Emerging Pathogens and a Current-Use Pesticide: Potential Impacts on Eastern Hellbenders.

Populations of the eastern hellbender Cryptobranchus alleganiensis alleganiensis have been declining for decades, and emerging pathogens and pesticides are hypothesized to be contributing factors. However, few empirical studies have attempted to test the potential effects of these factors on hellbenders. We simultaneously exposed subadult hellbenders to environmentally relevant concentrations of either Batrachochytrium dendrobatidis (Bd) or a frog virus 3-like ranavirus (RV), a combination of the pathogens, or each pathogen following exposure to a glyphosate herbicide (Roundup). Additionally, we measured the ability of the skin mucosome to inactivate Bd and RV in growth assays. We found that mucosome significantly inactivated RV by an average of 40% but had no negative effects on Bd growth. All treatments that included RV exposure experienced reduced survival compared to controls, and the combination of RV and herbicide resulted in 100% mortality. Histopathology verified RV as the cause of mortality in all RV-exposed treatments. No animals were infected with Bd or died in the Bd-only treatment. Our results suggest that RV exposure may be a significant threat to the survival of subadult hellbenders and that Roundup exposure may potentially exacerbate this threat.

Staphylococcus ursi sp. nov., a new member of the 'Staphylococcus intermedius group' isolated from healthy black bears.

Six Staphylococcus strains were isolated from healthy black bears (Ursus americanus) in the Great Smoky Mountains National Park, Tennessee, USA. Phylogenetic analysis based on complete genome, 16S rRNA, dnaJ, hsp60, rpoB and sodA genes, and MALDI-TOF-MS main spectral profiles revealed that the strains belonged to one species and showed the closest relatedness to members of the 'Staphylococcus intermedius group' (SIG), which include Staphylococcus intermedius, Staphylococcus pseudintermedius, Staphylococcus delphini and Staphyloccoccus cornubiensis. The strains were positive in SIG-specific and negative in individual species-specific PCR assays for the nuc gene. The strains can be differentiated from the other SIG species by the absence of sucrose fermentation, from S. intermedius DSM 20373T, S. pseudintermedius CCUG 49543T and S. cornubiensis DSM 105366T by the absence of methyl ß-d-glucopyranoside fermentation and from S. delphini DSM 20771T by fermentation of trehalose. DNA relatedness of the type strain MI 10-1553T with the type strains of S. delphini, S. pseudintermedius, S. intermedius and S. cornubiensis was ≤48.2 % by digital DNA-DNA hybridization and ≤92.3 % by average nucleotide identity calculations. Iso-C15:0, anteiso-C15 : 0 and anteiso-C17 : 0 were the most common fatty acids. Polar lipids consisted of phosphadidylglycerols, phospholipids, glycolipid, diphosphatidylglycerol and aminophospholipid. Cell-wall peptidoglycan was of type A3α l-Lys-Gly3 (Ser; similar to A11.2 and A11.3). The respiratory quinone belonged to menaquinone 7 (MK-7). The G+C content of MI 10-1553T was 39.3 mol%. The isolated strains represent a novel species of the genus Staphylococcus, for which we propose the name Staphylococcus ursi sp. nov. The type strain is MI 10-1553T (=ATCC TSD-55T=CCOS 1900T).

Staphylococcus pseudintermedius 5'-nucleotidase suppresses canine phagocytic activity.

Staphylococcus pseudintermedius is a major opportunistic bacterial pathogen and the leading cause of pyoderma in dogs. In canines it is also often associated with infections of the urinary system and wounds and occasionally infects people. Widespread antimicrobial resistance has made the development of alternative treatments a high priority. The development of a staphylococcal vaccine, however, has proven challenging. Identification of virulence factors that inhibit phagocytosis and avoid innate immunity may play a significant role in preventing or treating infection with S. pseudintermedius. In this study, we identified a putative 5'-nucleotidase provisionally named SpAdsA, a S. pseudintermedius cell- wall protein encoded by SpAdsA. SpAdsA shares approximately 52% identity with the orthologous protein of Staphylococcus aureus and 14.8% identity with that of Streptococcus suis type2. It catalyzes the dephosphorylation of adenosine triphosphate and attenuation of this enzyme with critical amino acid substitutions nearly eliminated its hydrolytic activity. Exogenous adenosine inhibited phagocytosis of S. pseudintermedius by canine neutrophils and monocytes. Conversely, the addition of SpAdsA inhibitor or A2A adenosine receptor antagonist impaired the capacity of S. pseudintermedius to escape from killing by phagocytic cells. The neutralizing ability of canine antibody produced against SpAdsA-M was determined. Taken together, these results suggest that SpAdsA likely plays an important role in S. pseudintermedius virulence and that attenuated SpAdsA may be a good candidate for inclusion in a vaccine against S. pseudintermedius.

