Establishing relative release kinetics of faecal indicator organisms from different faecal matrices

Aims: A laboratory assay for comparative characterization of various faecal matrices with respect to faecal indicator organism (FIO) release using, artificial rain water. Methods and Results: Fresh sheep and beef‐cattle faeces, dairy cattle slurry and beef cattle farm yard manure (FYM) were collected from commercial units in south‐west England and applied to 20 randomized 1 m² plots established on permanent grassland. Representative samples from each faecal matrix (n = 5) were collected on four occasions over 16 days. One gram of each sample was transferred to a sterile vial to which 9 ml of standard local rain was carefully pipetted. The vial was then rotated through 360°, 20 times in 60 s to ‘simulate’ a standardized interaction of the faecal material with rainfall, providing an assay of comparative release potential. Appropriate decimal dilutions were prepared from the eluent. Following agitation, with a sterile spatula, the remaining faecal material and eluent in the vials were vortex mixed for 60 s before decimal dilutions were prepared from the resulting mixture, providing a quantitative assessment of the total FIO in the sample from which percentage release could be determined. Bacterial concentrations were enumerated in duplicate by membrane filtration following standard methods for FIO. Significant differences in release kinetics of Escherichia coli and enterococci from each of the faecal matrices were determined. Conclusions: Differences in release from each faecal substrate and between FIO type (E. coli and intestinal enterococci) were observed in this laboratory study. The order of release of E. coli from the faecal matrices (greatest to least, expressed as a percentage of the total present) was dairy cattle slurry > beef cattle FYM > beef‐cattle faeces > sheep faeces. For intestinal enterococci the order of percentage release was dairy cattle slurry > beef‐cattle faeces > beef cattle FYM > sheep faeces. Significance and Impact of the Study: This laboratory‐based method provides the first data on the relative release kinetics of FIO from different faecal matrices in rain water. This is fundamental information needed to parameterize laboratory‐based microbial models and inform approaches to field and catchment risk assessment.     

rRNA‐based analysis to monitor succession of faecal bacterial communities in Holstein calves

Aims: To quantitatively analyse the faecal bacterial communities of Holstein calves and track their succession up to 12 weeks of age. Methods and Results: Faecal samples obtained from four female Holstein calves were analysed by the RNA‐based, sequence‐specific rRNA cleavage method. Twelve scissor probes covering major rumen bacterial groups were used, detecting c. 60–90% of the total 16S rRNAs. At 1 week of age, 16S rRNAs from members of the Bacteroides‐Prevotella group (40·0% of the total 16S rRNAs), Faecalibacterium (21·7%), the Clostridium coccoides–Eubacterium rectale group (16·7%) and the Atopobium cluster (10·9%) were detected at high levels. Throughout the 12‐week period, rRNAs of the Bacteroides‐Prevotella and the Cl. coccoides‐Eu. rectale groups constituted the major fraction of microbiota (c. 50–70% of the total). The relative abundances of the Atopobium cluster, Faecalibacterium, and some probiotic bacteria (such as those of the genera Lactobacillus and Bifidobacterium) decreased as the animal aged. Instead, an uncultivated rumen bacterial group, as well as Ruminococcus flavefaciens and Fibrobacter emerged at the detectable levels (1–2%) in the faeces sampled at a postweaning age. In addition, certain bacterial groups that were not covered by the probe suite increased as the animals aged. Conclusions: Young calves undergo dynamic changes in their intestinal bacterial community during the first 12 weeks of life. As young ruminants undergo metabolic and physiological development in their digestive tracts in the transition from a monogastric to a ruminant animal at an early age, the intestinal bacterial community may reflect such development. Significance and Impact of the Study: The succession of the bacterial communities in the faeces of calves was quantitatively monitored in the present study for the first time. The approach used here was demonstrated to be a useful means for determining the populations of predominant faecal bacterial groups in a variety of calf experiments in response to diet, stress and disease.     

