake Mjøsa is known to be contaminated by polybrominated diphenyl ethers (PBDEs), a subgroup of brominated flame retardants from local industrial activities. Fish from Lake Losna, a less contaminated lake located close to Lake Mjøsa, was used as reference (control). The sampling of burbot (Lota lota) was carried out between 2005 and 2008. Hepatic levels of POPs were analysed in burbot from the two lakes, and the fish were examined for bacterial- and parasite infection and histopathological changes. The levels of polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethanes (DDTs), and PBDEs were about 10, 15 and 300 times higher in fish from Lake Mjøsa compared to fish from Lake Losna. Mycobacterium salmoniphilum was present in 7% and 35% of the fish from Lake Losna and Lake Mjøsa respectively. Significantly higher number of external and visceral macroscopic lesions, histopathological diffuse changes and granulomas were seen in fish from Lake Mjøsa compared to Lake Losna. Furthermore the parasite infection was higher and the hepatic lipid content was significantly lower in burbot from Lake Mjøsa. The results of the present study suggest that the high level of contamination in Lake Mjøsa could have a negative impact on the health status of wild fish inhabiting the lake.

Caracterização microbiológica de filés de merluza comercializados em supermercados do Recife, PE / Microbiological characterization of fishes fillets commercialized in supermarkets of Recife, PE

A merluza Merluccius hubbsi é uma espécie típica da Região Sudeste-Sul do Brasil de grande importância econômica e comercialização em forma de filé. Desta forma, objetivou-se avaliar a qualidade microbiológica de amostras de filés de merluza congelados provenientes de quatro redes de supermercados da cidade do Recife, com relação à contagem de estafilococos coagulase positiva, contagem de coliformes totais, termotolerantes e Salmonella spp, seguindo-se o método descrito pelo Ministério da Agricultura Pecuária e Abastecimento. Foi analisado um total de 24 amostras para as quais foram obtidas contagens variando de 1,9 x 10² a 2,2 x 10³ UFC/g e de 1,0 x 10² a 6,0 x 10² UFC/g respectivamente para estafilococos coagulase positiva e coliformes termotolerantes. Não foi constatada a presença de Salmonella. Diante dos resultados obtidos, conclui-se que as amostras, na sua grande maioria, estão aptas para o consumo.

Branchiibius hedensis gen. nov., sp. nov., an actinobacterium isolated from a Japanese codling (Physiculus japonicus).

A novel, Gram-stain-positive bacterial strain, Mer 29717(T), was isolated from the branchia (gills) of a Japanese codling, Physiculus japonicus, collected from bottom waters of Suruga Bay in Shizuoka, Japan. Phylogenetic analysis based on 16S rRNA gene sequences indicated that this strain represents a distinct lineage within the family Dermacoccaceae and was related most closely to members of the genera Demetria and Yimella. It shared highest 16S rRNA gene sequence similarity (95.1 %) with Yimella lutea YIM 45900(T). Strain Mer 29717(T) contained MK-8(H(2)) and MK-8(H(4)) as menaquinones, and iso-C(16 : 0), C(16 : 0), C(17 : 1) cis-9, C(17 : 0), C(18 : 1) cis-9 and C(19 : 1) cis-10 were the major cellular fatty acids. The cell-wall peptidoglycan of strain Mer 29717(T) was composed of l-Lys, d-Ser, l-Ser, Gly, d-Glu and d-Ala. Polar lipids were phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol and one unidentified phospholipid. Mycolic acids were not detected. The G+C content of the DNA of strain Mer 29717(T) was 68 mol%. On the basis of differential chemotaxonomic, physiological and biochemical data, strain Mer 29717(T) is considered to represent a novel species of a new genus, for which the name Branchiibius hedensis gen. nov., sp. nov. is proposed. The type strain of Branchiibius hedensis is Mer 29717(T) ( = NBRC 106121(T)  = DSM 22951(T)).

Freshness characterisation of whiting (Merlangius merlangus) using an SPME/GC/MS method and a statistical multivariate approach.

BACKGROUND: The freshness of whiting was studied at five stages of ice storage by comparing the analysis of volatile compounds obtained through solid phase microextraction/gas chromatography/mass spectrometry (SPME/GC/MS) with two sensory methods. RESULTS: Of the volatile compounds identified, 38 were analysed using a statistical multivariate approach and classified according to their role in the estimation of freshness during storage as markers of freshness or spoilage. Regarding the evolution of the presence or absence of individual compounds, three categories were defined. For example, the volatile compounds propanal, hexanal, 1-penten-3-ol, pentanal, 2,3-pentanedione, 1-penten-3-one, heptanal, (E)-2-pentenal, 2,3-octanedione, (Z)-2-penten-1-ol, 1-pentanol, butanal, octanal, 3,5,5-trimethyl-2-hexene, 1-hexanol and 4,4-dimethyl-1,3-dioxane appeared highly relevant, because they are found throughout storage and can be divided into several categories that are directly related to the quality of fish. CONCLUSION: SPME/GC/MS combined with a statistical multivariate approach may be a useful method to identify volatile compounds and characterise fish freshness during storage.

