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Tumor necrosis factor-alpha (TNF-α) and interleukin-8 (IL-8/CXCL8) play pivotal roles in mediating inflammatory responses to invading pathogens. In this study, we identified and analyzed expressions of cobia TNF-α and IL-8 during Streptococcus dysgalactiae infection. The cloned cDNA transcript of cobia TNF-α comprised of 1281 base pairs (bp), with a 774 bp open reading frame (ORF) encoding 257 amino acids. The deduced amino acid sequence of cobia TNF-α showed a close relationship (84% similarity) with TNF-α of yellowtail amberjack. The cloned IL-8 cDNA sequence was 828 bp long, including a 300-bp ORF encoding 99 amino acids. The deduced amino acid sequence of cobia IL-8 shared 90% identity with IL-8 of striped trumpeter. Cobia challenged with a virulent S. dysgalactiae strain displayed an early significant up-regulation of TNF-α and IL-8 in head kidney, liver, and spleen. Notably, IL-8 expression level increased dramatically in the liver at the severe stage of infection (72 h). In conclusion, a better understanding of TNF-α and IL-8 allows more detailed investigation of immune responses in cobia and furthers study on controlling the infectious disease caused by S. dysgalactiae., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Here, we investigated the antibacterial effects of chemical changes induced by nonthermal atmospheric pressure plasma (NTAPP) on smooth and rough Ti. The morphologies of smooth and rough surfaces of Ti were examined using scanning electron microscopy (SEM). Both Ti specimens were then treated for 10 min by NTAPP with nitrogen gas. The surface roughness, chemistry, and wettability were examined by optical profilometry, x-ray photoelectron spectroscopy, and water contact angle analysis, respectively. Bacterial attachment was measured by determining the number of colony forming units and by SEM analysis. The rough Ti showed irregular micropits, whereas smooth Ti had a relatively regular pattern on the surface. There were no differences in morphology between samples before and after NTAPP treatment. NTAPP treatment resulted in changes from hydrophobic to hydrophilic properties on rough and smooth Ti; rough Ti showed relatively higher hydrophilicity. Before NTAPP treatment, Streptococcus sanguinis (S. sanguinis) showed greater attachment on rough Ti, and after NTAPP treatment, there was a significant reduction in bacterial attachment. Moreover, the bacterial attachment rate was significantly lower on rough Ti, and the structure of S. sanguinis colonies were significantly changed on NTAPP-treated Ti. NTAPP treatment inhibited bacterial attachment surrounding titanium implants, regardless of surface topography. Therefore, NTAPP treatment on Ti is a next-generation tool for antibacterial applications in the orthopaedic and dental fields.

Objective: To evaluate whether ethnicity is independently associated with vaginal microbiota (VMB) composition in women living in Amsterdam, the Netherlands, as has been shown for American women., Methods: Women (18-34 years, non-pregnant, N = 610) representing the six largest ethnic groups (Dutch, African Surinamese, South-Asian Surinamese, Turkish, Moroccan, and Ghanaian) were sampled from the population-based HELIUS study. Sampling was performed irrespective of health status or healthcare seeking behavior. DNA was extracted from self-sampled vaginal swabs and sequenced by Illumina MiSeq (16S rRNA gene V3-V4 region)., Results: The overall prevalence of VMBs not dominated by lactobacilli was 38.5%: 32.2% had a VMB resembling bacterial vaginosis and another 6.2% had a VMB dominated by Bifidobacteriaceae (not including Gardnerella vaginalis), Corynebacterium, or pathobionts (streptococci, staphylococci, Proteus or Enterobacteriaceae). The most prevalent VMB in ethnically Dutch women was a Lactobacillus crispatus-dominated VMB, in African Surinamese and Ghanaian women a polybacterial G. vaginalis-containing VMB, and in the other ethnic groups a L. iners-dominated VMB. After adjustment for sociodemographic, behavioral and clinical factors, African Surinamese ethnicity (adjusted odds ratio (aOR) 5.1, 95% confidence interval (CI) 2.1-12.0) and Ghanaian ethnicity (aOR 4.8, 95% CI 1.8-12.6) were associated with having a polybacterial G. vaginalis-containing VMB, and African Surinamese ethnicity with a L. iners-dominated VMB (aOR 2.8, 95% CI 1.2-6.2). Shorter steady relationship duration, inconsistent condom use with casual partners, and not using hormonal contraception were also associated with having a polybacterial G. vaginalis-containing VMB, but human papillomavirus infection was not. Other sexually transmitted infections were uncommon., Conclusions: The overall prevalence of having a VMB not dominated by lactobacilli in this population-based cohort of women aged 18-34 years in Amsterdam was high (38.5%), and women of sub-Saharan African descent were significantly more likely to have a polybacterial G. vaginalis-containing VMB than Dutch women independent of modifiable behaviors.

Interferon-inducible protein 56 (IFI56, also known as ISG56/IFIT1, interferon-induced protein with tetratricopeptide repeats 1) is strongly induced in response to interferon and a potent inhibitor of viral replication and translational initiation. Here, we describe the identification of IFI56 (OfIFI56) in olive flounder, its characteristic features, and expression levels in various tissues before and after viral hemorrhagic septicemia virus (VHSV) infection. The full-length OfIFI56 sequence was identified from rapid amplification of cDNA ends PCR. The complete coding sequence of OfIFI56 is 1971 bp in length and encodes 431 amino acids. The putative OfIFI56 protein has multiple tetratricopeptide (TPR) motifs, which regulate diverse biological processes, such as organelle targeting, protein import, and vesicle fusion. Based on sequence analysis, the Larimichthys crocea IFI56 protein (61%) had the highest sequence homology to OfIFI56. In healthy olive flounder, OfIFI56 mRNA expression was detected in many tissues such as intestine, gill, head kidney, heart, spleen, and trunk kidney tissues. After VHSV challenge, OfIFI56 mRNA was significantly up-regulated in these tissues. Additionally, OfIFI56 expression was induced by poly I:C but not by Streptococcus parauberis and S. iniae infection or lipopolysaccharide injection in kidney and spleen tissues of olive flounder. These results demonstrate that piscine OfIFI56 expression is not induced by bacterial infection but is selectively induced by viral infection, especially VHSV, and that OfIFI56 may play an important role in the host response against VHSV infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

An ultrasound/ultraviolet (US/UV) baffled reactor was developed to fill the gap in ultraviolet (UV) disinfection associated with disinfection efficiency. According to the previously selected operational condition, a continuous-flow US/UV baffled reactor was continuously operated in a wastewater treatment plant at a pilot scale for nearly three months, and the disinfection influent and effluent were analyzed, including fecal coliforms, Escherichia coli, and fecal streptococci. The US/UV baffled reactor could guarantee a high effluent disinfection performance in terms of fecal coliforms removal even with the fluctuation of the secondary effluent. All the disinfected effluents satisfied the requirement of the "Pollutants Discharge Standard of Municipal Wastewater Treatment Plant in China" (fecal coliforms below 1000CFU/L for class 1A), and 87% of the tested fecal coliforms concentration in the disinfected effluent was below 100CFU/L, nearly eliminating all fecal coliforms. Further analysis of the E. coli and fecal streptococci showed the broad disinfection ability and high disinfection efficiency of the US/UV baffled reactor. The flexibility of the specific energy consumption for the disinfection system depends on the water quality., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The main components of the quorum-sensing system are expected to be favorable targets for drug development to combat various chronic infectious diseases. ComA of Streptococcus is an ATP-binding cassette transporter containing a peptidase domain (PEP), which is essential for the quorum-sensing signal production. Using high-throughput screening, we found a potent small molecule that suppressed the S. mutans quorum-sensing pathway through inhibition of PEP activity. The compound effectively attenuated the biofilm formation and competence development of S. mutans without inhibiting cell growth. The kinetic and structural studies with this molecule and a related compound unexpectedly revealed an allosteric site of PEP. This relatively hydrophobic site is thought to undergo large structural changes during the catalytic process. These compounds inhibit PEP activity by binding to and suppressing the structural changes of this site. These results showed that PEP is a good target for inhibitors of the Streptococcus quorum-sensing system.

