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.

Safety issues of raw milk: evaluation of bacteriological and physicochemical characteristics of human milk from a bank in a teaching hospital, focusing on Staphylococcus species.

Many infants are nurtured with milk supplied by human banks, whose bacteriological and physical-chemical profiles are a major issue. We investigated the bacteriological and physical-chemical characteristics, as well as genotypic and phenotypic and profiles of Staphylococcus species isolated from 240 samples of breast milk from a bank in a teaching hospital. Dornic acidity of milk revealed that 95.4% (229/240) had acceptable limits (< 8.0 oD). Caloric intake showed a wide variation in cream content (4%), fat (4%) and energy values (559.81 Kcal/L). Staphylococcus (105/186 or 56.5%) and Enterobacter (25/186 or 13.4%) were the most prevalent genera, although other microorganisms were identified, including Klebsiella pneumoniae and Pseudomonas aeruginosa. Amoxicillin/clavulanic acid (125/157 or 79.6%), vancomycin (115/157 or 73.2%), and cephalexin (112/157 or 71.3%) were the most effective antimicrobials. High resistance rates of isolates were found to penicillin G (141/157 or 89.8%), ampicillin (135/157 or 86%), and oxacillin (118/157 or 75.2%). Multidrug resistance to ≥ 3 antimicrobials occurred in 66.2% (123/186) of the isolates. Residues of microbial multiplication inhibitory substances were found in 85% (204/240) of samples. Among the coagulase-positive-CPS and negative-CoNS staphylococci, the mecA gene was detected in 53.3% (8/15) and 75% (30/40), respectively. Genes sea, seb and sec were detected in 20% (3/15) of CPS, while tsst-1 was detected in 13.34% (2/15). In addition, 13.3% (2/15) of S. aureus were toxin-producers. Genes sea, seb and sec were detected in 90% (36/40), 5% (2/40) and 15% (6/40) CoNS, respectively. Enterotoxin production was identified in 5% (2/40) of CoNS. The identification of multidrug-resistant bacteria, staphylococci species toxin-producers harboring methicillin-resistance genes, and residues of microbial multiplication inhibitory substances reinforce the need for a continuous vigilance of milk quality offered to infant consumption by human banks.

Enterotoxin genes, multidrug resistance, and molecular typing of Staphylococcus spp. isolated from organic bovine milk / Genes de enterotoxinas, multirresistência a antimicrobianos e caracterização molecular de espécies de Staphylococcus spp. isoladas de leite bovino orgânico

The multidrug resistant and the emergence of methicillin-resistant staphylococci isolated from animals, food, and humans are public health concern. These microorganisms produce different toxins related to food poisoning in humans. This study aimed to characterize Staphylococcus spp. isolated from two organic milk farms in Brazil. A total of 259 milk samples were collected, from which 58 (22.4%) Staphylococcus spp. were isolated. The highest sensibility to ceftiofur and sulfamethoxazole/trimethoprim was observed in 96.6% of Staphylococcus spp., and whereas 89% were resistant to penicillin G. The mecA gene was detected in 13.8% of the isolates. SEA and SEC were the most common enterotoxins detected. PFGE revealed genetic heterogeneity from S. intermedius and S. warneri analyzed, while S. aureus presented similar profiles among isolates from the two studied herds. To the best of our knowledge, the current study describes for the first time presence of enterotoxins, mecA gene, and genetic diversity of staphylococci isolated from organic dairy farms in Brazil.(AU)

