In silico metatranscriptomic approach for tracking biofilm-related effectors in dairies and its importance for improving food safety.

Sessile microorganisms are usually recalcitrant to antimicrobial treatments, and it is possible that finding biofilm-related effectors in metatranscriptomics datasets helps to understand mechanisms for bacterial persistence in diverse environments, by revealing protein-encoding genes that are expressed in situ. For this research, selected dairy-associated metatranscriptomics bioprojects were downloaded from the public databases JGI GOLD and NCBI (eight milk and 45 cheese samples), to screen for sequences encoding biofilm-related effectors. Based on the literature, the selected genetic determinants were related to adhesins, BAP, flagellum-related, intraspecific QS (AHL, HK, and RR), interspecific QS (LuxS), and QQ (AHL-acylases, AHL-lactonases). To search for the mRNA sequences encoding for those effector proteins, a custom database was built from UniprotKB, yielding 1,154,446 de-replicated sequences that were indexed in DIAMOND for alignment. The results revealed that in all the dairy-associated metatranscriptomic datasets obtained, there were reads assigned to genes involved with flagella, adhesion, and QS/QQ, but BAP-reads were found only for milk. Significant Pearson correlations (p < 0.05) were observed for transcripts encoding for flagella, RR, histidine kinases, adhesins, and LuxS, although no other significant correlations were found. In conclusion, the rationale used in this study was useful to demonstrate the presence of biofilm-associated effectors in metatranscriptomics datasets, pointing out to possible regulatory mechanisms in action in dairy-related biofilms, which could be targeted in the future to improve food safety.

Role of the LytSR Two-Component Regulatory System in Staphylococcus lugdunensis Biofilm Formation and Pathogenesis.

Staphylococcus lugdunensis is a coagulase negative Staphylococcus recognized as a virulent pathogen. It is responsible for a wide variety of infections, some of which are associated with biofilm production, such as implanted medical device infections or endocarditis. However, little is known about S. lugdunensis regulation of virulence factor expression. Two-component regulatory systems (TCS) play a critical role in bacterial adaptation, survival, and virulence. Among them, LytSR is widely conserved but has variable roles in different organisms, all connected to metabolism or cell death and lysis occurring during biofilm development. Therefore, we investigated here the functions of LytSR in S. lugdunensis pathogenesis. Deletion of lytSR in S. lugdunensis DSM 4804 strain did not alter either susceptibility to Triton X-100 induced autolysis or death induced by antibiotics targeting cell wall synthesis. Interestingly, ΔlytSR biofilm was characterized by a lower biomass, a lack of tower structures, and a higher rate of dead cells compared to the wild-type strain. Virulence toward Caenorhabditis elegans using a slow-killing assay was significantly reduced for the mutant compared to the wild-type strain. By contrast, the deletion of lytSR had no effect on the cytotoxicity of S. lugdunensis toward the human keratinocyte cell line HaCaT. Transcriptional analyses conducted at mid- and late-exponential phases showed that lytSR deletion affected the expression of 286 genes. Most of them were involved in basic functions such as the metabolism of amino acids, carbohydrates, and nucleotides. Furthermore, LytSR appeared to be involved in the regulation of genes encoding known or putative virulence and colonization factors, including the fibrinogen-binding protein Fbl, the major autolysin AtlL, and the type VII secretion system. Overall, our data suggest that the LytSR TCS is implicated in S. lugdunensis pathogenesis, through its involvement in biofilm formation and potentially by the control of genes encoding putative virulence factors.

Behavior of Foodborne Pathogens Listeria monocytogenes and Staphylococcus aureus in Mixed-Species Biofilms Exposed to Biocides.

In nature and man-made environments, microorganisms reside in mixed-species biofilms, in which the growth and metabolism of an organism are different from these behaviors in single-species biofilms. Pathogenic microorganisms may be protected against adverse treatments in mixed-species biofilms, leading to health risk for humans. Here, we developed two mixed five-species biofilms that included one or the other of the foodborne pathogens Listeria monocytogenes and Staphylococcus aureus The five species, including the pathogen, were isolated from a single food-processing environmental sample, thus mimicking the environmental community. In mature mixed five-species biofilms on stainless steel, the two pathogens remained at a constant level of ∼105 CFU/cm2 The mixed five-species biofilms as well as the pathogens in monospecies biofilms were exposed to biocides to determine any pathogen-protective effect of the mixed biofilm. Both pathogens and their associate microbial communities were reduced by peracetic acid treatments. S. aureus decreased by 4.6 log cycles in monospecies biofilms, but the pathogen was protected in the five-species biofilm and decreased by only 1.1 log cycles. Sessile cells of L. monocytogenes were affected to the same extent when in a monobiofilm or as a member of the mixed-species biofilm, decreasing by 3 log cycles when exposed to 0.0375% peracetic acid. When the pathogen was exchanged in each associated microbial community, S. aureus was eradicated, while there was no significant effect of the biocide on L. monocytogenes or the mixed community. This indicates that particular members or associations in the community offered the protective effect. Further studies are needed to clarify the mechanisms of biocide protection and to identify the species playing the protective role in microbial communities of biofilms.IMPORTANCE This study demonstrates that foodborne pathogens can be established in mixed-species biofilms and that this can protect them from biocide action. The protection is not due to specific characteristics of the pathogen, here S. aureus and L. monocytogenes, but likely caused by specific members or associations in the mixed-species biofilm. Biocide treatment and resistance are a challenge for many industries, and biocide efficacy should be tested on microorganisms growing in biofilms, preferably mixed systems, mimicking the application environment.

