Population dynamics of lactobacilli in Grana cheese

A survey on the presence, microbial diversity, and population dynamics of lactobacilli in Grana cheese is presented. Evolution of thermophilic rod lactic acid bacteria within the first two days from cheese making and during ripening was different according to different bacterial groups, which were selectively enumerated and identified by molecular methods. Species-specific microbial counts indicated prevalence ofLactobacillus helveticus in both the whey starter and the cheese at moulding, and ofLactobacillus delbrueckii subsp.lactis in cheese after two months of ripening. In more advanced ripening, a decrease of total thermophilic lactobacilli and an increase of mesophilic lactobacilli (mostly belonging toLactobacillus casei/paracasei andLactobacillus rhamnosus) was observed. PCR fingerprinting of lactobacilli, which was performed by PCR-fingerprinting, indicated a marked microbial heterogeneity within theLactobacillus spp. populations, which enabled strain (or group)-specific fingerprints to be observed.     

Microbial Activation of Wooden Vats Used for Traditional Cheese Production and Evolution of Neoformed Biofilms

Three Lactococcus lactis subsp. cremoris strains were used to develop ad hoc biofilms on the surfaces of virgin wooden vats used for cheese production. Two vats (TZ) were tested under controlled conditions (pilot plant), and two vats (TA) were tested under uncontrolled conditions (industrial plant). In each plant, one vat (TA1 and TZ1) was used for the control, traditional production of PDO Vastedda della Valle del Belìce (Vastedda) cheese, and one (TA2 and TZ2) was used for experimental production performed after lactococcal biofilm activation and the daily addition of a natural whey starter culture (NWSC). Microbiological and scanning electron microscopy analyses showed differences in terms of microbial levels and composition of the neoformed biofilms. The levels of the microbial groups investigated during cheese production showed significant differences between the control trials and between the control and experimental trials, but the differences were not particularly marked between the TA2 and TZ2 productions, which showed the largest numbers of mesophilic lactic acid bacterium (LAB) cocci. LAB populations were characterized phenotypically and genotypically, and 44 dominant strains belonging to 10 species were identified. Direct comparison of the polymorphic profiles of the LAB collected during cheese making showed that the addition of the NWSC reduced their biodiversity. Sensory evaluation showed that the microbial activation of the wooden vats with the multistrain Lactococcus culture generated cheeses with sensory attributes comparable to those of commercial cheese. Thus, neoformed biofilms enable a reduction of microbial variability and stabilize the sensorial attributes of Vastedda cheese.     

Effect of Antimicrobial Dosage Regimen on Salmonella and Escherichia coli Isolates from Feeder Swine

A body of evidence exists that suggests that antimicrobial use in food animals leads to resistance in both pathogenic and commensal bacteria. This study focused on the impact of three different antimicrobial regimes (low-level continuous, pulse, and no antimicrobial) for two antimicrobials (chlortetracycline and tylosin) on the presence of Salmonella spp. and on the prevalence of antimicrobial resistance of both Salmonella spp. and nonspecific Escherichia coli in fecal samples from feeder swine. The prevalence of fecal samples positive for Salmonella spp. significantly decreased between the samples taken at feeder placement compared to samples taken when the animals were close to market weight. Differences in resistance of Salmonella spp. did not appear to be influenced by dosing treatment including the control. Analysis of antimicrobial resistance examining both susceptibility and resistance, as well as MIC outcomes, demonstrated that only resistance to cephalothin increased in E. coli under the pulse chlortetracycline treatment. These results suggest that the dosing regimes examined in this study did not lead to an increase in either the prevalence of Salmonella spp. or the prevalence of antimicrobial resistance in isolates of Salmonella spp. or E. coli.     