Antibacterial effect of N-acetylcysteine in combination with antimicrobials on common canine otitis externa bacterial isolates.

BACKGROUND: Approved treatments for canine otitis externa are limited in variety and may contain ototoxic ingredients. With bacterial resistance an ongoing concern, it would be ideal if non-ototoxic agents combined with antibiotics resulted in a synergistic effect, requiring lower antibiotic concentrations to treat infections. Evidence of synergism and antagonism between N-acetylcysteine (NAC) and various antibiotic classes has been reported; the present research group was interested in examining these interactions. HYPOTHESIS/OBJECTIVES: To determine if NAC, an otoprotective and antimicrobial compound, has synergistic activity when combined with enrofloxacin or gentamicin in vitro against bacterial isolates causing canine otitis externa. ANIMALS: Twenty-two isolates from canine clinical cases of otitis externa were identified and tested, including seven Staphylococcus pseudintermedius, 12 Pseudomonas aeruginosa and three Corynebacterium spp. isolates. METHODS AND MATERIALS: Each isolate was grown on blood agar for 24 h and transferred to Mueller-Hinton broth (MHB), with a final concentration of 5 × 105 cfu/mL. Each well was inoculated with 50 µL of bacterial suspension. N-acetylcysteine was diluted in MHB to a starting concentration of 160 mg/mL. Enrofloxacin and gentamicin were diluted to 64 µg/mL. Individual and checkerboard serial microdilution assays were performed in triplicate with negative controls for all isolates tested. RESULTS: Interactions observed for NAC and enrofloxacin were synergistic (4.5%), indifferent (77.3%) or antagonistic (18.2%). Interactions observed for NAC and gentamicin were synergistic (4.5%), indifferent (45.5%) or antagonistic (50%). CONCLUSIONS AND CLINICAL RELEVANCE: Most interactions between NAC and enrofloxacin or gentamicin were indifferent or antagonistic at the concentrations tested in vitro.

The effect of enterococci on feline Tritrichomonas foetus infection in vitro.

Tritrichomonas foetus is a common cause of large bowel diarrhea in cats. Probiotics have been suggested to be effective for many intestinal pathogens; however, there are a lack of studies evaluating the effect of probiotics in T. foetus infection. In vitro studies were performed to evaluate the effect of a probiotic containing Enterococcus faecium (Efm) SF68 and a novel probiotic, Enterococcus hirae, on the inhibition of T. foetus growth, adhesion to, and cytotoxicity towards the intestinal epithelium. The effect of enterococci on T. foetus proliferation during co-culture was evaluated throughout log phase T. foetus growth. The previously validated in vitro co-culture model system using porcine intestinal epithelial cells (IPEC-J2) was used to evaluate the effect of enterococci on T. foetus adhesion and cytotoxicity towards intestinal epithelial cells. Cytotoxicity was assessed using fluorescent microscopy and spectrophotometry. Interactions of T. foetus, enterococci, and intestinal epithelial cells were assessed using scanning electron microscopy and immunofluorescence assays (IFA). Enterococcus-induced inhibition of T. foetus growth was demonstrated at concentrations as low as 104 enterococci colony forming units (CFU)/mL and was dependent, in part, on environmental pH and the presence of viable enterococci organisms. T. foetus adhesion, including with a ronidazole-resistant strain, was reduced with pretreatment of intestinal epithelial cells with enterococci but was not significantly affected when enterococci were introduced simultaneously or following T. foetus infection. Compared to Efm, E. hirae more effectively decreased T. foetus adhesion, suggesting its superior potential as a novel probiotic for T. foetus infection. There was no effect of enterococci treatment on T. foetus-induced intestinal epithelial cell cytotoxicity. Our results support further study into the investigation of a possible benefit of enterococci-containing probiotic treatment for prevention of T. foetus infection in at-risk uninfected cats.

