The Impact of Low-Level Benzalkonium Chloride Exposure on Staphylococcus spp. Strains and Control by Photoinactivation.

Exposure of bacteria to low concentrations of biocides can facilitate horizontal gene transfer, which may lead to bacterial adaptive responses and resistance to antimicrobial agents. The emergence of antibacterial resistance not only poses a significant concern to the dairy industry but also adds to the complexity and cost of mastitis treatment. This study was aimed to evaluate how selective stress induced by benzalkonium chloride (BC) promotes antibiotic non-susceptibility in Staphylococcus spp. In addition, we investigated the efficacy of photodynamic inactivation (PDI) in both resistant and susceptible strains. The study determined the minimum inhibitory concentration (MIC) of BC using the broth microdilution method for different Staphylococcus strains. The experiments involved pairing strains carrying the qacA/qacC resistance genes with susceptible strains and exposing them to subinhibitory concentrations of BC for 72 h. The recovered isolates were tested for MIC BC and subjected to disc diffusion tests to assess changes in susceptibility patterns. The results demonstrated that subinhibitory concentrations of BC could select strains with reduced susceptibility and antibiotic resistance, particularly in the presence of S. pasteuri. The results of PDI mediated by toluidine blue (100 µM) followed by 60 min irradiation (total light dose of 2.5 J/cm2) were highly effective, showing complete inactivation for some bacterial strains and a reduction of up to 5 logs in others.

Multiclonal human origin and global expansion of an endemic bacterial pathogen of livestock.

Most new pathogens of humans and animals arise via switching events from distinct host species. However, our understanding of the evolutionary and ecological drivers of successful host adaptation, expansion, and dissemination are limited. Staphylococcus aureus is a major bacterial pathogen of humans and a leading cause of mastitis in dairy cows worldwide. Here we trace the evolutionary history of bovine S. aureus using a global dataset of 10,254 S. aureus genomes including 1,896 bovine isolates from 32 countries in 6 continents. We identified 7 major contemporary endemic clones of S. aureus causing bovine mastitis around the world and traced them back to 4 independent host-jump events from humans that occurred up to 2,500 y ago. Individual clones emerged and underwent clonal expansion from the mid-19th to late 20th century coinciding with the commercialization and industrialization of dairy farming, and older lineages have become globally distributed via established cattle trade links. Importantly, we identified lineage-dependent differences in the frequency of host transmission events between humans and cows in both directions revealing high risk clones threatening veterinary and human health. Finally, pangenome network analysis revealed that some bovine S. aureus lineages contained distinct sets of bovine-associated genes, consistent with multiple trajectories to host adaptation via gene acquisition. Taken together, we have dissected the evolutionary history of a major endemic pathogen of livestock providing a comprehensive temporal, geographic, and gene-level perspective of its remarkable success.

Growth conditions affect biofilms of Staphylococcus aureus producing mastitis: Contribution of MALDI-TOF-MS to strain characterization.

Bovine mastitis is a disease of dairy cattle prevalent throughout the world that causes alterations in the quality and composition of milk, compromising technological performance. Staphylococcus aureus is one of the most important pathogens that produce clinical, subclinical, and chronic mastitis. Biofilms are considered a virulence factor necessary for the survival of S. aureus in the mammary gland. Its zoonotic potential is important not only for the dairy industry sector but also for public health. This study aimed to evaluate the effect of different growing culture conditions on the biofilm formation of S. aureus isolated from mastitis and to test the MALDI-TOF-MS's ability to discriminate among different biofilm formation levels. Fluids commonly found in the dairy environment were incorporated to approach the pathogen's behavior in natural surroundings. PIA production was also evaluated. All strains were able to form high biofilms in TSB, TSBg, and milk. Milk changed the behavior of some strains which formed more biofilms in this medium than in TSBg. The free iron medium CTSBg and milk whey inhibited the biofilm formation of the most strains. MALDI-TOF-MS performance was an excellent tool to discriminate between high, moderate, and low biofilm producers strains of S. aureus in each media, confirming the results of crystal violet assay. PIA production was variable among the strains and showed a media-dependent behavior. Our data highlights the importance of considering the growing conditions that mimic the natural ones to the study of biofilm formation in vitro.

