Wound swabs versus biopsies to detect methicillin resistant Staphylococcus aureus in experimental equine wounds.

OBJECTIVE: To compare: (1) the load and diversity of cultivatable bacterial species isolated from tissue biopsies with cultures from surface swabs, and (2) the ability of each technique to detect methicillin-resistant Staphylococcus aureus (MRSA) in a model of MRSA-infected equine wounds. STUDY DESIGN: Experimental in vivo study. ANIMALS: Three light-breed adult horses. METHODS: Four 2.5 × 2.5 cm full-thickness skin wounds were created on the dorsolateral aspect of each forelimb. Five days later, each wound was inoculated with a pure culture of MRSA (ATCC 43300). One hundred microlitres of 0, 5 × 108 , 5 × 109 or 5 × 1010 colony forming units (CFU)/ml was used to inoculate each wound. Surface swabs (Levine technique) and tissue biopsy samples (3 mm punch biopsy) were obtained at 2, 7, 14, and 21 days after inoculation. Quantitative aerobic culture was performed using routine clinical techniques. RESULTS: A similar bacterial profile was identified from the culture of each wound-sampling technique and there was moderate correlation (R = 0.49, P < .001) between the bacterial bioburdens. Agreement was fair (κ = 0.31; 95% CI, 0.129-0.505) between the sampling techniques in identification of MRSA. Methicillin-resistant Staphylococcus aureus was isolated more frequently (P = .016) from cultures of tissue biopsies (79%; 76/96) than from surface swabs (62%; 60/96). CONCLUSION: Bacterial load and diversity did not differ between sampling techniques but MRSA was detected more often from the cultures of tissue biopsies. CLINICAL SIGNIFICANCE: Tissue biopsy should be preferred to culture swab in wounds where MRSA is suspected.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Analysis of Virulence and Antimicrobial Resistance Gene Carriage in Staphylococcus aureus Infections in Equids Using Whole-Genome Sequencing.

While Staphylococcus aureus is associated with significant morbidity and mortality in equids (horses, donkeys, and mules), few studies have performed whole-genome sequencing to fully categorize large collections of equine isolates. Such sequencing allows for a comprehensive analysis of the genetic lineage and relationships of isolates, as well as the virulence genes present in each, which can be important for understanding the epidemiology of strains and their range of infections. Seventy-two clinical Staphylococcus aureus isolates from equids were collected at the Texas A&M University Veterinary Medical Teaching Hospital between 2007 and 2017. Whole-genome sequencing was performed to characterize the isolates according to sequence typing, biofilm association, antimicrobial resistance, and toxin gene carriage. Of the 72 isolates, 19% were methicillin resistant, of which the majority belonged to clonal complex 8. Eighteen distinct sequence types (STs) were represented, with the most common being ST1, ST133, ST8, and ST97. Most isolates had weak or negative overall biofilm production. Toxin and antimicrobial resistance gene carriage was varied; of note, this study revealed that a large proportion of North American equine isolates carry the leucocidin PQ toxin (66% of isolates). One isolate (17-021) carried genes imparting lincosamide and high-level mupirocin resistance, a combination not previously reported in equine-derived S. aureus isolates. IMPORTANCE This is one of the first studies to perform whole-genome sequencing (WGS) of a large collection of Staphylococcus aureus isolates, both methicillin resistant and susceptible, collected from horses. A large proportion of the isolates carry leucocidin PQ (LukPQ), making this one of the first reports of such carriage in the United States. The presence of lincosamide and high-level mupirocin resistance in a methicillin-susceptible S. aureus (MSSA) isolate highlights the importance of MSSA as a reservoir of important antimicrobial resistance genes. As microbial resistance genes on mobile genetic elements can pass between S. aureus strains and livestock-associated strains can be transferred to humans, these findings have important public health implications.

Mucocutaneous nasal histoplasmosis in an immunocompetent dog.