Characterization of an extremely heat-resistant Escherichia coli obtained from a beef processing facility

Aims:  This study aimed to determine the survival of Escherichia coli strains during steam and lactic acid decontamination interventions currently used by the beef‐processing industry, and to determine their heat resistance. Methods and Results:  Strains were grouped into cocktails of five strains each differing in their RAPD patterns for subsequent identification. Steam and lactic acid treatments on meat reduced cell counts of E. coli strain cocktails by 90–99%. The 20 slaughter plant isolates exhibited only minor variation in their resistance to steam and lactic acid treatments but were more resistant than reference strains (three strains) or isolates from live cattle (seven strains). D₆₀ values of strains from live cattle, and reference strains ranged from 0·1 to 0·5 min, in keeping with literature data. However, D₆₀ values of current slaughter plant isolates ranged between 15 for E. coli DM18.3 and 71 min AW 1.7. Cell counts of E. coli AW 1.7 were reduced by <5 log₁₀ CFU g^?1 in ground beef patties cooked to an internal temperature of 71°C. Conclusions:  Strains of E. coli that survive cooking of ground beef to the recommended internal temperature of 71°C can be isolated from beef‐processing facilities. Significance and Impact of the Study:  Pathogen interventions in current commercial beef slaughter may select for extremely heat‐resistant strains of E. coli.     

Bovine recto-anal junction squamous epithelial (RSE) cell adhesion assay for studying Escherichia coli O157 adherence

Aim: To develop a new adherence assay, using cattle recto‐anal junction squamous epithelial (RSE) cells, for evaluating bacterial adherence to cells of bovine origin. Methods and Results: Proof of concept was demonstrated using the human gastrointestinal pathogen Escherichia coli O157:H7, for which cattle are reservoirs. Adherence assays were conducted using both RSE and HEp‐2 cells, in the presence and absence of D+Mannose. E. coli O157 specifically adhered in a type I fimbriae‐independent manner to RSE cells in significantly higher numbers and also bound significantly higher numbers of RSE cells than diverse laboratory strains of nonpathogenic E. coli. Conclusion: The RSE cell adhesion assay output highly reproducible and interpretable results that compared very well with those obtained using the more extensively used HEp‐2 cell adherence assay. Significance and Impact of the study: The RSE cell adhesion assay provides a convenient means of directly defining and evaluating pathogen factors operating at the bovine recto‐anal junction. The RSE cell adhesion assay further has the potential for extrapolation to diverse bacteria, including food‐borne pathogens that colonize cattle via adherence to this particular anatomical site.     

Detection of extended-spectrum beta-lactamase-producing Escherichia coli isolates in faecal samples of Iberian lynx

Aims: To characterize the diversity of extended‐spectrum beta‐lactamase (ESBL)‐producing Escherichia coli isolates recovered within the faecal microbiota of Iberian lynx. The identification of other associated resistance genes and the analysis of clonal relationship were also focused in this study. Methods and Results: From 2008 to 2010, 128 faecal samples of Iberian lynx (wild and captive animals) were collected. Eleven tested samples contained cefotaxime‐resistant E. coli isolates (all belonging to captive animals) and 10 ESBL‐producing isolates were showed. CTX‐M‐14 and SHV‐12 ESBL‐types were detected and seven different patterns were identified by pulsed‐field gel electrophoresis analysis. Conclusions: The occurrence of unrelated multiresistant E. coli in faecal flora of captive specimens of Iberian lynx, including the presence of ESBLs, resistant genes in integrons and virulence determinants was showed in this study. Significance and Impact of the Study: The results obtained in this study highlight the environmental problem as future reintroductions of Iberian lynx could lead to a spread of resistant bacteria. Additionally, ESBL‐producing bacteria can represent a health problem for this endangered species.     

