Identification of Escherichia coli isolated from flies (Insecta: Diptera) that inhabit the environment of dairy farms harboring extraintestinal virulence markers.

AIMS: We investigate extraintestinal pathogenic genes (ExPEC) related to virulence of Escherichia coli in flies from the dairy environment. METHODS AND RESULTS: We collected 217 flies from nine dairy farms, which were submitted to microbiological culture. Fifty-one E. coli were identified using mass spectrometry. Eleven dipteran families were identified, with a predominance of Muscidae, and a minor frequency of Tachinidae, Drosophilidae, Sphaeroceridae, Ulidiidae, Syrphidae, Chloropidae, Calliphoridae, Sarcophagidae, and Piophilidae. A panel of 16 virulence-encoding genes related to ExPEC infections were investigated, which revealed predominance of serum resistance (traT, 31/51 = 60.8%; ompT, 29/51 = 56.9%), iron uptake (irp2, 17/51 = 33.3%, iucD 11/51 = 21.6%), and adhesins (papC, 6/51 = 11.8%; papA, 5/51 = 9.8%). CONCLUSIONS: Our findings reveal Dipterans from milking environment carrying ExPEC virulence-encoding genes also identified in clinical bovine E. coli-induced infections.

Microbial Complexity of Oral Cavity of Healthy Dogs Identified by Mass Spectrometry and Next-Generation Sequencing.

The high complexity of the oral microbiota of healthy dogs and the close exposure of humans to companion animals represent a risk of the transmission of potential zoonotic microorganisms to humans, especially through dog bites, including multidrug-resistant ones. Nonetheless, a limited number of comprehensive studies have focused on the diversity of the microorganisms that inhabit the oral cavities of healthy dogs, particularly based on modern molecular techniques. We investigated bacterial and fungal organisms in the oral cavities of 100 healthy dogs based on a combination of conventional and selective microbiological culture, mass spectrometry (MALDI-TOF MS), and next-generation sequencing. In addition, in vitro antimicrobial susceptibility patterns of isolates and mecA resistance gene were assessed. A total of 213 bacteria and 20 fungi were isolated. Staphylococcus pseudintermedius (40/100 = 40%), α-hemolytic Streptococcus (37/100 = 37%), and Pasteurella stomatis (22/100 = 22%) were the most prevalent bacteria diagnosed by microbiological culture and MALDI-TOF MS, whereas Aspergillus (10/100 = 10%) was the most common fungi identified. Based on next-generation sequencing of selected 20 sampled dogs, Porphyromonas (32.5%), Moraxella (16.3%), Fusobacterium (12.8%), Conchiformibius (9.5%), Bergeyella (5%), Campylobacter (3.8%), and Capnocytophaga (3.4%) genera were prevalent. A high multidrug resistance rate was observed in Staphylococcus pseudintermedius isolates, particularly to azithromycin (19/19 = 100%), penicillin (15/19 = 78.9%), and sulfamethoxazole/trimethoprim (15/19 = 78.9%). In addition, the mecA resistance gene was detected in 6.1% (3/49) of coagulase-positive staphylococci. Here, we highlight the microbial complexity of the oral mucosa of healthy dogs, including potential zoonotic microorganisms and multidrug-resistant bacteria, contributing with the investigation of the microbiota and antimicrobial resistance patterns of the microorganisms that inhabit the oral cavity of healthy dogs.

Characterization of mammary pathogenic Escherichia coli reveals the diversity of Escherichia coli isolates associated with bovine clinical mastitis in Brazil.