BACKGROUND: Histoplasma (H.) capsulatum is a dimorphic fungus, and infection is typically via inhalation of microconidia. After conversion to the yeast phase within the lung, the organism is subsequently disseminated to other tissues by macrophages. Nasal histoplasmosis appears to be a rare condition in dogs. CASE PRESENTATION: We report the clinical case of a 4.5-year-old male neutered Cocker spaniel/Poodle mix, 7.7 kg, body condition score 6/9, that presented with a 3-month history of sneezing and left-sided mucoid nasal discharge. The history also included a mild swelling (transient) of the right carpus with a lameness (grade II-III/IV), coinciding with the onset of sneezing and nasal discharge. The dog lived primarily indoors in the Texas Gulf Coast area. On physical examination, the dog was febrile, and the left nostril was swollen, ulcerative, deformed, and hypopigmented. Mandibular lymph nodes were firm and mildly enlarged bilaterally. Mild lymphopenia, thrombocytopenia, and hyperglobulinemia were noted. Thoracic radiographs were unremarkable. Computed tomography and rhinoscopy revealed swelling of the rostral portion of the left and right nasal passages. Cytology and histology of biopsies of the affected nasal tissue showed pyogranulomatous inflammation and yeast organisms consistent with H. capsulatum. Weak antigenuria was detected on the MVista H. capsulatum antigen test. Treatment with oral itraconazole led to a resolution of the nasal signs and normalization of the appearance of the nostril over 13 weeks, and neither antigenuria nor antigenemia was detected on several recheck examinations. The dog remained in good general and physical condition and showed no signs of disease recurrence more than 6 years after the last examination. CONCLUSION: We report a rare case of nasal mucocutaneous histoplasmosis in an immunocompetent dog, with an excellent clinical response to oral itraconazole. This case documents that histoplasmosis in dogs can affect primarily the nasal cavity, which responds rapidly to triazole antifungal therapy and has a good prognosis. A similar case has only been reported in human medicine in a young adult.

Regulatory protein HilD stimulates Salmonella Typhimurium invasiveness by promoting smooth swimming via the methyl-accepting chemotaxis protein McpC.

In the enteric pathogen Salmonella enterica serovar Typhimurium, invasion and motility are coordinated by the master regulator HilD, which induces expression of the type III secretion system 1 (T3SS1) and motility genes. Methyl-accepting chemotaxis proteins (MCPs) detect specific ligands and control the direction of the flagellar motor, promoting tumbling and changes in direction (if a repellent is detected) or smooth swimming (in the presence of an attractant). Here, we show that HilD induces smooth swimming by upregulating an uncharacterized MCP (McpC), and this is important for invasion of epithelial cells. Remarkably, in vitro assays show that McpC can suppress tumbling and increase smooth swimming in the absence of exogenous ligands. Expression of mcpC is repressed by the universal regulator H-NS, which can be displaced by HilD. Our results highlight the importance of smooth swimming for Salmonella Typhimurium invasiveness and indicate that McpC can act via a ligand-independent mechanism when incorporated into the chemotactic receptor array.

Correction: Detection of vapN in Rhodococcus equi isolates cultured from humans.

[This corrects the article DOI: 10.1371/journal.pone.0190829.].

Bacillus subtilis PB6 Supplementation in Weaned Holstein Steers During an Experimental Salmonella Challenge.