Causal Relationship between Microbial Ecology Dynamics and Proteolysis during Manufacture and Ripening of Protected Designation of Origin (PDO) Cheese Canestrato Pugliese

Pyrosequencing of the 16S rRNA gene, community-level physiological profiles determined by the use of Biolog EcoPlates, and proteolysis analyses were used to characterize Canestrato Pugliese Protected Designation of Origin (PDO) cheese. The number of presumptive mesophilic lactococci in raw ewes'' milk was higher than that of presumptive mesophilic lactobacilli. The numbers of these microbial groups increased during ripening, showing temporal and numerical differences. Urea-PAGE showed limited primary proteolysis, whereas the analysis of the pH 4.6-soluble fraction of the cheese revealed that secondary proteolysis increased mainly from 45 to 75 days of ripening. This agreed with the concentration of free amino acids. Raw ewes'' milk was contaminated by several bacterial phyla: Proteobacteria (68%; mainly Pseudomonas), Firmicutes (30%; mainly Carnobacterium and Lactococcus), Bacteroidetes (0.05%), and Actinobacteria (0.02%). Almost the same microbial composition persisted in the curd after molding. From day 1 of ripening onwards, the phylum Firmicutes dominated. Lactococcus dominated throughout ripening, and most of the Lactobacillus species appeared only at 7 or 15 days. At 90 days, Lactococcus (87.2%), Lactobacillus (4.8%; mainly Lactobacillus plantarum and Lactobacillus sakei), and Leuconostoc (3.9%) dominated. The relative utilization of carbon sources by the bacterial community reflected the succession. This study identified strategic phases that characterized the manufacture and ripening of Canestrato Pugliese cheese and established a causal relationship between mesophilic lactobacilli and proteolysis.     

Dynamics of Bacterial Communities during the Ripening Process of Different Croatian Cheese Types Derived from Raw Ewe's Milk Cheeses

Microbial communities play an important role in cheese ripening and determine the flavor and taste of different cheese types to a large extent. However, under adverse conditions human pathogens may colonize cheese samples during ripening and may thus cause severe outbreaks of diarrhoea and other diseases. Therefore in the present study we investigated the bacterial community structure of three raw ewe''s milk cheese types, which are produced without the application of starter cultures during ripening from two production sites based on fingerprinting in combination with next generation sequencing of 16S rRNA gene amplicons. Overall a surprisingly high diversity was found in the analyzed samples and overall up to 213 OTU₉₇ could be assigned. 20 of the major OTUs were present in all samples and include mostly lactic acid bacteria (LAB), mainly Lactococcus, and Enterococcus species. Abundance and diversity of these genera differed to a large extent between the 3 investigated cheese types and in response to the ripening process. Also a large number of non LAB genera could be identified based on phylogenetic alignments including mainly Enterobacteriaceae and Staphylococcacae. Some species belonging to these two families could be clearly assigned to species which are known as potential human pathogens like Staphylococcus saprophyticus or Salmonella spp. However, during cheese ripening their abundance was reduced. The bacterial genera, namely Lactobacillus, Streptococcus, Leuconostoc, Bifidobacterium, Brevibacterium, Corynebacterium, Clostridium, Staphylococcus, Thermoanerobacterium, E. coli, Hafnia, Pseudomonas, Janthinobacterium, Petrotoga, Kosmotoga, Megasphaera, Macrococcus, Mannheimia, Aerococcus, Vagococcus, Weissella and Pediococcus were identified at a relative low level and only in selected samples. Overall the microbial composition of the used milk and the management of the production units determined the bacterial community composition for all cheese types to a large extend, also at the late time points of cheese ripening.     

