A Review on Bio- and Chemosensors for the Detection of Biogenic Amines in Food Safety Applications: The Status in 2022.

This article provides an overview on the broad topic of biogenic amines (BAs) that are a persistent concern in the context of food quality and safety. They emerge mainly from the decomposition of amino acids in protein-rich food due to enzymes excreted by pathogenic bacteria that infect food under inappropriate storage conditions. While there are food authority regulations on the maximum allowed amounts of, e.g., histamine in fish, sensitive individuals can still suffer from medical conditions triggered by biogenic amines, and mass outbreaks of scombroid poisoning are reported regularly. We review first the classical techniques used for selective BA detection and quantification in analytical laboratories and focus then on sensor-based solutions aiming at on-site BA detection throughout the food chain. There are receptor-free chemosensors for BA detection and a vastly growing range of bio- and biomimetic sensors that employ receptors to enable selective molecular recognition. Regarding the receptors, we address enzymes, antibodies, molecularly imprinted polymers (MIPs), and aptamers as the most recent class of BA receptors. Furthermore, we address the underlying transducer technologies, including optical, electrochemical, mass-sensitive, and thermal-based sensing principles. The review concludes with an assessment on the persistent limitations of BA sensors, a technological forecast, and thoughts on short-term solutions.

Community-wide changes reflecting bacterial interspecific interactions in multispecies biofilms.

Existence of most bacterial species, in natural, industrial, and clinical settings in the form of surface-adhered communities or biofilms has been well acknowledged for decades. Research predominantly focusses on single-species biofilms as these are relatively easy to study. However, microbiologists are now interested in studying multispecies biofilms and revealing interspecific interactions in these communities because of the existence of a plethora of different bacterial species together in almost all natural settings. Multispecies biofilms-led emergent properties are triggered by bacterial social interactions which have huge implication for research and practical knowledge useful for the control and manipulation of these microbial communities. Here, we discuss some important bacterial interactions that take place in multispecies biofilm communities and provide insights into community-wide changes that indicate bacterial interactions and elucidate underlying mechanisms.

Pseudomonas putida as a potential biocontrol agent against Salmonella Java biofilm formation in the drinking water system of broiler houses.

BACKGROUND: Environmental biofilms can induce attachment and protection of other microorganisms including pathogens, but can also prevent them from invasion and colonization. This opens the possibility for so-called biocontrol strategies, wherein microorganisms are applied to control the presence of other microbes. The potential for both positive and negative interactions between microbes, however, raises the need for in depth characterization of the sociobiology of candidate biocontrol agents (BCAs). The inside of the drinking water system (DWS) of broiler houses is an interesting niche to apply BCAs, because contamination of these systems with pathogens plays an important role in the infection of broiler chickens and consequently humans. In this study, Pseudomonas putida, which is part of the natural microbiota in the DWS of broiler houses, was evaluated as BCA against the broiler pathogen Salmonella Java. RESULTS: To study the interaction between these species, an in vitro model was developed simulating biofilm formation in the drinking water system of broilers. Dual-species biofilms of P. putida strains P1, P2, and P3 with S. Java were characterized by competitive interactions, independent of P. putida strain, S. Java inoculum density and application order. When equal inocula of S. Java and P. putida strains P1 or P3 were simultaneously applied, the interaction was characterized by mutual inhibition, whereas P. putida strain P2 showed an exploitation of S. Java. Lowering the inoculum density of S. Java changed the interaction with P. putida strain P3 also into an exploitation of S. Java. A further increase in S. Java inhibition was established by P. putida strain P3 forming a mature biofilm before applying S. Java. CONCLUSIONS: This study provides the first results showing the potential of P. putida as BCA against S. Java in the broiler environment. Future work should include more complex microbial communities residing in the DWS, additional Salmonella strains as well as chemicals typically used to clean and disinfect the system.

Quantification of Extracellular Proteases and Chitinases from Marine Bacteria.