Characterization of susceptibility and carrier status of burbot, Lota lota (L.), to IHNV, IPNV, Flavobacterium psychrophilum, Aeromonas salmonicida and Renibacterium salmoninarum.

In this study, susceptibility and potential carrier status of burbot, Lota lota, were assessed for five important fish pathogens. Burbot demonstrated susceptibility and elevated mortality following challenge with infectious haematopoietic necrosis virus (IHNV) by immersion and to Aeromonas salmonicida by intraperitoneal (i.p.) injection. IHNV persisted in fish for at least 28 days, whereas A. salmonicida was not re-isolated beyond 17 days post-challenge. In contrast, burbot appeared refractory to Flavobacterium psychrophilum following intramuscular (i.m.) injection and to infectious pancreatic necrosis virus (IPNV) by immersion. However, i.p injection of IPNV resulted in re-isolation of virus from fish for the duration of the 28 day challenge. Renibacterium salmoninarum appeared to induce an asymptomatic carrier state in burbot following i.p. injection, but overt manifestation of disease was not apparent. Viable bacteria persisted in fish for at least 41 days, and bacterial DNA isolated by diagnostic polymerase chain reaction was detected from burbot kidney tissue 90 days after initial exposure. This study is the first to investigate susceptibility of burbot to selected fish pathogens, and this information will aid in efforts to culture and manage this species.

Selection of candidate probionts by two different screening strategies from Atlantic cod (Gadus morhua L.) larvae.

Two primary selection criteria were used to collect a pool of nearly 500 candidate probiotic bacteria from Atlantic cod (Gadus morhua L.) larvae, i.e. the dominant intestinal bacterial flora and isolates with antagonistic activity against Vibrio anguillarum. Bacteria were isolated from cod larvae from five rearing groups with variable rearing technologies. The bacteria were brought to pure culture and characterized phenotypically. Based on properties such as uniqueness, dominance and fermentative ability, a selection of approximately 10% of the isolates were chosen from the initial pool of bacteria to reduce the number of candidates. These 55 isolates were characterized further in vitro regarding antagonism, adhesion to mucus, growth in mucus, production of extracellular enzymes, fish bile resistance and haemolytic properties. Based on the results of the in vitro tests, the number of probiotic candidates was further reduced to seven isolates. To evaluate the probiotic potential and to assure that the seven isolates were not harmful to the host, yolk sac larvae of cod were exposed to the isolates in a small-scale in vivo experiment. The in vivo experiment excluded two of the candidate bacteria due to increased mortality of cod larvae, whereas three isolates from the dominant (Vibrio and two different strains of Microbacterium) and two from the antagonistic (Ruegeria and Pseudoalteromonas) group improved the survival of larvae compared to the positive control. Thus, a combination of the two screening methods was suited for making multistrain probiotics with complementary modes of action.

Effect of gutting on microbial loads, sensory properties, and volatile and biogenic amine contents of European hake (Merluccius merluccius var. mediterraneus) stored in ice.

The effect of gutting on sensory, microbiological, and chemical properties of European hake (Merluccius merluccius var. mediterraneus) stored in ice was studied. Gutting of hake noticeably affected the development of gram-negative bacteria: counts of Enterobacteriaceae, Shewanella putrefaciens, and Pseudomonas throughout ice storage were higher in gutted than in ungutted samples. These differences in microbial loads were also reflected in the lower sensory scores of both raw and cooked hake, in the quicker trimethylamine accumulation, and in the higher contents of putrescine, cadaverine, tyramine, and histamine found in gutted hake. All of the fish quality indicators studied showed that gutting made hake more susceptible to spoilage during ice storage and decreased its shelf life by 4 days.

Identification of interleukin-22 in gadoids and examination of its expression level in vaccinated fish.

This paper reports the cloning and sequencing of interleukin (IL)-22 in two gadoid fish, cod (Gadus morhua) and haddock (Melanogrammus aeglefinus). The complete transcript of this gene was 1002 and 1154 bp respectively, of which 492 bp was the open reading frame (ORF) in both genes. High amino acid identity (88.3%) was found between these genes but was less than 50% aa identity to other known genes. The gene organisation of haddock IL-22 consisted of five exons and four introns, as with other IL-10 family members. Expression studies showed that IL-22 is constitutively expressed in gill, with low level expression also observed in gut, gonad and head kidney. In a vaccination experiment, haddock were injected intraperitoneally with formalin-killed Vibrio anguillarum or with PBS, and 2 months later challenged by immersion in 10(7)cfu/ml V. anguillarum for 30 min. Head kidney and gill samples were collected prior to challenge and 24, 48 and 72 h post-challenge (hpc) for Real-time PCR analysis of IL-22 expression. No significant changes in IL-22 expression were observed in head kidney tissue but vaccinated fish showed a significantly increased expression of IL-22 24 hpc in gill and no mortalities were seen in these fish. In contrast, control fish, which started to succumb to the disease from 72 hpc, showed no significant increase in gill expression after challenge.

