Evaluation of the main disorders and microbiota of the oral cavity of capuchin monkeys (Sapajus apella) under human care.

BACKGROUND: Although critical to the overall condition of animals under human care, there is still limited information about oral health in neotropical primates. METHODS: We analyzed the main oral conditions and microbiota using mass spectrometry from 13 capuchin monkeys (Sapajus apella) under human care. The findings were registered on odontograms following the Triadan system. RESULTS: The most prevalent conditions were dental fractures (n = 9), mainly enamel fractures, and periodontal disease (n = 8), mainly grade 1 calculi. When exanimating teeth, alterations were identified in 90 out of the 416 evaluated pieces, being periodontal disease the most common (n = 60), followed by enamel fracture (n = 15) and missing teeth (n = 10). In the oral microbiota analyses, Staphylococcus and Streptococcus species were the most prevalent, although no obvious association was observed between isolated organisms and oral conditions. CONCLUSIONS: These findings hold the potential to prevent oral disorders, including fractures and periodontal diseases, contribute to molecular identification of oral microbiota, and to improve the well-being of primates under human care.

Flies (Insecta, Diptera) collected in the environment of dairy farms as carriers of Rotavirus A and betacoronavirus.

AIMS: We aimed to investigate the prevalence of rotavirus and coronavirus in dipterans that commonly inhabit the environment of dairy farms. METHODS AND RESULTS: We collected 217 insect specimens from nine dairy farms, which were examined through hemi-nested RT-PCR followed by Sanger sequencing in search of VP1 and N genes for rotavirus and bovine coronavirus-BCoV, respectively. With a predominance of Muscidae (152/217 = 70%) 11 families of Diptera were identified. Rotavirus A (RVA) and betacoronavirus (BCoV) were detected in 14.7% (32/217) and 4.6% (10/217) of the dipterans, respectively. Sequencing of the amplicons was possible for 11.5% (25/217) of RVA and 0.5% (1/217) of BCoV, confirming the presence of these pathogens. CONCLUSIONS: Our findings highlight the role of dipterans as carriers of RVA and BCoV of great relevance for public and animal health.

Genome-Based Characterization of Multidrug-Resistant Escherichia coli Isolated from Clinical Bovine Mastitis.

Mastitis occurrence in dairy cows is a broad topic that involves several sectors, from antimicrobial resistance and virulence of strains to economic implications and cattle management practices. Here, we assessed the molecular characterization (antimicrobial resistance determinants, virulence genes, sequences type, serotypes, and plasmid types) of 178 Escherichia coli strains isolated from milk samples from cows with clinical mastitis using a genome-based k-mers approach. Of these, 53 (29.8%) showed multidrug resistance by disc diffusion. We selected eight multidrug-resistant mastitis-associated E. coli for whole-genome sequencing and molecular characterization based on raw data using k-mers. We assessed antimicrobial resistance genes, virulence factors, serotypes, Multilocus Sequence Typing (MLST), and plasmid types. The most antimicrobial resistance gene found were blaTEM-1B (7/8), tetA (6/8), strA (6/8), strB (6/8), and qnrB19 (5/8). A total of 25 virulence factors were detected encoding adhesins, capsule, enzymes/proteins, increased serum survival, hemolysin, colicins, and iron uptake. These virulence factors were associated with Extraintestinal Pathogenic E. coli. Three pandemic clones were found: ST10, ST101, and ST69. Two E. coli were assigned in the O117 serogroup and one in the O8:H25 serotype. The most common plasmid groups were IncFII (7/8) and IncFIB (6/8). Our findings contribute to the knowledge of virulence mechanisms, epidemiological aspects, and antimicrobial resistance determinants of E. coli strains obtained from clinical mammary infections of cows.

Cellulitis-related Rhodococcus equi in a cat harboring VAPA-type plasmid pattern.