Inhibitory effects of soluble MD‐2 and soluble CD14 on bacterial growth

The effects of the soluble forms of the endotoxin receptor molecules sMD‐2 and sCD14 on bacterial growth were studied. When Escherichia coli and Bacillus subtilis were incubated at 37°C for 18 hr with either sMD‐2 or sCD14, growth of these bacteria was significantly inhibited as evaluated by viable cell counts and NADPH/NADH activity. A mutant of sCD14 (sCD14d57‐64) lacking a region essential for LPS binding did not inhibit the growth of E. coli, whereas this mutant did inhibit the growth of B. subtilis. Addition of excess PG to the bacterial culture reversed the inhibitory effect of sMD‐2 on the growth of B. subtilis, but not on the growth of E. coli. Furthermore, when evaluated by ELISA, both sMD‐2 and sCD14 bound specifically to PG. Taken together, these results indicate that sMD‐2 and sCD14 inhibit the growth of both Gram‐positive and Gram‐negative bacteria and further suggest that binding to PG and LPS is involved in the inhibitory effect of sMD‐2 on Gram‐positive bacteria and of sCD14 on Gram‐negative bacteria, respectively.     

Effect of oxygen and temperature on the dynamic of the dominant bacterial populations of pig manure and on the persistence of pig-associated genetic markers, assessed in river water microcosms

Aims: The aim is to evaluate the dynamic of Bacteroides–Prevotella and Bacillus–Streptococcus–Lactobacillus populations originating from pig manure and the persistence of pig‐associated markers belonging to these groups according to temperature and oxygen. Methods and Results:  River water was inoculated with pig manure and incubated under microaerophilic and aerobic conditions, at 4 and 20°C over 43 days. The diversity of bacterial populations was analysed by capillary electrophoresis‐single‐strand conformation polymorphism. The persistence of the pig‐associated markers was measured by real‐time PCR and compared with the survival of Escherichia coli and enterococci. Decay was characterized by the estimation of the time needed to produce a 1‐log reduction (T90). The greatest changes were observed at 20°C under aerobic conditions, leading to a reduction in the diversity of the bacterial populations and in the concentrations of the Pig‐1‐Bac, Pig‐2‐Bac and Lactobacillus amylovorus markers with a T90 of 10·5, 8·1 and 17·2 days, respectively. Conclusions: Oxygen and temperature were found to have a combined effect on the persistence of the pig‐associated markers in river waters. Significance and Impact of the Study: The persistence profiles of the Pig‐1‐Bac, Pig‐2‐Bac and Lact. amylovorus markers in addition to their high specificity and sensitivity support their use as relevant markers to identify pig faecal contamination in river waters.     

Isolation of faecal coliform bacteria from the American alligator (Alligator mississippiensis)

Aims: To determine whether American alligators (Alligator mississippiensis) are an unrecognized poikilothermic source of faecal coliform and/or potential pathogenic bacteria in South Carolina’s coastal waters. Methods and Results: Bacteria from the cloaca of American alligators, as well as bacteria from surface water samples from their aquatic habitat, were isolated and identified. The predominant enteric bacteria identified from alligator samples using biochemical tests included Aeromonas hydrophila, Citrobacter braakii, Edwardsiella tarda, Escherichia coli, Enterobacter cloacae, Plesiomonas shigelloides and putative Salmonella, and these were similar to bacteria isolated from the surface waters in which the alligators inhabited. Based on most‐probable‐number enumeration estimates from captive alligator faeces, faecal coliform bacteria numbered 8·0 × 10⁹ g^?1 (wet weight) of alligator faecal material, a much higher concentration than many other documented endothermic animal sources. Conclusions: A prevalence of enteric bacteria, both faecal coliforms and potential pathogens, was observed in American alligators. The high faecal coliform bacterial density of alligator faeces may suggest that alligators are a potential source of bacterial contamination in South Carolina coastal waters. Significance and Impact of the Study: These findings help to increase our understanding of faecal coliform and potential pathogenic bacteria from poikilothermic reptilian sources, as there is the potential for these sources to raise bacterial water quality levels above regulatory thresholds.     