A comparative study of the ability of EMA and PMA to distinguish viable from heat killed mixed bacterial flora from fish fillets.

Ethidium bromide monoazide (EMA) and propidium monoazide (PMA) were utilized to selectively allow real-time PCR (Rti-PCR) amplification of target DNA from viable but not heat killed cells from the mixed bacterial flora derived from cod fillets. A linear range of DNA amplification was exhibited from 3.2x10(2) to 1.0x10(5) genomic targets per Rti-PCR. Following the heat treatment of cell suspensions the surviving populations with the EMA and PMA Rti-PCR method were evaluated by comparison with plate counts and MPN assays following different heat exposures (45 to 95 degrees C) for 5 min. The percent of erroneous survival with PMA Rti-PCR was higher than with EMA treatment. Cellular leakage was examined by following the extracellular increase of 260 and 280 nm absorbing materials. Initial leakage of protein and nucleic acids occurred at 50 degrees C, the maximal amount of leakage occurred at 70 degrees C.

Characterization of the bacterial community associated with the surface and mucus layer of whiting (Merlangius merlangus).

The bacterial community inhabiting the mucus layer and surface of whiting was examined to determine whether the bacteria present are a reflection of the surrounding water or an indigenous bacterial flora is present. The outer mucus, mouth mucus and gut of four whiting harvested from a site in the Irish Sea and the surrounding water were examined by terminal restriction fragment length polymorphism (tRFLP) analysis of the 16S rRNA gene and clone library construction. The water community was the most diverse, with only a small number of shared water-mucus phylotypes present. The bacterial flora associated with the outer mucus layer were more diverse than that of the mouth mucus and gut. All three mucus layers were characterized by the presence of a dominant phylotype, identified as clone wom-1, highly similar to Photobacterium iliopiscarium. In addition to other Photobacterium phylotypes, members of the CFB and Clostridia groups were also detected. Subsequently, whiting from 11 different sites along the east and south coast of Ireland were compared by tRFLP analysis. Strikingly, the mucus layer of whiting at all sites was characterized by the presence and dominance of a TRF corresponding to the clone wom-1 which was virtually absent from the water column.

Polymerase Chain Reaction assay for the detection of Kudoa paniformis and Kudoa thyrsites in Pacific Hake (Merluccius productus).

Myoliquefaction of Pacific hake has been attributed to proteolytic action associated with parasitic infection. Among the two infecting species of Kudoa, Kudoa paniformis and Kudoa thyrsites, the former is reported to be more virulent for the "soft flesh" phenomenon in Pacific hake. The objective of this research was to develop a sensitive and specific polymerase chain reaction (PCR) assay to detect infection of hake by K. paniformis. Primers based on specific regions ( approximately 1562 bp) of the small subunit ribosomal DNA of K. paniformis successfully amplified the target DNA segments from both spore and muscle extracted DNA templates. DNA sequencing confirmed the veracity of this method to distinguish parasitic infection by K. paniformis versus K. thyrsites. The established PCR method was applied to investigate Kudoa infection in 44 Pacific hake samples using DNA extracted from muscle and/or spores, and the results were compared to infection evaluated by microscopic examination of extracted spores.

Use of ethidium bromide monoazide for quantification of viable and dead mixed bacterial flora from fish fillets by polymerase chain reaction.

Ethidium bromide monoazide (EMA) was utilized to selectively allow conventional PCR amplification of target DNA from viable but not dead cells from a broth culture of bacterial mixed flora derived from cod fillets. The universal primers designated DG74 and RW01 that amplify a 370-bp sequence of a highly conserved region of all eubacterial 16S rDNA were used for the PCR. The use of 10 microg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The minimum amount of EMA to completely inhibit the PCR amplification of DNA derived from dead bacterial cells was 0.8 microg/ml. Amplification of target DNA from only viable cells in a suspension with dead cells was selectively accomplished by first treating the cells with 1 microg/ml of EMA. A standard curve was generated relating the intensity of fluorescence of DNA bands to the log of CFU of mixed bacterial cultures for rapidly assessing the number of genomic targets per PCR derived from the number of CFU. A linear range of DNA amplification was exhibited from 1 x 10(2) to 1 x 10(5) genomic targets per PCR. The viable/dead cell discrimination with the EMA-PCR method was evaluated by comparison with plate counts following freezing and thawing. Thawing frozen cell suspensions initially containing 1 x 10(5) CFU/ml at 4, 20, and 37 degrees C yielded a 0.8 log reduction in the number of viable cells determined by both plate counts and EMA-PCR. In contrast, thawing for 5 min at 70 degrees C resulted in a 5 log reduction in CFU derived from plate counts (no CFU detected) whereas the EMA-PCR procedure resulted in only a 2.8 log reduction in genomic targets, possibly reflecting greater damage to enzymes or ribosomes at 70 degrees C to a minority of the mixed population compared to membrane damage.