Adaptation of Campylobacter jejuni to biocides used in the food industry affects biofilm structure, adhesion strength, and cross-resistance to clinical antimicrobial compounds.

The emergence of biocide-adapted Campylobacter jejuni strains that developed into biofilms and their potential to develop clinical resistance to antimicrobial compounds was studied. C. jejuni was grown in sub-lethal concentrations of five biocides used in the food industry. C. jejuni exhibited adaptation to these biocides with increased minimum inhibitory concentrations. The 3-D structures of the biofilms produced by the biocide-adapted cells were investigated by atomic force microscopy (AFM). The results revealed marked variability in biofilm architecture, including ice-crystal-like structures. Adaptation to the biocides enhanced biofilm formation, with significant increases in biovolume, surface coverage, roughness, and the surface adhesion force of the biofilms. Adaptation to commercial biocides induced resistance to kanamycin and streptomycin. This study suggests that the inappropriate use of biocides may lead to cells being exposed to them at sub-lethal concentrations, which can result in adaptation of the pathogens to the biocides and a subsequent risk to public health.

Comparison of the major virulence-related genes of Listeria monocytogenes in internalin A truncated strain 36-25-1 and a clinical wild-type strain.

BACKGROUND: Internalin A (InlA) facilitates the invasion of Listeria monocytogenes into a host cell. Some strains of Listeria monocytogenes express truncated forms of InlA, which reduces invasiveness. However, few virulence-related genes other than inlA have been analyzed in InlA-truncated strains. In the present study, we sequenced the draft genome of strain 36-25-1, an InlA-truncated strain, with pyrosequencing and compared 36 major virulence-related genes in this strain and a clinical wild-type strain. RESULTS: Strain 36-25-1 possessed all of the virulence-related genes analyzed. Of the analyzed genes, only 4 genes (dltA, gtcA, iap, and inlA) differed when the nucleotide sequences of strain 36-25-1 and the clinical wild-type strain were compared. Analysis of the deduced amino acid sequences found no mutations that significantly influenced virulence in genes other than inlA. CONCLUSIONS: The virulence-associated genes in strain 36-25-1 differ little from those of the clinical wild-type strain, indicating that a slight mutation in the nucleotide sequence determines the virulence of the InlA-truncated strain. In addition, the results suggest that, aside from InlA-mediated cell invasiveness, there is almost no difference between the virulence of strain 36-25-1 and that of the clinical wild-type strain.

Genetic distance in the whole-genome perspective on Listeria monocytogenes strains F2-382 and NIHS-28 that show similar subtyping results.

BACKGROUND: Genome subtyping approaches could provide useful epidemiological information regarding food pathogens. However, the full genomic diversity of strains that show similar subtyping results has not yet been completely explored. Most subtyping methods are based on the differences of only a portion of the genome. We investigated two draft genome sequences of Listeria monocytogenes strain F2-382 and NIHS-28, which have been identified as closely related strains by subtyping (identical multi-virulence-locus sequence typing and multiple-locus variable number tandem repeat analysis sequence types and very similar pulsed-field gel electrophoresis patterns), despite their different sources. RESULTS: Two closely related strains were compared by genome structure analysis, recombination analysis, and single nucleotide polymorphism (SNP) analysis. Both genome structure analysis and recombination analysis showed that these two strains are more closely related than other strains, from a whole-genome perspective. However, the analysis of SNPs indicated that the two strains differ at the single nucleotide level. CONCLUSION: We show the relationship between the results of genome subtyping and whole-genome sequencing. It appears that the relationships among strains indicated by genome subtyping methods are in accord with the relationships indicated by whole-genome analysis. However, our results also indicate that the genetic distance between the closely related strains is greater than that between clonal strains. Our results demonstrate that subtyping methods using a part of the genome are reliable in assessing the genetic distance of the strains. Furthermore, the genetic differences in the same subtype strains may provide useful information to distinguish the bacterial strains.

Risk of Listeria monocytogenes contamination of raw ready-to-eat seafood products available at retail outlets in Japan.