Staphylococcus aureus in Some Brazilian Dairy Industries: Changes of Contamination and Diversity.

Staphylococcus aureus, a major food-poisoning pathogen, is a common contaminant in dairy industries worldwide, including in Brazil. We determined the occurrence of S. aureus in five dairies in Brazil over 8 months. Of 421 samples, 31 (7.4%) were positive for S. aureus and prevalence varied from 0 to 63.3% between dairies. Sixty-six isolates from the 31 samples were typed by Multi-Locus Sequence Typing to determine if these isolates were persistent or continuously reintroduced. Seven known sequence types (STs), ST1, ST5, ST30, ST97, ST126, ST188 and ST398, and four new ST were identified, ST3531, ST3540, ST3562 and ST3534. Clonal complex (CC) 1 (including the four new ST), known as an epidemic clone, was the dominant CC. However, there were no indications of persistence of particular ST. The resistance toward 11 antibiotic compounds was assessed. Twelve profiles were generated with 75.8% of strains being sensitive to all antibiotic classes and no Methicillin-resistant S. aureus (MRSA) strains were found. The enterotoxin-encoding genes involved in food-poisoning, e.g., sea, sed, see, and seg were targeted by PCR. The two toxin-encoding genes, sed and see, were not detected. Only three strains (4.5%) harbored seg and two of these also harbored sea. Despite the isolates being Methicillin-sensitive S. aureus (MSSA), the presence of CC1 clones in the processing environment, including some harboring enterotoxin encoding genes, is of concern and hygiene must have high priority to reduce contamination.

Listeria monocytogenes incidence changes and diversity in some Brazilian dairy industries and retail products.

Listeria monocytogenes can cause listeriosis, a severe foodborne disease. In Brazil, despite very few reported cases of listeriosis, the pathogen has been repeatedly isolated from dairies. This has led the government to implement specific legislation to reduce the hazard. Here, we determined the incidence of L. monocytogenes in five dairies and retail products in the Southeast and Midwest regions of Brazil over eight months. Of 437 samples, three samples (0.7%) from retail and only one sample (0.2%) from the dairies were positive for L. monocytogenes. Thus, the contamination rate was significantly reduced as compared to previous studies. MultiLocus Sequence Typing (MLST) was used to determine if contamination was caused by new or persistent clones leading to the first MLST profile of L. monocytogenes from the Brazilian dairy industry. The processing environment isolate is of concern being a sequence-type (ST) 2, belonging to the lineage I responsible for the majority of listeriosis outbreaks. Also, ST3 and ST8 found in commercialized cheese have previously been reported in outbreaks. Despite the lower incidence, dairy products still pose a potential health risk and the occurrence of L. monocytogenes in dairies and retail products emphasize the need for continuous surveillance of this pathogen in the Brazilian dairy industry.

Oligomerized backbone pilin helps piliated Lactococcus lactis to withstand shear flow.

The present work focuses on the role of pili present at the cell surface of Lactococcus lactis in bacterial adhesion to abiotic (hydrophobic polystyrene) and biotic (mucin-coated polystyrene) surfaces. Native pili-displaying strains and isogenic derivatives in which pilins or sortase C structural genes had been modified were used. Surface physico-chemistry, morphology and shear-flow-induced detachment of lactococcal cells were evaluated. The involvement of pili in L. lactis adhesion was clearly demonstrated, irrespective of the surface characteristics (hydrophobic/hydrophilic, presence or not of specific binding sites). The accessory pilin, PilC, and the backbone pilin, PilB, were revealed to play a major role in adhesion, provided that the PilB was present in its polymerized form. Within the population fraction that remained attached to the surface under increasing shear flow, different association behaviors were observed, showing that pili could serve as anchoring sites thus hampering the effect of shear flow on cell orientation and detachment.

A proteomic approach of SigX function in Pseudomonas aeruginosa outer membrane composition.

SigX is one of the 19 extracytoplasmic function sigma factors that have been predicted in the human opportunistic pathogen Pseudomonas aeruginosa genome. SigX is involved in the transcription of oprF, encoding the major outer membrane protein OprF, a pleiotropic porin that contributes to the maintaining of the wall structure, and is essential to P. aeruginosa virulence. This study aimed to get further insights into the functions of SigX. We performed here an outer membrane subproteome of a sigX mutant. Proteomic investigations revealed lower production of 8 porins among which 4 gated channels involved in iron or hem uptake, OprF, and the three substrate-specific proteins OprD, OprQ and OprE. On the other side, the glucose-specific porin OprB and the lipid A 3-O-deacylase that is involved in LPS modification were up-regulated. Our results indicate that SigX may be involved in the control and/or regulation of the outer membrane composition. BIOLOGICAL SIGNIFICANCE: A proteomic approach was used herein to get further insights into SigX functions in P. aeruginosa. The data presented here suggest that SigX is involved in the outer membrane protein composition, and could be linked to a regulatory network involved in OM homeostasis.