Salicylic acid stabilizes Staphylococcus aureus biofilm by impairing the agr quorum-sensing system.

Salicylic acid (SAL) has recently been shown to induce biofilm formation in Staphylococcus aureus and to affect the expression of virulence factors. This study was aimed to investigate the effect of SAL on the regulatory agr system and its impact on S. aureus biofilm formation. The agr quorum-sensing system, which is a central regulator in S. aureus pathogenicity, plays a pivotal role in the dispersal of S. aureus mature biofilms and contributes to the creation of new colonization sites. Here, we demonstrate that SAL impairs biofilm dispersal by interfering with agr expression. As revealed by our work, protease and surfactant molecule production is diminished, and bacterial cell autolysis is also negatively affected by SAL. Furthermore, as a consequence of SAL treatment, the S. aureus biofilm matrix revealed the lack of extracellular DNA. In silico docking and simulation of molecular dynamics provided evidence for a potential interaction of AgrA and SAL, resulting in reduced activity of the agr system. In conclusion, SAL stabilized the mature S. aureus biofilms, which may prevent bacterial cell dissemination. However, it may foster the establishment of infections locally and consequently increase bacterial persistence leading to therapeutic failure.

Acapsular Staphylococcus aureus with a non-functional agr regains capsule expression after passage through the bloodstream in a bacteremia mouse model.

Selection pressures exerted on Staphylococcus aureus by host factors during infection may lead to the emergence of regulatory phenotypes better adapted to the infection site. Traits convenient for persistence may be fixed by mutation thus turning these mutants into microevolution endpoints. The feasibility that stable, non-encapsulated S. aureus mutants can regain expression of key virulence factors for survival in the bloodstream was investigated. S. aureus agr mutant HU-14 (IS256 insertion in agrC) from a patient with chronic osteomyelitis was passed through the bloodstream using a bacteriemia mouse model and derivative P3.1 was obtained. Although IS256 remained inserted in agrC, P3.1 regained production of capsular polysaccharide type 5 (CP5) and staphyloxanthin. Furthermore, P3.1 expressed higher levels of asp23/SigB when compared with parental strain HU-14. Strain P3.1 displayed decreased osteoclastogenesis capacity, thus indicating decreased adaptability to bone compared with strain HU-14 and exhibited a trend to be more virulent than parental strain HU-14. Strain P3.1 exhibited the loss of one IS256 copy, which was originally located in the HU-14 noncoding region between dnaG (DNA primase) and rpoD (sigA). This loss may be associated with the observed phenotype change but the mechanism remains unknown. In conclusion, S. aureus organisms that escape the infected bone may recover the expression of key virulence factors through a rapid microevolution pathway involving SigB regulation of key virulence factors.

Photodynamic inactivation mediated by 5-aminolevulinic acid of bacteria in planktonic and biofilm forms.

Bacterial photodynamic inactivation (PDI) employing endogenous production of porphyrins from 5-aminolevulinic acid (ALA) - named ALA-PDI-, is a new promising tool to achieve bacteria control in non-spread infections. The technique combines the action of the porphyrins acting as photosensitisers with light, to produce reactive oxygen species to target the pathogen. To date, some clinical applications of ALA-PDI have been reported although variable responses ranging from total eradication to absence of photokilling were found. ALA-PDI conducted at suboptimal conditions may lead to misleading results and the complexity of haem synthesis in bacteria hinders the optimization of the treatment. The present work aimed to gain insight on the variables affecting ALA-PDI in Gram-positives and Gram-negatives bacteria growing on planktonic and biofilm cultures and to correlate the degree of the response with the amount and type of porphyrin synthesised. Staphylococcus epidermidis and Escherichia coli clinical isolates and Pseudomonas aeruginosa ATCC27853 and Staphylococcus aureus ATCC25923 strains were utilised, and the optimal conditions of concentration and time exposure of ALA, and light dose were set. In both Gram-positive species analysed, a peak of porphyrin synthesis was observed at 1-2 mM ALA in biofilm and planktonic cultures, which fairly correlated with the decrease in the number of CFU after PDI (5 to 7 logs) and porphyrin content was in the same order of magnitude. In addition, ALA-PDI was similarly effective for planktonic and biofilm S. aureus cultures, and more effective in S. epidermidis planktonic cultures at low light doses. Beyond a certain light dose, it was not possible to achieve further photosensitization. Similarly, a plateau of cell death was attained at a certain ALA incubation time. Accumulation of hydrophilic porphyrins at longer incubation periods was observed. The proportion of porphyrins changed as a function of ALA concentration and incubation time in the Gram-positive bacteria, though we did not find a clear correlation between the porphyrin type and PDI response. As a salient feature was the presence of isococroporphyrin isoforms in both Gram-positive and Gram-negative bacteria. Gram-negative bacteria were quite refractory to the treatment: P. aeruginosa was slightly inactivated (4-logs reduction) at 40 mM ALA, whereas E. coli was not inactivated at all. These species accumulated high ALA quantities and the amount of porphyrins did not correlate with the degree of photoinactivation. Our microscopy studies show that porphyrins are not located in the envelopes of Gram-negative bacteria, reinforcing the hypothesis that endogenous porphyrins fail to attack these structures.