Performance of Five Commercial Identification Platforms for Identification of Staphylococcus delphini.

The Staphylococcus intermedius group (SIG) is a collection of coagulase-positive staphylococci consisting of four distinct species, namely, Staphylococcus cornubiensis, Staphylococcus delphini, Staphylococcus intermedius, and Staphylococcus pseudintermedius SIG members are animal pathogens and rare causes of human infection. Accurate identification of S. pseudintermedius has important implications for interpretation of antimicrobial susceptibility testing data and may be important for other members of the group. Therefore, we sought to evaluate the performance of five commercially available identification platforms with 21 S. delphini isolates obtained from a variety of animal and geographic sources. Here, we show that automated biochemical platforms were unable to identify S. delphini to the species level, a function of its omission from their databases, but could identify isolates to the SIG level with various degrees of success. However, all automated systems misidentified at least one isolate as Staphylococcus aureus One matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system was able to identify S. delphini to the species level, suggesting that MALDI-TOF MS is the best option for distinguishing members of the SIG. With the exception of S. pseudintermedius, it is unclear if other SIG members should be routinely identified to the species level; however, as our understanding of their role in animal and human diseases increases, it may be necessary and important to do so.

Identification, cloning and characterization of SpEX exotoxin produced by Staphylococcus pseudintermedius.

Staphylococci have evolved numerous strategies to evade their hosts' immune systems. Some staphylococcal toxins target essential components of host innate immunity, one of the two main branches of the immune system. Analysis of the Staphylococcus pseudintermedius secretome using liquid chromatography mass spectrometry guided by genomic data, was used to identify an S. pseudintermedius exotoxin provisionally named SpEX. This exoprotein has low overall amino acid identity with the Staphylococcus aureus group of proteins named staphylococcal superantigen like proteins (SSLs) and staphylococcal enterotoxin- like toxin X (SEIX), but predictive modeling showed that it shares similar folds and domain architecture to these important virulence factors. In this study, we found SpEX binds to complement component C5, prevents complement mediated lysis of sensitized bovine red blood cells, kills polymorphonuclear leukocytes and monocytes and inhibits neutrophil migration at sub-lethal concentrations. A mutant version of SpEX, produced through amino acid substitution at selected positions, had diminished cytotoxicity. Anti-SpEX produced in dogs reduced the inhibitory effect of native SpEX on canine neutrophil migration and protected immune cells from the toxic effects of the native recombinant protein. These results suggest that SpEX likely plays an important role in S. pseudintermedius virulence and that attenuated SpEX may be an important candidate for inclusion in a vaccine against S. pseudintermedius infections.

Staphylococcus pseudintermedius Sbi paralogs inhibit complement and bind IgM, IgG Fc and Fab.

The success of staphylococci as pathogens has been attributed, in part, to their ability to evade their hosts' immune systems. Although the proteins involved in evasion have been extensively studied in staphylococci affecting humans little characterization has been done with Staphylococcus pseudintermedius, an important cause of pyoderma in dogs. Staphylococcus aureus binder of immunoglobulin (Sbi) interferes with innate immune recognition by interacting with multiple host proteins. In this study, a S. pseudintermedius gene that shares 38% similarity to S. aureus Sbi was cloned from S. pseudintermedius strains representative of major clonal lineages bearing two paralogs of the protein. Binding of immunoglobulins and Fab and Fc fragments as well as interaction with complement was measured. S. pseudintermedius Sbi protein bound IgG from multiple species and canine complement C3, neutralized complement activity and bound to canine IgM and B cells. Evidence from this work suggests Sbi may play an important role in S. pseudintermedius immune evasion.

Characterization of Fungal Colonization of Indwelling Esophagostomy Tubes.