Physical Covering for Control of Escherichia coli O157:H7 and Salmonella spp. in Static and Windrow Composting Processes

This study investigated the effect of a 30-cm covering of finished compost (FC) on survival of Escherichia coli O157:H7 and Salmonella spp. in active static and windrow composting systems. Feedstocks inoculated with E. coli O157:H7 (7.41 log CFU/g) and Salmonella (6.46 log CFU/g) were placed in biosentry tubes (7.5-cm diameter, 30-cm height) at three locations: (i and ii) two opposing sides at the interface between the FC cover layer (where present) and the feedstock material (each positioned approximately 10 cm below the pile''s surface) and (iii) an internal location (top) (approximately 30 cm below the surface). On specific sampling days, surviving populations of inoculated E. coli O157:H7 and Salmonella, generic E. coli, and coliforms in compost samples were determined. Salmonella spp. were reduced significantly within 24 h in windrow piles and were below the detection limit after 3 and 7 days at internal locations of windrow and static piles containing FC covering, respectively. Likewise, E. coli O157:H7 was undetectable after 1 day in windrow piles covered with finished compost. Use of FC as a covering layer significantly increased the number of days that temperatures in the windrows remained ≥55°C at all locations and in static piles at internal locations. These time-temperature exposures resulted in rapid reduction of inoculated pathogens, and the rate of bacterial reduction was rapid in windrow piles. The sample location significantly influenced the survival of these pathogens at internal locations compared to that at interface locations of piles. Finished compost covering of compost piles aids in the reduction of pathogens during the composting process.     

Environmental Factors Associated with High Fly Densities and Diarrhea in Vellore,India

Diarrhea causes significant morbidity and mortality in Indian children under 5 years of age. Flies carry enteric pathogens and may mediate foodborne infections. In this study, we characterized fly densities as a determinant of infectious diarrhea in a longitudinal cohort of 160 urban and 80 rural households with 1,274 individuals (27% under 5 years of age) in Vellore, India. Household questionnaires on living conditions were completed at enrollment. Fly abundance was measured during the wet and dry seasons using fly ribbons placed in kitchens. PCRs for enteric bacteria, viruses, and protozoa were performed on 60 fly samples. Forty-three (72%) fly samples were positive for the following pathogens: norovirus (50%), Salmonella spp. (46.7%), rotavirus (6.7%), and Escherichia coli (6.7%). Ninety-one episodes of diarrhea occurred (89% in children under 5 years of age). Stool pathogens isolated in 24 of 77 (31%) samples included E. coli, Shigella spp., Vibrio spp., Giardia, Cryptosporidium, and rotavirus. Multivariate log-linear models were used to explore the relationships between diarrhea and fly densities, controlling for demographics, hygiene, and human-animal interactions. Fly abundance was 6 times higher in rural than urban sites (P < 0.0001). Disposal of garbage close to homes and rural living were significant risk factors for high fly densities. The presence of latrines was protective against high fly densities and diarrhea. The adjusted relative risks of diarrheal episodes and duration of diarrhea, associated with fly density at the 75th percentile, were 1.18 (95% confidence interval [CI], 1.03 to 1.34) and 1.15 (95% CI, 1.02 to 1.29), respectively. Flies harbored enteric pathogens, including norovirus, a poorly documented pathogen on flies.     

Long-Term Monitoring of Waterborne Pathogens and Microbial Source Tracking Markers in Paired Agricultural Watersheds under Controlled and Conventional Tile Drainage Management

Surface waters from paired agricultural watersheds under controlled tile drainage (CTD) and uncontrolled tile drainage (UCTD) were monitored over 7 years in order to determine if there was an effect of CTD (imposed during the growing season) on occurrences and loadings of bacterial and viral pathogens, coliphages, and microbial source tracking markers. There were significantly lower occurrences of human, ruminant, and livestock (ruminant plus pig) Bacteroidales markers in the CTD watershed in relation to the UCTD watershed. As for pathogens, there were significantly lower occurrences of Salmonella spp. and Arcobacter spp. in the CTD watershed. There were no instances where there were significantly higher quantitative loadings of any microbial target in the CTD watershed, except for F-specific DNA (F-DNA) and F-RNA coliphages, perhaps as a result of fecal inputs from a hobby farm independent of the drainage practice treatments. There was lower loading of the ruminant marker in the CTD watershed in relation to the UCTD system, and results were significant at the level P = 0.06. The odds of Salmonella spp. occurring increased when a ruminant marker was present relative to when the ruminant marker was absent, yet for Arcobacter spp., the odds of this pathogen occurring significantly decreased when a ruminant marker was present relative to when the ruminant marker was absent (but increased when a wildlife marker was present relative to when the wildlife marker was absent). Interestingly, the odds of norovirus GII (associated with human and swine) occurring in water increased significantly when a ruminant marker was present relative to when a ruminant marker was absent. Overall, this study suggests that fecal pollution from tile-drained fields to stream could be reduced by CTD utilization.     