A total of 92 marine bacteria belonging to Pseudomonas, Pseudoalteromonas, Psychrobacter, and Shewanella were first screened for their proteolytic activity. In total, four Pseudomonas strains belonging to Ps. fluorescens, Ps. fragi, Ps. gessardii, and Ps. marginalis; 14 Pseudoalteromonas strains belonging to Psa. arctica, Psa. carrageenovora, Psa. elyakovii, Psa. issachenkonii, Psa. rubra, Psa. translucida, and Psa. tunicata; and two Shewanella strains belonging to S. baltica and S. putrefaciens were identified to have a weak to high proteolytic activity (from 478 to 4445 mU/mg trypsin equivalent) against skim milk casein as protein source. Further chitinolytic activity screening based on these 20 proteolytic strains using colloidal chitin yielded five positive strains which were tested against three different chitin substrates in order to determine the various types of chitinases. Among the strains that can produce both proteases and chitinases, Psa. rubra DSM 6842T expressed not only the highest proteolytic activity (2558 mU/mg trypsin equivalent) but also the highest activity of exochitinases, specifically, ß-N-acetylglucosaminidase (6.33 mU/107 cfu) as well. We anticipate that this strain can be innovatively applied to the valorization of marine crustaceans side streams.

Genomic and Toxigenic Heterogeneity of Bacillus cereus sensu lato Isolated from Ready-to-Eat Foods and Powdered Milk in Day Care Centers in Colombia.

Bacillus cereus sensu lato (s.l.) is a group of bacteria commonly found in diverse environments, including foods, with potential to cause emesis and diarrhea. In Colombia, it is one of the main foodborne pathogens. The aim of this study was to determine the genomic and toxigenic heterogeneity of B. cereus s.l. isolated from ready-to-eat foods and powdered milk collected in day care centers of Medellin, Colombia. Of 112 B. cereus s.l. isolates obtained, 94% were ß-hemolytic. Toxigenic heterogeneity was established by the presence of nheABC, hblCDAB, cytK2, entFM, and cesB toxigenic genes. The nheABC operon and entFM gene were most frequently detected in the isolates, whereas the cesB gene was not found. According to the toxin genes content, nine toxigenic profiles were identified. A 44% of isolates had profiles with all genes for nonhemolytic enterotoxin, hemolysin BL, and enterotoxin FM production (profiles II and IV). Pulsed-field gel electrophoresis analysis indicated a high genomic heterogeneity among the B. cereus s.l., with 68 isolates grouping into 16 clusters and 33 placed separately in the dendrogram. This study provides useful information on the safety of ready-to-eat foods and powdered milk in day care centers where children, a susceptible population, are exposed and it should incentive for more studies to understand the distribution of different toxin-encoding genes among B. cereus s.l. isolates, enabling detailed risk assessment.

Occurrence and characterisation of biofilms in drinking water systems of broiler houses.

BACKGROUND: Water quality in the drinking water system (DWS) plays an important role in the general health and performance of broiler chickens. Conditions in the DWS of broilers are ideal for microbial biofilm formation. Since pathogens might reside within these biofilms, they serve as potential source of waterborne transmission of pathogens to livestock and humans. Knowledge about the presence, importance and composition of biofilms in the DWS of broilers is largely missing. In this study, we therefore aim to monitor the occurrence, and chemically and microbiologically characterise biofilms in the DWS of five broiler farms. RESULTS: The bacterial load after disinfection in DWSs was assessed by sampling with a flocked swab followed by enumerations of total aerobic flora (TAC) and Pseudomonas spp. The dominant flora was identified and their biofilm-forming capacity was evaluated. Also, proteins, carbohydrates and uronic acids were quantified to analyse the presence of extracellular polymeric substances of biofilms. Despite disinfection of the water and the DWS, average TAC was 6.03 ± 1.53 log CFU/20cm2. Enumerations for Pseudomonas spp. were on average 0.88 log CFU/20cm2 lower. The most identified dominant species from TAC were Stenotrophomonas maltophilia, Pseudomonas geniculata and Pseudomonas aeruginosa. However at species level, most of the identified microorganisms were farm specific. Almost all the isolates belonging to the three most abundant species were strong biofilm producers. Overall, 92% of all tested microorganisms were able to form biofilm under lab conditions. Furthermore, 63% of the DWS surfaces appeared to be contaminated with microorganisms combined with at least one of the analysed chemical components, which is indicative for the presence of biofilm. CONCLUSIONS: Stenotrophomonas maltophilia, Pseudomonas geniculata and Pseudomonas aeruginosa are considered as opportunistic pathogens and could consequently be a potential risk for animal health. Additionally, the biofilm-forming capacity of these organisms could promote attachment of other pathogens such as Campylobacter spp. and Salmonella spp.