Rhodococcus equi is a well-known intracellular facultative bacterium that is opportunistic in nature, and a contagious disease-causing agent of pyogranulomatous infections in humans and multihost animals. Feline rhodococcosis is an uncommon or unnoticed clinical condition, in which the organism is usually refractory to conventional antimicrobial therapy. The pathogenicity of the agent is intimately associated with plasmid-governed infectivity, which is attributed to the presence of plasmid-encoded virulence-associated proteins (Vap). Three host-adapted virulence plasmid types (VAPs) have been distinguished to date: pVAPA, pVAPB, and pVAPN, whose infections are related to equine, pig, and bovine or caprine origin, respectively, while humans are infected by all three VAP types. Most virulence studies with R. equi plasmid types in animals involve livestock species. Conversely, data on the pathogenicity and human relevance of the virulence plasmid profile of R. equi isolated from cats remains unclear. This report describes a case of cellulitis-related R. equi that harbors the pVAPA-type in a cat with cutaneous lesion. Long-term therapy of the cat using marbofloxacin, a broad-spectrum third-generation fluoroquinolone, resulted effectiveness. pVAPA is a host-adapted virulent type that has been associated predominantly with pulmonary foal infections. Our cat had a history of contact with other cats, livestock (including horses), and farm environment that could have favored the transmission of the pathogen. Besides no clear evidence of cat-to-humans transmission of the pathogen, the identification of R. equi harboring pVAPA-type in a cat with cutaneous abscessed lesion represent relevance in human health because this virulent type has been described in people worldwide with clinical rhodococcal disorders.

In vitro algicidal effect of polypyrrole on Prototheca species isolates from bovine mastitisAlgicidal activity of polypyrrole on Prototheca spp.

Algae of the genus Prototheca are microorganisms involved in the occurrence of diseases in humans and animals. In bovine species, Prototheca spp. cause environmental mastitis, productive losses in dairy herds, mainly leading to the discard of infected cows. Currently, there are no effective anti-Prototheca spp. drugs to combat this infection. Thus, the search for an efficacious therapy for Prototheca spp. infections have become essential. Highly soluble polypyrrole (Ppy) is a molecule with known antimicrobial activity. This study aimed to characterize Prototheca spp. isolates from bovine mastitis as well as to evaluate the susceptibility profile and to verify the morphological alterations on Prototheca spp. isolates treated with Ppy. In this research, 36 Brazilian isolates of Prototheca spp. were characterized by restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) assay for the mitochondrial cytB gene. Additionally, Ppy algicidal activity against these isolates of Prototheca spp. was assessed by minimal microbicidal concentration method in microplates. Further, scanning electron microscopy (SEM) was performed in order to verify the morphological alterations on Prototheca spp. isolates in response to Ppy. The isolates were characterized as belonging to Prototheca zopfii genotype 2 (35/36) and Prototheca blaschkeae (1/36). Ppy had an algicidal effect on all isolates tested at concentrations ranging from 15.625 µg ml-1 to 62.5 µg ml-1. SEM showed changes on planktonic and sessile P. zopfii, including a decrease of the number of cells with the presence of an amorphous substance involving the cells. The algicidal activity of Ppy suggests the therapeutic potential of this molecule in the prevention and treatment of Prototheca spp. in bovine mastitis.

Isolation of Clostridium perfringens and Clostridioides difficile in diarrheic and nondiarrheic cats.

The occurrence and characteristics of Clostridioides (previously Clostridium) difficile and Clostridium perfringens in the feces of diarrheic and non-diarrheic cats was investigated. Apparently healthy animals were more likely to be positive for C. perfringens type A (p = 0.009). Two isolates (0.7%), one each from a diarrheic and an apparently healthy cat, were positive for the enterotoxin-encoding gene but negative for the NetF-encoding gene. Six toxigenic C. difficile isolates were isolated, all RT106 and ST42, which is commonly reported in humans with C. difficile infection.

Identification of Mycobacterium species and Rhodococcus equi in peccary lymph nodes.