Within-host evolution of bovine Staphylococcus aureus selects for a SigB-deficient pathotype characterized by reduced virulence but enhanced proteolytic activity and biofilm formation.

Staphylococcus aureus is a major cause of bovine mastitis, commonly leading to long-lasting, persistent and recurrent infections. Thereby, S. aureus constantly refines and permanently adapts to the bovine udder environment. In this work, we followed S. aureus within-host adaptation over the course of three months in a naturally infected dairy cattle with chronic, subclinical mastitis. Whole genome sequence analysis revealed a complete replacement of the initial predominant variant by another isogenic variant. We report for the first time within-host evolution towards a sigma factor SigB-deficient pathotype in S. aureus bovine mastitis, associated with a single nucleotide polymorphism in rsbU (G368A → G122D), a contributor to SigB-functionality. The emerged SigB-deficient pathotype exhibits a substantial shift to new phenotypic traits comprising strong proteolytic activity and poly-N-acetylglucosamine (PNAG)-based biofilm production. This possibly unlocks new nutritional resources and promotes immune evasion, presumably facilitating extracellular persistence within the host. Moreover, we observed an adaptation towards attenuated virulence using a mouse infection model. This study extends the role of sigma factor SigB in S. aureus pathogenesis, so far described to be required for intracellular persistence during chronic infections. Our findings suggest that S. aureus SigB-deficiency is an alternative mechanism for persistence and underpin the clinical relevance of staphylococcal SigB-deficient variants which are consistently isolated during human chronic infections.

The anti MRSA biofilm activity of Thymus vulgaris essential oil in nanovesicles.

BACKGROUND: Thymus vulgaris essential oil (T) could be an alternative to classical antibiotics against bacterial biofilms, which show increased tolerance to antibiotics and host defence systems and contribute to the persistence of chronic bacterial infections. HYPOTHESIS: A nanovesicular formulation of T may chemically protect the structure and relative composition of its multiple components, potentially improving its antibacterial and antibiofilm activity. STUDY DESIGN: We prepared and structurally characterized T in two types of nanovesicles: nanoliposomes (L80-T) made of Soybean phosphatidylcholine (SPC) and Polysorbate 80 (P80) [SPC:P80:T 1:0.75:0.3 w:w], and nanoarchaeosomes (A80-T) made of SPC, P80 and total polar archaeolipids (TPA) extracted from archaebacteria Halorubrum tebenquichense [SPC:TPA:P80:T 0.5:0.50.75:0.7 w:w]. We determined the macrophage cytotoxicity and the antibacterial activity against Staphylococcus aureus ATCC 25,923 and four MRSA clinical strains. RESULTS: L80-T (Z potential -4.1 ±â€¯0.6 mV, ∼ 115 nm, ∼ 22 mg/ml T) and A80-T (Z potential -6.6 ±â€¯1.5 mV, ∼ 130 nm, ∼ 42 mg/ml T) were colloidally and chemically stable, maintaining size, PDI, Z potential and T concentration for at least 90 days. While MIC90 of L80-T was > 4 mg/ml T, MIC90 of A80-T was 2 mg/ml T for all S. aureus strains. The antibiofilm formation activity was maximal for A80-T, while L80-T did not inhibit biofilm formation compared to untreated control. A80-T significantly decreased the biomass of preformed biofilms of S. aureus ATCC 25,923 strain and of 3 of the 4 clinical MRSA isolates at 4 mg/ml T. It was found that the viability of J774A.1 macrophages was decreased significantly upon 24 h incubation with A80-T, L80-T and T emulsion at 0.4 mg/ml T. These results show that from 0.4 mg/ml T, a value lower than MIC90 and the one displaying antibiofilm activity, with independence of its formulation, T significantly decreased the macrophages viability. CONCLUSION: Overall, because of its lower MIC90 against planktonic bacteria, higher antibiofilm formation capacity and stability during storage, A80-T resulted better antibacterial agent than T emulsion and L80-T. These results open new avenues to explode the A80-T antimicrobial intracellular activity.