Multiplex PCR and DNA array for the detection of Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. targeting virulence-related genes

A multiplex PCR and DNA array for quick detection of Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. was developed using specific genetic markers derived from virulence-related genes. The genetic markers of cytK, sei, prfA, rfB, and hilA gene specifically amplified DNA fragments of 320 bp, 500 bp, 700 bp, 1.0 kb and 1.2 kb from B. cereus, S. aureus, L. monocytogenes, E. coli O157:H7, and Salmonella spp., respectively. These markers are specific for the detection of the corresponding target pathogens. The sensitivity of the genetic markers was down to ～0.5 fg genomic DNA and ～10¹ CFU/ml (one bacterial cell per reaction) of bacterial culture. The combination of mPCR and DNA macroarray hybridization sensitively and specifically detected B. cereus, S. aureus, L. monocytogenes, E. coli O157:H7, and Salmonella spp., in complex mixed cultures and food matrices. Thus, this mPCR and macroarray-based approach serves as rapid and reliable diagnostic tool for the detection of these five pathogens.     

Prevalence and Characterization of Escherichia coli and Salmonella Strains Isolated from Stray Dog and Coyote Feces in a Major Leafy Greens Production Region at the United States-Mexico Border

In 2010, Romaine lettuce grown in southern Arizona was implicated in a multi-state outbreak of Escherichia coli O145:H28 infections. This was the first known Shiga toxin-producing E. coli (STEC) outbreak traced to the southwest desert leafy green vegetable production region along the United States-Mexico border. Limited information exists on sources of STEC and other enteric zoonotic pathogens in domestic and wild animals in this region. According to local vegetable growers, unleashed or stray domestic dogs and free-roaming coyotes are a significant problem due to intrusions into their crop fields. During the 2010–2011 leafy greens growing season, we conducted a prevalence survey of STEC and Salmonella presence in stray dog and coyote feces. Fresh fecal samples from impounded dogs and coyotes from lands near produce fields were collected and cultured using extended enrichment and serogroup-specific immunomagnetic separation (IMS) followed by serotyping, pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing. A total of 461 fecal samples were analyzed including 358 domestic dog and 103 coyote fecals. STEC was not detected, but atypical enteropathogenic E. coli (aEPEC) strains comprising 14 different serotypes were isolated from 13 (3.6%) dog and 5 (4.9%) coyote samples. Salmonella was cultured from 33 (9.2%) dog and 33 (32%) coyote samples comprising 29 serovars with 58% from dogs belonging to Senftenberg or Typhimurium. PFGE analysis revealed 17 aEPEC and 27 Salmonella distinct pulsotypes. Four (22.2%) of 18 aEPEC and 4 (6.1%) of 66 Salmonella isolates were resistant to two or more antibiotic classes. Our findings suggest that stray dogs and coyotes in the desert southwest may not be significant sources of STEC, but are potential reservoirs of other pathogenic E. coli and Salmonella. These results underscore the importance of good agriculture practices relating to mitigation of microbial risks from animal fecal deposits in the produce production area.     