Mycobacterium species and the virulence-associated proteins (vapA, vapB, and vapN genes) of Rhodococcus equi isolated from 330 lymph nodes of collared peccaries (Tayassu tajacu) and white-lipped peccaries (Tayassu pecari) intended for human consumption were investigated. Thirty-six (10.9%) R. equi strains were isolated; 3.3% (n = 11/330) were from white-lipped peccary lymph nodes, and 7.6% (25/330) were from collared peccary lymph nodes. Among the 11 isolates of R. equi from the white-lipped peccaries, 90.9% (n = 10/11) were obtained from the mesenteric lymph nodes, and only 9.1% (n = 1/10) were obtained from the mediastinal lymph nodes. In the 25 isolates of R. equi obtained from the collared peccaries, 40.0% (n = 10/25) were recovered from the mesenteric lymph nodes, 36% (n = 9/25) from the submandibular lymph nodes, and 24.0% (n = 6/25) from the mediastinal lymph nodes. No vapA, vapB, or vapN genes (plasmidless) or three host-associated types (pVAPA, pVAPB, and pVAPN) were identified among the R. equi isolates. Mycobacterium species were isolated in 3.03% (n = 10/330) of all the lymph nodes analyzed. Among the 10 mycobacterial isolates, 60% (n = 6/10) were from the white-lipped peccary lymph nodes, and 40% (n = 4/10) were from the collared peccary lymph nodes. Ten Mycobacterium species were detected by PCR-PRA with a predominance of M. avium type 1. Sequencing of the hsp65 and rpob genes revealed mycobacteria that were saprophytic (M. sinense and M. kumamotonense) and potentially pathogenic (M. colombiense and M. intracellulare) to humans and animals. To our knowledge, this is the first description of R. equi and/or mycobacterial species identified in the lymph nodes of peccary specimens. R. equi (plasmidless) and the mycobacterial species described here have been reported as causes of pulmonary and extrapulmonary infections in both immunocompetent and immunocompromised humans.

Prototheca blaschkeae subsp. brasiliensis subsp. nov., isolated from cow milk.

A strain of an achlorophyllic alga, named PR24T, was isolated from cow milk samples from the state of Minas Gerais, Brazil. Based on 18S rDNA, 28S rRNA, D1/D2 region of the LSU rDNA and SSU rRNA gene sequence similarities, this strain was found to be a member of the genus Prototheca and closely related to Protothecablaschkeae SAG2064T. However, the novel strain could easily be distinguished from recognized Prototheca species by internal transcribed spacer, species-specific PCR, single-strand conformation polymorphism-PCR analysis and phenotypic characteristics. The inability to grow in Sabouraud broth at pH 4.0 and the different cellular fatty acid composition clearly distinguished PR24T from the reference strain of P. blaschkeae. The combination of genotypic and phenotypic data indicates that strain PR24T represents a subspecies of P. blaschkeae, for which the name Prototheca blaschkeae subsp. brasiliensis subsp. nov. is proposed. The respective type strain is PR24T (=DSM 103592T=IHEM 26958T).

Clostridium perfringens and C. difficile in parvovirus-positive dogs.

The aim of this study was to investigate Clostridium difficile and Clostridium perfringens in 82 diarrheic dogs positive for canine parvovirus type 2 (CPV). Enterotoxigenic C. perfringens type A was isolated from three (3.6%) dogs. One (1.2%) strain was also positive for NetE- and NetF-encoding genes, which are commonly associated with diarrhea in dogs. Toxigenic C. difficile was isolated from one animal (1.2%), which was also positive for A/B toxins. The present study identified C. difficile and C. perfringens infection in CPV-positive dogs. Further studies are necessary to clarify if clostridial infections may predispose or potentiate CPV-infection in dogs or vice versa.

Methicillin-resistant Staphylococcus spp. investigation in hospitalized horses and contacting personnel in a teaching veterinary hospital.