To evaluate the effects of a patented Bacillus subtilis probiotic, weaned Holstein steers, not shedding Salmonella (n = 40; ∼90 kg), were supplemented (CLO) or not (CON) with CLOSTAT® (13 g/hd per day; Kemin Industries, Des Moines, IA) in a starter ration for 35 d. The calves were assigned to one of four treatments in a 2 × 2 factorial design with CLO and CON calves that were orally administered Salmonella (STM) or not (NoSTM). Calves were challenged with 1.6 × 106 colony-forming unit (CFU) Salmonella Typhimurium (resistant to 50 µg/mL nalidixic acid) in 1 L of milk replacer on day 0. Blood samples were collected through jugular catheters every 6 h for 96 h, and body temperature was measured every 5 min through indwelling rectal temperature recording devices. Five calves from each treatment were harvested 48 h postchallenge, and the remaining calves were harvested 96 h postchallenge. During necropsy, tissues were collected for the isolation and quantification of the inoculated STM from various tissues. The CLOSTM group had reduced STM concentrations in the jejunum, ileum, and transverse colon 48 h after the challenge (p ≤ 0.03), but were not different 96 h postchallenge (p > 0.05). Decreased (p < 0.01) pyrexia was observed after the challenge in CLOSTM calves when compared with CONSTM calves. White blood cells and lymphocyte counts were increased (p ≤ 0.05) in CLOSTM calves after the challenge in comparison with other treatments. In calves given STM, the CLO group had greater feed intake before and after the challenge (p < 0.01) compared with the CON group. Increased serum IL-6 and IFN-γ concentrations were observed in the CONSTM group compared with other treatments. Overall, CLO reduced Salmonella presence and concentrations in gastrointestinal tissues while simultaneously reducing the severity of the challenge as indicated by blood parameters and the reduced febrile response.

NSAIDs disrupt intestinal homeostasis by suppressing macroautophagy in intestinal epithelial cells.

Small intestinal damage induced by nonsteroidal anti-inflammatory drugs (NSAIDs) remains an under-recognized clinical disorder. The incomplete understanding of the pathophysiology has hampered the development of prevention and treatment strategies leading to the high morbidity and mortality rates. NSAIDs are known to modulate macroautophagy, a process indispensable for intestinal homeostasis. Whether NSAIDs stimulate or repress macroautophagy and how this correlates with the clinical manifestations of NSAID enteropathy, however, remains unknown. The objectives of this study were to determine whether NSAIDs impaired macroautophagy and how this affects macroautophagy-regulated intestinal epithelial cell (IEC) processes essential for intestinal homeostasis (i.e., clearance of invading pathogens, secretion and composition of mucus building blocks, and inflammatory response). We show that NSAID treatment of IECs inhibits macroautophagy in vitro and in vivo. This inhibition was likely attributed to a reduction in the area and/or distribution of lysosomes available for degradation of macroautophagy-targeted cargo. Importantly, IEC regulatory processes necessary for intestinal homeostasis and dependent on macroautophagy were dysfunctional in the presence of NSAIDs. Since macroautophagy is essential for gastrointestinal health, NSAID-induced inhibition of macroautophagy might contribute to the severity of intestinal injury by compromising the integrity of the mucosal barrier, preventing the clearance of invading microbes, and exacerbating the inflammatory response.

Whole-Genome Sequences of Staphylococcus pseudintermedius Isolates from Canine and Human Bacteremia Infections.

Here, we report the complete and draft genome sequences of 8 Staphylococcus pseudintermedius isolates, 4 from human bacteremia infections and 4 from canine bacteremia infections. This species is recognized primarily as an important canine pathogen, but it is increasingly being identified in human infections.

Ulcerative, granulomatous glossitis and enteritis caused by Rhodococcus equi in a heifer.

Rhodococcus equi infection in horses is common and is characterized by pyogranulomatous pneumonia and ulcerative enterocolitis. R. equi clinical disease in cattle, however, is rare and typically manifests as granulomatous lymphadenitis discovered in the abattoir. A 19-mo-old female Santa Gertrudis had a history of intermittent inappetence and weight loss for a 3-mo period before euthanasia. Gross and histologic examination revealed severe, chronic, ulcerative, and granulomatous inflammation in the tongue, pharynx, and small intestine. Also, the heifer had severe, granulomatous pharyngeal and mesenteric lymphadenitis. Bacterial cultures from the ileum, tongue, and liver yielded numerous-to-moderate numbers of R. equi. PCR analysis of the isolate detected the linear virulence plasmid vapN, which is often identified in bovine isolates (traA- and vapN-positive). The bacteria also lack the circular plasmids vapA and vapB that are associated with virulence in horses and swine, respectively. We report herein an atypical and unusual clinical presentation of R. equi infection in cattle, which has zoonotic potential.