Fungal colonization of feeding tubes occurs rapidly in people, resulting in decreased structural integrity and complications such as luminal obstruction and tube failure. Esophagostomy tubes (E-tubes) are commonly used in dogs and cats for enteral support, but data are lacking regarding colonizing fungi and the impact of colonization on tube integrity. In this study, esophagostomy tubes were collected in lieu of disposal from dogs and cats undergoing feeding tube exchange. Fungi were isolated with culture and identified using morphological characteristics. Scanning electron microscopy was used to evaluate the surface characteristics of the tubes. Two silicone and one polyurethane E-tube were evaluated. Fungi associated with the normal microbiota, including Candida sp. and Penicillium sp., as well as environmental fungi were identified. This case series represents the first documentation of fungal colonization of silicone and polyurethane E-tubes in dogs and cats. Additionally, this is the first report to document degenerative changes in a silicone E-tube.

First reported human isolation of Staphylococcus delphini.

The Staphylococcus intermedius group is a collection of coagulase-positive staphylococci composed of 5 members, including Staphylococcus pseudintermedius, a zoonotic pathogen often associated with exposure to dogs, and Staphylococcus delphini, which has not previously been recovered from humans. Here, we describe the first human case of S. delphini infection.

Complete Genome Sequences of Four Staphylococcus aureus Sequence Type 398 Isolates from Four Goats with Osteomyelitis.

Staphylococcus aureus is the causative agent of multiple infections, including bacteremia, infective endocarditis, osteomyelitis, septic arthritis, and prosthetic device infections. We report here the first whole-genome sequence for four S. aureus sequence type 398 isolates from clinical cases of osteomyelitis in four goats with a history of orthopedic surgery.

Long-Term Treatment and Survival in Three Apparently Immunocompetent Dogs with Disseminated Fungal Infection Caused by Phialosimplex caninus.

Disseminated fungal infections cause morbidity and mortality in dogs. The prognosis varies depending on the infecting agent. Phialosimplex caninus is a recently recognized type of hyalohyphomyces. Knowledge regarding the clinical course of P caninus infection in dogs is limited to two previous case reports. The clinical features, diagnostic findings, responses to medical therapy, and long-term outcomes of three dogs with disseminated P caninus are presented in this study. All dogs had improved quality of life once itraconazole administration, with or without terbinafine, was instituted. Long-term disease remission was maintained even after discontinuation of antifungal therapy in a single dog.

Characterization of a leukocidin identified in Staphylococcus pseudintermedius.

Bacterial infections from Staphylococcus pseudintermedius are the most common cause of skin infections (pyoderma) affecting dogs. Two component pore-forming leukocidins are a family of potent toxins secreted by staphylococci and consist of S (slow) and F (fast) components. They impair the innate immune system, the first line of defense against these pathogens. Seven different leukocidins have been characterized in Staphylococcus aureus, some of which are host and cell specific. Through genome sequencing and analysis of the S. pseudintermedius secretome using liquid chromatography mass spectrometry we identified two proteins, named "LukS-I" and "LukF-I", encoded on a degenerate prophage contained in the genome of S. pseudintermedius isolates. Phylogenetic analysis of LukS-I components in comparison to the rest of the leukocidin family showed that LukS-I was most closely related to S. intermedius LukS-I, S. aureus LukE and LukP, whereas LukF-I was most similar to S. intermedius LukF-I S. aureus gamma hemolysin subunit B. The killing effect of recombinant S. pseudintermedius LukS-I and LukF-I on canine polymorphonuclear leukocytes was determined using a flow cytometry cell permeability assay. The cytotoxic effect occurred only when the two recombinant proteins were combined. Engineered mutant versions of the two-component pore-forming leukocidins, produced through amino acids substitutions at selected points, were not cytotoxic. Anti-Luk-I produced in dogs against attenuated proteins reduced the cytotoxic effect of native canine leukotoxin which highlights the importance of Luk-I as a promising component in a vaccine against canine S. pseudintermedius infections.

Characterization of recombinant wild-type and nontoxigenic protein A from Staphylococcus pseudintermedius.