Characterization of rumen bacterial diversity and fermentation parameters in concentrate fed cattle with and without forage

Aims: To determine the effects of the removal of forage in high‐concentrate diets on rumen fermentation conditions and rumen bacterial populations using culture‐independent methods. Methods and Results: Detectable bacteria and fermentation parameters were measured in the solid and liquid fractions of digesta from cattle fed two dietary treatments, high concentrate (HC) and high concentrate without forage (HCNF). Comparison of rumen fermentation conditions showed that duration of time spent below pH 5·2 and rumen osmolality were higher in the HCNF treatment. Simpson’s index of 16S PCR‐DGGE images showed a greater diversity of dominant species in the HCNF treatment. Real‐time qPCR showed populations of Fibrobacter succinogenes (P = 0·01) were lower in HCNF than HC diets. Ruminococcus spp., F. succinogenes and Selenomonas ruminantium were at higher (P ≤ 0·05) concentrations in the solid vs the liquid fraction of digesta regardless of diet. Conclusions: The detectable bacterial community structure in the rumen is highly diverse. Reducing diet complexity by removing forage increased bacterial diversity despite the associated reduction in ruminal pH being less conducive for fibrolytic bacterial populations. Quantitative PCR showed that removal of forage from the diet resulted in a decline in the density of some, but not all fibrolytic bacterial species examined. Significance and Impact of the Study: Molecular techniques such as DGGE and qPCR provide an increased understanding of the impacts of dietary changes on the nature of rumen bacterial populations, and conclusions derived using these techniques may not match those previously derived using traditional laboratory culturing techniques.     

Isolation of catechin-converting human intestinal bacteria

Aims: To isolate and characterize bacteria from the human intestine that are involved in the conversion of catechins, a class of bioactive polyphenols abundant in the human diet. Methods and Results: Two bacterial strains, rK3 and aK2, were isolated from an epicatechin‐converting human faecal suspension. The isolates catalysed individual steps in the degradation of (?)‐epicatechin and (+)‐catechin. Based on their phenotypic characteristics and 16S rRNA gene sequences, the isolates were identified as Eggerthella lenta and Flavonifractor plautii (formerly Clostridium orbiscindens). Eggerthella lenta rK3 reductively cleaved the heterocyclic C‐ring of both (?)‐epicatechin and (+)‐catechin giving rise to 1‐(3,4‐dihydroxyphenyl)‐3‐(2,4,6‐trihydroxyphenyl)propan‐2‐ol. The conversion of catechin proceeded five times faster than that of epicatechin. Higher (epi)catechin concentrations led to an accelerated formation of the ring fission product without affecting the growth of Eg. lenta rK3. Flavonifractor plautii aK2 further converted 1‐(3,4‐dihydroxyphenyl)‐3‐(2,4,6‐trihydroxyphenyl)propan‐2‐ol to 5‐(3,4‐dihydroxyphenyl)‐γ‐valerolactone and 4‐hydroxy‐5‐(3,4‐dihydroxyphenyl)valeric acid. Flavonifractor plautii DSM 6740 catalysed the identical reaction indicating it is not strain specific. Conclusions: The conversion of dietary catechins by the isolated Eg. lenta and F. plautii strains in the human intestine may affect their bioavailability. Significance and Impact of the Study: The majority of catechin metabolites are generated by the intestinal microbiota. The identification of catechin‐converting gut bacteria therefore contributes to the elucidation of the bioactivation and the health effects of catechins.     

Role of the probiotic strain Lactobacillus paracasei LMGP22043 carried by artichokes in influencing faecal bacteria and biochemical parameters in human subjects

Aims: To evaluate the positive influence of the probiotic strain Lactobacillus paracasei LMGP22043 carried by artichokes into the human gut with special reference to faecal bacterial balance, short‐chain fatty acid concentrations and enzyme activities in a randomized, double‐blind human trial in comparison with probiotic‐free artichokes (control). Methods: Twenty subjects were randomized into two groups, which consumed daily 180 g of the artichoke product (probiotic or control) during two 15‐day study periods (periods 1 and 2) separated by a 15‐day washout in a crossover manner. Faecal samples were subjected to microbiological and biochemical analyses, and a strain‐specific PCR was performed to monitor the probiotic strain. Results: The probiotic strain, transported by the vegetable matrix, transiently colonized the gut of 17/20 subjects (median 6·87 log CFU g^?1 faeces), antagonized Escherichia coli and Clostridium spp. and increased the genetic diversity of lactic population based on REP‐PCR profiles, mainly after period 1. Conclusions: The probiotic L. paracasei LMGP22043 successfully colonized the human gut and positively influenced faecal bacteria and biochemical parameters. Significance and Impact of the Study: The association of the probiotic L. paracasei with a food carrier rich in fibre can represent a new strategy for favouring a daily supply of probiotics and attracting more consumers to vegetable food fortified with probiotic strains.     