Characterization of agr Groups of Staphylococcus pseudintermedius Isolates from Dogs in Texas.

Staphylococcus pseudintermedius is an important canine pathogen implicated in an increasing number of human infections. Along with rising levels of methicillin and multidrug resistance, staphylococcal biofilms are a complicating factor for treatment and contribute to device, implant, and surgical infections. Staphylococcal virulence, including biofilm formation, is regulated in part by the quorum sensing accessory gene regulator system (agr). The signal molecule for agr, known as the autoinducing peptide molecule, contains polymorphisms that result in the formation of distinct groups. In S. pseudintermedius, 4 groups (i.e., groups I, II, III, and IV) have been identified but not comprehensively examined for associations with infection type, virulence factor carriage, or phylogenetic relationships-all of which have been found to be significant in S. aureus In this study, 160 clinical canine isolates from Texas, including isolates from healthy dogs (n = 40) and 3 different infection groups (pyoderma, urinary tract, and surgical, n = 40 each), were sequenced. The agr group, biofilm-producing capabilities, toxin gene carriage, antimicrobial resistance, and sequence type (ST) were identified for all isolates. While no significant associations were discovered among the clinical infection types and agr groups, agr II isolates were significantly less common than any other group in diseased dogs. Furthermore, agr II isolates were less likely than other agr groups to be multidrug resistant and to carry toxin genes expA and sec-canine Fifty-two (33%) of the 160 isolates were methicillin resistant, and the main sequence types (ST64, ST68, ST71, ST84, ST150, and ST155) of methicillin-resistant strains of S. pseudintermedius (MRSP) were identified for the geographic region.IMPORTANCEStaphylococcus pseudintermedius is an important disease-causing bacterium in dogs and is recognized as a growing threat to human health. Due to increasing multidrug resistance, discovery of alternative methods for treatment of these infections is vital. Interference with one target for alternative treatment, the quorum sensing system agr, has demonstrated clinical improvement of infections in S. aureus animal models. In this study, we sequenced and characterized 160 clinical S. pseudintermedius isolates and their agr systems in order to increase understanding of the epidemiology of the agr group and clarify its associations with types of infection and antimicrobial resistance. We found that isolates with agr type II were significantly less common than other agr types in diseased dogs. This provides valuable information to veterinary clinical microbiologists and clinicians, especially as less research has been performed on infection associations of agr and its therapeutic potential in S. pseudintermedius than in S. aureus.

Cost of Treatment and Care for People with Alzheimer's Disease: A Meta- Analysis.

There is now a general attempt in developed countries to implement strategic plans to fight against Alzheimer's disease, for which treatment represents an increasing economic burden for the ageing society. At present, the costs of treatment and care for Alzheimer's Disease (AD) patients are not consistently tracked and logged, therefore, the economic burden is calculated based on the records kept by individual countries. The aim of this paper is to conduct a meta-analysis of the available data on the total costs of treatment and care for elderly AD patients with respect to the stage of the disease determined by the Mini Mental State Examination (MMSE). The Web of Science and PubMed databases were used for a systematic search. Two independent reviewers screened the identified records and selected relevant articles published in the period from 2007 to 2017. A meta-analysis of costs is performed in three categories related to the stages of Alzheimer's disease (mild, moderate, and severe). The resulting estimation of total costs per patient per year determined by the meta-analysis is 20,461$ total costs. The total costs in relation to the stage of the disease according to the MMSE scale are 14,675 $ for the mild stage, 19,975 $ for the moderate stage, and 29,708 $ for the severe stage. The meta- analysis confirms that the costs rise significantly with the severity of AD. These findings therefore, emphasize the severity of the economic burden carried out by the AD patients, their families, and the healthcare system, and this fact must be taken into account when planning health policy strategies for the years to come.