Examination of Listeria monocytogenes prevalence among ready-to-eat foods in Japan revealed frequent (5.7 to 12.1%) contamination of minced tuna and fish roe products, and the isolates had the same virulence levels as clinical isolates in terms of invasion efficiency and infectivity in cell cultures and a murine infection model, respectively. Premature stop codons in inlA were infrequent (1 out of 39 isolates). Cell numbers of L. monocytogenes in minced tuna and salmon roe increased rapidly under inappropriate storage temperatures (from a most probable number [MPN] of 10(0) to 10(1)/g to an MPN of 10(3) to 10(4)/g over the course of 2 days at 10 degrees C). Thus, regulatory guidelines are needed for acceptable levels of L. monocytogenes in these foods.

Biofilm formation ability of Listeria monocytogenes isolates from raw ready-to-eat seafood.

Listeria monocytogenes is of great concern as a foodborne pathogen. Many ready-to-eat foods are widely contaminated with this organism and have caused listeriosis outbreaks and sporadic cases in many countries. In Japan, there is a high incidence of L. monocytogenes contamination, specifically in raw ready-to-eat seafood. Identical L. monocytogenes subtypes have been isolated repeatedly from samples of food manufactured at a given store or processing plant, and researchers suspected that certain L. monocytogenes isolates have formed biofilms at these sites. A microtiter plate biofilm formation assay was conducted, and all raw ready-to-eat seafood isolates tested were able to form biofilms to various degrees. Biofilm formation by L. monocytogenes isolates of lineage I was significantly greater (P = 0.000) than that by isolates of lineage II. However, isolates of clonal lineages formed different levels of biofilms, indicating that the ability to form a biofilm is affected positively or negatively by environmental factors.

Whole-Genome Sequence of a Serotype 1/2b Listeria monocytogenes Strain Isolated from Raw Seafood in Japan.

Listeria monocytogenes is a pathogen typically acquired through the ingestion of foods. It has been specifically reported that the pathogen is widely distributed in raw seafood in Japan. Here, we report the whole-genome sequence and sequence type (ST) of a Listeria monocytogenes strain isolated from salmon roe sold in the Japanese retail market.

Development of a multilocus variable-number of tandem repeat typing method for Listeria monocytogenes serotype 4b strains.

Listeria monocytogenes serotype 4b strains have been identified as the causative agent in many human listeriosis epidemics as well as in a considerable number of sporadic cases. Due to the genetic homogeneity of serotype 4b isolates, development of rapid subtyping methods with high discriminatory power for serotype 4b isolates is required to allow for improved outbreak detection and source tracking. In this study, multilocus variable-number tandem repeat analysis (MLVA) was developed and used to characterize 60 serotype 4b isolates from various sources. All isolates were also characterized by automated EcoRI ribotyping, single enzyme pulsed-field gel electrophoresis (PFGE) with ApaI, and a multilocus sequence typing (MLST) scheme targeting six virulence and virulence-associated genes. Discriminatory power of MLVA (as determined by Simpson Index of Discrimination) was higher than the discriminatory power of any of the other three methods. MLVA markers targeted were found to be stable and did not change when three isolates were passaged daily for 70 days. Cluster analyses of MLVA, PFGE and MLST consistently grouped the same isolates into three major clusters, each of which includes one of the three major L. monocytogenes epidemic clones (i.e., ECI, ECIa and ECII). We conclude that the MLVA method described here (i) provides for more discriminatory subtyping of L. monocytogenes serotype 4b strains than the other three methods, (ii) identifies three major groups within the serotype 4b, which are consistent with the groups identified by other subtyping methods, and (iii) is easy to interpret. Use of MLVA may thus be recommended for subtyping of serotype 4b isolates, including as a secondary more discriminatory subtyping method that could be used after initial isolate characterization by PFGE or ribotyping.

Growth and toxin production of proteolytic Clostridium botulinum in aseptically steamed rice products at pH 4.6 to 6.8, packed under modified atmosphere, using a deoxidant pack.