Distinct phenotypic traits of Staphylococcus aureus are associated with persistent, contagious bovine intramammary infections.

Staphylococcus aureus causing persistent, recurrent bovine intramammary infections are still a major challenge to dairy farming. Generally, one or a few clonal lineages are predominant in dairy herds, indicating animal-to-animal transfers and the existence of distinct pathotypic traits. The aim of this study was to determine if long term persistence and spreading of S. aureus are associated with specific phenotypic traits, including cellular invasion, cytotoxicity and biofilm formation. Mastitis isolates were collected over a 3-years period from a single dairy herd, resulting in two persistent subtypes, the high within-herd prevalent subtype ST9 (CC9)-methicillin-susceptible S. aureus (MSSA), designated HP/ST9, and the low within-herd prevalent subtype ST504 (CC705)-MSSA, designated LP/ST504. Characterization of the two different coexisting persistent subtypes showed that the following phenotypic traits are particularly associated with high within-herd prevalence: lack of capsular polysaccharide expression, high cellular invasiveness, low cytotoxicity and high biofilm/ poly-N-acetylglucosamine (PNAG) production, which may concomitantly contribute to the spreading of HP/ST9 within the herd. By contrast to HP/ST9, LP/ST504 is characterized by the formation of colony dendrites, which may help the bacteria to access deeper tissues as niches for persistence in single animals. Thus, within a single herd, two different types of persistence can be found in parallel, allowing longtime persistence of S. aureus in dairy cattle. Furthermore, this study indicates that ST9 (CC9)-MSSA strains, which are currently thought to have their primary reservoir in swine and humans, can also successfully spread to new hosts and persist in dairy herds for years.

Mutation of Agr Is Associated with the Adaptation of Staphylococcus aureus to the Host during Chronic Osteomyelitis.

Selection pressures exerted on Staphylococcus aureus by host factors may lead to the emergence of mutants better adapted to the evolving conditions at the infection site. This study was aimed at identifying the changes that occur in S. aureus exposed to the host defense mechanisms during chronic osteomyelitis and evaluating whether these changes affect the virulence of the organism. Genome assessment of two S. aureus isolates collected 13 months apart (HU-85a and HU-85c) from a host with chronic osteomyelitis was made by whole genome sequencing. Agr functionality was assessed by qRT-PCR. Isolates were tested in a rat model of osteomyelitis and the bacterial load (CFU/tibia) and the morphometric osteomyelitic index (OI) were determined. The ability of the isolates to trigger the release of proinflammatory cytokines was determined on macrophages in culture. Persistence of S. aureus within the host resulted in an agrC frameshift mutation that likely led to the observed phenotype. The capacity to cause bone tissue damage and trigger proinflammatory cytokines by macrophages of the agr-deficient, unencapsulated derivative (HU-85c) was decreased when compared with those of the isogenic CP8-capsulated parental strain (HU-85a). By comparison, no significant differences were found in the bacterial load or the OI from rats challenged with isogenic Reynolds strains [CP5, CP8, and non-typeable (NT)], indicating that lack of CP expression alone was not likely responsible for the reduced capacity to cause tissue damage in HU-85c compared with HU-85a. The production of biofilm was significantly increased in the isogenic derivative HU-85c. Lack of agr-dependent factors makes S. aureus less virulent during chronic osteomyelitis and alteration of the agr functionality seems to permit better adaptation of S. aureus to the chronically infected host.