Ventilator-associated pneumonia (VAP) is a serious complication of mechanical ventilation leading to high morbidity and mortality among intubated neonates in neonatal intensive care units (NICUs). Endotracheal tube (ETT) biofilm flora were considered to be responsible for the occurrence of VAP as a reservoir of pathogens. However, regarding neonates with VAP, little is known about the complex microbial signatures in ETT biofilms. In the present study, a culture-independent approach based on next generation sequencing was performed as an initial survey to investigate the microbial communities in ETT biofilms of 49 intubated neonates with and without VAP. Our results revealed a far more complex microflora in ETT biofilms from intubated neonates compared to a previous culture-based study. The abundance of Streptococci in ETT biofilms was significantly related to the onset of VAP. By isolating Streptococci in ETT biofilms, we found that Streptococci enhanced biofilm formation of the common nosocomial pathogen Pseudomonas aeruginosa PAO1 and decreased IL-8 expression of airway epithelia cells exposed to the biofilm conditioned medium of PAO1. This study provides new insight into the pathogenesis of VAP among intubated neonates. More studies focusing on intubated neonates are warranted to develop strategies to address this important nosocomial disease in NICUs.

In recent years, mandarin fish had a high mortality rate associated with abnormal swimming, exophthalmia, corneal opacity and eye hemorrhage on a fish farm located at Foshan city, Guangdong province, China. Three isolates of Gram-positive, chain-forming cocci were recovered from moribund fish, and designated as SS131025-1, SS131025-2, and SS131025-3. These isolates were identified as Streptococcus uberis according to their morphologic and physio-biochemical characteristics as well as phylogenetic analysis based on their 16S rRNA and GapC gene sequences. The pathogenicity of S. uberis to mandarin fish was determined by challenge experiments. Results of artificial challenge showed S. uberis infected healthy mandarin fish and lead to death by eyeball injection or immersion route, and the LD 50 of SS131025-1 with eyeball injection was 2.0 × 10 6.42  CFU per fish. Moreover extracellular product (ECP) of the isolated S.uberis induced CPB cell apoptosis and cause death of mandarin fish. In addition, these S. uberis strains could also infect tilapia, but not grass carp and crucian carp, and grew in brain-heart infusion broth with an optimal temperature of 37 °C, pH of 7.0, and salinity of 0%. Antibiotic sensitivity testing indicated that these isolates were susceptible to rifampicin and furazolidone but resistant to 20 kinds of antibiotics. Histopathologically, infection with S. uberis could cause serious pathological changes in brain tissues such as vacuoles in matrix, swollen mitochondria with lysis of cristae and disintegration, and lots of coccus was observed both under electron and light microscope. These results shed some light on the pathogenicity of the isolates and how to prevent and control S. uberis infection in mandarin fish., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

An extract prepared from cranberry juice by dialysis known as nondialyzable material (NDM) has been shown previously to possess anti-adhesion activity toward microbial species including oral bacteria, uropathogenic Escherichia coli and Helicobacter pylori. Bioassay-guided fractionation of cranberry NDM was therefore undertaken to identify the anti-adhesive constituents. An aqueous acetone-soluble fraction (NDMac) obtained from Sephadex LH-20 inhibited adhesion-linked activities by oral bacteria, including co-aggregation of oral bacteria Fusobacterium nucleatum with Streptococcus sanguinis or Porphyromonas gingivalis, and biofilm formation by Streptococcus mutans. Analysis of NDMac and subsequent subfractions by MALDI-TOF MS and 1 H NMR revealed the presence of A-type proanthocyanidin oligomers (PACs) of 3-6 degrees of polymerization composed of (epi)catechin units, with some (epi)gallocatechin and anthocyanin units also present, as well as quercetin derivatives. Subfractions containing putative xyloglucans in addition to the mixed polyphenols also inhibit biofilm formation by S. mutans (MIC = 125-250 μg mL -1 ). These studies suggest that the anti-adhesion activities of cranberry NDM on oral bacteria may arise from a combination of mixed polyphenol and non-polyphenol constituents.

Mastitis is an inflammation of mammary gland parenchyma that adversely affects bovine health and dairy production worldwide despite significant efforts to eradicate it. The aim of this work was to characterize the antimicrobial activity of 7-epiclusianone (7-epi), a compound extracted from the Rheedia brasiliensis fruit, its complex with copper against Streptococcus spp. isolated from bovine mastitis, and to assess their cytotoxicity to bovine mammary alveolar cells (MAC-T). The complex 7-epiclusianone-Cu (7-epi-Cu) was an amorphous green solid with optical activity. Its vibrational spectrum in the infrared region showed absorption bands in the high-frequency region, as well as bands that can be attributed to the unconjugated and conjugated stretching of the free ligand. The complex was anhydrous. One of the tested bacterial strains was not sensitive to the compounds, while the other three had MIC values of 7.8 µg mL -1 and minimum bactericidal concentration (MBC) values between 15.6 and 31.3 µg mL -1 . These two compounds are bacteriostatic, did not cause damage to the cell wall and, at sub-inhibitory concentrations, did not induce bacterial adhesion. The compounds were not cytotoxic. Based on these results, 7-epi and 7-epi-Cu exhibited desirable antimicrobial properties and could potentially be used in bovine mastitis treatment., Competing Interests: The authors declare no conflict of interest.

This Research Communication describes the study of in vitro biofilm formation of mastitis causing pathogens. Biofilms are communities of bacteria that are attached to a surface and to each other and are embedded in a self-produced matrix of extracellular polymeric substances. Biofilm formation is an important virulence factor that may result in recurrent or persistent udder infections and treatment failure through increased resistance to antibiotics and protection against host defences. In the present study 252 bacterial isolates from milk samples from bovine udder quarters with intramammary infections were examined with Congo Red agar (CRA) method and tube method (TM) for their ability to form biofilms. Both tests revealed a high number of biofilm-positive strains. Literature reports that the cure rates for Staphylococcus aureus infected udders are lower (27%) in comparison to cure rates of Streptococcus uberis (64-81%) or coagulase-negative staphylococci (CNS) mastitis (80-90%). The findings of the present study suggest that biofilm formation is not the main factor for the differences in cure rates of the various bacteria genera, because all tested pathogen groups showed a similarly high proportion of biofilm formation. Further research is needed to detect microbial biofilms on bovine udder epithelia.

Chitinases are hydrolytic enzymes which have been employed to breakdown chitin coats of pathogenic microorganisms, thereby weaken the defense system of several pathogens and insects. In this regard, we identified the chitinase genes of turbot and characterized their expression patterns in mucosal tissues following Vibrio anguillarum and Streptococcus iniae challenge. In present study, transcripts of three chitinase genes (CHIT1, CHIT2 and CHIT3) were captured, as well as their protein structures and expression patterns following different bacterial infection were also characterized. The chitinases were widely expressed in all tested tissues with the highest expression levels of CHIT1 and CHIT2 in intestine, and CHIT3 in skin. Finally, these three genes showed different expression patterns following bacterial challenge. The significant quick induction of chitinases in mucosal surfaces against infection indicated their key roles to prevent pathogen attachment and entry in mucosal immunity. Functional studies should further characterize the chitinases and avail utilization of their function to increase the disease resistance in maintaining the integrity of the mucosal barriers against infection and facilitating the disease resistant family/strain selection in turbot., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Streptococcus dysgalactiae subsp. equisimilis (SDSE) is a pyogenic, Lancefield C or G streptococcal pathogen. Until recently, it has been considered as an exclusive animal pathogen. Nowadays, it is responsible for both animal infections in wild animals, pets, and livestock and human infections often clinically similar to the ones caused by group A streptococcus (Streptococcus pyogenes). The risk of zoonotic infection is the most significant in people having regular contact with animals, such as veterinarians, cattlemen, and farmers. SDSE is also prevalent on skin of healthy dogs, cats, and horses, which pose a risk also to people having contact with companion animals. The main aim of this study was to evaluate if there are features differentiating animal and human SDSE isolates, especially in virulence factors involved in the first stages of pathogenesis (adhesion and colonization). Equal groups of human and animal SDSE clinical strains were obtained from superficial infections (skin, wounds, abscesses). The presence of five virulence genes (prtF1, prtF2, lmb, cbp, emm type) was evaluated, as well as ability to form bacterial biofilm and produce BLIS (bacteriocin-like inhibitory substances) which are active against human skin microbiota. The study showed that the presence of genes coding for fibronectin-binding protein and M protein, as well as BLIS activity inhibiting the growth of Corynebacterium spp. strains might constitute the virulence factors which are necessary to colonize human organism, whereas they are not crucial in animal infections. Those virulence factors might be horizontally transferred from human streptococci to animal SDSE strains, enabling their ability to colonize human organism.