The extra-cytoplasmic function sigma factor sigX modulates biofilm and virulence-related properties in Pseudomonas aeruginosa.

SigX, one of the 19 extra-cytoplasmic function sigma factors of P. aeruginosa, was only known to be involved in transcription of the gene encoding the major outer membrane protein OprF. We conducted a comparative transcriptomic study between the wildtype H103 strain and its sigX mutant PAOSX, which revealed a total of 307 differentially expressed genes that differed by more than 2 fold. Most dysregulated genes belonged to six functional classes, including the "chaperones and heat shock proteins", "antibiotic resistance and susceptibility", "energy metabolism", "protein secretion/export apparatus", and "secreted factors", and "motility and attachment" classes. In this latter class, the large majority of the affected genes were down-regulated in the sigX mutant. In agreement with the array data, the sigX mutant was shown to demonstrate substantially reduced motility, attachment to biotic and abiotic surfaces, and biofilm formation. In addition, virulence towards the nematode Caenorhabditis elegans was reduced in the sigX mutant, suggesting that SigX is involved in virulence-related phenotypes.

Pilus biogenesis in Lactococcus lactis: molecular characterization and role in aggregation and biofilm formation.

The genome of Lactococcus lactis strain IL1403 harbors a putative pilus biogenesis cluster consisting of a sortase C gene flanked by 3 LPxTG protein encoding genes (yhgD, yhgE, and yhhB), called here pil. However, pili were not detected under standard growth conditions. Over-expression of the pil operon resulted in production and display of pili on the surface of lactococci. Functional analysis of the pilus biogenesis machinery indicated that the pilus shaft is formed by oligomers of the YhgE pilin, that the pilus cap is formed by the YhgD pilin and that YhhB is the basal pilin allowing the tethering of the pilus fibers to the cell wall. Oligomerization of pilin subunits was catalyzed by sortase C while anchoring of pili to the cell wall was mediated by sortase A. Piliated L. lactis cells exhibited an auto-aggregation phenotype in liquid cultures, which was attributed to the polymerization of major pilin, YhgE. The piliated lactococci formed thicker, more aerial biofilms compared to those produced by non-piliated bacteria. This phenotype was attributed to oligomers of YhgE. This study provides the first dissection of the pilus biogenesis machinery in a non-pathogenic Gram-positive bacterium. Analysis of natural lactococci isolates from clinical and vegetal environments showed pili production under standard growth conditions. The identification of functional pili in lactococci suggests that the changes they promote in aggregation and biofilm formation may be important for the natural lifestyle as well as for applications in which these bacteria are used.

The GBS PI-2a pilus is required for virulence in mice neonates.

BACKGROUND: Streptococcus agalactiae (Group B Streptococcus) is a leading cause of sepsis and meningitis in newborns. Most bacterial pathogens, including gram-positive bacteria, have long filamentous structures known as pili extending from their surface. Although pili are described as adhesive organelles, they have been also implicated in many other functions including thwarting the host immune responses. We previously characterized the pilus-encoding operon PI-2a (gbs1479-1474) in strain NEM316. This pilus is composed of three structural subunit proteins: PilA (Gbs1478), PilB (Gbs1477), and PilC (Gbs1474), and its assembly involves two class C sortases (SrtC3 and SrtC4). PilB, the bona fide pilin, is the major component whereas PilA, the pilus associated adhesin, and PilC the pilus anchor are both accessory proteins incorporated into the pilus backbone. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the role of the major pilin subunit PilB was tested in systemic virulence using 6-weeks old and newborn mice. Notably, the non-piliated ΔpilB mutant was less virulent than its wild-type counterpart in the newborn mice model. Next, we investigated the possible role(s) of PilB in resistance to innate immune host defenses, i.e. resistance to macrophage killing and to antimicrobial peptides. Phagocytosis and survival of wild-type NEM316 and its isogenic ΔpilB mutant in immortalized RAW 264.7 murine macrophages were not significantly different whereas the isogenic ΔsodA mutant was more susceptible to killing. These results were confirmed using primary peritoneal macrophages. We also tested the activities of five cationic antimicrobial peptides (AMP-1D, LL-37, colistin, polymyxin B, and mCRAMP) and found no significant difference between WT and ΔpilB strains whereas the isogenic dltA mutant showed increased sensitivity. CONCLUSIONS/SIGNIFICANCE: These results question the previously described role of PilB pilus in resistance to the host immune defenses. Interestingly, PilB was found to be important for virulence in the neonatal context.

Functionality of sortase A in Lactococcus lactis.

Lactococcus lactis IL1403 harbors a putative sortase A (SrtA) and 11 putative sortase substrates that carry the canonical LPXTG signature of such substrates. We report here on the functionality of SrtA to anchor five LPXTG substrates to the cell wall, thus suggesting that SrtA is the housekeeping sortase in L. lactis IL1403.