Bovine mastitis is a polymicrobial disease requiring a polydiagnostic approach

peer-reviewedBovine mastitis, an inflammation of the udder, is associated with increases in milk somatic cell count usually resulting from bacterial infection. We analysed 50 mastitic milk samples via cultivation, 16S rRNA sequencing and a combination of the two (culturomics) to define the complete microbial content of the milk. Most samples contained over 10,000 cfu mL-1 total bacterial counts including isolates that were haemolysin positive (n = 36). Among colonies isolated from blood agar plates, Streptococcus uberis was dominant (11/50) followed by Streptococcus dysgalactiae (6/50), Pseudomonas (6/50), Enterococcus faecalis (6/50), Escherichia coli (6/50), Staphylococcus argenteus (4/50), Bacillus (4/50) and Staphylococcus aureus (2/50). 16S rRNA profiling revealed that amplicons were dominated by Rhodococcus, Staphylococcus, Streptococcus and Pseudomonas. A higher inter-sample diversity was noted in the 16S rRNA readouts, which was not always reflected in the plating results. The combination of the two methods highlights the polymicrobial complexity of bovine mastitis

Vancomycin and nisin A are effective against biofilms of multi-drug resistant Staphylococcus aureus isolates from human milk

Human milk provides complete nutrition for infants and at the same time promotes the growth of specific bacteria in the infant gastrointestinal tract. Breastfeeding can often be discontinued due to mastitis which is an inflammation of the breast tissue. We isolated 18 Staphylococcus aureus strains from milk donated by healthy (n = 6), subclinical (n = 6), and mastitic (n = 6) mothers, two strains of which were VISA (Vancomycin Intermediate S. aureus). All tested strains (n = 12) were able to form biofilms. We then examined the impact of nisin A and vancomycin alone and in combination on biofilm formation and eradication of selected strains (n = 8). We observed strain-specific responses, with the combinatorial treatment at 1/4X MIC (for both singularly) significantly inhibiting biofilm formation for seven out of eight strains when compared with nisin A or vancomycin alone. None of the selected treatments were able to eradicate pre-formed biofilms. Finally, we selected two strains, namely a VISA (APC3814H) and a strong biofilm former (APC3912CM) and used confocal microscopy to evaluate the effects of the antimicrobial agents at 1X MIC on biofilm inhibition and eradication. All treatments inhibited biofilm formation of APC3814H but were ineffective in eradicating a pre-formed biofilm. Single treatments at 1X MIC against APC3912CM cells did not prevent biofilm formation whereas combination treatment caused increased death of APC3912CM cells. Finally, the combination treatment reduced the thickness of the pre-formed APC3912CM biofilm as compared with the single treatments

Bacillus cereus spore damage recovery and diversity in spore germination and carbohydrate utilisation