Staphylococci are well-known opportunistic pathogens associated with suppurative diseases in humans and animals. Antimicrobial resistance is an emergent threat to humans and animals worldwide. This study investigated the prevalence of methicillin-resistant Staphylococcus spp. (MRS) in hospitalized horses and contacting personnel (veterinarians and staff), and assessed possible interspecies transmission in a teaching veterinary hospital. Nasal swabs from horses (n = 131) and humans (n = 35) were collected. The microorganisms were identified by traditional biochemical tests and genotypic methods, i.e., PCR, internal transcript spacer PCR (ITS-PCR), and gene sequencing. Staphylococcal species were isolated in 18% (23/131) of the horses, of which 8% (11/131) were S. hyicus, 4 % (5/131) were S. aureus, 4% (5/131) were S. pseudintermedius, and 2% (2/131) were S. schleiferi subsp. coagulans. The mecA gene was detected in an S. pseudintermedius isolate. Staphylococcus spp. was isolated in 40% (14/35) of the human samples, all of which were S. aureus. In four samples of S. aureus, the clonal profile ST398 was identified; among them, a clonal similarity of 98.1% was observed between a horse and a contacting human. This finding supports the need for biosecurity measures to avoid the spread of multidrug-resistant staphylococci in humans and horses.

Occurrence of mycobacteria in bovine milk samples from both individual and collective bulk tanks at farms and informal markets in the southeast region of Sao Paulo, Brazil.

BACKGROUND: Mycobacterium spp. is one of the most important species of zoonotic pathogens that can be transmitted from cattle to humans. The presence of these opportunistic, pathogenic bacteria in bovine milk has emerged as a public-health concern, especially among individuals who consume raw milk and related dairy products. To address this concern, the Brazilian control and eradication program focusing on bovine tuberculosis, was established in 2001. However, bovine tuberculosis continues to afflict approximately 1,3 percent of the cattle in Brazil. In the present study, 300 samples of milk from bovine herds, obtained from both individual and collective bulk tanks and informal points of sale, were cultured on Löwenstein-Jensen and Stonebrink media. Polymerase chain reaction (PCR)-based tests and restriction-enzyme pattern analysis were then performed on the colonies exhibiting phenotypes suggestive of Mycobacterium spp., which were characterized as acid-fast bacilli. RESULTS: Of the 300 bovine milk samples that were processed, 24 were positively identified as Mycobacterium spp.Molecular identification detected 15 unique mycobacterial species: Mycobacterium bovis, M. gordonae, M. fortuitum, M. intracellulare, M. flavescens, M. duvalii, M. haemophilum, M. immunogenum, M. lentiflavum, M. mucogenicum, M. novocastrense, M. parafortuitum, M. smegmatis, M. terrae and M. vaccae. The isolation of bacteria from the various locations occurred in the following proportions: 9 percent of the individual bulk-tank samples, 7 percent of the collective bulk-tank samples and 8 percent of the informal-trade samples. No statistically significant difference was observed between the presence of Mycobacterium spp. in the three types of samples collected, the milk production profiles, the presence of veterinary assistance and the reported concerns about bovine tuberculosis prevention in the herds. CONCLUSION: The microbiological cultures associated with PCR-based identification tests are possible tools for the investigation of the presence of Mycobacterium spp. in milk samples. Using these methods, we found that the Brazilian population may be regularly exposed to mycobacteria by consuming raw bovine milk and related dairy products. These evidences reinforces the need to optimize quality programs of dairy products, to intensify the sanitary inspection of these products and the necessity of further studies on the presence of Mycobacterium spp. in milk and milk-based products.

Multiplex polymerase chain reaction to identify and determine the toxigenicity of Corynebacterium spp with zoonotic potential and an overview of human and animal infections.

Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis constitute a group of potentially toxigenic microorganisms that are related to different infectious processes in animal and human hosts. Currently, there is a lack of information on the prevalence of disease caused by these pathogens, which is partially due to a reduction in the frequency of routine laboratory testing. In this study, a multiplex polymerase chain reaction (mPCR) assay that can simultaneously identify and determine the toxigenicity of these corynebacterial species with zoonotic potential was developed. This assay uses five primer pairs targeting the following genes: rpoB (Corynebacterium spp), 16S rRNA (C. ulcerans and C. pseudotuberculosis), pld (C. pseudotuberculosis), dtxR (C. diphtheriae) and tox [diphtheria toxin (DT) ]. In addition to describing this assay, we review the literature regarding the diseases caused by these pathogens. Of the 213 coryneform strains tested, the mPCR results for all toxigenic and non-toxigenic strains of C . diphtheriae, C. ulcerans and C. pseudotuberculosis were in 100% agreement with the results of standard biochemical tests and PCR-DT. As an alternative to conventional methods, due to its advantages of specificity and speed, the mPCR assay used in this study may successfully be applied for the diagnosis of human and/or animal diseases caused by potentially toxigenic corynebacterial species.

Short review: Geographical distribution of equine-associated pVAPA plasmids in Rhodococcus equi in the world.

Virulent Rhodococcus equi strains expressing virulence-associated 15-17 kDa protein (VapA) and having a large virulence plasmid (pVAPA) of 85-90 kb containing vapA gene are pathogenic for horses. In the last two decades, following pVAPA, two host-associated virulence plasmid types of R. equi have been discovered: a circular plasmid, pVAPB, associated with porcine isolates in 1995, and a recently detected linear plasmid, pVAPN, related to bovine and caprine isolates. Molecular epidemiological studies of R. equi infection in foals on horse-breeding farms in Japan and many countries around the world have been conducted in the last three decades, and the epidemiological studies using restriction enzyme digestion patterns of plasmid DNAs from virulent isolates have shown 14 distinct pVAPA subtypes and their geographical preference. This short review summarizes previous reports regarding equine-associated pVAPA subtypes in the world and discusses their geographic distribution from the standpoint of horse movements.

Cutaneous protothecosis in a dog successfully treated with oral itraconazole in pulse dosing.

BACKGROUND: Protothecosis is a rare infectious disease caused by unicellular, achlorophyllous, microalgae of the genus Prototheca, ubiquitously distributed in nature. The algae are emerging pathogens, whose incidence is increasing in both human and animal populations and serious systemic infections related to this pathogen have been increasingly described in humans in recent years. After mastitis in dairy cows, canine protothecosis is the second most prevalent form of the protothecal disease in animals. Here, we report the first case of chronic cutaneous protothecosis due to P. wickerhamii in a dog in Brazil, successfully treated with a long-term therapy with itraconazole in pulse. CASE PRESENTATION: Upon clinical examination, exudative nasolabial plaque, ulcered, and painful lesions in central and digital pads and lymphadenitis were observed in a 2-year-old mixed-breed dog, with a 4-month history of cutaneous lesions and contact with sewage water. Histopathological examination revealed intense inflammatory reaction, with numerous spherical to oval, encapsulated structures stained with Periodic Acid Schiff, compatible with Prototheca morphology. Tissue culture on Sabouraud agar revealed yeast-like, greyish-white colonies after 48 h of incubation. The isolate was subjected to mass spectrometry profiling and PCR-sequencing of the mitochondrial cytochrome b (CYTB) gene marker, leading to identification of the pathogen as P. wickerhamii. The dog was initially treated with oral itraconazole at a dosage of 10 mg/kg once daily. After six months, the lesions resolved completely, yet recurred shortly after cessation of therapy. The dog was then treated with terbinafine at a dose of 30 mg/kg, once daily for 3 months, with no success. The resolution of clinical signs, with no recurrence over a 36-months follow-up period, was achieved after 3 months of treatment with itraconazole (20 mg/kg) in pulse intermittently on two consecutive days a week. CONCLUSIONS: This report highlights the refractoriness of skin infections by Prototheca wickerhamii with therapies proposed in the literature and suggests a new treatment option with oral itraconazole in pulse dosing for long-term disease control successfully performed in a dog with skin lesions.

Pleural Empyema in Six Horses: A Retrospective Case Series.