Demand for aseptically steamed rice products has been increasing rapidly in Japan over the past 10 years. In our previous study, we showed that proteolytic Clostridium botulinum produce toxins in steamed rice products packaged under a modified atmosphere of < or =0.3% oxygen. In the present study, we examined the effect of pH to control botulism risk in steamed rice products packaged under modified atmosphere (5% CO2 and 95% N2 as the balance) with the inclusion of a deoxidant pack to produce an oxygen concentration of < or =0.3%. A mixture of 10 strains of proteolytic C. botulinum (5 type A strains and 5 type B strains) was inoculated into steamed rice products at pH values between 4.6 and 6.8 prior to packaging. All samples were stored at 30 degrees C for 24 weeks. Samples at higher pH showed earlier starts of neurotoxin production. Neurotoxin was detected after 2 weeks of incubation in samples at pH 5.4 or above, whereas it took 4 weeks for the toxin to be detected in samples at pH 5.2 to 5.3 and 12 weeks in samples at pH 5.0 to 5.1. In samples at pH 4.9 or below, no toxin was detected during the experimental period. Apparent sample spoilage did not occur before C. botulinum produced neurotoxin in most of the samples. Based on these results, we conclude that aseptically steamed rice products must be packaged at pH 4.9 or below under modified atmosphere containing < or =0.3% oxygen, with the inclusion of a deoxidant pack.

Efficacy of Propidium Monoazide on Quantitative Real-Time PCR-Based Enumeration of Staphylococcus aureus Live Cells Treated with Various Sanitizers.

Propidium monoazide (PMA) has been used together with quantitative real-time PCR (qPCR) to enumerate live bacteria, while discriminating against the residual DNA of dead bacterial cells. Although the effectiveness of PMA at increasing the accuracy of enumeration of live bacteria treated with heat has been investigated in a number of studies, few studies have involved bacteria treated with sanitizers. In this study, dead Staphylococcus aureus cells were prepared by treatment with six kinds of sanitizers (ethanol, isopropyl alcohol, benzalkonium chloride, sodium hypochlorite, hydrogen peroxide, and nisin) and were mixed with a culture of live bacteria in different ratios. PMA-qPCR was able to accurately enumerate live bacteria with a <0.5 CFU/500 µL difference with that of plate counts for cultures treated with ethanol, isopropyl alcohol, and nisin. For ethanol and isopropyl alcohol treatments, live cells were accurately enumerated for live/dead cell ratios of 10/1 to 0.01/1, while live cells for the nisin treatment were accurately enumerated for live/dead cell ratios of 10/1 to 0.1/1. In contrast, PMA-qPCR was not able to accurately enumerate live cells in bacterial cultures treated with benzalkonium chloride and hydrogen peroxide. In addition, qPCR without PMA was able to enumerate live cells as consistently as plate counts with a bacterial culture treated with sodium hypochlorite. The results of this study show that the use of PMA for qPCR-based enumeration of live cells is not always recommended, and its effectiveness depends on the treatment used on the cells.

Nonsense-mutated inlA and prfA not widely distributed in Listeria monocytogenes isolates from ready-to-eat seafood products in Japan.

InlA is a surface protein participating in the entry of Listeria monocytogenes into mammalian non-phagocytic cells. PrfA is a positive regulatory factor that regulates the expression of a set of virulence genes. Recent studies revealed that some L. monocytogenes strains have a truncated form of these proteins because of nonsense mutations in their sequences, and these truncations contribute to the significant reduction in virulence of this pathogen. In this study, sequence analyses of inlA and prfA among L. monocytogenes isolated from ready-to-eat seafood revealed that only one out of 59 isolates had a nonsense-mutated inlA and all had non-mutated prfA. This indicated that these strains could be fully virulent based on the sizes of these proteins.

Development of multilocus single strand conformation polymorphism (MLSSCP) analysis of virulence genes of Listeria monocytogenes and comparison with existing DNA typing methods.

Development of rapid and simple typing methods is required for analyzing the distribution and contamination routes of food-borne pathogens. We established a simple typing method for Listeria monocytogenes using MLSSCP (Multilocus Single Strand Conformation Polymorphism) analysis. Four virulence genes, hlyA, iap, actA and inlB were amplified by PCR, digested with endonucleases and applied to gels for SSCP. As banding patterns have been shown to reflect even a single nucleotide difference, this method has a potential discriminatory power comparable to that of sequencing analysis. The 64 strains isolated from five meat processing plants were divided into 18 groups by this MLSSCP. Additionally, clustering obtained with this method showed strong correspondence with phylogenetic lineages I and II, and was achieved with much less expenditure in time and cost than is required for other methods, such as MLST. The validity of the MLSSCP lineage classification was confirmed by PFGE, AFLP and ribotyping results. This newly developed MLSSCP method is suitable when obtaining accurate results quickly and simply is crucial.