Multiple‐locus variable‐nucleotide tandem repeat subtype analysis implicates European starlings as biological vectors for Escherichia coli O157:H7 in Ohio,USA

Aims: To provide molecular epidemiological evidence of avian transmission of Escherichia coli O157:H7 between dairy farms in Ohio, this study was designed to identify genetic relatedness between isolates originating from bovine faecal samples and intestinal contents of European starlings captured on these farms. Methods and Results: During a three‐year period (2007–2009), cattle (n = 9000) and starlings (n = 430) on 150 different dairy farms in northern Ohio were sampled for the presence of E. coli O157:H7. Isolates were subjected to multiple‐locus variable‐nucleotide tandem repeat analysis (MLVA). Distinct allelic groups were identified on most farms; however, isolates clustering into three MLVA groups originated from both cattle and birds on different farms. Conclusions: Sharing of indistinguishable epidemiologically linked E. coli O157 MLVA subtypes between starlings and cattle on different farms supports the hypothesis that these birds contribute to the transmission of E. coli O157:H7 between dairy farms. Significance and Impact of Study: A continued need exists to identify and to improve preharvest measures for controlling E. coli O157:H7. Controlling wildlife intrusion, particularly European starlings, on livestock operations, may be an important strategy for reducing dissemination of E. coli O157:H7 between farms and thereby potentially decreasing the on‐farm prevalence of E. coli O157:H7 and enhancing the safety of the food supply.     

Bacterial survival studies to assess the efficacy of static pile composting and above ground burial for disposal of bovine carcases

Aim: To assess the survival of bacteria during two alternative means of cattle carcase disposal in windrows: static pile composting (SPC) and above ground burial in soil (AGB), under temperate climate conditions on agricultural land, compared to surface disposal as the control method. Methods and Results: Bacteriological reference materials (pooled bovine faeces in permeable nylon bags and lyophilized cultures of Escherichia coli in glass ampoules) were positioned above and below each of 33 beef cattle carcases (250–300 kg). Temperatures at these sites were monitored with data loggers, while temperature and CO₂ probes were applied repeatedly at varying depths along the windrows. Aliquots of each reference material were cultured from three randomly selected animals from the SPC and AGB group and from all three control animals on five occasions (at 28, 56, 84, 126 and 182 days). SPC was highly efficacious in the destruction of coliforms in faeces and E. coli in ampoules within 28 days, while AGB was not significantly better than controls until 84 days, and bacteria in reference materials above the AGB carcases were still viable after 182 days. Temperature probes and loggers showed SPC provided sustained temperatures of 55–70°C, while AGB did not reach temperatures of 30°C, and the temperature differences correlated with bacteriological findings. Conclusions: In relation to emergency disease management, SPC can be successfully applied to eliminate pathogenic bacteria in cattle carcases, but AGB is unsuitable for carcase disposal. Significance and Impact of the Study: In emergency, animal disease outbreaks in temperate climates requiring large‐scale ruminant carcase disposal, SPC can be successfully applied for the destruction of micro‐organisms.     