The clinical findings of pleural empyema in six horses were retrospectively studied using epidemiological, clinical pathology, microbiological, ultrasound, and post-mortem data. The clinical findings included tachycardia (n = 3/6), tachypnea (n = 6/6), cyanotic mucosa (n = 2/6), hyperthermia (n = 4/6), inspiratory or mixed dyspnea (n = 6/6), presence of fluid and/or pleural rubbing (n = 2/6) and coarse crackling on auscultation (n = 4/6). Horses demonstrated leukocytosis (16.22 × 103/µL) with neutrophilia (12.32 × 103/µL) and hyperfibrinogenemia (633.33 mg/dL) and an increase in urea (69.80 mg/kg) and globulins (5.22 g/dL). The pleural fluid exhibited exudate (n = 5/6). The pathogens isolated from transtracheal wash (TTW) and/or pleural effusion included Aspergillus fumigatus, Enterobacter cloacae, alpha and beta hemolytic Streptococcus, Pseudomonas aeruginosa, Salmonella sp., Streptococcus equi subsp. zooepidemicus, and beta hemolytic Staphylococcus. The in vitro tests of microbial sensitivity of the isolates revealed that ceftiofur (5/6) and penicillin (3/6) were the most effective drugs. The fatality rate was 83% (5/6). The main post-mortem finding was the presence of fibrin in the pleural cavity with adhesion between the parietal and visceral pleura. These results show that pleural empyema is a complex disease pathophysiology that is refractory to conventional treatment.

Potential in vitro action of an adenosine analog and synergism with penicillin against Corynebacterium pseudotuberculosis.

Caseous lymphadenitis is a well-known disease caused by Corynebacterium pseudotuberculosis affecting small ruminants with small significance to human health because of its minor zoonotic potential. In both cases, few treatment options are available and conventional antimicrobial therapy is commonly refractory due to development of pyogranulomatous reactions, bringing great interest in discovering novel therapeutics for more suitable approaches. Dideoxynucleotides presented antibacterial action against various bacteria but were never described for C. pseudotuberculosis. Hypothesizing the antimicrobial action of 2',3'-dideoxiadenosine (ddATP) against C. pseudotuberculosis, we performed for the first time an investigation of its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in the ATCC® 19,410 strain and a well-characterized clinical isolate of C. pseudotuberculosis. We also assessed potential synergism with penicillin. ddATP showed a growth delay effect for C. pseudotuberculosis at 2 µmol/mL and a MIC and MBC of 4 µmol/mL against the ATCC® 19,410 strain, but not for the clinical strain. An antimicrobial effect was observed when using concentrations lower than the MIC of ddATP associated with penicillin for both strains tested. Our data suggest the potential of nucleotide analogs, especially adenosine, and its combination with penicillin, as a possible novel treatment for C. pseudotuberculosis-induced infections, and contributes with knowledge regarding alternative drugs to treat C. pseudotuberculosis infections.

Molecular identification and antimicrobial resistance pattern of Nocardia isolated from 14 diseased dogs and cats.

Nocardia are ubiquitous, saprophytic and opportunistic bacteria. They cause a set of pyogenic clinical infections in animals and humans, particularly immunocompromised patients, mostly affecting the skin and respiratory tract, with refractoriness to conventional therapy. The most descriptions of nocardial infections in companion animals involve case reports, and there are scarce case series studies focused on canine and feline nocardiosis in which diagnosis has been based on molecular techniques. We investigated epidemiological aspects, clinical findings, in vitro susceptibility profile, and molecular identification of Nocardia using PCR-based method targeted 16S rRNA gene in twelve dogs and two cats. Among dogs were observed cutaneous lesions (8/12 = 67%), pneumonia (3/12 = 25%), and encephalitis (2/12 = 17%), whereas cats developed cutaneous lesions and osteomyelitis. Nocardia and canine morbillivirus coinfection was described in six dogs (6/12 = 50%). A high mortality rate (6/8 = 75%) was seen among dogs. Three dogs (3/4 = 75%) and one cat (1/2 = 50%) with systemic signs (pneumonia, encephalitis, osteomyelitis), and 83% (5/6) of dogs with a history of concomitant morbillivirus infection died. N. nova (5/12 = 42%), N. cyriacigeorgica (3/12 = 25%), N. farcinica (2/12 = 17%), N. veterana (1/12 = 8%), and N. asteroides (1/12 = 8%) species were identified in dogs, whereas N. africana and N. veterana in cats. Among the isolates from dogs, cefuroxime (12/12 = 100%), amikacin (10/12 = 83%), gentamycin (10/12 = 83%), and imipenem (10/12 = 83%) were the most effective antimicrobials, whereas cefuroxime, cephalexin, amoxicillin/clavulanic acid, imipenem, and gentamycin were efficient against isolates from cats. Multidrug resistance was observed in 36% (5/14) of isolates. We describe a variety of Nocardia species infecting dogs and cats, multidrug-resistant ones, and a high mortality rate, highlighting a poor prognosis of nocardiosis in companion animals, particularly among animals systemically compromised or coinfected by canine morbillivirus. Our study contributes to species identification, in vitro antimicrobial susceptibility profile, clinical-epidemiological aspects, and outcome of natural Nocardia-acquired infections in dogs and cats.