Mammary pathogenic Escherichia coli (MPEC) is one of the most common pathogens associated with clinical mastitis. We analyzed isolates obtained from milk samples of cows with clinical mastitis, collected from 10 farms in Brazil, to verify molecular and phenotypic characteristics. A total of 192 (4.5%) mammary pathogenic E. coli isolates were obtained from 4,275 milk samples analyzed, but we tested 161. We assigned most of these isolates to E. coli phylogroups B1 (52.8%) and A (36.6%), although phylogroups B2, C, D, E, and unknown also occurred. All isolates were assessed for the presence of several genes encoding virulence factors, such as adhesins (sfaDE, papC, afaBC III, ecpA, fimH, papA, and iha), toxins (hlyA, cnf1, sat, vat, and cdt), siderophores (iroN, irp2, iucD, ireA, and sitA), an invasion protein (ibeA), and serum resistance proteins (traT, KpsMTII, and ompT), and isolates from phylogroups B1, B2, and E showed up to 8 genes. Two isolates harbored the locus of enterocyte effacement (escN+) and lack the bundle-forming pilus (bfpB-) operon, which corresponds to a molecular profile of a subgroup of diarrheagenic E. coli (aEPEC), thus being classified as hybrid MPEC/aEPEC isolates. These isolates displayed a localized adherence-like pattern of adherence in HeLa cells and were able to promote F-actin polymerization underneath adherent bacteria. Based on the pulsed-field gel electrophoresis analyses, considerable genetic variability was observed. A low index of antimicrobial resistance was observed and 2 extended-spectrum ß-lactamase-producing E. coli were identified, both harboring blaCTX-M15 gene, and were classified as ST10 and ST993 using multilocus sequence typing. A total of 148 (91.2%) isolates were weak biofilm producers or formed no biofilm. Because raw milk is still frequently consumed in Brazil, the occurrence of virulence factor-encoding genes from extraintestinal or diarrheagenic E. coli added to the presence of extended-spectrum ß-lactamase-producing isolates can turn this veterinary medicine problem into a public health concern.

Virulence-encoding genes related to extraintestinal pathogenic E. coli and multidrug resistant pattern of strains isolated from neonatal calves with different severity scores of umbilical infections.

Umbilical infections in calves comprise a major cause of neonatal mortality and have been related to a variety of microorganisms. E. coli is an opportunistic enteropathogen characterized by a diversity of virulence factors (VF). Nonetheless, the gene profiles that encode VF associated with umbilical infections in calves and their effect on the clinical severity remains unclear. In this scenario, microbial identification (with an emphasis on E. coli), was carried out among 150 neonatal calves (≤30 days of age) with umbilical infections, where the omphalopathies were clinically scored as mild, moderate, or severe. Also, a panel of 16 virulence-encoding genes related to extraintestinal pathogenic E. coli (ExPEC) were investigated, i.e., fimbriae/adhesins (sfa/focDEa, papA, papC, afaBC), toxins (hlyA, sat, cnf1, cdt), siderophores (iroN, irp2, iucD, ireA), invasins (ibeA), and serum resistance (ompT, traT, kpsMT II). Bacteria and yeasts isolates were identified using mass spectrometry. Bacteria, yeasts, and fungi were isolated in 94.7% (142/150) of neonatal calves sampled. E. coli was the agent most frequently isolated (59/150 = 39.3%), in pure culture (27/59 = 45.8%) and combined infections (32/59 = 54.2%), although a great variety (n = 83) of other species of microorganisms were identified. Clinical severity scores of 1, 2, and 3 were observed in 32.2% (19/59), 23.7% (14/59), and 44.1% (26/59) of E. coli infections, respectively. The ExPEC genes detected were related to serum resistance (traT, 42/59 = 72.2%; ompT, 35/59 = 59.3%, kpsMTII, 10/59 = 17%), invasins (ibeA, 11/59 = 18.6%), siderophores (iucD, 9/59 = 15.3%; iroN, 8/59 = 13.6%), and adhesins/fimbriae (papA, 8/59 = 13.6%; papC, 15/59 = 9.6%). The presence of each virulence gene was not associated with the case's clinical score. Among all isolates, 89.8% (53/59) showed in vitro resistance to sulfamethoxazole/trimethoprim and 59.3% to ampicillin (35/59), while 94.1% (55/59) revealed a multidrug resistant profile. Great complexity of bacteria, yeast, and fungi species was identified, reinforcing the umbilical infections of neonatal calves as a polymicrobial disorder. The high occurrence of E. coli (39.3%) highlights the role of this pathogen in the etiology of umbilical infections in calves. Furthermore, a panel of ExPEC genes was investigated for the first time among calves that were clinically scored for case severity. The high prevalence of traT and ompT indicates that these serum resistance-related genes could be used as biomarkers for further investigations of ExPEC isolates from umbilical infections. Our results contribute to the etiological investigation, clinical severity scoring, antimicrobial resistance pattern, and virulence-related to ExPEC genes involved in umbilical infections of neonatal calves.