The effects of dietary additives on faecal levels of Lactobacillus spp., coliforms,and Escherichia coli,and faecal prevalence of Salmonella spp. and Campylobacter spp. in US production nursery swine1

Aims: In the United States, carbadox and copper sulfate are growth promoters commonly used in combination in nursery swine diets. Our aim was to determine how selected dietary additives affect selected bacterial populations and pathogens in nursery swine, and compare to larch extract, which contains potential antibacterial activities. Methods and Results: Piglets were weaned and sorted into one of the four treatments: (i) basal diet without antimicrobials; (ii) basal diet with carbadox + copper sulfate; (iii) basal diet + 1000 ppm larch extract; or (iv) basal diet + 2000 ppm larch extract. Diets were fed for a 4‐week period after weaning. In both trials, the carbadox + copper sulfate group consumed more feed over the 4‐week period relative to the other three diet groups (P < 0·05), but did not gain significantly more weight. Faecal shedding of Salmonella spp. was not affected by dietary supplement in either trial, but faecal shedding of Campylobacter spp. was the lowest for the carbadox + copper sulfate diet. In faecal samples collected at the end of each trial, Lactobacillus spp. cell counts for the basal and larch extract diets were nearly 1·0 log₁₀ g^?1 faeces greater (P < 0·05) than the carbadox + copper sulfate group, whereas the coliforms and Escherichia coli were nearly 1·0 log₁₀ g^?1 faeces lower (P < 0·05). Conclusions: Compared to basal fed animals, supplementation with carbadox + copper sulfate significantly altered faecal E. coli, coliform bacteria and Lactobacillus spp. Larch extract has no benefit up to 0·2% of diet in regard to pathogen shedding, whereas carbadox + copper sulfate decreased faecal shedding of Campylobacter spp. Significance and Impact of the Study: Current swine management practices in the United States may be beneficial to managing Campylobacter spp. shedding in nursery swine, but also result in significant changes in the resident gastrointestinal microflora.     

Effects of the acid–base treatment of corn on rumen fermentation and microbiota,inflammatory response and growth performance in beef cattle fed high-concentrate diet

Beef cattle are often fed high-concentrate diet (HCD) to achieve high growth rate. However, HCD feeding is strongly associated with metabolic disorders. Mild acid treatment of grains in HCD with 1% hydrochloric acid (HA) followed by neutralization with sodium bicarbonate (SB) might modify rumen fermentation patterns and microbiota, thereby decreasing the negative effects of HCD. This study was thus aimed to investigate the effects of treatment of corn with 1% HA and subsequent neutralization with SB on rumen fermentation and microbiota, inflammatory response and growth performance in beef cattle fed HCD. Eighteen beef cattle were randomly allocated to three groups and each group was fed different diets: low-concentrate diet (LCD) (concentrate : forage = 40 : 60), HCD (concentrate : forage = 60 : 40) or HCD based on treated corn (HCDT) with the same concentrate to forage ratio as the HCD. The corn in the HCDT was steeped in 1% HA (wt/wt) for 48 h and neutralized with SB after HA treatment. The animal trial lasted for 42 days with an adaptation period of 7 days. At the end of the trial, rumen fluid samples were collected for measuring ruminal pH values, short-chain fatty acids, endotoxin (or lipopolysaccharide, LPS) and bacterial microbiota. Plasma samples were collected at the end of the trial to determine the concentrations of plasma LPS, proinflammatory cytokines and acute phase proteins (APPs). The results showed that compared with the LCD, feeding the HCD had better growth performance due to a shift in the ruminal fermentation pattern from acetate towards propionate, butyrate and valerate. However, the HCD decreased ruminal pH and increased ruminal LPS release and the concentrations of plasma proinflammatory cytokines and APPs. Furthermore, feeding the HCD reduced bacterial richness and diversity in the rumen. Treatment of corn increased resistant starch (RS) content. Compared with the HCD, feeding the HCDT reduced ruminal LPS and improved ruminal bacterial microbiota, resulting in decreased inflammation and improved growth performance. In conclusion, although the HCD had better growth performance than the LCD, feeding the HCD promoted the pH reduction and the LPS release in the rumen, disturbed the ruminal bacterial stability and increased inflammatory response. Treatment of corn with HA in combination with subsequent SB neutralization increased the RS content and helped counter the negative effects of feeding HCD to beef steers.     