A emergência de estafilococos multirresistentes e resistentes à meticilina, isolados de animais, alimentos e humanos é uma preocupação em saúde pública. Esses micro-organismos produzem diferentes toxinas relacionadas à intoxicação alimentar em humanos. Este estudo caracterizou Staphylococcus spp. isolados em duas fazendas orgânicas no Brasil. Foram coletadas 259 amostras de leite em duas propriedades leiteiras orgânicas, nas quais 58 (22,4%) estirpes de Staphylococcus spp. foram isoladas. A maior sensibilidade dos isolados foi observada para ceftiofur e sulfametoxazol/trimetoprim em 96,6%. Em contraste, acima de 89% de resistência dos estafilicocos foi encontrada para penicilina G. O gene mecA foi identificado em 13,8% dos isolados. SEA e SEC foram as enterotoxinas mais comumente detectadas. PFGE revelou heterogeneidade genética entre S. intermedius e S. warneri, enquanto S. aureus demonstraram perfis semelhantes entre isolados dos dois rebanhos estudados. Relata-se pela primeira vez no Brasil a detecção de enterotoxinas, o gene mecA e diversidade genética em estafilococos isolados de vacas em produção orgânica.(AU)

MOLECULAR IDENTIFICATION AND ANTIMICROBIAL RESISTANCE PATTERN OF SEVEN CLINICAL ISOLATES OF Nocardia spp. IN BRAZIL.

Nocardia is a ubiquitous microorganism related to pyogranulomatous infection, which is difficult to treat in humans and animals. The occurrence of the disease is on the rise in many countries due to an increase in immunosuppressive diseases and treatments. This report of cases from Brazil presents the genotypic characterization and the antimicrobial susceptibility pattern using the disk-diffusion method and inhibitory minimal concentration with E-test® strips. In summary, this report focuses on infections in young adult men, of which three cases were cutaneous, two pulmonary, one neurological and one systemic. The pulmonary, neurological and systemic cases were attributed to immunosuppressive diseases or treatments. Sequencing analysis of the 16S rRNA segments (1491 bp) identified four isolates of Nocardia farcinica, two isolates of Nocardia nova and one isolate of Nocardia asiatica. N. farcinica was involved in two cutaneous, one systemic and other pulmonary cases; N. nova was involved in one neurological and one pulmonary case; and Nocardia asiatica in one cutaneous case. The disk-diffusion antimicrobial susceptibility test showed that the most effective antimicrobials were amikacin (100%), amoxicillin/clavulanate (100%), cephalexin (100%) and ceftiofur (100%), while isolates had presented most resistance to gentamicin (43%), sulfamethoxazole/trimethoprim (43%) and ampicillin (29%). However, on the inhibitory minimal concentration test (MIC test), only one of the four isolates of Nocardia farcinica was resistant to sulfamethoxazole/trimethoprim.

MOLECULAR IDENTIFICATION AND ANTIMICROBIAL RESISTANCE PATTERN OF SEVEN CLINICAL ISOLATES OF Nocardia spp. IN BRAZIL / Identificação molecular e perfil de sensibilidade a antimicrobianos de sete isolados clínicos de Nocardia spp. no Brasil

Nocardia is a ubiquitous microorganism related to pyogranulomatous infection, which is difficult to treat in humans and animals. The occurrence of the disease is on the rise in many countries due to an increase in immunosuppressive diseases and treatments. This report of cases from Brazil presents the genotypic characterization and the antimicrobial susceptibility pattern using the disk-diffusion method and inhibitory minimal concentration with E-test® strips. In summary, this report focuses on infections in young adult men, of which three cases were cutaneous, two pulmonary, one neurological and one systemic. The pulmonary, neurological and systemic cases were attributed to immunosuppressive diseases or treatments. Sequencing analysis of the 16S rRNA segments (1491 bp) identified four isolates of Nocardia farcinica, two isolates of Nocardia nova and one isolate of Nocardia asiatica. N. farcinica was involved in two cutaneous, one systemic and other pulmonary cases; N. nova was involved in one neurological and one pulmonary case; and Nocardia asiatica in one cutaneous case. The disk-diffusion antimicrobial susceptibility test showed that the most effective antimicrobials were amikacin (100%), amoxicillin/clavulanate (100%), cephalexin (100%) and ceftiofur (100%), while isolates had presented most resistance to gentamicin (43%), sulfamethoxazole/trimethoprim (43%) and ampicillin (29%). However, on the inhibitory minimal concentration test (MIC test), only one of the four isolates of Nocardia farcinica was resistant to sulfamethoxazole/trimethoprim.