Collaborating genomic, transcriptomic and microbiomic alterations lead to canine extreme intestinal polyposis.

Extreme intestinal polyposis in pet dogs has not yet been reported in literature. We identified a dog patient who developed numerous intestinal polyps, with the severity resembling human classic familial adenomatous polyposis (FAP), except the jejunum-ileum junction being the most polyp-dense. We investigated this dog, in comparison with 22 other dogs with spontaneous intestinal tumors but no severe polyposis, and with numerous published human cancers. We found, not APC mutation, but three other alteration pathways as likely reasons of this canine extreme polyposis. First, somatic truncation mutation W411X of FBXW7, a component of an E3 ubiquitin ligase, over-activates MYC and cell cycle-promoting network, accelerating crypt cell proliferation. Second, genes of protein trafficking and localization are downregulated, likely associated with germline mutation G406D of STAMBPL1, a K63-deubiquitinase, and MYC network activation. This inhibits epithelial apical-basolateral polarity establishment, preventing crypt cell differentiation. Third, Bacteroides uniformis, a commensal gut anaerobe, thrives and expresses abundantly thioredoxin and nitroreductase. These bacterial products could reduce oxidative stress linked to host germline mutation R51X of CYB5RL, a cytochrome b5 reductase homologue, decreasing cell death. Our work emphasizes the close collaboration of alterations across the genome, transcriptome and microbiome in promoting tumorigenesis.

Detection of vapN in Rhodococcus equi isolates cultured from humans.

Rhodococcus equi can cause severe infections in people, particularly in immunocompromised individuals. The R. equi virulence plasmids (vap) encoding vapA and vapB are linked to development of infections in domestic animals. Recently, a novel virulence plasmid, vapN, was identified in isolates cultured from cattle, but its prevalence or significance in human R. equi infections has not been extensively studied. To determine the prevalence of vapN in a diverse collection of human-derived isolates from different countries, 65 R. equi isolates collected by various institutions from 1984 to 2002 were screened for the presence of vapN and other virulence plasmids through polymerase chain reaction (PCR) using redesigned primer sets. Of the isolates that carried plasmids, 43% (16/37) were vapN-positive and fewer were vapB or vapA-positive (30 and 16%, respectively). This is the first report of vapN carriage in R. equi isolated from human infections in the United States. One isolate (H-30) carried vapN but did not amplify the conjugal plasmid transfer gene traA associated with carriage of vap, which could be explained by sequence variation within the traA gene. Another isolate (H-55) amplified traA, but did not amplify vapA, B, or N (traA+ vapABN-) with previously described primer sets or those developed for this study. The H-55 traA sequence had 98% identity to traA sequences in vapA plasmids, which suggests that it may carry a variant of previously characterized virulence plasmids or a novel virulence plasmid. Carriage of vapN in R. equi isolates derived from people is not uncommon and more research is needed to determine its significance in the epidemiology and pathogenesis of human R. equi infections.

Rhodococcus equi Infections in Goats: Characterization of Virulence Plasmids.

Rhodococcus equi is an uncommon cause of systemic pyogranulomatous infections in goats with macroscopic similarities to caseous lymphadenitis caused by Corynebacterium pseudotuberculosis. Caprine cases have previously been reported to be caused by avirulent R. equi strains. Six cases of R. equi infection in goats yielding 8 R. equi isolates were identified from 2000 to 2017. Lesions varied from bronchopneumonia, vertebral and humeral osteomyelitis, and subcutaneous abscesses, to disseminated infection involving the lungs, lymph nodes, and multiple visceral organs. Isolates of R. equi from infected goats were analyzed by polymerase chain reaction for R. equi virulence-associated plasmid ( vap) genes. Seven of 8 isolates carried the VapN plasmid, originally characterized in bovine isolates, while 1 isolate lacked virulence plasmids and was classified as avirulent. The VapN plasmid has not been described in isolates cultured from goats.