Prevalence and diversity of class 1 integrons and resistance genes in antimicrobial-resistant Escherichia coli originating from beef cattle administered subtherapeutic antimicrobials

Aims: To characterize class 1 integrons and resistance genes in tetracycline‐resistant Escherichia coli originating from beef cattle subtherapeutically administered chlortetracycline (A44), chlortetracycline and sulfamethazine (AS700), or no antimicrobials (control). Methods and Results: Tetracycline‐resistant E. coli (control, n = 111; AS700, n = 53; A44, n = 40) were studied. Class 1 integrons, inserted gene cassettes and the presence of other antimicrobial resistance genes, as well as phylogenetic analysis, were performed by PCR, restriction enzyme analysis and sequencing. Susceptibilities to 11 antimicrobials were conducted on all isolates. Prevalence of class 1 integrase was higher (P < 0·001) in isolates from AS700 (33%) and A44 (28%) steers as compared to control (7%). Most integron gene cassettes belonged to the aad or dfr families. Correlations were found between the tet(A) gene and the genetic elements sul1 (r = 0·44), aadA1 (r = 0·61), cat (r = 0·58) and intI1(r = 0·37). Both closely and distantly related isolates harboured integrons with identical gene cassette arrays. Conclusions: Subtherapeutic administration of chlorotetracycline alone or in combination with sulfamethazine may select for class 1 integrons in bovine tetracycline‐resistant E. coli isolates. Vertical spread and horizontal transfer are responsible for the dissemination of a particular type of class 1 integron, but this study could not differentiate if this phenomenon occurred within or outside of the feedlot. Tetracycline‐resistant E. coli strains with sul1 and tet(A) genes were more likely to harbour class 1 integrons. Significance and Impact of the Study: Subtherapeutic use of chlortetracycline and sulfamethazine may promote the presence of class 1 integrons in tetracycline‐resistant E. coli isolated from feedlot cattle.     

Diet shapes the ability of human intestinal microbiota to degrade phytate – in vitro studies

Aims

Investigation of intestinal bacterial groups involved in phytate degradation and the impact of diets with different phytate contents on phytase activity.

Methods and Results

Faecal samples of adults on conventional (n = 8) or vegetarian (n = 8) diets and breastfed infants (n = 6) were used as an inoculum for modified media supplemented with phytate. Populations of Gram‐positive anaerobes (GPA), lactic acid bacteria (LAB), Proteobacteria–Bacteroides (P‐B), coliforms and anaerobes were studied. The PCR‐DGGE analysis revealed a random distribution of DGGE profiles in the dendrograms of GPA, P‐B and coliforms, and a partially diet‐specific distribution in the DGGE dendrograms of LAB and anaerobes. The degradation of phytic acid (PA) was determined with HPLC method in supernatants of the cultures. Regardless of the diet, the Gram‐positive anaerobes and LAB displayed the lowest ability to degrade phytate, whereas the coliforms and P‐B cultures produced higher amounts of intermediate myo‐inositol phosphates. Bacterial populations grown in a nonselective medium were the most effective ones in phytate degradation. It was the vegetarians' microbiota that particularly degraded up to 100% phytate to myo‐inositol phosphate products lower than InsP₃.

Conclusions

A diet rich in phytate increases the potential of intestinal microbiota to degrade phytate. The co‐operation of aerobic and anaerobic bacteria is essential for the complete phytate degradation.

Significance and Impact of the Study

This study provides insights on the effect of diet on specific metabolic activity of human intestinal microbiota.     

Comparison of the diversity of the bacterial communities in the intestinal tract of adult Bactrocera dorsalis from three different populations