Streptococcus sanguinis, a naturally competent opportunistic human pathogen, is a Gram-positive workhorse for genomics. It has recently emerged as a model for the study of type IV pili (Tfp)-exceptionally widespread and important prokaryotic filaments. To enhance genetic manipulation of Streptococcus sanguinis, we have developed a cloning-independent methodology, which uses a counterselectable marker and allows sophisticated markerless gene editing in situ. We illustrate the utility of this methodology by answering several questions regarding Tfp biology by (i) deleting single or mutiple genes, (ii) altering specific bases in genes of interest, and (iii) engineering genes to encode proteins with appended affinity tags. We show that (i) the last six genes in the pil locus harbouring all the genes dedicated to Tfp biology play no role in piliation or Tfp-mediated motility, (ii) two highly conserved Asp residues are crucial for enzymatic activity of the prepilin peptidase PilD and (iii) that pilin subunits with a C-terminally appended hexa-histidine (6His) tag are still assembled into functional Tfp. The methodology for genetic manipulation we describe here should be broadly applicable., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Since the introduction of modern dental implants in the 1980s, the number of inserted implants has steadily increased. Implant systems have become more sophisticated and have enormously enhanced patients' quality of life. Although there has been tremendous development in implant materials and clinical methods, bacterial infections are still one of the major causes of implant failure. These infections involve the formation of sessile microbial communities, called biofilms. Biofilms possess unique physical and biochemical properties and are hard to treat conventionally. There is a great demand for innovative methods to functionalize surfaces antibacterially, which could be used as the basis of new implant technologies. Present, there are few test systems to evaluate bacterial growth on these surfaces under physiological flow conditions. We developed a flow chamber model optimized for the assessment of dental implant materials. As a result it could be shown that biofilms of the five important oral bacteria Streptococcus gordonii, Streptococcus oralis, Streptococcus salivarius, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans, can be reproducibly formed on the surface of titanium, a frequent implant material. This system can be run automatically in combination with an appropriate microscopic device and is a promising approach for testing the antibacterial effect of innovative dental materials., Competing Interests: The authors have declared that no competing interests exist.

This manuscript presents novel approaches to grow and evaluate Streptococcal biofilm formation using the human respiratory pathogen Streptococcus pneumoniae (the pneumococcus) as the main model organism on biological surfaces in vitro and in vivo. Most biofilm models are based on growth on abiotic surfaces, which is relevant for many pathogens whose growth on surfaces or medical devices is a major cause of disease transmission and infections, especially in hospital environments. However, most infections with commensal organisms require biofilm formation on biological surfaces in the host at the site of colonization or infection. In vitro model systems incorporating biological components from the host and taking into account the host environment of the infectious site are not well described.In a series of publications, we have shown that S. pneumoniae form complex biofilms in the nasopharynx of mice and have devised methodology to evaluate the biofilm structure and function in this environment. We have also been able to recapitulate this biofilm phenotype in vitro by incorporating crucial factors associated with the host environment. Although the protocols presented in this manuscript are focused on S. pneumoniae, the same methodology can and has been used for other Streptococcal species that form biofilms on mucosal surfaces.

Biofilms are aggregates of attached microbial organisms whose existence on tissues is often recognised as a mechanism for the establishment of most chronic diseases. Herein we investigated the ability of piscine Streptococcus agalactiae, an important aquatic pathogen, for adaptation to this sessile lifestyle in vitro and in the brain of a tilapia fish model. Piscine S. agalactiae exhibited a weak attachment to polystyrene plates and expressed a low biofilm phenotype under the study conditions. Furthermore, fluorescent in situ hybridization and confocal laser scanning microscopy revealed discrete aggregates of attached S. agalactiae within brain tissues and around meningeal surfaces. They were embedded in an exopolysaccharide containing matrix, intractable to inflammatory response and showed some level of resistance to penicillin despite proven susceptibility on sensitivity test. Intracellular bacterial aggregates were also observed, moreover, antibody mediated response was not demonstrated during infection. Nucleated erythrocytes appear to facilitate brain invasion possibly via the Trojan horse mechanism leading to a granulomatous inflammation. We have demonstrated that biofilm is associated with persistence of S. agalactiae and the development of chronic meningoencephalitis in fish., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The discovery that Streptococcus pneumoniae uses a competence-stimulating peptide (CSP) to induce competence for natural transformation, and that other species of the mitis and the anginosus streptococcal groups use a similar system, has expanded the tools to explore gene function and regulatory pathways in streptococci. Two other classes of pheromones have been discovered since then, comprising the bacteriocin-inducing peptide class found in Streptococcus mutans (also named CSP, although different from the former) and the SigX-inducing peptides (XIP), in the mutans, salivarius, bovis, and pyogenes groups of streptococci. The three classes of peptide pheromones can be ordered from peptide synthesis services at affordable prices, and used in transformation assays to obtain competent cultures consistently at levels usually higher than those achieved during spontaneous competence. In this chapter, we present protocols for natural transformation of oral streptococci that are based on the use of synthetic pheromones, with examples of conditions optimized for transformation of S. mutans and Streptococcus mitis.

Aims: To compare the rate of growth of four microbial strains that cause disease in the horse, on four commonly used types of bedding. The moisture-holding capacity of each bedding type was also tested., Methods and Results: Microbial strains included Streptococcus equi, Streptococcus zooepidemicus, Fusobacterium necrophorum, Dichelobacter nodosus and Dermatophilus congolensis. The bedding types tested were Pinus sylvestris (Scots pine shavings), Pinus nigra (Corsican pine shavings), Picea sitchensis (Sitka spruce shavings), Cannabis sativa (hemp) and chopped wheat straw. A suspension of each microbial strain was spread in triplicate on agar media and incubated in its optimal growth conditions. The viable count (colony-forming unit per ml) was determined for each bacterial strain for the five different bedding types. Pinus sylvestris bedding resulted in significantly less (P = 0·001) bacterial growth of all strains tested., Conclusions: Factors resulting in the inhibition of bacterial growth include the antibacterial effects reported in the Pinacea family and the physical properties of the bedding substrate. Research is currently focussed on the diagnosis and management of disease. Prevention of disease is also important for matters of biosecurity. Strategies should include the provision of a hygienic environment and the use of specific types of bedding., Significance and Impact of the Study: Bedding choice has implications for global equine health and disease prevention as well as potential benefits in other animal species., (© 2016 The Society for Applied Microbiology.)

Natural transformation, or competence, is an ability inherent to bacteria for the uptake of extracellular DNA. This process is central to bacterial evolution and allows for the rapid acquirement of new traits, such as antibiotic resistance in pathogenic microorganisms. For the Gram-positive bacteria genus Streptococcus, genes required for competence are under the regulation of quorum sensing (QS) mediated by peptide pheromones. One such system, ComRS, consists of a peptide (ComS) that is processed (XIP), secreted, and later imported into the cytoplasm, where it binds and activates the transcription factor ComR. ComR then engages in a positive feedback loop for the expression of ComS and the alternative sigma-factor SigX. Although ComRS are present in the majority of Streptococcus species, the sequence of both ComS/XIP and ComR diverge significantly, suggesting a mechanism for species-specific communication. To study possible cross-talk between streptococcal species in the regulation of competence, and to explore in detail the molecular interaction between ComR and XIP we undertook an interdisciplinary approach. We developed a 'test-bed' assay to measure the activity of different ComR proteins in response to cognate and heterologous XIP peptides in vivo, revealing distinct ComR classes of strict, intermediate, and promiscuous specificity among species. We then solved an X-ray crystal structure of ComR from S. suis to further understand the interaction with XIP and to search for structural features in ComR proteins that may explain XIP recognition. Using the structure as a guide, we probed the apo conformation of the XIP-binding pocket by site-directed mutagenesis, both in test-bed cultures and biochemically in vitro. In alignments with ComR proteins from other species, we find that the pocket is lined by a variable and a conserved face, where residues of the conserved face contribute to ligand binding and the variable face discriminate among XIP peptides. Together, our results not only provide a model for XIP recognition and specificity, but also allow for the prediction of novel XIP peptides that induce ComR activity., Competing Interests: The authors have declared that no competing interests exist.