The interaction of the antimicrobial peptide cLEAP-2 and the bacterial membrane

Chicken Liver Expressed Antimicrobial Peptide-2 (cLEAP-2) is known to have killing activities against Salmonella spp., but the mechanism by which killing occurs remains to be elucidated. The ability of cLEAP-2 to disrupt the outer membrane of several Salmonella spp. was assessed using the fluorescent probe N-phenyl-1-naphthylamine (NPN). A rapid dose-dependent permeabilization of the outer membranes of Salmonella enterica serovar Typhimurium phoP, and S. enterica serovar Typhimurium SL1344 was observed although no significant permeabilisation of the S. enteriditis membrane was detected. These data suggested that the ability of the mature cLEAP-2 peptide to permeabilise the Salmonella outer membrane is important in mediating its killing activities. The ability of the peptide to kill Gram-positive bacteria, specifically Streptococcus spp. and Staphylococcus spp. was also investigated using recombinant peptide and a time-kill assay. Of the strains analysed the Streptococcus pyogenes M1 strain appeared the most resistant to LEAP-2 killing although S. pyogenes mutants deficient in Sortase and M1 activities showed increased sensitivity to the mature peptide. This suggested the involvement of specific Streptococcus cell wall proteins including M1 in the resistance of the bacteria to cLEAP-2 killing. cLEAP-2 showed no significant toxicity towards mammalian erythrocytes indicating selectivity for bacterial over eukaryote cell membranes. These data provide further support for mature cLEAP-2 functioning in protecting the chicken against microbial attack.     

Antibiotic Resistance in Food-Borne Bacterial Contaminants in Vietnam

This study was conducted to examine the rate of contamination and the molecular characteristics of enteric bacteria isolated from a selection of food sources in Vietnam. One hundred eighty raw food samples were tested; 60.8% of meat samples and 18.0% of shellfish samples were contaminated with Salmonella spp., and more than 90% of all food sources contained Escherichia coli. The isolates were screened for antibiotic resistance against 15 antibiotics, and 50.5% of Salmonella isolates and 83.8% of E. coli isolates were resistant to at least one antibiotic. Isolates were examined for the presence of mobile genetic elements conferring antibiotic resistance. Fifty-seven percent of E. coli and 13% of Salmonella isolates were found to contain integrons, and some isolates contained two integrons. Sequencing results revealed that the integrons harbored various gene cassettes, including aadA1, aadA2, and aadA5 (resistance to streptomycin and spectinomycin), aacA4 (resistance to aminoglycosides), the dihydrofolate reductase gene cassettes dhfrXII, dfrA1, and dhfrA17 (trimethoprim resistance), the beta-lactamase gene bla_PSE1 (ampicillin resistance), and catB3 (chloramphenicol resistance). Plasmids were also detected in all 23 antibiotic-resistant Salmonella isolates and in 33 E. coli isolates. Thirty-five percent of the Salmonella isolates and 76% of the E. coli isolates contained plasmids of more than 95 kb, and some of the isolates contained two large plasmids. Conjugation experiments showed the successful transfer of all or part of the antibiotic resistance phenotypes among the Salmonella and E. coli food isolates. Our results show that enteric bacteria in raw food samples from Vietnam contain a pool of mobile genetic elements and that the transfer of antibiotic resistance can readily occur between similar bacteria.     

Development of antigen-delivery systems,based on the Escherichia coli hemolysin secretion pathway

We describe the development of plasmid vectors carrying the expression sites, an hlyA cassette and the secretion genes of Escherichia coli hemolysin. These allow the synthesis and secretion of heterologous microbial antigens in E. coli and attenuated Salmonella aroA strains. Genes or gene fragments encoding microbial antigens are inserted in-frame into a residual part of the hlyA gene which essentially encodes the HlyA secretion signal (HlyA₈). In general, the fused genes, carrying the hlyA_s sequence at the 3' terminus, are efficiently expressed, and the synthesized antigens are secreted into the culture supernatant of the producing strain. Attenuated Salmonella strains synthesizing either HlyA_s-fused listeriolysin or p60 of Listeria monocytogenes were constructed by this procedure and shown to provide protective immunity against L. monocytogenes in mice. The most effective protection was obtained when these microbial antigens were secreted by the attenuated Salmonella strains. We further present new approaches which may allow the application of this antigen-delivery system to any microbial antigen.     