Presence and fate of antibiotic residues, antibiotic resistance genes and zoonotic bacteria during biological swine manure treatment.

The presence and dissemination of antibiotic residues, antibiotic resistance genes and zoonotic bacteria in the environment is of growing concern worldwide. Manure management practices, such as biological removal of nitrogen from swine manure, may help to decrease levels of antibiotic residues, antibiotic resistance genes and zoonotic bacteria present in manure before fertilization, thereby reducing environmental contamination. Therefore, the aim of this study was to monitor the presence and fate of seven antibiotic residues (colistin, sulfadiazine, trimethoprim, doxycycline, oxytetracycline, ceftiofur and tylosin A), nine antibiotic resistance genes (tet(B), tet(L), tet(M), tet(O), tet(Q), tet(W), erm(B), erm(F) and sul2) and two zoonotic bacteria (Salmonella Typhimurium and Campylobacter coli) during biological nitrogen removal from swine manure over time. Samples from the raw manure, the solid fraction, the liquid fraction and the storage lagoon were analyzed on two farms at six time points with an interval of two weeks. Only the antibiotics which were used during the three months preceding the first sampling could be detected before and after biological nitrogen removal from swine manure. Of all the antibiotics studied, doxycycline was recovered in all of the samples and sulfadiazine was recovered in most samples on both farms. For both antibiotics, there appears to be a reduction of the amount of residues present in the storage lagoon compared to the liquid fraction, however, this reduction was not statistically significant. A significant reduction of the relative abundances of most of the antibiotic resistance genes studied was observed when comparing the liquid fraction and the storage lagoon. For tet(L), no differences were observed between the fractions sampled and for sul2 and erm(F), a significant increase in relative abundances was observed on the second farm sampled. For the zoonotic bacteria, a reduction of at least 1 log was observed after biological nitrogen removal from swine manure. The results indicate that the concentration of certain antibiotic residues and several antibiotic resistance genes and the amount of zoonotic bacteria present in the manure may be reduced in the end product of the biological nitrogen removal from swine manure.

Bacillus cereus NVH 0500/00 Can Adhere to Mucin but Cannot Produce Enterotoxins during Gastrointestinal Simulation.

Adhesion to the intestinal epithelium could constitute an essential mechanism of Bacillus cereus pathogenesis. However, the enterocytes are protected by mucus, a secretion composed mainly of mucin glycoproteins. These may serve as nutrients and sites of adhesion for intestinal bacteria. In this study, the food poisoning bacterium B. cereus NVH 0500/00 was exposed in vitro to gastrointestinal hurdles prior to evaluation of its attachment to mucin microcosms and its ability to produce nonhemolytic enterotoxin (Nhe). The persistence of mucin-adherent B. cereus after simulated gut emptying was determined using a mucin adhesion assay. The stability of Nhe toward bile and pancreatin (intestinal components) in the presence of mucin agar was also investigated. B. cereus could grow and simultaneously adhere to mucin during in vitro ileal incubation, despite the adverse effect of prior exposure to a low pH or intestinal components. The final concentration of B. cereus in the simulated lumen at 8 h of incubation was 6.62 ± 0.87 log CFU ml(-1). At that point, the percentage of adhesion was approximately 6%. No enterotoxin was detected in the ileum, due to either insufficient bacterial concentrations or Nhe degradation. Nevertheless, mucin appears to retain B. cereus and to supply it to the small intestine after simulated gut emptying. Additionally, mucin may play a role in the protection of enterotoxins from degradation by intestinal components.

Residues of chlortetracycline, doxycycline and sulfadiazine-trimethoprim in intestinal content and feces of pigs due to cross-contamination of feed.