Background: During a 27 month period, we detected four incidents of penicillin-resistant (PR) Streptococcus dysgalactiae subsp. equisimilis (SDSE) isolated from blood cultures of three patients., Methods: The 4 PR-SDSE were compared phenotypically and molecularly (using WGS) with 36 penicillin-susceptible SDSE from blood cultures obtained in the same catchment area and time period., Results: Phylogenetic analysis showed that the four PR-SDSE belonged to a single clone and a possible epidemiological link between the three patients was identified to be a dermatology department. MICs of penicillin were determined to be 0.5-2 mg/L using Etest and 0.5 mg/L when tested by a broth microdilution method. The four PR-SDSE were unrelated to the 36 penicillin-susceptible isolates, which could suggest that they did not evolve locally from a susceptible clone, but have been introduced into the region. In silico genome-based resistome analysis revealed identical PBP mutations in all four isolates. We detected mutations in multiple PBPs, including two amino acid substitutions within the active sites of the transpeptidase domain of PBP2x (T341P and Q555E), which have also been detected in other PR streptococci. The remaining mutations were, however, all located outside the active-site motifs of the transpeptidase domain., Conclusions: To the best of our knowledge, this is the first description and characterization of invasive PR-SDSE. The resistant isolates had several amino acid changes in various PBPs compared with penicillin-susceptible SDSE. The observation that SDSE also can become PR emphasizes the importance of performing antimicrobial susceptibility testing., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Direct metal laser sintering is a technology that allows the fabrication of titanium (Ti) implants with a functional gradation of porosity and surface roughness according to three-dimensional (3D) computer data. The surface roughness of direct metal laser sintered titanium (DMLS-Ti) implants may provide abundant binding sites for bacteria. Bacterial colonization and subsequent biofilm formation can cause unsatisfactory cell adhesion and implant-related infections. To prevent such infections, a novel phase-transited lysozyme (PTL) was utilized as an initial functional layer to simply and effectively prime DMLS-Ti surfaces for subsequent coating with antibacterial multilayers. The purpose of the present study was to establish a surface with dual biological functionality. The minocycline-loaded polyelectrolyte multilayers of hyaluronic acid (HA) and chitosan (CS) formed via a layer-by-layer (LbL) self-assembly technique on PTL-functionalized DMLS-Ti were designed to inhibit pathogenic microbial infections while allowing the DMLS-Ti itself and the modified coatings to retain acceptable biocompatibility. The experimental results indicate that the DMLS-Ti and the hydrogel treated surfaces can inhibit early bacterial adhesion while completely preserving osteoblast functions. This design is expected to gain considerable interest in the medical field and to have good potential for applications in multifunctional DMLS-Ti implants.

Balanced bacterial biofilm communities help to maintain host health. Disturbance of such balance can lead to bacterial dysbiosis and pathogenesis. However, complex and dynamic bacterial interactions within the biofilm communities are poorly understood. In this study, we used a dual-species biofilm consisting of the periodontal pathogen Aggregatibacter actinomycetemcomitans, and a commensal Streptococcus parasanguinis to investigate bacterial interactions since the two organisms have been found to coexist during the development of localized aggressive periodontal disease. We report that A. actinomycetemcomitans promoted biofilm formation of S. parasanguinis in vitro and in vivo. Protein profiling of S. parasanguinis co-cultured with A. actinomycetemcomitans revealed a significant decrease in the protein level of pyruvate oxidase(PoxL), an enzyme required for the generation of hydrogen peroxide (H 2 O 2 ). Consistently, the H 2 O 2 concentration was concurrently decreased. However, the complete removal of H 2 O 2 impaired the biofilm formation. H 2 O 2 at a low concentration range regulated by A. actinomycetemcomitans enhanced the biofilm formation. These results demonstrate that A. actinomycetemcomitans promotes the S. parasanguinis biofilm formation through modulating the production of H 2 O 2 by fine-tuning the expression of poxL, indicating that H 2 O 2 functions as a signaling molecule. Taken together, this report revealed a previously unknown bacteria-bacteria interaction mechanism., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Unlabelled: This paper describes a high-throughput method that relies upon a microplate reader to score coaggregation 60 min postmixing, and use of a high-speed real-time imaging technology to describe the rate of coaggregation over time. The results of visual, microplate, and FlowCam(™) aggregation scores for oral bacteria Streptococcus gordonii, Streptococcus oralis, and Actinomyces oris, whose ability to coaggregate are well characterized, are compared. Following mixing of all possible pairs, the top fraction of the supernatant was added to a microplate to quantify cell-density. Pairs were also passed through a flow cell within a FlowCam(™) to quantify the rate of coaggregation of each pair. Results from both the microplate and FlowCam(™) approaches correlated with corresponding visual coaggregation scores and microscopic observations. The microplate-based assay enables high-throughput screening, whereas the FlowCam(™) -based assay validates and quantifies the extent that autoaggregation and coaggregation occur. Together these assays open the door for future in-depth studies of autoaggregation and coaggregation among large panels of test strains., Significance and Impact of the Study: Coaggregation between bacterial species is integral to multi-species biofilm development. Difficulties in rapidly and reproducibly identifying and quantifying coaggregation have limited mechanistic studies. This paper demonstrates two complementary quantitative methods to screen for coaggregation. The first approach uses a microplate-based high-throughput approach and the other uses a FlowCam(™) device. The microplate-based approach enables rapid detection of coaggregation between candidate coaggregating pairs of strains simultaneously while controlling for variation between replicates. The FlowCam(™) approach allows for in-depth analysis of the rates of coaggregation and size of aggregates formed., (© 2016 The Society for Applied Microbiology.)

Treatment of mastitis is the most common reason for use of antimicrobial agents in dairy cattle. The responsible use of antimicrobials could be strengthened by knowledge of predictors for cure, which would help to tailor treatment decisions. Ideally, to allow for widespread uptake, this would be achieved using data that are routinely available. To assess whether this is feasible in practice, farmers were invited to submit milk samples from mastitis cases to their veterinary practice for bacteriological culture. Among 624 culture-positive samples, 251 were positive for Streptococcus uberis. Using cow-level data, cases were classified as severe, first nonsevere, repeat, or subclinical. Additional data were collected at the cow level [somatic cell count (SCC), parity, lactation stage, milk yield, fat and protein contents, treatment] and at the herd level (housing, bedding, premilking teat disinfection, postmilking teat disinfection). Severe cases were overrepresented among heifers and animals in early lactation, and repeat cases were overrepresented in cows with 3 or more lactations. The probability of cure was higher among first- and second-parity animals than among older cows, and was higher in animals with a single elevated cow-level SCC than in animals with multiple high SCC records. Results obtained in the current study are similar to those previously described for Staphylococcus aureus mastitis. Thus, routinely available cow-level information can help to predict the outcome of antimicrobial treatment of the most common causes of gram-positive mastitis., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Previous studies have shown that the pro-inflammatory cytokine IL-1β has a crucial role in host defenses against group B streptococcus (GBS), a frequent human pathogen, by recruiting neutrophils to infection sites. We examined here the cell types and mechanisms involved in IL-1β production during infection. Using a GBS-induced peritonitis model in mice, we first found that a large proportion of exudate cells contain intracellular IL-1β by immunofluorescence. Of the IL-1β positive cells, 82 and 7% were neutrophils and macrophages, respectively, suggesting that the former cell type might significantly contribute to IL-1β production. Accordingly, depletion of neutrophils with anti-Ly6G antibodies resulted in a significant reduction in the levels of IL-1β, but not of TNF-α or IL-6. We next found that neutrophils are capable of releasing mature IL-1β and TNF-α directly in response to in vitro stimulation with GBS. The production of pro-IL-1β and TNF-α in these cells required the Toll-like receptor (TLR) adaptor MyD88 and the chaperone protein UNC93B1, which is involved in mobilization of a subfamily of TLRs to the endosomes. Moreover, pro-IL-1β processing and IL-1β release was triggered by GBS hemolysin and required components of the canonical inflammasome, including caspase-1, ASC and NLRP3. Collectively our findings indicate that neutrophils make a significant contribution to IL-1β production during GBS infection, thereby amplifying their own recruitment. These cells directly recognize GBS by means of endosomal TLRs and cytosolic sensors, leading to activation of the caspase-1 inflammasome.