Enumeration of Escherichia coli Cells on Chicken Carcasses as a Potential Measure of Microbial Process Control in a Random Selection of Slaughter Establishments in the United States

To evaluate whether the number of Escherichia coli bacteria in carcass rinses from chicken slaughter establishments could be monitored for the purpose of microbial process control, we drew a random sample from 20 of 127 large USDA-inspected operations. In 2005, every 3 months, two sets of 10 carcass rinses, 100 ml each, were collected from establishments, netting 80 sample sets from the rehang and postchill stages. E. coli and Campylobacter numbers and Salmonella prevalence were measured. Mixed-effect models were used to estimate variance of mean log₁₀ E. coli cell numbers of 10-carcass rinse sample sets. Relationships between E. coli and Campylobacter and Salmonella were examined. For 10-carcass rinse sets, at both the rehang and postchill stages the mean log₁₀ E. coli CFU/ml fit the logistic distribution better than the normal distribution. The rehang overall mean log₁₀ E. coli was 3.3 CFU/ml, with a within-sample set standard deviation of 0.6 CFU/ml. The overall postchill mean log₁₀ E. coli was 0.8 CFU/ml, with 13 establishments having mean log₁₀ E. coli CFU/ml values of less than 1.0 and 7 having mean values of 1.2 or more. At the midpoint separating these establishments, a mean log₁₀ E. coli CFU/ml of 1.1, the within-sample set standard deviation was 0.5 CFU/ml, with smaller standard deviations as means increased. Postchill sample sets with mean log₁₀ E. coli counts less than or equal to 1.1 CFU/ml had lower overall prevalence of Salmonella and mean log₁₀ Campylobacter CFU/ml than sample sets with higher means. These findings regarding reductions in E. coli numbers provide insight relevant to microbial process control.Regulatory food microbiology standards are defined and enforced with the intent of protecting public health and maintaining consumer confidence in the safety of the food supply. Resource demands (22) and legal constraints (21) have hindered the U.S. Department of Agriculture (USDA) from enforcing its current Salmonella performance standard (3). For this reason, in 2004 the USDA requested guidance from its national scientific advisory committee on the possible use of E. coli numbers to monitor sanitary conditions during poultry slaughter (12). The committee acknowledged that, if valid, such a performance standard could facilitate inspection of slaughter processing establishments. The committee recommended studies to define how E. coli numbers vary in poultry carcass rinses during poultry processing by processing stage, time of year, and geographic region and with respect to food-borne pathogens.The widespread presence and high numbers of generic E. coli bacteria on poultry entering the slaughter establishment (2, 5, 14) are suitable characteristics for an indicator organism used to monitor microbial control processes. The ease and lower cost (5, 13) of E. coli enumeration also allow more observations than can be made when comparable resources are allocated for Campylobacter or Salmonella testing (15).Regulatory agencies and food manufacturers have recognized the potential utility of E. coli numbers as a measure of slaughter process control. For example, USDA''s hazard analysis and critical control point rule (3) specifies two criteria for evaluating process control: establishments are to maintain less than 100 CFU of E. coli per ml in 80% of poultry carcass rinses and never exceed 1,000 CFU/ml. Surveys have been performed to define precise E. coli performance criteria for poultry (5), to monitor microbial reduction during slaughter processing (6), and to validate interventions to reduce microbial numbers on poultry (20).If generic E. coli numbers on poultry carcasses fit a parametric distribution, with a predictable mean and standard deviation, then carcasses could be monitored using a statistical process control plan. For example, if E. coli numbers decrease by an acceptable amount during processing to a reasonable level, then the process could be considered to be under control. Or a plan could be designed to monitor for acceptable occurrences of small, medium, and large deviations above a target E. coli number (7). If relationships were found between E. coli and Campylobacter numbers during chicken slaughter as well as Salmonella prevalence, they would further support the use of E. coli numbers as a measure of process control.This study of a random sample of 20 large chicken slaughter operations located throughout the United States measured microbial numbers at two processing line locations. Once a quarter, 10 carcass rinse samples were collected from both the post-feather-pick (rehang) and postchill locations. Rinses were examined to estimate mean Salmonella prevalence and E. coli and Campylobacter numbers by location within establishments. The primary objective was to assess whether the reduction in E. coli numbers between the rehang and postchill stages or numbers at the postchill location might have utility as a measure of process control during chicken slaughter. A related objective was to estimate values of parameters that could be used to design statistical process control plans (7).     