Nocardia é um microorganismo ubiquitário relacionado a infecções piogranulomatosas, com difícil resolução tecidual em humanos e animais. A doença é mundialmente emergente devido ao aumento de doenças e tratamentos imunossupressores. Este relato de casos ocorridos no Brasil visa apresentar a identificação molecular dos isolados e o padrão de sensibilidade a antimicrobianos por disco-difusão e concentração inibitória mínima (CIM) através de fitas E-test®. Os casos ocorreram em homens, em idade adulta. Três quadros foram cutâneos, dois pulmonares, um neurológico e um sistêmico. O quadro respiratório, o neurológico e um sistêmico estavam associados à doença ou terapia imunossupressoras. O sequenciamento do gene 16S rRNA (1491pb) possibilitou a identificação de quatro isolados de Nocardia farcinica, dois de Nocardia nova e um de Nocardia asiatica. N. farcinica foi observada em dois casos dermatológicos, um pulmonar e um quadro sistêmico, N. nova foi isolada de um caso neurológico e outro pulmonar; e N. asiatica em um caso dermatológico. O teste de disco-difusão mostrou que amicacina (100%), amoxicilina/clavulanato (100%), cefalexina (100%) e ceftiofur (100%) foram mais efetivos; enquanto gentamicina (43%), sulfametoxazol/trimetoprim (43%) e ampicilina (29%) foram menos efetivos. No entanto, no teste de concentração inibitória mínima (CIM), apenas um dos quatro isolados da espécie Nocardia farcinica mostrou-se resistente a sulfametoxazole-trimetropina.

Uncommon mandibular osteomyelitis in a cat caused by Nocardia africana.

BACKGROUND: Nocardiosis is an unusual infection in companion animals characterized by suppurative to pyogranulomatous lesions, localized or disseminated. Cutaneous-subcutaneous, pulmonary and systemic signs are observed in feline nocardiosis. However, osteomyelitis is a rare clinical manifestation in cats. Nocardia cyriacigeorgica (formerly N. asteroides sensu stricto), Nocardia brasiliensis, Nocardia otitidiscaviarum, and Nocardia nova are the most common pathogenic species identified in cats, based on recent molecular classification (16S rRNA gene). The present report is, to our knowledge, the first case of mandibular osteomyelitis in a cat caused by Nocardia africana, diagnosed based upon a combination of methods, including molecular techniques. CASE PRESENTATION: A one-year-old non-neutered female cat, raised in a rural area, was admitted to the Companion Animal Hospital-PUCPR, São José dos Pinhais, State of Paraná, Brazil, with a history a progressive facial lesion, difficulty apprehending food, loss of appetite, apathy and emaciation. Clinical examination showed fever, submandibular lymphadenitis, and a painless, 8 cm diameter mass, which was irregularly-shaped, of firm consistency, and located in the region of the left mandible. The skin around the lesion was friable, with diffuse inflammation (cellulitis), multiple draining sinuses, and exudation of serosanguinous material containing whitish "sulfur" granules.Diagnosis was based initially in clinical signs, microbiological culture, cytological, and histopathological findings, and radiographic images. Molecular sequencing of 16S rRNA of isolate allowed diagnosis of Nocardia africana. Despite supportive care and antimicrobial therapy based on in vitro susceptibility testing the animal died. CONCLUSION: The present report describes a rare clinical case of feline osteomyelitis caused by Nocardia africana, diagnosed based upon a combination of clinical signs, microbiological culture, cytological and histopathological findings, radiographic images, and molecular methods. The use of modern molecular techniques constitutes a quick and reliable method for Nocardia species identification, and may contribute to identification to new species of Nocardia that are virulent in cats.

Paracoccidioidomycosis in a dog: case report of generalized lymphadenomegaly.