Enhancement of photodynamic inactivation of Staphylococcus aureus biofilms by disruptive strategies.

Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers and visible light. On the one hand, near-infrared treatment (NIRT) has also bactericidal and dispersal effects on biofilms. In addition, dispersal biological tools such as enzymes have also been employed in antibiotic combination treatments. The aim of this work was to use alternative approaches to increase the PDI efficacy, employing combination therapies aimed at the partial disruption of the biofilms, thus potentially increasing photosensitizer or oxygen penetration and interaction with bacteria. To that end, we applied toluidine blue (TB)-PDI treatment to Staphylococcus aureus biofilms previously treated with NIRT or enzymes and investigated the outcome of the combined therapies. TB employed at 0.5 mM induced per se 2-log drop in S. aureus RN6390 biofilm viability. Each NIRT (980-nm laser) and PDI (635-nm laser) treatment induced a further reduction of 1-log of viable counts. The combination of successive 980- and 635-nm laser treatments on TB-treated biofilms induced additive effects, leading to a 4.5-log viable count decrease. Proteinase K treatment applied to S. aureus of the Newman strain induced an additive effect on PDI mortality, leading to an overall 4-log decrease in S. aureus viability. Confocal scanning laser microscopy after biofilm staining with a fluorescent viability test and scanning electron microscopy observations were correlated with colony counts. The NIRT dose employed (227 J/cm2) led to an increase from 21 to 47 °C in the buffer temperature of the biofilm system, and this NIRT dose also induced 100% keratinocyte death. Further work is needed to establish conditions under which biofilm dispersal occurs at lower NIRT doses.

The Active Component of Aspirin, Salicylic Acid, Promotes Staphylococcus aureus Biofilm Formation in a PIA-dependent Manner.

Aspirin has provided clear benefits to human health. But salicylic acid (SAL) -the main aspirin biometabolite- exerts several effects on eukaryote and prokaryote cells. SAL can affect, for instance, the expression of Staphylococcus aureus virulence factors. SAL can also form complexes with iron cations and it has been shown that different iron chelating molecules diminished the formation of S. aureus biofilm. The aim of this study was to elucidate whether the iron content limitation caused by SAL can modify the S. aureus metabolism and/or metabolic regulators thus changing the expression of the main polysaccharides involved in biofilm formation. The exposure of biofilm to 2 mM SAL induced a 27% reduction in the intracellular free Fe2+ concentration compared with the controls. In addition, SAL depleted 23% of the available free Fe2+ cation in culture media. These moderate iron-limited conditions promoted an intensification of biofilms formed by strain Newman and by S. aureus clinical isolates related to the USA300 and USA100 clones. The slight decrease in iron bioavailability generated by SAL was enough to induce the increase of PIA expression in biofilms formed by methicillin-resistant as well as methicillin-sensitive S. aureus strains. S. aureus did not produce capsular polysaccharide (CP) when it was forming biofilms under any of the experimental conditions tested. Furthermore, SAL diminished aconitase activity and stimulated the lactic fermentation pathway in bacteria forming biofilms. The polysaccharide composition of S. aureus biofilms was examined and FTIR spectroscopic analysis revealed a clear impact of SAL in a codY-dependent manner. Moreover, SAL negatively affected codY transcription in mature biofilms thus relieving the CodY repression of the ica operon. Treatment of mice with SAL induced a significant increase of S aureus colonization. It is suggested that the elevated PIA expression induced by SAL might be responsible for the high nasal colonization observed in mice. SAL-induced biofilms may contribute to S. aureus infection persistence in vegetarian individuals as well as in patients that frequently consume aspirin.

Sae regulator factor impairs the response to photodynamic inactivation mediated by Toluidine blue in Staphylococcus aureus.