Genetic and Antimicrobial Resistance Profiles of Mammary Pathogenic E. coli (MPEC) Isolates from Bovine Clinical Mastitis.

Mammary pathogenic E. coli (MPEC) is one of the main pathogens of environmental origin responsible for causing clinical mastitis worldwide. Even though E. coli are strongly associated with transient or persistent mastitis and the economic impacts of this disease, the virulence factors involved in the pathogenesis of MPEC remain unknown. Our aim was to characterize 110 MPEC isolates obtained from the milk of cows with clinical mastitis, regarding the virulence factor-encoding genes present, adherence patterns on HeLa cells, and antimicrobial resistance profile. The MPEC isolates were classified mainly in phylogroups A (50.9%) and B1 (38.2%). None of the isolates harbored genes used for diarrheagenic E. coli classification, but 26 (23.6%) and 4 (3.6%) isolates produced the aggregative or diffuse adherence pattern, respectively. Among the 22 genes investigated, encoding virulence factors associated with extraintestinal pathogenic E. coli pathogenesis, fimH (93.6%) was the most frequent, followed by traT (77.3%) and ompT (68.2%). Pulsed-field gel electrophoresis analysis revealed six pulse-types with isolates obtained over time, thus indicating persistent intramammary infections. The genes encoding beta-lactamases detected were as follows: blaTEM (35/31.8%); blaCTX-M-2/blaCTX-M-8 (2/1.8%); blaCTX-M-15 and blaCMY-2 (1/0.9%); five isolates were classified as extended spectrum beta-lactamase (ESBL) producers. As far as we know, papA, shf, ireA, sat and blaCTX-M-8 were detected for the first time in MPEC. In summary, the genetic profile of the MPEC studied was highly heterogeneous, making it impossible to establish a common genetic profile useful for molecular MPEC classification. Moreover, the detection of ESBL-producing isolates is a serious public health concern.

MALDI-TOF MS identification of Prototheca algae associated with bovine mastitis.

We evaluated the use of MALDI-TOF MS for the identification of 3 major, dairy-associated Prototheca species, namely, Prototheca bovis (formerly P. zopfii genotype 2), P. blaschkeae, and P. ciferrii (formerly P. zopfii genotype 1). The MALDI-TOF MS spectra established for those species were introduced into the reference spectra library of the Bruker Biotyper MALDI-TOF MS analysis software. Next, 31 Prototheca isolates from Holstein cows with mastitis, from herds located in the midwestern area of São Paulo State, Brazil, were subjected to MALDI-TOF MS profiling. MALDI-TOF MS allowed identification of 22 of 27 P. bovis and 3 of 4 P. blaschkeae isolates with scores >2.0, with 5 of 27 P. bovis and 1 of 4 P. blaschkeae isolates identified only to the genus level. With our extended algae database, MALDI-TOF MS can contribute to quick and effective speciation of Prototheca from mastitis cases.

Plasma and Peritoneal Ceftriaxone Concentrations After Intraperitoneal Administration in Horses With Septic Peritonitis.

Intraperitoneal ceftriaxone administration in healthy horses results in high and prolonged peritoneal concentrations. Recent findings suggest that intraperitoneal ceftriaxone might increase survival rates in horses affected by peritonitis. The present study aimed to evaluate plasma and peritoneal concentrations of ceftriaxone after intraperitoneal administration in horses with septic peritonitis. Twenty-six horses presenting clinical, laboratorial, and sonographic findings compatible with the disease were included. All horses received daily intraperitoneal ceftriaxone (25 mg/kg bwt) in addition or not with other antibiotics and support therapies. High-performance liquid chromatography was used to determine plasma and peritoneal ceftriaxone concentrations before and after 12 and 24 hours of ceftriaxone administration. Mean plasma concentrations 12 and 24 hours after administration were, respectively, 1.84 ± 0.43 and 0.37 ± 0.07 µg/mL, and mean peritoneal concentrations were 5.7 ± 2.84 and 0.42 ± 0.13 µg/mL. Ceftriaxone concentration was lower in comparison with previous studies in healthy horses and presented under the minimal inhibitory concentration for enterobacteria (≤1 µg/mL) and for gram-positive isolates (≤0.5 µg/mL) at 24 hours. The variation of the results obtained between healthy horses and with septic peritonitis demonstrated that pharmacokinetics/dynamics are different between these patients and suggests the use of an interval of dose of 12 hours.