Paracoccidioidomycosis (PCM) is a severe systemic mycosis, endemic in Latin America and highly prevalent in Brazil, where it ranks eighth as a mortality cause among infectious and parasitic diseases in humans. The disease in animals has been little explored. It is observed that armadillos can harbor the fungus at high frequencies, although the active disease has not been well documented in this wild mammal. Dogs are susceptible to experimental infection, and the naturally acquired PCM-disease was reported only recently in a dog from Brazil. The present work reports the second case of naturally acquired PCM in a 6-year-old female dog that presented emaciation, lymphadenomegaly, and hepatosplenomegaly. Biochemical and pulmonary radiographic evaluation did not reveal any abnormalities. PCM was diagnosed by clinical findings, culturing, immunohistochemistry, and histopathology of popliteal lymph node. The fungus was recovered from popliteal lymph node, and the molecular analysis showed respective sequencing similarities of 99 and 100% for 803 nucleotides of the Gp43 gene and 592 nucleotides from the ITS-5.8S region of Paracoccidioides brasiliensis. Immunohistochemistry revealed severe lymphadenitis and presented numerous yeasts, which reacted against the gp43 antibody. Histopathology revealed a severe granulomatous lymphadenitis associated with numerous single or multiple budding yeasts. After diagnosis, the dog was successfully treated with itraconazol for 2 years. Veterinarians should be aware of the importance of considering PCM for differential diagnosis, especially in dogs from PCM-endemic areas, whose monophagocytic system involvement is evident.

Virulence genes and plasmid profiles in Rhodococcus equi isolates from domestic pigs and wild boars (Sus scrofa) in Brazil.

The virulence genes and plasmid profiles of 23 Rhodococcus equi isolates from 258 lymph nodes from domestic pigs (129 nodes with lesions and 129 without lesions) and 120 lymph nodes from slaughtered wild boars (60 nodes with lesions and 60 without) were characterized. R. equi was obtained from 19 lymph nodes of domestic pigs, 17 with, and two without lesions, and from four lymph nodes with lesions, from wild boars. The 23 isolates were tested for the presence of vapA and vapB genes, responsible for the 15-17 and 20 kDa virulence-associated proteins, respectively, by PCR in order to characterize as virulent (VapA), intermediately virulent (VapB) and avirulent. Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases to estimate size and compare their polymorphisms. Of the 19 domestic pigs strains, seven (36.8%) were avirulent and 12 (63.2%) were intermediately virulent, with the intermediately virulent isolates being plasmid types 8 (8 isolates), 10 (2 isolates), 1 (1 isolate) and 29 (1 isolate). The plasmid type of four strains isolated from wild boars was also intermediately virulent type 8. None of the domestic pigs and wild boar isolates showed the vapA gene. These findings demonstrate a high occurrence of plasmid type 8 in isolates from pigs and wild boars, and the similarity of plasmid types in the domestic pigs, wild boars and human isolates in Brazil.

Cryptococcus neoformans Isolated from Passerine and Psittacine bird excreta in the state of Paraná, Brazil.

Cryptococcus neoformans is an opportunistic basidiomycete yeast that causes life-threatening infections as meningoencephalitis primarily in immunocompromised hosts, generally associated with AIDS. The source of this organism is mainly pigeon excreta; however, other avian species' excreta are implicated as a source of this yeast. The occurrence of C. neoformans and Cryptococcus gattii in bird excreta in the state of Paraná in Brazil was determined in this study. A total of 141 samples of Passerine and Psittacine excreta from captive birds were collected. Additionally, 25 clinical samples from Hospital de Clínicas, in the state of Paraná were also analyzed. The determination of molecular and mating type of the isolates was performed by PCR fingerprinting, multiplex PCR, and mating type PCR. Cryptococcus neoformans var. grubii (VNI) was isolated from 36 (25.53%) of Passerine and Psittacine excreta samples. Almost all clinical samples, except one (C. gattii VGI), were classified as C. neoformans var. grubii (VNI). All environmental and clinical isolates were mating type alpha. These findings reinforce that, besides pigeon excreta, the excreta of these birds can also be a reservoir of C. neoformans in domestic and public environments and is of zoonotic importance to immunocompromised patients.