BACKGROUND: Cross-contamination of feed with low concentrations of antimicrobials can occur at production, transport and/or farm level. Concerns are rising about possible effects of this contaminated feed on resistance selection in the intestinal microbiota. Therefore, an experiment with pigs was set up, in which intestinal and fecal concentrations of chlortetracycline (CTC), doxycycline (DOX) and sulfadiazine-trimethoprim (SDZ-TRIM) were determined after administration of feed containing a 3 % carry-over level of these antimicrobials. RESULTS: The poor oral bioavailability of tetracyclines resulted in rather high concentrations in cecal and colonic content and feces at steady-state conditions. A mean concentration of 10 mg/kg CTC and 4 mg/kg DOX in the feces was reached, which is higher than concentrations that were shown to cause resistance selection. On the other hand, lower mean levels of SDZ (0.7 mg/kg) and TRIM (< limit of detection of 0.016 mg/kg) were found in the feces, corresponding with the high oral bioavailability of SDZ and TRIM in pigs. CONCLUSIONS: The relation between the oral bioavailability and intestinal concentrations of the tested antimicrobials, may be of help in assessing the risks of cross-contaminated feed. However, future research is needed to confirm our results and to evaluate the effects of these detected concentrations on resistance selection in the intestinal microbiota of pigs.

Genetic diversity of Shiga toxin-producing Escherichia coli O157 : H7 recovered from human and food sources.

The aim of this study was to identify an epidemiological association between Shiga toxin-producing Escherichia coli O157 : H7 strains associated with human infection and with food sources. Frequency distributions of different genetic markers of E. coli O157 : H7 strains recovered from human and food sources were compared using molecular assays to identify E. coli O157 : H7 genotypes associated with variation in pathogenic potential and host specificity. Genotypic characterization included: lineage-specific polymorphism assay (LSPA-6), clade typing, tir (A255T) polymorphism, Shiga toxin-encoding bacteriophage insertion site analysis and variant analysis of Shiga toxin 2 gene (stx2a and stx2c) and antiterminator Q genes (Q933 and Q21). The intermediate lineage (LI/II) dominated among both food and human strains. Compared to other clades, clades 7 and 8 were more frequent among food and human strains, respectively. The tir (255T) polymorphism occurred more frequently among human strains than food strains. Q21 and Q933 + Q21 were found at significantly higher frequencies among food and human strains, respectively. Moreover, stx2a and stx2a+c were detected at significantly higher frequencies among human strains compared to food strains. Bivariate analysis revealed significant concordance (P<0.05) between the LSPA-6 assay and the other typing methods. Multivariable regression analysis suggested that tir (255T) was the most distinctive genotype that can be used to detect bacterial clones with potential risk for human illness from food sources. This study supported previous reports of the existence of diversity in genetic markers among different isolation sources by including E. coli O157 : H7 strains from both food and human sources. This might enable tracking genotypes with potential risk for human illness from food sources.

Bacillus cereus Adhesion to Simulated Intestinal Mucus Is Determined by Its Growth on Mucin, Rather Than Intestinal Environmental Parameters.

Adhesion of pathogenic bacteria to intestinal mucus, the protective layer of the gastrointestinal epithelium, is often considered a virulence factor. The ability of food-poisoning Bacillus cereus strains to attach to mucus and the factors affecting this interaction have not yet been investigated. Therefore, the role of adhesion in pathogenesis of B. cereus still remains unknown. In the present study, an in vitro assay based on mucin agar was used to simulate adhesion of B. cereus to mucus. Bacterial-associated factors (e.g., strain specificity and microbial competition) known to influence adhesion to different surfaces and a variety of environmental conditions (e.g., pH and oxygen) encountered in the gastrointestinal tract were investigated. The effect of these parameters on B. cereus NVH 0500/00 mucin adhesion was generally limited even in the presence of microbial competition. This suggests that B. cereus NVH 0500/00 is a versatile pathogen. Inoculation of 4 to 5 log colony-forming units (CFU) per milliliter. B. cereus NVH 0500/00 resulted in 5-6 log CFU/mL mucin-associated bacteria after a short incubation period. This indicates that this pathogenic strain could grow in the presence of mucin agar. This growth may potentially mask the effect of the studied conditions. Yet, extensive attachment of B. cereus to mucin is not necessarily a prerequisite for virulence, because other pathogenic strains do not adhere with the same efficiency to mucin. Nevertheless, adhesion may contribute to the disease by providing close contact to nutrient sources, such as mucin, which would not only result in bacterial proliferation, but also in disruption of the protective host mucus surface.

Thermotolerant Campylobacter during Broiler Rearing: Risk Factors and Intervention.