Photodynamic inactivation (PDI) involves the combined use of light and a photosensitizer, which, in the presence of oxygen, originates cytotoxic species capable of inactivating bacteria. Since the emergence of multi-resistant bacterial strains is becoming an increasing public health concern, PDI becomes an attractive choice. The aim of this work was to study the differential susceptibility to Toluidine blue (TB) mediated PDI (TB-PDI) of S. aureus mutants (RN6390 and Newman backgrounds) for different key regulators of virulence factors related to some extent to oxidative stress. Complete bacteria eradication of planktonic cultures of RN6390 S. aureus photosensitized with 13µM TB was obtained upon illumination with a low light dose of 4.2J/cm2 from a non-coherent light source. Similarly, complete cell death was achieved applying 1.3µM TB and 19J/cm2 light dose, showing that higher light doses can lead to equal cell death employing low photosensitizer concentrations. Interestingly, RN6390 in planktonic culture responded significantly better to TB-PDI than the Newman strain. We showed that deficiencies in rsbU, mgrA (transcription factors related to stress response) or agr (quorum sensing system involved in copper resistance to oxidative stress) did not modify the response of planktonic S. aureus to PDI. On the other hand, the two component system sae impaired the response to TB-PDI through a mechanism not related to the Eap adhesin. More severe conditions were needed to inactivate S. aureus biofilms (0.5mM TB, 157J/cm2 laser light). In mutant sae biofilms, strain dependant differential susceptibilities are not noticed.

Genotipificación y estudio de los genes pauA y sua de aislamientos de Streptococcus uberis de mastitis bovina / Genotyping and study of the pauA and sua genes of Streptococcus uberis isolates from bovine mastitis

This study aimed to determine the clonal relationship among 137 Streptococcus uberis isolates from bovine milk with subclinical or clinical mastitis in Argentina and to assess the prevalence and conservation of pauA and sua genes. This information is critical for the rational design of a vaccine for the prevention of bovine mastitis caused by S. uberis. The isolates were typed by random amplified polymorphic DNA (RAPD) analysis and by pulsed-field gel electrophoresis (PFGE). The 137 isolates exhibited 61 different PFGE types and 25 distinct RAPD profiles. Simpson's diversity index was calculated both for PFGE (0.983) and for RAPD (0.941), showing a high discriminatory power in both techniques. The analysis of the relationship between pairs of isolates showed 92.6 % concordance between both techniques indicating that any given pair of isolates distinguished by one method tended to be distinguished by the other. The prevalence of the sua and pauA genes was 97.8 % (134/137) and 94.9 % (130/137), respectively. Nucleotide and amino acid sequences of the sua and pauA genes from 20 S. uberis selected isolates, based on their PFGE and RAPD types and geographical origin, showed an identity between 95 % and 100 % with respect to all reference sequences registered in GenBank. These results demonstrate that, in spite of S. uberis clonal diversity, the sua and pauA genes are prevalent and highly conserved, showing their importance to be included in future vaccine studies to prevent S. uberis bovine mastitis.

Este estudio pretendió determinar la relación clonal entre 137 aislamientos de S. uberis obtenidos de leche de bovinos con mastitis clínica o subclínica en la Argentina, como así también la prevalencia y la conservación de los genes sua y PauA entre dichos aislamientos. Esta información es crítica para el diseño racional de una vacuna que prevenga la mastitis bovina por S. uberis. Los aislamientos se tipificaron molecularmente por amplificación al azar del ADN polimórfico (RAPD) y mediante electroforesis de campos pulsados (PFGE). Los 137 aislamientos mostraron 61 pulsotipos mediante PFGE y 25 tipos de RAPD diferentes. Los índices de Simpson calculados fueron 0,983 por PFGE y 0,941 por RAPD; esto evidencia el elevado poder discriminatorio de ambas técnicas. El análisis de la relación entre pares de aislamientos mostró un 92,6 % de concordancia entre ambas técnicas, lo que indica que cualquier par de aislamientos que fue distinguido por un método tendió a ser distinguido por el otro. La prevalencia de los genes sua y puaA fue del 97,8 % (134/137) y 94,9 % (130/137), respectivamente. Las secuencias de nucleótidos y de aminoácidos codificados por los genes sua y pauA de los 20 aislamientos de S. uberis seleccionados sobre la base de su tipo de PFGE y RAPD y origen geográfico tuvieron un porcentaje de identidad de entre 95 % y 100 % con respecto a todas las secuencias de referencia registradas en GenBank. Estos resultados demuestran que, a pesar de la diversidad clonal de S. uberis, los genes sua y pauA son prevalentes y están altamente conservados y deberían ser incluidos en futuros estudios de vacunas para prevenir mastitis bovina causada por S. uberis.