Alphitobius diaperinus Panzer (Insecta,Coleoptera) in a single house of a broiler production facility as a potential source of pathogenic bacteria for broilers and humans

Pest infestation in any stage can lead to a quality reduction in the finished products. This study aimed to detect Escherichia coli, Salmonella spp., Campylobacter spp., and Staphylococcus aureus in Alphitobius diaperinus adults, and in samples from broiler swabs, administered water and feed collected in a single house from a broiler production facility in central Italy. Three samplings were carried out, each collecting ninety adult beetles for microbial detection in the external, faecal and internal content; ten cloacal swab samples; and one sample of both administered feed and water. Microbiological cultures and biochemical identification were performed on suspected cultures and confirmed by species-specific PCRs. A. diaperinus was abundantly found near the windows, under the manger and in the corners of the facility. Salmonella enterica serovar Cholerasuis was found at the external surface of the beetles, while Staphylococcus xylosus and E. coli were in the faecal content. The latter micro-organism together with Staphylococcus lentus, S. xylosus and other staphylococcal species were detected in the internal microbiota. E. coli and Campylobacter spp. were observed in cloacal swabs, and S. xylosus in one feed sample. The study findings support evidence for Salmonella spp. and E. coli, and remark that adherence to sanitation rules and biosecurity procedures are required.     

Change Is Good: Variations in Common Biological Mechanisms in the Epsilonproteobacterial Genera Campylobacter and Helicobacter

Summary: Microbial evolution and subsequent species diversification enable bacterial organisms to perform common biological processes by a variety of means. The epsilonproteobacteria are a diverse class of prokaryotes that thrive in diverse habitats. Many of these environmental niches are labeled as extreme, whereas other niches include various sites within human, animal, and insect hosts. Some epsilonproteobacteria, such as Campylobacter jejuni and Helicobacter pylori, are common pathogens of humans that inhabit specific regions of the gastrointestinal tract. As such, the biological processes of pathogenic Campylobacter and Helicobacter spp. are often modeled after those of common enteric pathogens such as Salmonella spp. and Escherichia coli. While many exquisite biological mechanisms involving biochemical processes, genetic regulatory pathways, and pathogenesis of disease have been elucidated from studies of Salmonella spp. and E. coli, these paradigms often do not apply to the same processes in the epsilonproteobacteria. Instead, these bacteria often display extensive variation in common biological mechanisms relative to those of other prototypical bacteria. In this review, five biological processes of commonly studied model bacterial species are compared to those of the epsilonproteobacteria C. jejuni and H. pylori. Distinct differences in the processes of flagellar biosynthesis, DNA uptake and recombination, iron homeostasis, interaction with epithelial cells, and protein glycosylation are highlighted. Collectively, these studies support a broader view of the vast repertoire of biological mechanisms employed by bacteria and suggest that future studies of the epsilonproteobacteria will continue to provide novel and interesting information regarding prokaryotic cellular biology.     