Thermotolerant Campylobacters are one of the most important bacterial causative agents of human gastrointestinal illness worldwide. In most European Union (EU) member states human campylobacteriosis is mainly caused by infection with Campylobacter jejuni or Campylobacter coli following consumption or inadequate handling of Campylobacter-contaminated poultry meat. To date, no effective strategy to control Campylobacter colonization of broilers during rearing is available. In this review, we describe the public health problem posed by Campylobacter presence in broilers and list and critically review all currently known measures that have been researched to lower the numbers of Campylobacter bacteria in broilers during rearing. We also discuss the most promising measures and which measures should be investigated further. We end this review by elaborating on readily usable measures to lower Campylobacter introduction and Campylobacter numbers in a broiler flock.

Genetic diversity of livestock-associated MRSA isolates obtained from piglets from farrowing until slaughter age on four farrow-to-finish farms.

During a previous longitudinal study, performed on four farrow-to-finish farms (A to D), samples were taken from twelve sows, their offspring, and the environment on various occasions over six months to study the MRSA presence. During the present study, a selection of the obtained MRSA isolates were typed by multiple-locus variable-number tandem-repeat analysis (MLVA), Pulsed Field Gel Electrophoresis (PFGE), spa typing, and SCCmec typing to study the genetic diversity of LA-MRSA isolates and to determine possible MRSA sources for pig(let)s. PFGE, spa typing, and SCCmec typing revealed the presence of one or few dominant genotype(s) per farm. In contrast, 212 MLVA types were detected on the four farms, forming one cluster on farm A, three on farm B, four on farm C and two on farm D. The genotype, found on farm A was unique for this farm. Farms B, C and D shared one cluster. In general, MLVA types from these clusters were isolated from piglets, sows, and the environment on various sampling events. Piglets carried MLVA types both related and unrelated to their mother sows' MLVA types at farrowing and onwards. In conclusion, molecular typing revealed that within a farm one or a few dominant strain(s) are widespread. Potential MRSA sources for piglets were mother sows, the environment and other piglets.

Passive immunization to reduce Campylobacter jejuni colonization and transmission in broiler chickens.

Campylobacter jejuni is the most common cause of bacterium-mediated diarrheal disease in humans worldwide. Poultry products are considered the most important source of C. jejuni infections in humans but to date no effective strategy exists to eradicate this zoonotic pathogen from poultry production. Here, the potential use of passive immunization to reduce Campylobacter colonization in broiler chicks was examined. For this purpose, laying hens were immunized with either a whole-cell lysate or the hydrophobic protein fraction of C. jejuni and their eggs were collected. In vitro tests validated the induction of specific ImmunoglobulinY (IgY) against C. jejuni in the immunized hens' egg yolks, in particular. In seeder experiments, preventive administration of hyperimmune egg yolk significantly (P < 0.01) reduced bacterial counts of seeder animals three days after oral inoculation with approximately 104 cfu C. jejuni, compared with control birds. Moreover, transmission to non-seeder birds was dramatically reduced (hydrophobic protein fraction) or even completely prevented (whole-cell lysate). Purified IgY promoted bacterial binding to chicken intestinal mucus, suggesting enhanced mucosal clearance in vivo. Western blot analysis in combination with mass spectrometry after two-dimensional gel-electrophoresis revealed immunodominant antigens of C. jejuni that are involved in a variety of cell functions, including chemotaxis and adhesion. Some of these (AtpA, EF-Tu, GroEL and CtpA) are highly conserved proteins and could be promising targets for the development of subunit vaccines.

Interspecies interactions in dairy biofilms drive community structure and response against cleaning and disinfection.