BACKGROUND: Staphylococcus pseudintermedius is an opportunistic pathogen that is the major cause of pyoderma affecting dogs. Conventional antimicrobial treatment for infections caused by this organism have failed in recent years due to widespread resistance and alternative treatment strategies are a high priority. Protein A encoded in Staphylococcus aureus by spa protects the bacterium by binding IgG and acts as a superantigen. Staphylococcus pseudintermedius possess two genes orthologous to S. aureus spa, spsP, and spsQ. METHODS: SpsQ and SpsQ-M, a non-toxigenic SpsQ, were cloned and expressed as recombinant proteins and their cytotoxic effect on canine B cells was measured. The neutralizing ability of antibody raised against them in clinically healthy dogs was evaluated. RESULTS: S. pseudintermedius SpsQ induced apoptosis of canine B cells. Specific amino acid substitutions diminished SpsQ-M binding to immunoglobulin and its super-antigenic activity, while its antigenicity was maintained. This recombinant, non-toxigenic S. pseudintermedius SpsQ stimulated the production of antibodies in dogs that specifically reacted with SpsQ and greatly diminished its cytotoxic effect on canine B cells. CONCLUSIONS: The production of neutralizing antibody suggests that attenuated, non-toxic SpsQ produced in this study is a good candidate for inclusion in a vaccine for use in the treatment and prevention of S. pseudintermedius infections. ABBREVIATIONS: SpA: Staphylococcus aureus protein A; SpsP: Staphylococcus pseudintermedius protein A; SpsQ: Staphylococcus pseudintermedius protein A; SpsQ-M: attenuated Staphylococcus pseudintermedius protein A; MRSP: methicillin resistant Staphylococcus pseudintermedius; IgA: immunoglobulin A; IgG: immunoglobulin G; IgM: immunoglobulin M; VH: variable region of immunoglobulin heavy chain; IgBD: immunoglobulin binding domains; MFI: mean fluorescent intensity; SEM: standard error of the mean; PBMC: Peripheral blood mononuclear cells; CD21: complement receptor type 2; ST: Sequence type; OD: Optical density; ORF: open reading frame; PBS: Phosphate buffered saline; Tween 20: Polyethylene glycol sorbitan monolaurate 20; HRP: horseradish peroxidase; TMB- 3,3',5,5'-Tetramethylbenzidine.

Identification, Cloning, and Characterization of Staphylococcus pseudintermedius Coagulase.

Coagulase activation of prothrombin by staphylococcus induces the formation of fibrin deposition that facilitates the establishment of infection by Staphylococcus species. Coagulase activity is a key characteristic of Staphylococcus pseudintermedius; however, no coagulase gene or associated protein has been studied to characterize this activity. We report a recombinant protein sharing 40% similarity to Staphylococcus aureus coagulase produced from a putative S. pseudintermedius coagulase gene. Prothrombin activation by the protein was measured with a chromogenic assay using thrombin tripeptide substrate. Stronger interaction with bovine prothrombin than with human prothrombin was observed. The S. pseudintermedius coagulase protein also bound complement C3 and immunoglobulin. Recombinant coagulase facilitated the escape of S. pseudintermedius from phagocytosis, presumably by forming a bridge between opsonizing antibody, complement, and fibrinogen. Evidence from this work suggests that S. pseudintermedius coagulase has multifunctional properties that contribute to immune evasion that likely plays an important role in virulence.

Temporal dynamics of gut microbiota in triclocarban-exposed weaned rats.