Bacterial spores are extremely robust survival vehicles that are highly resistant towards environmental stress conditions including heat, UV radiation and other stresses commonly applied during food production and preservation. Spores, including those of the toxin-producing food-borne human pathogen Bacillus cereus, are ubiquitously present in a wide range of environmental niches such as soil, plant rhizosphere, intestinal tract of insects and animals, and it is virtually impossible to prevent contamination at the primary production level. Heat treatments are conventionally applied in food processing to reduce the microbial load of food products, however, to comply with consumer desire for products with higher sensory and nutritional values, the treatment intensity may become milder. Consequently, subpopulations of spores may emerge that are sublethally damaged rather than inactivated conceivably causing quality and safety issues following repair and outgrowth. In this thesis, a functional genomics approach was used in combination with subpopulation and single spore analysis to identify factors involved in recovery of heat damaged spores, and to link B. cereus genotypes to nutrient-induced germination capacity and carbohydrate utilisation capacity. Using comparative analysis of B. cereus ATCC 14579 wild type and targeted mutants, putative damage repair factors were identified such as putative transcriptional regulator CdnL, that supported recovery of spores in a range of conditions including model foods. The majority of identified genes encoding putative damage repair factors appeared to be unique for B. cereus group members. This novel information on spore recovery adds to further insights in versatility of survival strategies of B. cereus. Different types of foods may contain different types and levels of nutrients including amino acids and carbohydrates, that can affect spore germination capacity and subsequent outgrowth performance of vegetative B. cereus cells. Nutrient germinants present in food products can trigger specific germinant receptors (GRs) located in the spore inner membrane leading to spore germination, a critical step before growth resumes. Combined analysis of genotypes and nutrient-induced germination phenotypes using high throughput flow cytometry analysis at the level of individual spores, revealed substantial diversity in germination capacity with a subset of strains showing a very weak germination response even in nutrient-rich media containing high levels of amino acids. Phylogenetically, these B. cereus strains grouped in subgroup IIIA encompassing strains containing pseudogenes or variants of some of the Ger clusters and two strains containing the recently identified SpoVA2mob transposon, that induced heat resistance with concomitant reduced germination response in Bacillus subtilis spores. The same B. cereus isolates were also used to link genotypes with carbohydrate utilisation clusters present on the genomes, and this revealed representatives of subgroup IIIA to lack specific carbohydrate utilisation clusters (starch, glycogen, aryl beta-glucosides; salicin, arbutin and esculin) suggesting a reduced capacity to utilise plant-associated carbohydrates for growth. Since these B. cereus subgroup IIIA representatives contain host-associated carbohydrate utilisation gene clusters and a subset of unique Ger clusters, their qualification as poor germinators may require revision following assessment of spore germination efficacy using host-derived compounds as germinants. The research described in this thesis has added novel insights in B. cereus capacity to cope with spore damage and provided novel overviews of the distribution and putative functionality of (sub)clusters of GRs and carbohydrate utilisation clusters. Knowledge on spore damage repair, germination and metabolism capacity adds to further understanding of B. cereus ecology including niche occupation and transmission capacity

Identification of CdnL, a putative transcriptional regulator involved in repair and outgrowth of heat-damaged bacillus cereus spores

Spores are widely present in the environment and are common contaminants in the food chain, creating a challenge for food industry. Nowadays, heat treatments conventionally applied in food processing may become milder to comply with consumer desire for products with higher sensory and nutritional values. Consequently subpopulations of spores may emerge that are sublethally damaged rather than inactivated. Such spores may germinate, repair damage, and eventually grow out leading to uncontrolled spoilage and safety issues. To gain insight into both the behaviour of damaged Bacillus cereus spores, and the process of damage repair, we assessed the germination and outgrowth performance using OD595 measurements and microscopy combined with genome-wide transcription analysis of untreated and heat-Treated spores. The first two methods showed delayed germination and outgrowth of heat-damaged B. cereus ATCC14579 spores. A subset of genes uniquely expressed in heat-Treated spores was identified with putative roles in the outgrowth of damaged spores, including cdnL (BC4714) encoding the putative transcriptional regulator CdnL. Next, a B. cereus ATCC14579 cdnL (BC4714) deletion mutant was constructed and assessment of outgrowth from heat-Treated spores under food relevant conditions showed increased damage compared to wild type spores. The approach used in this study allows for identification of candidate genes involved in spore damage repair. Further identification of cellular parameters and characterisation of the molecular processes contributing to spore damage repair may provide leads for better control of spore outgrowth in foods.</p

Vancomycin and nisin A are effective against biofilms of multi-drug resistant Staphylococcus aureus isolates from human milk.

Human milk provides complete nutrition for infants and at the same time promotes the growth of specific bacteria in the infant gastrointestinal tract. Breastfeeding can often be discontinued due to mastitis which is an inflammation of the breast tissue. We isolated 18 Staphylococcus aureus strains from milk donated by healthy (n = 6), subclinical (n = 6), and mastitic (n = 6) mothers, two strains of which were VISA (Vancomycin Intermediate S. aureus). All tested strains (n = 12) were able to form biofilms. We then examined the impact of nisin A and vancomycin alone and in combination on biofilm formation and eradication of selected strains (n = 8). We observed strain-specific responses, with the combinatorial treatment at 1/4X MIC (for both singularly) significantly inhibiting biofilm formation for seven out of eight strains when compared with nisin A or vancomycin alone. None of the selected treatments were able to eradicate pre-formed biofilms. Finally, we selected two strains, namely a VISA (APC3814H) and a strong biofilm former (APC3912CM) and used confocal microscopy to evaluate the effects of the antimicrobial agents at 1X MIC on biofilm inhibition and eradication. All treatments inhibited biofilm formation of APC3814H but were ineffective in eradicating a pre-formed biofilm. Single treatments at 1X MIC against APC3912CM cells did not prevent biofilm formation whereas combination treatment caused increased death of APC3912CM cells. Finally, the combination treatment reduced the thickness of the pre-formed APC3912CM biofilm as compared with the single treatments