Genotyping and study of the pauA and sua genes of Streptococcus uberis isolates from bovine mastitis.

This study aimed to determine the clonal relationship among 137 Streptococcus uberis isolates from bovine milk with subclinical or clinical mastitis in Argentina and to assess the prevalence and conservation of pauA and sua genes. This information is critical for the rational design of a vaccine for the prevention of bovine mastitis caused by S. uberis. The isolates were typed by random amplified polymorphic DNA (RAPD) analysis and by pulsed-field gel electrophoresis (PFGE). The 137 isolates exhibited 61 different PFGE types and 25 distinct RAPD profiles. Simpson's diversity index was calculated both for PFGE (0.983) and for RAPD (0.941), showing a high discriminatory power in both techniques. The analysis of the relationship between pairs of isolates showed 92.6% concordance between both techniques indicating that any given pair of isolates distinguished by one method tended to be distinguished by the other. The prevalence of the sua and pauA genes was 97.8% (134/137) and 94.9% (130/137), respectively. Nucleotide and amino acid sequences of the sua and pauA genes from 20 S. uberis selected isolates, based on their PFGE and RAPD types and geographical origin, showed an identity between 95% and 100% with respect to all reference sequences registered in GenBank. These results demonstrate that, in spite of S. uberis clonal diversity, the sua and pauA genes are prevalent and highly conserved, showing their importance to be included in future vaccine studies to prevent S. uberis bovine mastitis.

Salmonella enterica serovar Enteritidis enterocolitis during late stages of gestation induces an adverse pregnancy outcome in the murine model.

Foodborne diseases caused by Salmonella enterica serovar Enteritidis (S. Enteritidis) are a significant health problem. Pregnancy, state of immunological tolerance, is a predisposing condition for the development of infections with intracellular pathogens. Salmonella species can cause pregnancy complications such as chorioamnionitis, transplacental fetal infection, pre term labor, abortions, neonatal and maternal septicemia. However, the specific mechanisms by which Salmonella infections trigger these alterations are not clear. In the present work, using a self-limiting enterocolitis murine model, we show that the ingestion of a low dose of S. Enteritidis at late stages of pregnancy (day 15 of gestation) is sufficient to induce massive maternal infection. We found that Salmonella infection leads to 40% of pre term delivery, 33% of abortion and fetal growth restriction. Placental dysfunction during S. Enteritidis enterocolitis was confirmed through cellular infiltration and hypoxia markers (MPO activity and COX-1 and COX-2 expression, respectively). Apoptosis in placental tissue due to Salmonella infection was also evident at day 18 of gestation when investigated by morphometric procedure, DNA fragmentation and Fas/FasL expression. Also, the expression of IFN-γ, TNF-α, IL-17 and IL-10 was up regulated in response to Salmonella not only in placenta, but also in amniotic fluid and maternal serum. Altogether, our results demonstrate that S. Enteritidis enterocolitis during late stages of gestation causes detrimental effect on pregnancy outcome.

Dam methylation regulates the expression of SPI-5-encoded sopB gene in Salmonella enterica serovar Typhimurium.

DNA adenine methylation is an essential factor in Salmonella virulence. Here, we investigate the involvement of DNA adenine methylase (Dam) in the expression and translocation of a SPI-5-encoded effector of S. Typhimurium. SopB expression and secretion were determined using SopB-FLAG-tagged wild type and dam strains of S. Typhimurium. Western blot and quantitative reverse transcriptase PCR analysis showed that the dam mutant expresses lower levels of SopB protein and sopB mRNA than the wild type strain under SPI-1 and SPI-2 inducing conditions in vitro. SopB secretion was also considerably impaired in the absence of dam. In agreement with in vitro experiments, SopB synthesis in dam mutants recovered from infected epithelial cells and from murine mesenteric lymph nodes was reduced by 40% respect to the wild type strain (p < 0.05). SopB translocation was neither detected in the cytosol of epithelial cells nor in the cytosol of cells isolated from mesenteric lymph nodes infected with the dam mutant. Taken together, our results demonstrate that, in S. Typhimurium, Dam methylation modulates the expression and translocation of SPI-5-encoded SopB effector.

AvrA effector protein of Salmonella enterica serovar Enteritidis is expressed and translocated in mesenteric lymph nodes at late stages of infection in mice.