Interspecies interactions within a biofilm community influence population dynamics and community structure, which in turn may affect the bacterial stress response to antimicrobials. This study was conducted to assess the impact of interactions between Kocuria salsicia and a three-species biofilm community (comprising Stenotrophomonas rhizophila, Bacillus licheniformis, and Microbacterium lacticum) on biofilm mass, the abundance of individual species, and their survival under a laboratory-scale cleaning and disinfection (C&D) regime. The presence of K. salsicia enhanced the cell numbers of all three species in pairwise interactions. The outcomes derived from summing up pairwise interactions did not accurately predict the bacterial population dynamics within communities of more than two species. In four-species biofilms, we observed the dominance of S. rhizophila and B. licheniformis, alongside a concurrent reduction in the cell counts of K. salsicia and M. lacticum. This pattern suggests that the underlying interactions are not purely non-transitive; instead, a more complex interplay results in the dominance of specific species. We observed that bacterial spatial organization and matrix production in different mixed-species combinations affected survival in response to C&D. Confocal microscopy analysis of spatial organization showed that S. rhizophila localized on the biofilm formed by B. licheniformis and M. lacticum, and S. rhizophila was more susceptible to C&D. Matrix production in B. licheniformis, evidenced by alterations in biofilm mass and by scanning electron microscopy, demonstrated its protective role against C&D, not only for this species itself, but also for neighbouring species. Our findings emphasise that various social interactions within a biofilm community not only affect bacterial population dynamics but also influence the biofilm community's response to C&D stress.

Detection of antibiotic residues in groundwater with a validated multiresidue UHPLC-MS/MS quantification method.

The occurrence of antibiotic residues in the environment has received considerable attention because of their potential to select for bacterial resistance. The overuse of antibiotics in human medicine and animal production results in antibiotic residues entering the aquatic environment, but concentrations are currently not well determined. This study investigates the occurrence of antibiotics in groundwater in areas strongly related to agriculture and the antibiotic treatment of animals. A multiresidue method was validated according to EU Regulation 2021/808, to allow (semi-)quantitative analysis of 78 antibiotics from 10 different classes: ß-lactams, sulfonamides, tetracyclines, lincosamides, amphenicols, (fluoro)quinolones, macrolides, pleuromutilins, ansamycins and diaminopyrimidines using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). This method was used to test different storage conditions of these water samples during a stability study over a period of 2 weeks. Sulfonamides, lincosamides and pleuromutilins were the most stable. Degradation was most pronounced for ß-lactam antibiotics, macrolides and ansamycins. To maintain stability, storage of samples at -18 °C is preferred. With the validated method, antibiotic residues were detected in groundwater, sampled from regions associated with intensive livestock farming in Flanders (Belgium). Out of 50 samples, 14% contained at least one residue. Concentrations were low, ranging from < LOD to 0.03 µg/L. Chloramphenicol, oxolinic acid, tetracycline and sulfonamides (sulfadiazine, sulfadoxine, sulfamethazine and sulfisoxazole) were detected. This study presents a new method for the quantification of antibiotic residues, which was applied to investigate the presence of antibiotic residues in groundwater in Flanders.

Rapid evolutionary tuning of endospore quantity versus quality trade-off via a phase-variable contingency locus.

The UV resistance of bacterial endospores is an important quality supporting their survival in inhospitable environments and therefore constitutes an essential driver of the ecological success of spore-forming bacteria. Nevertheless, the variability and evolvability of this trait are poorly understood. In this study, directed evolution and genetics approaches revealed that the Bacillus cereus pdaA gene (encoding the endospore-specific peptidoglycan-N-acetylmuramic acid deacetylase) serves as a contingency locus in which the expansion and contraction of short tandem repeats can readily compromise (PdaAOFF) or restore (PdaAON) the pdaA open reading frame. Compared with B. cereus populations in the PdaAON state, populations in the PdaAOFF state produced a lower yield of viable endospores but endowed them with vastly increased UV resistance. Moreover, selection pressures based on either quantity (i.e., yield of viable endospores) or quality (i.e., UV resistance of viable endospores) aspects could readily shift populations between PdaAON and PdaAOFF states, respectively. Bioinformatic analysis also revealed that pdaA homologs within the Bacillus and Clostridium genera are often equipped with several short tandem repeat regions, suggesting a wider implementation of the pdaA-mediated phase variability in other sporeformers as well. These results for the first time reveal (1) pdaA as a phase-variable contingency locus in the adaptive evolution of endospore properties and (2) bet-hedging between what appears to be a quantity versus quality trade-off in endospore crops.

Mechanistic insights into the adaptive evolvability of spore heat resistance in Bacillus cereus sensu lato.