Effects of Nondigestible Oligosaccharides on Salmonella enterica Serovar Typhimurium and Nonpathogenic Escherichia coli in the Pig Small Intestine In Vitro

An in vitro intestinal tissue model was developed for the investigation of bacterial association in the pig small intestine under different dietary regimes. In preliminary experiments, jejunal and ileal tissue was taken from Danish Landrace pigs fed standard diet and inoculated with either Salmonella or nonpathogenic Escherichia coli strains. Higher numbers of salmonellae associated with the ileal tissues, but the numbers did not reach significance. Hence, jejunal sections were inoculated with nonpathogenic E. coli and ileal sections were inoculated with salmonellae in the presence of mannose or commercial nondigestible oligosaccharides (NDO) at 2.5%. There was a significant decrease in E. coli associated with the jejunum in the presence of mannose (P < 0.05). Furthermore, in pigs fed a diet supplemented with commercial NDO at 4% there was a significant reduction in the numbers of E. coli in jejunal organ cultures of pigs fed the FOS diet (P < 0.05). There was a reduction, though not a significant one, in the association of Salmonella sp. to the ileal sections of pigs fed the commercial FOS diet. The feeding of commercial GOS or its addition to organ cultures did not affect E. coli or Salmonella numbers.     

The Microbial Diversity of Traditional Spontaneously Fermented Lambic Beer

Lambic sour beers are the products of a spontaneous fermentation that lasts for one to three years before bottling. The present study determined the microbiota involved in the fermentation of lambic beers by sampling two fermentation batches during two years in the most traditional lambic brewery of Belgium, using culture-dependent and culture-independent methods. From 14 samples per fermentation, over 2000 bacterial and yeast isolates were obtained and identified. Although minor variations in the microbiota between casks and batches and a considerable species diversity were found, a characteristic microbial succession was identified. This succession started with a dominance of Enterobacteriaceae in the first month, which were replaced at 2 months by Pediococcus damnosus and Saccharomyces spp., the latter being replaced by Dekkera bruxellensis at 6 months fermentation duration.     

Nonlethal Freezing Injury to Metabolism and Motility of Pseudomonas fluorescens and Escherichia coli

Some specific effects of the conditions of freezing on death and metabolic injury of a psychrophilic and a mesophilic species of bacteria were investigated, with particular respect to changes in motility. It was found that the extent of nonlethal metabolic freezing injury and the changes in motility are apparently correlated and are affected by the time and temperature of storage and the nature and pH of the suspending fluid. There were significant differences between the resistance to cold of the test organisms, the mesophilic being the more sensitive. The motility of the psychrophilic Pseudomonas fluorescens decreased considerably less after low-temperature exposure than did that of the mesophilic Escherichia coli. The percentage of restoration in motility of the subcultures was lower after freezing in water than in other suspending fluids.     

Temporal and Spatial Differences in Microbial Composition during the Manufacture of a Continental-Type Cheese

We sought to determine if the time, within a production day, that a cheese is manufactured has an influence on the microbial community present within that cheese. To facilitate this, 16S rRNA amplicon sequencing was used to elucidate the microbial community dynamics of brine-salted continental-type cheese in cheeses produced early and late in the production day. Differences in the microbial composition of the core and rind of the cheese were also investigated. Throughout ripening, it was apparent that cheeses produced late in the day had a more diverse microbial population than their early equivalents. Spatial variation between the cheese core and rind was also noted in that cheese rinds were initially found to have a more diverse microbial population but thereafter the opposite was the case. Interestingly, the genera Thermus, Pseudoalteromonas, and Bifidobacterium, not routinely associated with a continental-type cheese produced from pasteurized milk, were detected. The significance, if any, of the presence of these genera will require further attention. Ultimately, the use of high-throughput sequencing has facilitated a novel and detailed analysis of the temporal and spatial distribution of microbes in this complex cheese system and established that the period during a production cycle at which a cheese is manufactured can influence its microbial composition.