Panfungal Polymerase Chain Reaction for Identification of Fungal Pathogens in Formalin-Fixed Animal Tissues.

Identification of fungal organisms often poses a problem for pathologists because the histomorphology of some fungal organisms is not specific, fresh tissues may not be available, and isolation and identification in culture may take a long time. The purpose of this study was to validate the use of panfungal polymerase chain reaction (PCR) to identify fungal organisms from formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed paraffin-embedded curls were tested from 128 blocks containing canine, feline, equine, and bovine tissues with cutaneous, nasal, pulmonary, and systemic fungal infections, identified by the presence of fungi in histologic sections. Quantitative scoring of histologic sections identified rare (11.9%), occasional (17.5%), moderate (17.5%), or abundant (53.1%) fungal organisms. DNA was isolated from FFPE tissues and PCR was performed targeting the internal transcribed spacer 2 (ITS-2) region, a segment of noncoding DNA found in all eukaryotes. Polymerase chain reaction products were sequenced and identified at ≥97% identity match using the Basic Local Alignment Search Tool and the NCBI database of ITS sequences. Of the 128 blocks, 117 (91.4%) yielded PCR products and high-quality sequences were derived from 89 (69.5%). Sequence and histologic identifications matched in 79 blocks (61.7%). This assay was capable of providing genus- and species-level identification when histopathology could not and, thus, is a beneficial complementary tool for diagnosis of fungal diseases.

Weaned pigs experimentally infected with Salmonella display sexually dimorphic innate immune responses without affecting pathogen colonization patterns.

Sexually dimorphic innate immune responses have been observed in several species, but have not been studied in response to a live pathogen challenge in pigs. This study aimed to elucidate sexually dimorphic innate immune responses along with Salmonella translocation patterns in newly weaned pigs orally inoculated with Salmonella. Newly weaned pigs (n = 8 gilts and 12 barrows; 6.2 ± 0.2 kg BW) were obtained from a commercial swine facility and were maintained in an environmentally-controlled facility in individual pens equipped with feeders and nipple waterers. Pigs were allowed ad libitum access to a commercial non-medicated starter ration and water throughout the study. On d 12 post-weaning, pigs were anesthetized to allow placement of a temperature measuring device in the abdominal cavity for measurement of intraperitoneal temperature (TEMP). On d 17, pigs were anesthetized and fitted with indwelling jugular vein catheters. On the following day (d 18), pigs were orally inoculated with 4.7×109 Salmonella typhimurium. Blood samples were collected at 0.5-h intervals from -2 to 8 h, and at 8-h intervals from 8 to 72 h post-challenge. Whole blood was analyzed for complete blood cell counts. Serum was isolated for measurement of cortisol. Following collection of the 72 h sample, pigs were humanely euthanized and tissues were collected for Salmonella isolation. There was a sex × time interaction (P < 0.001) for TEMP such that gilts had a greater TEMP response to the Salmonella challenge compared to barrows. There was also a sex × time interaction (P = 0.03) for serum cortisol with gilts having decreased cortisol at 16 h yet greater cortisol at 32 h than barrows. Barrows had greater total white blood cells (17.8 vs. 16.2 ± 0.4 103 cells/µL; P < 0.01; respectively) and neutrophils (7.8 vs. 6.1 ± 0.4 103 cells/µL; P < 0.01; respectively) than gilts. However, gilts had greater lymphocytes (9.6 vs. 9.0 ± 0.2 103 cells/µL; P = 0.05; respectively) than barrows. While immune parameters were influenced by sex, there was no effect of sex (P > 0.05) on Salmonella concentrations from fecal shedding 3 d post-inoculation in the cecum, mesenteric and subiliac lymph nodes, liver, spleen, gallbladder, or kidney tissues. These data demonstrate that weaned gilts appear to produce a stronger acute phase response to a Salmonella challenge compared to barrows, without affecting the tissue translocation or shedding of Salmonella.