Surface display of proteins by sortases in Gram-positive bacteria is crucial for bacterial fitness and virulence. We found a unique gene locus encoding an amylase-binding adhesin AbpA and a sortase B in oral streptococci. AbpA possesses a new distinct C-terminal cell wall sorting signal. We demonstrated that this C-terminal motif is required for anchoring AbpA to cell wall. In vitro and in vivo studies revealed that SrtB has dual functions, anchoring AbpA to the cell wall and processing AbpA into a ladder profile. Solution structure of AbpA determined by NMR reveals a novel structure comprising a small globular α/β domain and an extended coiled-coil heliacal domain. Structural and biochemical studies identified key residues that are crucial for amylase binding. Taken together, our studies document a unique sortase/adhesion substrate system in streptococci adapted to the oral environment rich in salivary amylase.

Introduction: The purpose of this study was to observe platelet aggregation promoted by biofilms of Streptococcus sanguinis and Porphyromonas gingivalis and to evaluate the effect of two different mouth rinses on this process., Methodology: In the first experiment, the same amount of S. sanguinis, P. gingivalis, and the S. sanguinis + P. gingivalis mixed solution was added to an equivalent amount of platelet-rich plasma (PRP). Aggregation was measured using a recording platelet aggregometer. In the second experiment, S. sanguinis, P. gingivalis, S sanguinis + P. gingivalis mixed solutions were pretreated with either Listerine antiseptic mouth rinse or Xipayi mouth rinse for 3 minutes, 6 minutes, and 10 minutes, respectively. The same amount of solution was added to the PRP, and the inhibition of aggregation was measured., Results: In the first experiment, S. sanguinis and P. gingivalis were able to induce platelet aggregation. The aggregation rate of S. sanguinis + P. gingivalis was significantly lower than that of either S. sanguinis or P. gingivalis. In the second experiment, when S. sanguinis, P. gingivalis, and the S. sanguinis + P. gingivalis mixed solutions were pretreated with Listerine antiseptic mouth rinse for 3 minutes and Xipayi mouth rinse for 10 minutes, there was no significant platelet aggregation., Conclusions: Platelets could adhere to S. sanguinis or P. gingivalis, but when S. sanguinis was mixed with P. gingivalis, the aggregation rate was reduced significantly. Treatment with Listerine antiseptic mouth rinse or Xipayi mouth rinse inhibited the ability of the bacteria to induce platelet aggregation.

Lactic acid bacteria (LAB) are important starter, commensal, or pathogenic microorganisms. The stress physiology of LAB has been studied in depth for over 2 decades, fueled mostly by the technological implications of LAB robustness in the food industry. Survival of probiotic LAB in the host and the potential relatedness of LAB virulence to their stress resilience have intensified interest in the field. Thus, a wealth of information concerning stress responses exists today for strains as diverse as starter (e.g., Lactococcus lactis), probiotic (e.g., several Lactobacillus spp.), and pathogenic (e.g., Enterococcus and Streptococcus spp.) LAB. Here we present the state of the art for LAB stress behavior. We describe the multitude of stresses that LAB are confronted with, and we present the experimental context used to study the stress responses of LAB, focusing on adaptation, habituation, and cross-protection as well as on self-induced multistress resistance in stationary phase, biofilms, and dormancy. We also consider stress responses at the population and single-cell levels. Subsequently, we concentrate on the stress defense mechanisms that have been reported to date, grouping them according to their direct participation in preserving cell energy, defending macromolecules, and protecting the cell envelope. Stress-induced responses of probiotic LAB and commensal/pathogenic LAB are highlighted separately due to the complexity of the peculiar multistress conditions to which these bacteria are subjected in their hosts. Induction of prophages under environmental stresses is then discussed. Finally, we present systems-based strategies to characterize the "stressome" of LAB and to engineer new food-related and probiotic LAB with improved stress tolerance., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Organisms previously classified as Streptococcus bovis (i.e., the S. bovis/S. equinus complex) are common in cattle feces, but may also act as opportunistic pathogens. In the current work, Streptococcus infantarius subsp. coli, a member of this complex, was associated of a cluster of calves that died within hours of injection with a modified live viral vaccine. Within 12 h of vaccination of 46 calves at a cow/calf operation, 4 calves had died, 3 calves were ill, and 1 unvaccinated cow was dead. Autopsies were performed on the cow, 2 dead calves, and 1 affected surviving calf, which was euthanized ~24 h after vaccine administration. The animals had similar gross anatomic and microscopic lesions, including subcutaneous and intramuscular dark hemorrhage on the caudal neck, multiorgan ecchymosis and petechiation, and alveolitis to interstitial pneumonia. Gram-positive cocci were in the vasculature of the lung and skeletal muscle, and S. infantarius subsp. coli was cultured from tissues and from the vaccines used on affected animals, but not in vials used on unaffected animals. Together, these findings suggest death caused by streptococcal septicemia and toxemia as a result of contamination., (© 2016 The Author(s).)

Bovine mastitis is a disease that causes great economic losses per year, being Streptococcus uberis the main environmental pathogen involved. The aim of the present study was to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of Minthostachys verticillata essential oil and limonene for S. uberis strains isolated from bovine mastitis. In addition, the effect of MIC on biofilm formation was analyzed. MIC values for the essential oil ranged from 14.3 to 114.5mg/ml (1.56-12.5%v/v) and MBC between 114.5 and 229mg/ml (12.5-25%v/v). MICs for limonene ranged from 3.3 to 52.5mg/ml (0.39-6.25%v/v) and MBC was 210mg/ml (25%v/v). Both compounds showed antibacterial activity and affected the biofilm formation of most of the strains tested. In conclusion, these compounds could be used as an alternative and/or complementary therapy for bovine mastitis caused by S. uberis., (Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Beta-defensins are important part of innate immunity of fish, which are the first defense line against invading pathogens. In this study, the β-defensin (Lhβ-defensin) gene was cloned from spleen tissue of soiny mullet (Liza haematocheila). Lhβ-defensin cDNA was 747 bp in length, encoding 63 amino acids. Sequence alignment revealed that Lhβ-defensin contained six conserved cysteine residues and shared 97.5% sequence identities with grouper (Epinephelus coioides) β-defensin. Realtime PCR revealed that Lhβ-defensin was highest expressed in the immune related organs, such as spleen, kidney and gut of healthy fish. Following Streptococcus dysgalactiae infection, Lhβ-defensin was up-regulated in immune related organs, e.g. 17.6-fold in spleen and 10.87-fold in gut at 24 h post infection (hpi). Lhβ-defensin possessed a monomeric structure of a three-stranded anti-parallel β-sheet and an α-helix stabilized by three disulfide bonds formed by Cys30-Cys58, Cys36-Cys52, and Cys40-Cys59. In addition to the experimental work, computer simulation was also carried out to determine the possible conformation of β-defensin and its interaction with palmitoyloleoylphosphatidylglycerol (POPG), a model of bacteria membrane. The Lhβ-defensin was found to form dimeric structure stabilized by the van der Waals contacts of Leu35 and Cys37 in two anti-parallel β1-strands and the cation-π interaction between Tyr32 and Arg54 respectively in the two β1-strands. The most important interactions between β-defensin and membrane are the electrostatic interactions between Arg residues in β-defensin and head group of POPG bilayer as well as hydrogen bond interactions between them. Our results were useful for further understanding the potential mechanism of antimicrobial property of fish β-defensins.