Analysis of germination capacity and germinant receptor (sub)clusters of genomesequenced Bacillus cereus environmental isolates and model strains

Spore germination of 17 Bacillus cereus food isolates and reference strains was evaluated using flow cytometry analysis in combination with fluorescent staining at a single-spore level. This approach allowed for rapid collection of germination data under more than 20 conditions, including heat activation of spores, germination in complex media (brain heart infusion [BHI] and tryptone soy broth [TSB]), and exposure to saturating concentrations of single amino acids and the combination of alanine and inosine. Whole-genome sequence comparison revealed a total of 11 clusters of operons encoding germinant receptors (GRs): GerK, GerI, and GerL were present in all strains, whereas GerR, GerS, GerG, GerQ, GerX, GerF, GerW, and GerZ (sub)clusters showed a more diverse presence/absence in different strains. The spores of tested strains displayed high diversity with regard to their sensitivity and responsiveness to selected germinants and heat activation. The two laboratory strains, B. cereus ATCC 14579 and ATCC 10987, and 11 food isolates showed a good germination response under a range of conditions, whereas four other strains (B. cereus B4085, B4086, B4116, and B4153) belonging to phylogenetic group IIIA showed a very weak germination response even in BHI and TSB media. Germination responses could not be linked to specific (combinations of) GRs, but it was noted that the four group IIIA strains contained pseudogenes or variants of subunit C in their gerL cluster. Additionally, two of those strains (B4086 and B4153) carried pseudogenes in the gerK and gerRI (sub)clusters that possibly affected the functionality of these GRs

Physiological bioactivity of a postbiotic consisting of heat-treated lactobacilli on mouse small intestine

Lactobacillus LB is a postbiotic generated following fermentation by Limosilactobacillus fermentum and Lactobacillus delbrueckii. Lactobacillus LB alleviates acute diarrhoea and ameliorates the symptoms of irritable bowel syndrome. Here, we investigated whether modulation of intestinal ion transport and motility contributes to these beneficial effects and whether the postbiotic produced with both strains contributes to a unique biophysiological profile. In Ussing chamber studies, low lactose-Lactobacillus LB (LL-LB) significantly increased baseline short-circuit current, and this was partially mediated by sodium-D-glucose transporter 1. In organ baths, LL-LB significantly decreased ileal tone and increased carbachol-induced contractility. Relative to LL-LB, preparations produced using a single strain fermentate generated from L. fermentum significantly increased baseline short-circuit current and inhibited carbachol-induced contractility. Our data demonstrate a unique biophysiological profile for the dual strain postbiotic and support a direct and immediate effect of LL-LB on host physiology ex vivo which could contribute to the clinical efficacy of Lactobacillus LB

Array expression ratios (log2 values) of candidate genes displaying specific upregulation during germination and outgrowth of heat-treated <i>B</i>. <i>cereus</i> ATCC14579 spores and verified by qPCR.

<p>False Discovery Rates below 0.05 are indicated in bold.</p

Surviving and mildly and severely heat damaged spore fractions in <i>B</i>. <i>cereus</i> ATCC1457 and its mutant derivative strain Δ<i>cdnL</i> (BC4714) upon exposure to wet heat, hydrogen peroxide and sodium hypochlorite treatments.

<p>Averages of three independent experiments are represented.</p

Impact of heat-treatment on germination and outgrowth of <i>B</i>. <i>cereus</i> ATCC14579 spores.

<p>(A) Relative change in OD₅₉₅ for untreated (circles) and heat-treated for 1 min at 95°C (squares) dormant spores was monitored in time in BHI broth at 30°C. Closed symbols indicate the sampling points selected for transcriptome analysis. The starting OD₅₉₅ was 0.15–0.2 (B) Microscopy analysis of samples taken before initiation of germination (t0) and at indicated time points (10 up to 150 min) thereafter.</p