Aims: To (i) identify the bacterial communities in the gut of oriental fruit fly (Bactrocera dorsalis) adult and (ii) determine whether the different surroundings and diets influence the bacteria composition. Methods and Results: Polymerase chain reaction‐denaturing gradient gel electrophoresis (DGGE) fingerprinting was used to investigate bacterial diversity in the oriental fruit fly adult gut. The 16S rDNA cloned libraries from the intestinal tract of laboratory‐reared (LR), laboratory sterile sugar‐reared (LSSR) and field‐collected (FC) populations of oriental fruit fly were compared. Phylogenetic analysis of 16S rDNA revealed that Gammaproteobacteria were dominant in the all samples (73·0–98·3%). Actinobacteria and Firmicutes were judged to be major components of a given library as they constituted 10% or more of the total clones of such library. The Flavobacteria, Deltaproteobacteria, Bacteroidetes and Alphaproteobacteria were observed in small proportions in various libraries. Further phylogenetic analyses indicated common bacterial phylotypes for all three libraries, e.g. those related to Klebsiella, Citrobacter, Enterobacter, Pectobacterium and Serratia. libshuff analysis showed that the bacterial communities of B. dorsalis from the three populations were significantly different from each other (P < 0·0085). Conclusions: (i) The intestinal tract of B. dorsalis adult contains a diverse bacterial community, some of which are stable. (ii) Different environmental conditions and food supply could influence the diversity of the harboured bacterial communities and increase community variations. Significance and Impact of the Study: Comparison of the microbial compositions and common bacterial species found in this paper may be very important for the biocontrol of B. dorsalis.     

In vitro measurement of the impact of human milk oligosaccharides on the faecal microbiota of weaned formula-fed infants compared to a mixture of prebiotic fructooligosaccharides and galactooligosaccharides

Aims: To investigate the impact of human milk oligosaccharides (HMOs) from a single donor (SO), HMOs from multiple donors (PO), a fructooligosaccharides and galactooligosaccharides mixture (FG) on the composition of a batch culture inoculated with faecal microbiota from formula‐fed infants. Methods and Results: Three substrates were compared using 24‐h pH‐controlled anaerobic batch cultures inoculated with infant faecal slurries. Changes in bacterial populations, short‐chain fatty acids (SCFA) production and bacterial 16S rRNA gene profiles were determined. All three substrates significantly increased numbers of bifidobacteria, bacteroides and those aligning with the clostridial cluster XIVa. Neither the FG nor the HMOs substrates supported the growth of the Clostridium perfringens–histolyticum group. SCFA production corresponded to changes observed in bacterial populations. Denaturing gradient gel electrophoresis fingerprint analysis showed a distinct profile of faecal bacteria present in each infant. Conclusions: HMOs modulated infant faecal culture composition in a similar manner to the prebiotic mixture FG in vitro. Significance and Impact of the Study: This is the first demonstration of the impact of pure HMOs on the mixed culture of infant faecal bacteria. HMOs induced the growth of several saccharolytic bacterial groups and may thus play a role in the health‐promoting attributes of human breast milk and have an extended significance in infant diet during/after weaning.     

Structure of bacteriophage SPN1S endolysin reveals an unusual two‐module fold for the peptidoglycan lytic and binding activity

Bacteriophage SPN1S infects the pathogenic Gram‐negative bacterium Salmonella typhimurium and expresses endolysin for the release of phage progeny by degrading peptidoglycan of the host cell walls. Bacteriophage SPN1S endolysin exhibits high glycosidase activity against peptidoglycans, resulting in antimicrobial activity against a broad range of outer membrane‐permeabilized Gram‐negative bacteria. Here, we report a crystal structure of SPN1S endolysin, indicating that unlike most endolysins from Gram‐negative bacteria background, the α‐helical protein consists of two modular domains, a large and a small domain, with a concave groove between them. Comparison with other structurally homologous glycoside hydrolases indicated a possible peptidoglycan binding site in the groove, and the presence of a catalytic dyad in the vicinity of the groove, one residue in a large domain and the other in a junction between the two domains. The catalytic dyad was further validated by antimicrobial activity assay against outer membrane‐permeabilized Escherichia coli. The three‐helix bundle in the small domain containing a novel class of sequence motif exhibited binding affinity against outer membrane‐permeabilized E. coli and was therefore proposed as the peptidoglycan‐binding domain. These structural and functional features suggest that endolysin from a Gram‐negative bacterial background has peptidoglycan‐binding activity and performs glycoside hydrolase activity through the catalytic dyad.