Detection and genomic characterization of a multidrug-resistant Salmonella Newport co-harbouring blaCMY-2, qnrB19 and mcr-9 from the diarrheic faeces of a foal.

OBJECTIVES: This study reports the genomic characterization of the multidrug resistant Salmonella Newport strain 195_20 recovered from the diarrheic faeces of a foal in Brazil and co-harbouring the mcr-9, blaCMY-2 and qnrB19 antibiotic resistance genes. METHODS: Bacterial isolate positive for mobile colistin resistance gene (mcr-9) was submitted to antimicrobial susceptibility testing by disk diffusion and broth microdilution for colistin and polymyxin B. The isolate was submitted to whole genome sequencing by Illumina technology and Nanopore Sequencing. Conjugation assays, plasmid sizes determined by S1-PFGE and plasmid content were investigated by hybrid assembly after MinIon long reads sequencing. RESULTS: Isolate 195_20 was identified as sequence type ST45, resistant to penicillin and cephalosporins (ampicillin, ceftazidime, ceftriaxone and cefotaxime), aminoglycosides (streptomycin and gentamicin), phenicol (chloramphenicol), quinolones and fluoroquinolones (nalidixic acid, ciprofloxacin, and pefloxacin), folate pathway antagonists (sulfonamides and trimethoprim-sulfamethoxazole), and tetracycline. A transferable IncHI2/IncHI2A plasmid sized ca. 262kb was found to carry the mcr-9 gene in a module consisting of IS903-mcr-9-wbuC-IS26. In addition, an 174kb IncC and a 48kb IncN plasmid were also identified in the 195_20 isolate, carrying blaCMY-2 and qnrB19, respectively. CONCLUSIONS: Not surprisingly, isolate 195_20 was susceptible to polymyxins, possibly due to absence of qseBC regulatory operon. Presence of mobile colistin resistance (mcr-9), third-generation cephalosporins (blaCMY-2) and quinolone (qnrB19) resistance determinants in zoonotic pathogens from animals in close contact with humans alerts for the possible route of transmission between these different reservoirs.

In Vitro Activity of Quaternary Ammonium in Prototheca Isolated from Clinical Bovine Mastitis Identified by Mass Spectrometry and PCR Sequencing of the cytb Gene Marker.