One of the main requirements for probiotics is their ability to survive during passage through gastrointestinal tract and to maintain their activity at different adverse conditions. The aim of the study was to look for the strains of lactobacilli and streptococci with high adhesive properties even affected by inhibitory substances, such as nitrates (NO3 (-)). To study the adhesion properties hemagglutination reaction of bacterial cells with red blood cells of different animals and humans was used. The acid formation ability of bacteria was determined by the method of titration after 7 days of incubation in the sterile milk. These properties were investigated at different concentrations of NO3 (-). The high concentration (mostly ≥2.0 %) NO3 (-) inhibited the growth of both lactobacilli and streptococci, but compared with streptococcal cultures lactobacilli, especially Lactobacillus acidophilus Ep 317/402, have shown more stability and higher adhesive properties. In addition, the concentrations of NO3 (-) of 0.5-2.0 % decreased the acid-forming activity of the strains, but even under these conditions they coagulated milk and, in comparison to control, formed low acidity in milk. Thus, the L. acidophilus Ep 317/402 with high adhesive properties has demonstrated a higher activity of NO3 (-) transformation.

The association of the pioneer organisms Streptococcus mutans ATCC 25175 or Streptococcus oralis ATCC 9811 with secondary colonizers Fusobacterium nucleatum ATCC 25586 or Porphyromonas gingivalis ATCC 33277 during biofilm development on titanium surfaces was evaluated by flow cytometry (FCM) using specific polyclonal antibodies. ELISA and FCM were employed, revealing high antibody sensitivity and specificity. Biofilm formation of four dual-species combinations was analyzed by crystal violet staining, while the association between streptococci and periodontal pathogens was assessed using FCM. Dual-species association between S. oralis and P. gingivalis or F. nucleatum showed a proportional decrease in S. oralis during biofilm development, with a concomitant increase in P. gingivalis or F. nucleatum. This trend was not observed in either of the dual-species associations of S. mutans with the periodontal pathogens. Our dual-species microbial model, which employed FCM, proved to be useful in the study of partnerships between bacteria in oral associations, showing that the presence of primary colonizers is required for the establishment of secondary colonizers in biofilms. The proposed experimental approach is technically simple to prepare and analyze, and also proved to be reproducible; hence, it is well-suited for investigating the development and dynamics of oral communities., (Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Streptococcus dysgalactiae ssp. dysgalactiae is an important causative agent of bovine mastitis worldwide. Lactoferrin is an innate immune protein that is associated with many functions including immunomodulatory, antiproliferative, and antimicrobial properties. This study aimed to investigate the interactions between lactoferrin and a clinical bovine mastitis isolate, Strep. dysgalactiae ssp. dysgalactiae DPC5345. Initially a deliberate in vivo bovine intramammary challenge was performed with Strep. dysgalactiae DPC5345. Results demonstrated a significant difference in lactoferrin mRNA levels in milk cells between the control and infused quarters 7h postinfusion. Milk lactoferrin levels in the Strep. dysgalactiae DPC5345 infused quarters were significantly increased compared with control quarters at 48h postinfusion. In vitro studies demonstrated that lactoferrin had a bacteriostatic effect on the growth of Strep. dysgalactiae DPC5345 and significantly decreased the ability of the bacteria to internalize into HC-11 mammary epithelial cells. Confocal microscopy images of HC-11 cells exposed to Strep. dysgalactiae and lactoferrin further supported this effect by demonstrating reduced invasion of bacteria to HC-11 cells. The combined data suggest that a bovine immune response to Strep. dysgalactiae infection includes a significant increase in lactoferrin expression in vivo, and based on in vitro data, lactoferrin limits mammary cell invasion of this pathogen by binding to the bacteria and preventing its adherence., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Microbial colonization of the infant gastrointestinal tract (GIT) begins at birth, is shaped by the maternal microbiota, and is profoundly altered by antibiotic treatment. Antibiotic treatment of mothers during pregnancy influences colonization of the GIT microbiota of their infants. The role of the GIT microbiota in regulating adaptive immune function against systemic viral infections during infancy remains undefined. We used a mouse model of perinatal antibiotic exposure to examine the effect of GIT microbial dysbiosis on infant CD8(+) T cell-mediated antiviral immunity. Maternal antibiotic treatment/treated (MAT) during pregnancy and lactation resulted in profound alterations in the composition of the GIT microbiota in mothers and infants. Streptococcus spp. dominated the GIT microbiota of MAT mothers, whereas Enterococcus faecalis predominated within the MAT infant GIT. MAT infant mice subsequently exhibited increased and accelerated mortality following vaccinia virus infection. Ag-specific IFN-γ-producing CD8(+) T cells were reduced in sublethally infected MAT infant mice. MAT CD8(+) T cells from uninfected infant mice also demonstrated a reduced capacity to sustain IFN-γ production following in vitro activation. We additionally determined that control infant mice became more susceptible to infection if they were born in an animal facility using stricter standards of hygiene. These data indicate that undisturbed colonization and progression of the GIT microbiota during infancy are necessary to promote robust adaptive antiviral immune responses., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Dental biofilm formation is not only a precursor to tooth decay, but also induces more serious systematic health problems such as cardiovascular disease and diabetes. Understanding the conditions promoting colonization and subsequent biofilm development involving complex bacteria coaggregation is particularly important. In this paper, we report a high-throughput microfluidic 'artificial teeth' device offering controls of multiple microenvironmental factors (e.g. nutrients, growth factors, dissolved gases, and seeded cell populations) for quantitative characteristics of long-term dental bacteria growth and biofilm development. This 'artificial teeth' device contains multiple (up to 128) incubation chambers to perform parallel cultivation and analyses (e.g. biofilm thickness, viable-dead cell ratio, and spatial distribution of multiple bacterial species) of bacteria samples under a matrix of different combinations of microenvironmental factors, further revealing possible developmental mechanisms of dental biofilms. Specifically, we applied the 'artificial teeth' to investigate the growth of two key dental bacteria, Streptococci species and Fusobacterium nucleatum, in the biofilm under different dissolved gas conditions and sucrose concentrations. Together, this high-throughput microfluidic platform can provide extended applications for general biofilm research, including screening of the biofilm properties developing under combinations of specified growth parameters such as seeding bacteria populations, growth medium compositions, medium flow rates and dissolved gas levels.

As titanium (Ti) continues to be utilized in great extent for the fabrication of artificial implants, it is important to understand the crucial bacterium-Ti interaction occurring during the initial phases of biofilm formation. By employing a single-cell force spectroscopy technique, the nanoadhesive interactions between the early-colonizing Streptococcus sanguinis and a clinically analogous smooth Ti substrate were explored. Mean adhesion forces between S. sanguinis and Ti were found to be 0.32±0.00, 1.07±0.06, and 4.85±0.56 nN for 0, 1, and 60 seconds contact times, respectively; while adhesion work values were reported at 19.28±2.38, 104.60±7.02, and 1,317.26±197.69 aJ for 0, 1, and 60 seconds, respectively. At 60 seconds surface delays, minor-rupture events were modeled with the worm-like chain model yielding an average contour length of 668±12 nm. The mean force for S. sanguinis minor-detachment events was 1.84±0.64 nN, and Poisson analysis decoupled this value into a short-range force component of -1.60±0.34 nN and a long-range force component of -0.55±0.47 nN. Furthermore, a solution of 2 mg/mL chlorhexidine was found to increase adhesion between the bacterial probe and substrate. Overall, single-cell force spectroscopy of living S. sanguinis cells proved to be a reliable way to characterize early-bacterial adhesion onto machined Ti implant surfaces at the nanoscale.