Development of quantitative real-time PCR for detection and enumeration of Enterobacteriaceae.

The family Enterobacteriaceae, members of which are widely distributed in the environment, includes many important human pathogens. In this study, a rapid real-time PCR method targeting rplP, coding for L16 protein, a component of the ribosome large subunit, was developed for enumerating Enterobacteriaceae strains, and its efficiency was evaluated using naturally contaminated food products. The rplP-targeted real-time PCR amplified Enterobacteriaceae species with Ct values of 14.0-22.8, whereas the Ct values for non-Enterobacteriaceae species were >30, indicating the specificity of this method for the Enterobacteriaceae. Using a calibration curve of Ct=-3.025 (log CFU/g)+37.35, which was calculated from individual plots of the cell numbers in different concentrations of 5 Enterobacteriaceae species, the rplP-targeted real-time PCR was applied to 51 food samples. A <1log difference between the real-time PCR and culture methods was obtained in a majority of the food samples (81.8%), with good correlation (r2=0.8285). This study demonstrated that the rplP-targeted real-time PCR method could detect and enumerate Enterobacteriaceae species in foods rapidly and accurately, and therefore, it can be used for the microbiological risk analysis of foods.

Genetic characteristics of Japanese clinical Listeria monocytogenes isolates.

Listeria monocytogenes causes foodborne illnesses through consumption of ready-to-eat foods. Although 135-201annual listeriosis cases have been estimated in Japan, the details regarding the clinical isolates such as infection source, virulence level, and other genetic characteristics, are not known. In order to uncover the trends of listeriosis in Japan and use the knowledge for prevention measures to be taken, the genetic characteristics of the past human clinical isolates needs to be elucidated. For this purpose, multilocus tandem-repeat sequence analysis (MLTSA) and multi-virulence-locus sequence typing (MVLST) were used in this study. The clinical isolates showed a variety of genetically distant genotypes, indicating they were from sporadic cases. However, the MVLST profiles of 7 clinical isolates were identical to those of epidemic clone (EC) I isolates, which have caused several serious outbreaks in other countries, suggesting the possibility that they have strong virulence potential and originated from a single outbreak. Moreover, 6 Japanese food isolates shared their genotypes with ECI isolates, indicating that there may be risks for listeriosis outbreak in Japan. This is the first investigational study on genetic characteristics of Japanese listeriosis isolates. The listeriosis cases happened in the past are presumably sporadic, but it is still possible that some isolates with strong virulence potential have caused listeriosis outbreaks, and future listeriosis risks also exist.

Highly discriminatory typing method for Listeria monocytogenes using polymorphic tandem repeat regions.

Tandem repeats (TR), which are repetitive nucleotide sequences in DNA, are polymorphic both in repeat number and sequence. In this study, we developed a new typing method, multilocus TR sequence analysis (MLTSA), for the foodborne pathogen Listeria monocytogenes using sequence polymorphisms in three tandem repeat regions. The obtained dendrogram clustered L. monocytogenes strains of lineage I and lineage II separately, and formed three groups within the lineage I cluster, each of which included one of the three major L. monocytogenes epidemic clones (ECI, ECIa, and ECII). These results were consistent with a previously established virulence-gene-based MLST method. In comparison, our method grouped some epidemiologically related isolates together, which virulence-gene-based MLST did not. Moreover, our method, using three tandem repeat regions, showed a higher discriminatory power than the MLST method, which uses six virulence gene regions. This MLTSA approach using sequence polymorphisms in TR regions could be a useful tool in the epidemiological study of L. monocytogenes.

Modeling and predicting the simultaneous growth of Listeria monocytogenes and natural flora in minced tuna.