Widely used as an antimicrobial in antibacterial bar soaps, triclocarban (3,4,4'-trichlorocarbanilide; TCC) is effective against Gram-positive bacteria but shows little efficacy against Gram-negative strains, potentially altering the composition of indigenous microflora within and on the human body. To date, the consequence of continuous or previous nonprescription antimicrobial exposure from compounds in personal care products on commensal microflora is still elusive. Previous research has shown that TCC exposure during gestation and lactation induced dysbiosis of gut microbial communities among exposed dams and neonates. However, the impact of antimicrobial exposure specifically after discontinuation of the use of TCC on the gut microbiota has not been investigated. In this study, weaned Sprague Dawley rats (postnatal day, PND 22) were provided ad lib access to TCC-supplemented diet (0.2% w/w or 0.5% w/w) for 4 weeks (phase I) followed by a 4-week washout period (phase II) to determine gut microflora changes both during continuous exposure to TCC and to determine the potential rebound following TCC withdrawal. Fecal samples were collected at baseline (PND 22) prior to TCC exposure and throughout phase I and phase II. The V4 region of 16S rDNA was sequenced from extracted total fecal DNA with the MiSeq platform. Exposure to both 0.2% w/w and 0.5% w/w TCC was sufficient to alter diversity of microbiota during phase I of treatment. This effect was further prolonged into phase II, even when TCC exposure was discontinued. Collectively, these data highlight the impact of both continuous and prior TCC exposure on gut microbial ecology and shed light onto the potential long-term health risk of daily nonprescription antimicrobial personal care product use.

Complete Genome Sequences of Three Staphylococcus pseudintermedius Strains Isolated from Botswana.

We report here the first whole-genome sequences for 3 strains of Staphylococcus pseudintermedius (112N, 113N, and 114N) isolated in Africa. Samples of this opportunistic pathogen were collected from nasal swabs obtained from healthy carrier dogs in Botswana. The sequence information will facilitate spatial phylogenetic comparisons of staphylococcal species and other bacteria at the genome level.

Expression and function of protein A in Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius is an opportunistic pathogen in dogs and the most frequent cause of canine pyoderma. Protein A, a potent virulence factor in S. aureus is encoded by the spa gene. S. pseudintermedius possesses genes seemingly analogous to spa, but the expression and the characteristics of their products have not been directly determined. The purpose of this study was to test isolates from major clonal groups for the presence of spa gene orthologs, quantitate their expression levels, and to characterize protein A in S. pseudintermedius. From the data, it was observed that S. pseudintermedius isolates express genes analogous to spa in S. aureus. Isolates representing major clonal populations in the United States and Europe, ST68 and ST71 respectively, bound significantly higher amounts of canine IgG than isolates with other genetic backgrounds, suggesting that these isolates have a higher density of protein A on their surface. Also, canine IgG bound to protein A on S. pseudintermedius via its Fc region similar to protein A from S. aureus. The mRNA profile differed based on the bacterial sequence types and correlated to the density of protein A on the bacterial surface. Protein A was also found to be secreted during the exponential growth phase. Phagocytosis experiments with S. pseudintermedius show that blocking of protein A enhanced phagocytosis in whole blood, neutrophils and in DH82 canine macrophage-like cell line. Taken together, the results demonstrate that S. pseudintermedius produces protein A that shares S. aureus protein A's ability to bind the Fc region of immunoglobulins and may serve as a potential virulence factor by evading the host immune system.

Clonal Complexes and Antimicrobial Susceptibility Profiles of Staphylococcus pseudintermedius Isolates from Dogs in the United States.

Staphylococcus pseudintermedius is the primary cause of canine pyoderma and has been associated with diseases in other animals, including human beings. A high prevalence of methicillin and multidrug resistance has been reported in this bacterium in some geographic regions of the United States. Multilocus sequence type (MLST) 68 was implicated, initially, as the major clonal genotype based on a limited number of samples. The objectives of this study were to determine the population genetics of S. pseudintermedius isolated from a cross-section of the United States using a seven-locus multilocus sequence typing method, to identify clonal complexes (CCs), and to correlate sequence types with antimicrobial susceptibility profiles. A total of 190 S. pseudintermedius with 86 different MLSTs were detected and the constituents of three major CCs of methicillin-resistant S. pseudintermedius (MRSP), CC68, CC71, and CC84, were identified. Different patterns of resistance were associated with each CC. CC71 from the United States had notable differences with CC71 studied on other continents with chloramphenicol, tetracycline, and trimethoprim/sulfamethoxazole resistance. Some isolates with resistance to the broadest range of drugs tested, including that to chloramphenicol, had STs unrelated to the major CCs, suggesting the potential for the emergence of new clonal populations of MRSP that are resistant to most therapeutically useful antimicrobials.