Wet heat treatment is a commonly applied method in the food and medical industries for the inactivation of microorganisms, and bacterial spores in particular. While many studies have delved into the mechanisms underlying wet heat killing and spore resistance, little attention has so far been dedicated to the capacity of spore-forming bacteria to tune their resistance through adaptive evolution. Nevertheless, a recent study from our group revealed that a psychrotrophic strain of the Bacillus cereus sensu lato group (i.e. Bacillus weihenstephanensis LMG 18989) could readily and reproducibly evolve to acquire enhanced spore wet heat resistance without compromising its vegetative cell growth ability at low temperatures. In the current study, we demonstrate that another B. cereus strain (i.e. the mesophilic B. cereus sensu stricto ATCC 14579) can acquire significantly increased spore wet heat resistance as well, and we subjected both the previously and currently obtained mutants to whole genome sequencing. This revealed that five out of six mutants were affected in genes encoding regulators of the spore coat and exosporium pathway (i.e. spoIVFB, sigK and gerE), with three of them being affected in gerE. A synthetically constructed ATCC 14579 ΔgerE mutant likewise yielded spores with increased wet heat resistance, and incurred a compromised spore coat and exosporium. Further investigation revealed significantly increased spore DPA levels and core dehydration as the likely causes for the observed enhanced spore wet heat resistance. Interestingly, deletion of gerE in Bacillus subtilis 168 did not impose increased spore wet heat resistance, underscoring potentially different adaptive evolutionary paths in B. cereus and B. subtilis.

Molecular identification of the microbiota of peeled and unpeeled brown shrimp (Crangon crangon) during storage on ice and at 7.5 °C.

The dominant microbiota of brown shrimp (Crangon crangon) were systematically identified during storage under different conditions. Freshly caught shrimp were processed on board the fishing vessel under the best possible hygienic conditions (IDEAL), unpeeled and manually (sterile) peeled, then stored on ice and at 7.5 °C until microbiologically spoiled. Results were compared with industrially processed (INDUSTRIAL) shrimp. Isolates grown on various media were identified by 16S rRNA and gyrB gene sequencing. We examined the total microbiota and microbial population shifts of shrimp under various storage conditions using denaturant gradient gel electrophoresis (DGGE). The microbiota differed somewhat during storage and among the various storage conditions; however, members of the genera Psychrobacter and Pseudoalteromonas were found to dominate the microbiota of all shrimp samples regardless of processing procedures or storage conditions. Most isolates could be identified by gyrB gene sequencing as Psychrobacter immobilis or Psychrobacter cibarius. Also Pseudoalteromonas nigrifaciens, Pseudoalteromonas elyakovii or Pseudoalteromonas paragorgicola dominated the microbiota of brown shrimp during storage. Also species from the genera Planocuccus, Exiguobacterium, Carnobacterium, Pseudomonas, Chryseobacterium and Staphylococcus were detected during storage of brown shrimp. Culture-dependent and culture-independent DGGE analysis produced different results in band patterns. Both methods are therefore required to accurately identify the microbiota and bacterial population shifts on seafood during storage.

Survival of enteric pathogens during butterhead lettuce growth: crop stage, leaf age, and irrigation.

The survival of Salmonella enterica serovar Thompson and Escherichia coli O157 was investigated on growing butterhead lettuce plants in the plant-growth chamber and greenhouse. All inoculation tests were made under conditions that approximate the greenhouse conditions for butterhead lettuce cultivation in Flanders (Belgium). The survival and proliferation of the pathogens on the leaves was determined at days 0, 4, and 8 after inoculation using standard plating techniques on selective medium. In the growth chamber, the extent to which both pathogens were able to multiply on the lettuce leaves was influenced by crop stage and leaf age. On young plants, the older leaves supported pathogen survival better. On nearly mature plants, pathogen population sizes were significantly higher on the old and young leaves compared with middle-aged leaves (p<0.001). In the greenhouse, the environmental regimen with high fluctuations in temperature and relative humidity was less conducive to the survival of E. coli O157, though its survival on nearly mature lettuce was enhanced by overhead irrigation. The moist conditions between the folded inner leaves are likely contributing to the survival of enteric pathogens in the lettuce head. Butterhead lettuce grown in greenhouses with a sprinkle irrigation system may present a potential health hazard when contaminated near harvest. Experimental design (growth chamber versus greenhouse) largely influences enteric pathogen behavior on growing lettuce plants.