Objectives: The objectives of this study are to identify oral commensal species which can inhibit the growth of the main periodontopathogens, to determine the antimicrobial substances involved in these inhibitory activities and to evaluate the influence of environmental factors on the magnitude of these inhibitions., Methods: The spotting technique was used to quantify the capacity of 13 commensal species to inhibit the growth of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia. By altering experimental conditions (distance between spots and size of spots and concentration of commensal and pathogen) as well as environmental factors (inoculation sequence, oxygen and nutrition availability) the influence of these factors was evaluated. Additionally, the mechanism of inhibition was elucidated by performing inhibition experiments in the presence of peroxidase, trypsin and pepsin and by evaluating acid production., Results: Streptococcus sanguinis, Streptococcus cristatus, Streptococcus gordonii, Streptococcus parasanguinis, Streptococcus mitis and Streptococcus oralis significantly inhibit the growth of all pathogens. The volume of the spots and concentration of the commensal have a significant positive correlation with the amount of inhibition whereas distance between the spots and concentration of the pathogen reduced the amount of inhibition. Inhibition is only observed when the commensal species are inoculated 24h before the pathogen and is more pronounced under aerobic conditions. Hydrogen peroxide production by the commensal is the main mechanism of inhibition., Conclusion: Bacterial antagonism is species specific and depending on experimental as well as environmental conditions. Blocking hydrogen peroxide production neutralizes the inhibitory effect., Clinical Significance: Identifying beneficial oral bacteria and understanding how they inhibit pathogens might help to unravel the mechanisms behind dysbiotic oral diseases. In this context, this study points towards an important role for hydrogen peroxide. The latter might lead in the future to novel preventive strategies for oral health based on improving the antimicrobial properties of commensal oral bacteria., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Streptococcus phocae subsp. salmonis is a Gram-positive bacterium that causes mortality only in Atlantic salmon (Salmo salar) farmed in Chile, even when this species is co-cultured with rainbow trout (Oncorhynchus mykiss). This susceptibility could be determined by innate immune response components and their responses to bacterial infection. This fish pathogen shares subspecies status with Streptococcus phocae subsp. phocae isolated from seals. The present study compared innate immune system mechanisms in Atlantic salmon and rainbow trout when challenged with different S. phocae, including two isolates from Atlantic salmon (LM-08-Sp and LM-13-Sp) and two from seal (ATCC 51973(T) and P23). Streptococcus phocae growth was evaluated in the mucus and serum of both species, with rainbow trout samples evidencing inhibitory effects. Lysozyme activity supported this observation, with significantly higher (p < 0.01) expression in rainbow trout serum and mucus as compared to Atlantic salmon. No differences were found in phagocytic capacity between fish species when stimulated with ATCC 51973(T) and P23. Against all S. phocae strains, rainbow trout and Atlantic salmon showed up to two-fold increased bactericidal activity, and rainbow trout demonstrated up to three-fold greater reactive oxygen species production in macrophages. In conclusion, the non-specific humoral and cellular barriers of Atlantic salmon were immunologically insufficient against S. phocae subsp. salmonis, thereby facilitating streptococcosis. Moreover, the more robust response of rainbow trout to S. phocae could not be attributed to any specific component of the innate immune system, but was rather the consequence of a combined response by the evaluated components., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Phenotypic, genotypic, and antimicrobial characteristics of six phenotypically distinct human clinical isolates that most closely resembled the type strain of Streptococcus halichoeri isolated from a seal are presented. Sequencing of the 16S rRNA, rpoB, sodA, and recN genes; comparative whole-genome analysis; conventional biochemical and Rapid ID 32 Strep identification methods; and antimicrobial susceptibility testing were performed on the human isolates, the type strain of S. halichoeri, and type strains of closely related species. The six human clinical isolates were biochemically indistinguishable from each other and showed 100% 16S rRNA, rpoB, sodA, and recN gene sequence similarity. Comparative 16S rRNA gene sequencing analysis revealed 98.6% similarity to S. halichoeri CCUG 48324(T), 97.9% similarity to S. canis ATCC 43496(T), and 97.8% similarity to S. ictaluri ATCC BAA-1300(T). A 3,530-bp fragment of the rpoB gene was 98.8% similar to the S. halichoeri type strain, 84.6% to the S. canis type strain, and 83.8% to the S. ictaluri type strain. The S. halichoeri type strain and the human clinical isolates were susceptible to the antimicrobials tested based on CLSI guidelines for Streptococcus species viridans group with the exception of tetracycline and erythromycin. The human isolates were phenotypically distinct from the type strain isolated from a seal; comparative whole-genome sequence analysis confirmed that the human isolates were S. halichoeri. On the basis of these results, a novel subspecies, Streptococcus halichoeri subsp. hominis, is proposed for the human isolates and Streptococcus halichoeri subsp. halichoeri is proposed for the gray seal isolates. The type strain of the novel subspecies is SS1844(T) = CCUG 67100(T) = LMG 28801(T)., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The objectives of study were to determine the transmission parameters (β), durations of infection, and basic reproductive numbers (R0) of both Streptococcus agalactiae and Streptococcus uberis as pathogens causing mastitis outbreaks in dairy herds. A 10-mo longitudinal study was performed using 2 smallholder dairy herds with mastitis outbreaks caused by Strep. agalactiae and Strep. uberis, respectively. Both herds had poor mastitis control management and did not change their milking management during the entire study period. Quarter milk samples were collected at monthly intervals from all lactating animals in each herd for bacteriological identification. The durations of infection for Strep. uberis intramammary infection (IMI) and Strep. agalactiae IMI were examined using Kaplan-Meier survival curves, and the Kaplan-Meier survival functions for Strep. uberis IMI and Strep. agalactiae IMI were compared using log rank survival-test. The spread of Strep. uberis and Strep. agalactiae through the population was determined by transmission parameter, β, the probability per unit of time that one infectious quarter will infect another quarter, assuming that all other quarters are susceptible. For the Strep. uberis outbreak herd (31 cows), 56 new infections and 28 quarters with spontaneous cure were observed. For the Strep. agalactiae outbreak herd (19 cows), 26 new infections and 9 quarters with spontaneous cure were observed. The duration of infection for Strep. agalactiae (mean=270.84 d) was significantly longer than the duration of infection for Strep. uberis (mean=187.88 d). The transmission parameters (β) estimated (including 95% confidence interval) for Strep. uberis IMI and Strep. agalactiae IMI were 0.0155 (0.0035-0.0693) and 0.0068 (0.0008-0.0606), respectively. The R0 (including 95% confidence interval) during the study were 2.91 (0.63-13.47) and 1.86 (0.21-16.61) for Strep. uberis IMI and Strep. agalactiae IMI, respectively. In conclusion, the transmission parameter and R0 values were not different between both pathogens; however, the duration of infection for Strep. agalactiae was longer than Strep. uberis. These suggest that Strep. uberis may have a different transmission dynamic compared with Strep. agalactiae., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Unlabelled: Inhibitory CD33-related Siglec receptors regulate immune cell activation upon engaging ubiquitous sialic acids (Sias) on host cell surface glycans. Through molecular mimicry, Sia-expressing pathogen group B Streptococcus binds inhibitory human Siglec-9 (hSiglec-9) to blunt neutrophil activation and promote bacterial survival. We unexpectedly discovered that hSiglec-9 also specifically binds high molecular weight hyaluronan (HMW-HA), another ubiquitous host glycan, through a region of its terminal Ig-like V-set domain distinct from the Sia-binding site. HMW-HA recognition by hSiglec-9 limited neutrophil extracellular trap (NET) formation, oxidative burst, and apoptosis, defining HMW-HA as a regulator of neutrophil activation. However, the pathogen group A Streptococcus (GAS) expresses a HMW-HA capsule that engages hSiglec-9, blocking NET formation and oxidative burst, thereby promoting bacterial survival. Thus, a single inhibitory lectin receptor detects two distinct glycan "self-associated molecular patterns" to maintain neutrophil homeostasis, and two leading human bacterial pathogens have independently evolved molecular mimicry to exploit this immunoregulatory mechanism., Key Message: HMW-HA is the first example of a non-sialic acid containing glycan to be recognized by CD33-related Siglecs. HMW-HA engagement of hSiglec-9 attenuates neutrophil activation. Group A Streptococcus exploits hSiglec-9 recognition via its polysaccharide HMW-HA capsule to subvert neutrophil killing.

