Mary C. Rea

Characterization of the lactic acid bacteria in artisanal dairy products

The European Union is thanked for partly financing this project under ECLAIR contract CT-91-0064.

Thuricin CD, a posttranslationally modified bacteriocin with a narrow spectrum of activity against Clostridium difficile

The last decade has seen numerous outbreaks of Clostridium difficile-associated disease (CDAD), which presented significant challenges for healthcare facilities worldwide. We have identified and purified thuricin CD, a two-component antimicrobial that shows activity against C. difficile in the nanomolar range. Thuricin CD is produced by Bacillus thuringiensis DPC 6431, a bacterial strain isolated from a human fecal sample, and it consists of two distinct peptides, Trn-α and Trn-β, that act synergistically to kill a wide range of clinical C. difficile isolates, including ribotypes commonly associated with CDAD (e.g., ribotype 027). However, this bacteriocin thuricin CD has little impact on most other genera, including many gastrointestinal commensals. Complete amino acid sequencing using infusion tandem mass spectrometry indicated that each peptide is posttranslationally modified at its respective 21st, 25th, and 28th residues. Solution NMR studies on [13C,15N] Trn-α and [13C,15N]Trn-β were used to characterize these modifications. Analysis of multidimensional NOESY data shows that specific cysteines are linked to the α-carbons of the modified residues, forming three sulfur to α-carbon bridges. Complete sequencing of the thuricin CD gene cluster revealed genes capable of encoding two S′-adenosylmethionine proteins that are characteristically associated with unusual posttranslational modifications. Thuricin CD is a two-component antimicrobial peptide system with sulfur to α-carbon linkages, and it may have potential as a targeted therapy in the treatment of CDAD while also reducing collateral impact on the commensal flora.

Effect of broad- and narrow-spectrum antimicrobials on Clostridium difficile and microbial diversity in a model of the distal colon

Vancomycin, metronidazole, and the bacteriocin lacticin 3147 are active against a wide range of bacterial species, including Clostridium difficile. We demonstrate that, in a human distal colon model, the addition of each of the three antimicrobials resulted in a significant decrease in numbers of C. difficile. However, their therapeutic use in the gastrointestinal tract may be compromised by their broad spectrum of activity, which would be expected to significantly impact on other members of the human gut microbiota. We used high-throughput pyrosequencing to compare the effect of each antimicrobial on the composition of the microbiota. All three treatments resulted in a decrease in the proportion of sequences assigned to the phyla Firmicutes and Bacteroidetes, with a corresponding increase in those assigned to members of the Proteobacteria. One possible means of avoiding such “collateral damage” would involve the application of a narrow-spectrum antimicrobial with specific anti-C. difficile activity. We tested this hypothesis using thuricin CD, a narrow-spectrum bacteriocin produced by Bacillus thuringiensis, which is active against C. difficile. The results demonstrated that this bacteriocin was equally effective at killing C. difficile in the distal colon model but had no significant impact on the composition of the microbiota. This offers the possibility of developing a targeted approach to eliminating C. difficile in the colon, without collateral damage.

Biochemical properties of enterococci relevant to their technological performance

A total of 129 E. faecium, E. faecalis and E. durans strains of food, veterinary and human origin were screened for biochemical properties relevant to their technological performance. Strains exhibited low milk acidifying ability and low extracellular proteolytic activity, with food origin and E. faecalis strains being generally more active. Their peptidase activities were low and mainly specific against glycine-proline- and glutamate-4-nitroanilide, while only food origin and E. durans strains showed broader substrate specificity. In contrast, their lipolytic activities were relatively higher; food and veterinary origin and E. faecalis strains were the most lipolytic. The post-electrophoretic detection of esterase activities showed that the esterolytic system of enterococci was rather complex. All species showed strain-to-strain variation in their ability to metabolise citrate and pyruvate, with E. faecalis strains being generally more active. The main volatile compounds produced in milk were acetaldehyde, ethanol and acetoin; generally, E. faecalis strains produced the highest concentrations. None of the strains decarboxylated histidine, lysine and ornithine, but the majority produced tyramine from tyrosine, independently of origin and species. In respect of most biochemical properties considered in this study, E. faecalis strains were generally more active compared to E. faecium and E. durans. This was also the case for the isolates of food origin compared to those of veterinary and human origin. Results obtained allow the selection of enterococci strains to be used as adjunct starters in food fermentations. However, a final selection should take into account the potential virulence factors of enterococci.

Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera

Lactobacilli are a diverse group of species that occupy diverse nutrient-rich niches associated with humans, animals, plants and food. They are used widely in biotechnology and food preservation, and are being explored as therapeutics. Exploiting lactobacilli has been complicated by metabolic diversity, unclear species identity and uncertain relationships between them and other commercially important lactic acid bacteria. The capacity for biotransformations catalysed by lactobacilli is an untapped biotechnology resource. Here we report the genome sequences of 213 Lactobacillus strains and associated genera, and their encoded genetic catalogue for modifying carbohydrates and proteins. In addition, we describe broad and diverse presence of novel CRISPR-Cas immune systems in lactobacilli that may be exploited for genome editing. We rationalize the phylogenomic distribution of host interaction factors and bacteriocins that affect their natural and industrial environments, and mechanisms to withstand stress during technological processes. We present a robust phylogenomic framework of existing species and for classifying new species.

The vexed relationship between clostridium difficile and inflammatory bowel disease: An assessment of carriage in an outpatient setting among patients in remission

OBJECTIVES:Comorbidity with Clostridium difficile may cause diagnostic delay in newly presenting inflammatory bowel disease (IBD) patients, trigger relapse in established disease, confound therapies, and serve as an indicator of an underlying defect in innate immunity. Retrospective analyses have suggested community acquisition; to address this we conducted a prospective analysis of C. difficile carriage in IBD patients using molecular methods specifically in an outpatient setting.METHODS:Recruited participants had long-standing diagnoses of ulcerative colitis (n=64) and Crohns disease (n=58), were in clinical remission, and had no recent exposure to antibiotics, corticosteroids, immunomodulatory drugs or recent hospitalization. Isolates were cultured from stools and confirmed by 16S sequencing. The antibiotic susceptibilities of the isolates were tested followed by further strain characterization by toxinotyping, ribotyping, and pulsed-field gel electrophoresis (PFGE).RESULTS:The frequency of toxigenic C. difficile was higher in IBD patients than in healthy volunteers at 8.2 and 1.0%, respectively (P=0.02 Fishers exact test). All strains belonged to toxinotype 0 with rare subtypes of this group noted in five isolates and represented by an altered repressor genotype. Patients harbored a diverse range of toxigenic ribotype groups, including those previously associated with C. difficile–associated disease (CDAD) (R015, R005, and R020) and the rarer types R062, R050, and R003. Interestingly, common nosocomial groups were not identified. The considerable nonclonal distribution of distinct strains was further demonstrated by PFGE genomic fingerprinting. None of the study subjects experienced a clinical episode of CDAD during a 6-month period of follow-up.CONCLUSIONS:Detection of C. difficile is increased in IBD outpatients in remission, and strain diversity is consistent with community acquisition from a multitude of sources.

Isolation and analysis of bacteria with antimicrobial activities from the marine sponge haliclona simulans collected from irish waters

Samples of the marine sponge Haliclona simulans were collected from Irish coastal waters, and bacteria were isolated from these samples. Phylogenetic analyses of the cultured isolates showed that four different bacterial phyla were represented; Bacteriodetes, Actinobacteria, Proteobacteria, and Firmicutes. The sponge bacterial isolates were assayed for the production of antimicrobial substances, and biological activities against Gram-positive and Gram-negative bacteria and fungi were demonstrated, with 50% of isolates showing antimicrobial activity against at least one of the test strains. Further testing showed that the antimicrobial activities extended to the important pathogens Pseudomonas aeruginosa, Clostridium difficile, multi-drug-resistant Staphylococcus aureus, and pathogenic yeast strains. The Actinomycetes were numerically the most abundant producers of antimicrobial activities, although activities were also noted from Bacilli and Pseudovibrio isolates. Surveys for the presence of potential antibiotic encoding polyketide synthase and nonribosomal peptide synthetase genes also revealed that genes for the biosynthesis of these secondary metabolites were present in most bacterial phyla but were particularly prevalent among the Actinobacteria and Proteobacteria. This study demonstrates that the culturable fraction of bacteria from the sponge H. simulans is diverse and appears to possess much potential as a source for the discovery of new medically relevant biological active agents.