The growth kinetics of Listeria monocytogenes and natural flora (NF) in minced tuna from 2 to 30 °C were examined, and a simultaneous growth model was developed. The inhibiting effect of the NF on the growth of L. monocytogenes was examined by inoculating different levels of NF isolated from the minced tuna. The kinetic data were fitted to the Baranyi model and estimated the growth parameters such as specific growth rate (µ(max)), maximum population density (N(max)), and lag time. The temperature and inoculated NF dependency on the µ(max) of L. monocytogenes and NF were described by modified Ratkowsky's square-root model. As the initial NF level increased, the slopes of the square-root models were decreased for both L. monocytogenes and NF. The N(max) of L. monocytogenes was described as a function of temperature and inoculated NF level. Simultaneous growth prediction of L. monocytogenes and NF under constant temperature conditions was examined by using the differential equations based on the Baranyi model with the effect of interspecies competition substituted into the developed µ(max) and N(max) models. The root mean square errors between the model prediction and the observation for L. monocytogenes and NF were 0.42 and 0.34, respectively. Predictive simulation under fluctuating temperature conditions also demonstrated a high accuracy of simultaneous prediction for both L. monocytogenes and NF, representing the root mean square errors of 0.19 and 0.34, respectively. These results illustrate that the developed model permits accurate estimation of the behavior of L. monocytogenes in minced tuna under real temperature history until consumption.

Use of commercially available antimicrobial compounds for prevention of Listeria monocytogenes growth in ready-to-eat minced tuna and salmon roe during shelf life.

Listeria monocytogenes found in minced tuna and fish roe can cause listeriosis. These products are classified in category B according to the Codex Alimentarius Commission, i.e., ready-to-eat foods in which L. monocytogenes growth can occur. We investigated the effectiveness of nisin and other commercially available antimicrobial compounds (lysozyme, ε-polylysine, and chitosan) for prevention of L. monocytogenes growth during the expected shelf life of raw minced tuna and salmon roe products. Food samples inoculated with L. monocytogenes were incubated with each antimicrobial at 10°C for 7 days or at 25°C for 12 h. Nisaplin (an antimicrobial containing nisin) effectively inhibited L. monocytogenes growth in minced tuna at 500 ppm and in salmon roe at 250 ppm within their standard shelf lives. The effective concentration of each antimicrobial was determined: 2,000 ppm for ART FRESH 50/50 (containing lysozyme) and SAN KEEPER No. 381 (containing ε-polylysine) and 10,000 ppm for SAN KEEPER K-3 (containing chitosan).

Effect of food residues on norovirus survival on stainless steel surfaces.

BACKGROUND: In households and food processing plants, minute food residues left behind from improper cleaning may influence the survivability of human norovirus on surfaces. In this study, the survivability of norovirus on desiccated food residue-attached stainless steel coupons was investigated. METHODOLOGY/PRINCIPAL FINDINGS: Using murine norovirus-1 (MNV-1) as a surrogate of human norovirus, the survivability of norovirus was investigated on lettuce, cabbage, or ground pork-attached stainless steel coupons. A 6.2 log MPN/ml of MNV-1 infectivity was completely lost at day 30 in residue-free coupons, whereas only a 1.4 log MPN/ml reduction was observed in coupons with residues. Moreover, the disinfective effect of sodium hypochlorite was reduced when residues were present on the coupons. CONCLUSIONS/SIGNIFICANCE: This study revealed that the food residues increased the survivability and the resistance to chemicals of norovirus, indicating the need of thorough cleaning in food processing plants and household settings.

Difference of genotypic and phenotypic characteristics and pathogenicity potential of Photobacterium damselae subsp. damselae between clinical and environmental isolates from Japan.

Photobacterium damselae subsp. damselae has been known as an opportunistic pathogen in fish and mammals. Human infectious cases are often very serious and occasionally fatal. We previously reportedtwo fatal cases caused by this subspecies where the patients developed multiple organ failure within 20-36 h after the onset of initial symptoms. Despite its ability to cause serious infections in humans, this subspecies has not been well studied because human infectious cases caused by this subspecies are very rare. However, this subspecies has been reported to be present in a wide range with high incidence rate in aquatic environments. Thus, we investigated the genotypic and phenotypic differences between clinical and environmental strains of Photobacterium damselae subsp. damselae. Using molecular typing methods, such as ribotyping, AFLP (Amplified Fragment Length Polymorphism), and PFGE (Pulsed-Field Gel Electrophoresis) and sequencing analysis, we determined that thetwo clinical strains were genetically similar yet distinguishable from environmental strains, but not significantly so. On the other hand, phenotypic differences were clear; moreover, mouse assay and hemolytic assay indicated strong pathogenicity of only clinical isolates. Based on these data, we concluded that there are differences in pathogenicity potential among isolates of this subspecies, and some environmental isolates have the potential to become highly pathogenic.