Salmonellosis is a major health problem worldwide. Salmonella enterica serovar Enteritidis (S. Enteritidis) has been a primary cause of Salmonella outbreaks in many countries. AvrA is an SPI-1 effector protein involved in the enteritis pathway, with critical roles in inhibiting inflammation and apoptosis. In this work, we constructed an AvrA-FLAG-tagged strain of S. Enteritidis to analyse the expression profile of AvrA in vitro, in cell culture and in vivo. AvrA expression and secretion were observed in vitro under culture conditions that mimicked intestinal and intracellular environments. In agreement, bacteria isolated from infected cell monolayers expressed and translocated AvrA for at least 24 h post-inoculation. For in vivo experiments, BALB/c mice were inoculated by the natural route of infection with the AvrA-FLAG strain. Infecting bacteria and infected cells were recovered from mesenteric lymph nodes (MLN). Our results showed that AvrA continues to be synthesized in vivo up to day 8 post-inoculation. Moreover, AvrA translocation was detected in the cytosol of cells isolated from MLN 8 days after infection. Interestingly, we observed that AvrA is secreted by both type three secretion system (T3SS)-1 and T3SS-2. In summary, these findings indicate that AvrA expression is not constrained to the initial host-bacteria encounter in the intestinal environment as defined previously. The AvrA effector may participate also in systemic S. Enteritidis infection.

Protein antigens increase the protective efficacy of a capsule-based vaccine against Staphylococcus aureus in a rat model of osteomyelitis.

Staphylococcus aureus is an invasive bacterial pathogen, and antibiotic resistance has impeded adequate control of infections caused by this microbe. Moreover, efforts to prevent human infections with single-component S. aureus vaccines have failed. In this study, we evaluated the protective efficacy in rats of vaccines containing both S. aureus capsular polysaccharides (CPs) and proteins. The serotypes 5 CP (CP5) and 8 CP (CP8) were conjugated to tetanus toxoid and administered to rats alone or together with domain A of clumping factor A (ClfA) or genetically detoxified alpha-toxin (dHla). The vaccines were delivered according to a preventive or a therapeutic regimen, and their protective efficacy was evaluated in a rat model of osteomyelitis. Addition of dHla (but not ClfA) to the CP5 or CP8 vaccine induced reductions in bacterial load and bone morphological changes compared with immunization with either conjugate vaccine alone. Both the prophylactic and therapeutic regimens were protective. Immunization with dHla together with a pneumococcal conjugate vaccine used as a control did not reduce staphylococcal osteomyelitis. The emergence of unencapsulated or small-colony variants during infection was negligible and similar for all of the vaccine groups. In conclusion, addition of dHla to a CP5 or CP8 conjugate vaccine enhanced its efficacy against S. aureus osteomyelitis, indicating that the inclusion of multiple antigens will likely enhance the efficacy of vaccines against both chronic and acute forms of staphylococcal disease.

Rapid and reliable identification of Staphylococcus aureus capsular serotypes by means of artificial neural network-assisted Fourier transform infrared spectroscopy.

Staphylococcus aureus capsular polysaccharides (CP) are important virulence factors and represent putative targets for vaccine development. Therefore, the purpose of this study was to develop a high-throughput method to identify and discriminate the clinically important S. aureus capsular serotypes 5, 8, and NT (nontypeable). A comprehensive set of clinical isolates derived from different origins and control strains, representative for each serotype, were used to establish a CP typing system based on Fourier transform infrared (FTIR) spectroscopy and chemometric techniques. By combining FTIR spectroscopy with artificial neuronal network (ANN) analysis, a system was successfully established, allowing a rapid identification and discrimination of all three serotypes. The overall accuracy of the ANN-assisted FTIR spectroscopy CP typing system was 96.7% for the internal validation and 98.2% for the external validation. One isolate in the internal validation and one isolate in the external validation failed in the classification procedure, but none of the isolates was incorrectly classified. The present study demonstrates that ANN-assisted FTIR spectroscopy allows a rapid and reliable discrimination of S. aureus capsular serotypes. It is suitable for diagnostic as well as large-scale epidemiologic surveillance of S. aureus capsule expression and provides useful information with respect to chronicity of infection.