Biofilms are etiologically important in the development of chronic medical and dental infections. The biofilm extracellular polymeric substance (EPS) determines biofilm structure and allows bacteria in biofilms to adapt to changes in mechanical loads such as fluid shear. However, EPS components are difficult to visualize microscopically because of their low density and molecular complexity. Here, we tested potassium permanganate, KMnO4, for use as a non-specific EPS contrast-enhancing stain using confocal laser scanning microscopy in reflectance mode. We demonstrate that KMnO4 reacted with EPS components of various strains of Pseudomonas, Staphylococcus and Streptococcus, yielding brown MnO2 precipitate deposition on the EPS, which was quantifiable using data from the laser reflection detector. Furthermore, the MnO2 signal could be quantified in combination with fluorescent nucleic acid staining. COMSTAT image analysis indicated that KMnO4 staining increased the estimated biovolume over that determined by nucleic acid staining alone for all strains tested, and revealed non-eDNA EPS networks in Pseudomonas aeruginosa biofilm. In vitro and in vivo testing indicated that KMnO4 reacted with poly-N-acetylglucosamine and Pseudomonas Pel polysaccharide, but did not react strongly with DNA or alginate. KMnO4 staining may have application as a research tool and for diagnostic potential for biofilms in clinical samples., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The ability of certain oral biofilm bacteria to moderate pH through arginine metabolism by the arginine deiminase system (ADS) is a deterrent to the development of dental caries. Here, we characterize a novel Streptococcus strain, designated strain A12, isolated from supragingival dental plaque of a caries-free individual. A12 not only expressed the ADS pathway at high levels under a variety of conditions but also effectively inhibited growth and two intercellular signaling pathways of the dental caries pathogen Streptococcus mutans. A12 produced copious amounts of H2O2 via the pyruvate oxidase enzyme that were sufficient to arrest the growth of S. mutans. A12 also produced a protease similar to challisin (Sgc) of Streptococcus gordonii that was able to block the competence-stimulating peptide (CSP)-ComDE signaling system, which is essential for bacteriocin production by S. mutans. Wild-type A12, but not an sgc mutant derivative, could protect the sensitive indicator strain Streptococcus sanguinis SK150 from killing by the bacteriocins of S. mutans. A12, but not S. gordonii, could also block the XIP (comX-inducing peptide) signaling pathway, which is the proximal regulator of genetic competence in S. mutans, but Sgc was not required for this activity. The complete genome sequence of A12 was determined, and phylogenomic analyses compared A12 to streptococcal reference genomes. A12 was most similar to Streptococcus australis and Streptococcus parasanguinis but sufficiently different that it may represent a new species. A12-like organisms may play crucial roles in the promotion of stable, health-associated oral biofilm communities by moderating plaque pH and interfering with the growth and virulence of caries pathogens., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Periodontal disease is associated with changes in the composition of the oral microflora, where health-associated oral streptococci decrease while Gram-negative anaerobes predominate in disease. A key feature of periodontal disease-associated anaerobes is their ability to produce hydrogen sulfide (H2S) abundantly as a by-product of anaerobic metabolism. So far, H2S has been reported to be either cytoprotective or cytotoxic by modulating bacterial antioxidant defense systems. Although oral anaerobes produce large amounts of H2S, the potential effects of H2S on oral streptococci are currently unknown. The aim of this study was to determine the effects of H2S on the survival and biofilm formation of oral streptococci. The growth and biofilm formation of Streptococcus mitis and Streptococcus oralis were inhibited by H2S. However, H2S did not significantly affect the growth of Streptococcus gordonii or Streptococcus sanguinis. The differential susceptibility of oral streptococci to H2S was attributed to differences in the intracellular concentrations of reduced glutathione (GSH). In the absence of GSH, H2S elicited its toxicity through an iron-dependent mechanism. Collectively, our results showed that H2S exerts antimicrobial effects on certain oral streptococci, potentially contributing to the decrease in health-associated plaque microflora., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Platelets have been reported to become activated in response to bacteria and this is proposed to contribute to the acute response to bacterial infection. In the present study, we investigated platelet aggregation in response to group G streptococci (GGS) in vitro in healthy human donors and in vivo in a mouse model of streptococcal sepsis. Platelet aggregation by GGS was dependent on the bacterial surface protein FOG and engagement of the platelet fibrinogen receptor; however, it was independent of IgG and the platelet Fc receptor. Platelets exerted no antibacterial effects on the bacteria, and aggregates formed were markedly unstable, allowing bacteria to rapidly return to the plasma and grow post-aggregation. Thrombocytopenia and platelet activation occurred during invasive infection with GGS, and platelets were demonstrated to contribute to bacterial dissemination during infection. These findings reveal an important role for bacteria-platelet interactions during the pathogenesis of streptococcal infection.

The role of host-microbe interactions in the pathobiology of oral mucositis is still unclear; therefore, this study aimed to unravel the effect of irradiation on behavioral characteristics of oral microbial species in the context of mucositis. Using various experimental in vitro setups, the effects of irradiation on growth and biofilm formation of two Candida spp., Streptococcus salivarius and Klebsiella oxytoca in different culture conditions were evaluated. Irradiation did not affect growth of planktonic cells, but reduced the number of K. oxytoca cells in newly formed biofilms cultured in static conditions. Biofilm formation of K. oxytoca and Candida glabrata was affected by irradiation and depended on the culturing conditions. In the presence of mucins, these effects were lost, indicating the protective nature of mucins. Furthermore, the Galleria melonella model was used to study effects on microbial virulence. Irradiated K. oxytoca microbes were more virulent in G. melonella larvae compared to the nonirradiated ones. Our data indicate that low-dose irradiation can have an impact on functional characteristics of microbial species. Screening for pathogens like K. oxytoca in the context of mucosits could be useful to allow early detection and immediate intervention., (© 2016 by the Society for Experimental Biology and Medicine.)

Background: Natural products display numerous therapeutic properties (e.g., antibacterial activity), providing the population with countless benefits. Therefore, the search for novel biologically active, naturally occurring compounds is extremely important. The present paper describes the antibacterial action of the Copaifera langsdorffii oleoresin and ten compounds isolated from this oleoresin against multiresistant bacteria; it also reports the antiproliferative activity of the Copaifera langsdorffii oleoresin and (-)-copalic acid., Methods: MICs and MBCs were used to determine the antibacterial activity. Time-kill curve assays provided the time that was necessary for the bacteria to die. The Minimum Inhbitory Concentration of Biofilm (CIMB50) of the compounds that displayed the best results was calculated. Cytotoxicity was measured by using the XTT assay., Results: The diterpene (-)-copalic acid was the most active antibacterial and afforded promising Minimum Inhibitory Concentration (MIC) values for most of the tested strains. Determination of the bactericidal kinetics against some bacteria revealed that the bactericidal effect emerged within six hours of incubation for Streptococcus pneumoniae. Concerning the antibiofilm action of this diterpene, its MICB50 was twofold larger than its CBM against S. capitis and S. pneumoniae. The XTT assay helped to evaluate the cytotoxic effect; results are expressed as IC50. The most pronounced antiproliferative effect arose in tumor cell lines treated with (-)-copalic acid; the lowest IC50 value was found for the human glioblastoma cell line., Conclusions: The diterpene (-)-copalic acid is a potential lead for the development of new selective antimicrobial agents to treat infections caused by Gram-positive multiresistant microorganisms, in both the sessile and planktonic mode. This diterpene is also a good candidate to develop anticancer drugs.

Streptococcus gallolyticus subspecies gallolyticus (S. gallolyticus) can colonise the gastrointestinal tract of humans and animals and is known to cause similar infections in both humans and animals. Data about the spread or prevalence in farm animals are missing. In this study, Trypton Soya Agar was modified to a selective medium enabling the isolation and quantification of S. gallolyticus from faecal samples. The bacterium was observed in 82 out of 91 faecal samples obtained from 18 different organic turkey flocks. The prevalence of shedding birds was estimated by the number of positive fresh droppings and reached up to 100% on most farms. Furthermore, for the first time S. gallolyticus was quantified in faeces from poultry flocks. The median of colony forming units (CFU) per gramme faeces was 3.6 x 10(5) CFU/g. Typing of one isolate from each positive faecal sample by multilocus sequence typing delivered 24 sequence types (STs). Most of the isolates belonged to the clonal complex CC58. The same STs of this complex were detected in up to six different flocks. Partly, these flocks were located in various regions and stocked with varying breeding lines. Regarding the biochemical profiles of the same STs from different farms, the results did not contradict a spread of specific STs in the organic turkey production. Moreover, checking the pubMLST database revealed that STs found in this study were also found in other animal species and in humans. The high detection rate and the number of S. gallolyticus in turkey faeces indicate that this bacterium probably belongs to the common microbiota of the gastrointestinal tract of turkeys from organic flocks. Furthermore, the findings of this study support the suggestion of a possible interspecies transmission.