The in vitro algaecide activity of quaternary ammonium (QA) against Prototheca isolated from bovine clinical mastitis was investigated, in which the clinical severity was scored, milk samples were subjected to microbiological culture, and algal species were identified by molecular typing. A total of 4275 milk clinical samples of different cows from ten large dairy farms were used. Forty-four (1%) samples of cows from three dairy farms yielded growth of Prototheca, of which 88.6% (39/44) were identified as Prototheca bovis and 11.3% (5/44) as Prototheca sp. by MALDI-TOF MS, whereas 100% of the isolates were identified as P. bovis using PCR sequencing of the cytb gene. Among cows for which clinical severity scoring was available, 78.8% (26/33) and 21.2% (7/33) had mild and moderate infections, respectively, whereas no animal showed severe clinical signs. The algaecide activity of QA in Prototheca was observed in low concentrations among all isolates, in 20.4% (9/44) at 35 ppm, 36.4% (16/44) at 17 ppm, and 43.2% (19/44) at an 8 ppm, in addition to activity on three reference Prototheca strains. Overall, the study highlights the predominance of P. bovis as the causative agent of algal mastitis in bovines. Prototheca induced abnormalities preponderantly in the milk and mammary gland tissue of cows, and to our knowledge, our study is the first to apply clinical severity scoring in protothecal mastitis. In addition, the study underlines the activity of QA in low concentrations against Prototheca, indicating its potential use as an antiseptic/disinfectant in milking facilities and dairy environments.

Bacterial identification in cerebrospinal fluid of domestic species with neurologic signs: a retrospective case-series study in 136 animals (2005-2021).

Central nervous system (CNS) infections comprise life-threatening clinical conditions in domestic species, and are commonly related to severe sequelae, disability, or high fatality rates. A set of bacterial pathogens have been identified in central nervous infections in livestock and companion animals, although the most of descriptions are restricted to case reports and a lack of comprehensive studies involving CNS-related bacterial infections have been focused on a great number of domestic species. In this scenario, we retrospectively investigated selected epidemiological data, clinical findings, bacteriological culture, and in vitro susceptibility patterns of 136 nonrepetitive neurologic cases in domestic species (2005-2021). Bacterial isolates were recovered from 25% (34/136) of the cerebrospinal fluid (CSF) sampled. The isolates were obtained from cattle (9/136 = 6.6%), dogs (7/136 = 5.1%), horses (6/136 = 4.4%), goats (3/136 = 2.2%), pigs (3/136 = 2.2%), sheep (3/136 = 2.2%), cats (2/136 = 1.5%), and asinine (1/136 = 0.7%). Among animals with bacterial isolation, Staphylococcus aureus (6/34 = 17.6%), Escherichia coli (5/34 = 14.7%), Staphylococcus beta-hemolytic (5/34 = 14.7%), and Trueperella pyogenes (3/34 = 8.8%) were predominant, in addition to a miscellaneous of other bacteria isolated in minor frequency, e.g., Corynebacterium pseudotuberculosis, Enterobacter cloacae, Mannheimia haemolytica, Pseudomonas aeruginosa, and Streptococcus equi subsp. equi. In vitro susceptibility tests of isolates revealed that amoxicillin/clavulanic acid (11/13 = 84.6%), cephalexin (9/11 = 81.8%), and florfenicol (9/12 = 75%) were the most effective antimicrobials. Conversely, isolates exhibited resistance mainly to tetracycline (6/10 = 60%), penicillin (6/11 = 54.5%), and trimethoprim/sulfamethoxazole (5/11 = 45.5%). Also, multidrug resistance to ≥ 3 classes of antimicrobials was found in 23.5% (8/34) strains. Data relative to the outcome was available in 79.4% (27/34) of animals that had bacterial isolation, and from these, the lethality rate was 92.6% (25/27). Incoordination (14/34 = 41.2%), recumbency (11/34 = 32.4%), apathy (10/34 = 29.4%), anorexia (9/34 = 26.5%), blindness (7/34 = 20.6%), seizure (6/34 = 17.6%), limb paresis (5/34 = 14.7%), head-pressing (4/34 = 11.8%), and nystagmus (3/34 = 8.8%) were the most frequent clinical signs. A variety of bacterial pathogens were identified in the CSF of domestic species showing neurologic signs, with a predominance of staphylococci, streptococci, and enterobacteria. High lethality of cases, poor in vitro efficacy of conventional antimicrobials, and a high in vitro multidrug resistance pattern of isolates were seen. Our results contribute to etiological characterization, antimicrobial resistance patterns, and clinical-epidemiological findings of bacterial infections in domestic species with neurological signs.