Clostridium difficile Carriage in Elderly Subjects and Associated Changes in the Intestinal Microbiota

ABSTRACT Clostridium difficile is an important nosocomial pathogen associated particularly with diarrheal disease in elderly individuals in hospitals and long-term care facilities. We examined the carriage rate of Clostridium difficile by culture as a function of fecal microbiota composition in elderly subjects recruited from the community, including outpatient, short-term respite, and long-term hospital stay subjects. The carriage rate ranged from 1.6% (n = 123) for subjects in the community, to 9.5% (n = 43) in outpatient settings, and increasing to 21% (n = 151) for patients in short- or long-term care in hospital. The dominant 072 ribotype was carried by 43% (12/28) of subjects, while the hypervirulent strain R027 (B1/NAP1/027) was isolated from 3 subjects (11%), 2 of whom displayed C. difficile associated diarrhea (CDAD) symptoms at the time of sampling. Emerging ribotypes with enhanced virulence (078 and 018) were also isolated from two asymptomatic subjects. Pyrosequencing of rRNA gene amplicons was used to determine the composition of the fecal microbiota as a surrogate for the microbial population structure of the distal intestine. Asymptomatic subjects (n = 20) from whom C. difficile was isolated showed no dramatic difference at the phylum or family taxonomic level compared to those that were culture negative (n = 252). However, in contrast, a marked reduction in microbial diversity at genus level was observed in patients who had been diagnosed with CDAD at the time of sampling and from whom C. difficile R027 was isolated.

Applicability of a bacteriocin-producing Enterococcus faecium as a co-culture in Cheddar cheese manufacture

Two strains, Enterococcus faecium RZS C5 and E. faecium DPC 1146, produce listericidal bacteriocins, so-called enterocins. E. faecium RZS C5 was studied during batch fermentation in both a complex medium (MRS) and in milk to understand the influence of environmental factors, characteristic for milk and cheese, on both growth and bacteriocin production. Fermentation conditions were chosen in view of the applicability of in situ enterocin production during Cheddar cheese production. Enterocin production by E. faecium RZS C5 in MRS started in the early logarithmic growth phase, and enterocin activity decreased during the stationary phase. The effect of pH on enterocin production and decrease of activity was as intense as the effect on bacterial growth. Higher enterocin production took place at pH 5.5 compared with pH 6.5. The use of lactose instead of glucose increased the production of enterocin, and at higher lactose concentration, production increased more and loss of activity decreased. The production in skimmed milk compared to MRS was lower and was detected mainly in the stationary phase. When casein hydrolysate was added to the milk, enterocin production was higher and started earlier, indicating the importance of an additional nitrogen source for growth of E. faecium in milk. For co-cultures of E. faecium RZS C5 with the starters used during Cheddar cheese manufacture, no enterocin activity was detected during the milk fermentation. Furthermore, the applicability of E. faecium RZS C5 and E. faecium DPC 1146 strains was tested in Cheddar cheese manufacture on pilot scale. Enterocin production took place from the beginning of the cheese manufacturing and was stable during the whole ripening phase of the cheese. This indicates that both an early and late contamination of the milk or cheese can be combated with a stable, in situ enterocin production. The use of such a co-culture is an additional safety provision beyond good manufacturing practices.

A rapid PCR based method to distinguish between Lactococcus and Enterococcus.

Phenotypic characterisation of Lactococcus and Enterococcus species remains unreliable as strains of both genera have been isolated which do not conform to the traditional criteria for separation of these genera. A bank of 131 isolates was phenotypically characterised by three methods: (a) traditional broth tests, (b) API Rapid ID 32 Strep and (c) BBL Crystal ID kits. Differences in genus designation between commercial kits were evident for 12 strains (9%), while 7 strains (5%) remained unidentified by either kit. Published 16S rRNA sequences were aligned and used to design genus-specific primers which, when used in separate PCR reactions, were capable of distinguishing all type strains of Lactococcus and Enterococcus. These primers did not react with known species of Streptococcus, Pediococcus, Lactobacillus, Leuconostoc or Tetragenococcus. Isolates which could not be identified by phenotype were assigned to either genus on the basis of the gene primers.