Short communication: Investigation of extra-intestinal pathogenic Escherichia coli virulence genes, bacterial motility, and multidrug resistance pattern of strains isolated from dairy cows with different severity scores of clinical mastitis.

Escherichia coli is a major pathogen involved in the etiology of environmentally derived bovine mastitis and is characterized by a variety of virulence factors (VF). Mammary infections with E. coli have shown a wide range of clinical signs, causing changes in milk (score 1, or mild), abnormal appearance of milk and udder inflammation (score 2, or moderate), and abnormalities in milk, udder inflammation, and systemic signs of illness (score 3, or severe). Nevertheless, to date, the profile of the genes related to the virulence of the pathogen in mammary infections and the severity scores of cases have not been thoroughly elucidated. Therefore, a panel of 18 virulence-encoding genes associated with extra-enteric pathogenicity of E. coli (ExPEC) were investigated in addition to in vitro swimming and swarming motility profiles and antimicrobial susceptibility/resistance patterns among 114 E. coli strains isolated from cows with clinical mastitis and different severity scores. Of 114 clinical cases, 39.5, 54.4, and 6.1% were mild, moderate, and severe, respectively. The main genes related to VF harbored by isolates were adhesins (fimH 100%; ecpA 64.0%, fimA 31.6%), serum resistance (traT 81.6%; ompT 35.1%), siderophores (irp2 9.6%), and hemolysin (hlyA 7%). Among the isolates studied, 99.1% showed in vitro resistance to bacitracin and cloxacillin, and 98.2% to lincosamin. Of the total isolates, 98.2% were considered multidrug resistant based on the multiple antimicrobial resistance index. No significant difference was observed between mean swimming (13.8 mm) and swarming (13.5 mm) motility, as well as severity scores of clinical mastitis and the ExPEC genes studied. The isolation of strains resistant to various antimicrobials, even though tested only in vitro, highlights the importance of rational use of antimicrobials for mastitis treatment. The high prevalence of the genes related to serum resistance (traT and ompT) and adhesion (ecpA) of the pathogen, in addition to main associations between the genes fimH, ecpA, and traT among cows with severity scores of 1 (15%) and 2 (22.6%), indicates that the genes traT, ecpA, and ompT could be further studied as biomarkers of ExPEC for clinical intramammary infections. In addition, the ExPEC genes ompT (protectin), ibe10 (invasin), and ecpA (adhesin) were investigated for the first time among cows with mastitis, where scores of clinical severity were assessed. Results of this study contribute to the characterization of virulence mechanisms and antimicrobial resistance profile of ExPEC variants that affect dairy cows with different scores of clinical mastitis.

Comparative genomics of Staphylococcus aureus associated with subclinical and clinical bovine mastitis.

Many efforts have been made to understand the pathogenesis of bovine mastitis to reduce losses and promote animal welfare. Staphylococcus aureus may cause bovine clinical mastitis, but it is mainly associated with subclinical infection, which is usually persistent and can easily reoccur. Here, we conducted a comparative genomic analysis between strains of S. aureus causing subclinical infection (Sau170, 302, 1269, 1364), previously sequenced by our group, and two well-characterized strains causing clinical mastitis (N305 and RF122) to find differences that could be linked to mastitis outcome. A total of 146 virulence-associated genes were compared and no appreciable differences were found between the bacteria. However, several nonsynonymous single nucleotide polymorphisms (SNPs) were identified in genes present in the subclinical strains when compared to RF122 and N305, especially in genes encoding host immune evasion and surface proteins. The secreted and surface proteins predicted by in silico tools were compared through multidimensional scaling analysis (MDS), revealing a high degree of similarity among the strains. The comparison of orthologous genes by OrthoMCL identified a membrane transporter and a lipoprotein as exclusive of bacteria belonging to the subclinical and clinical groups, respectively. No hit was found in RF122 and N305 for the membrane transporter using BLAST algorithm. For the lipoprotein, sequences of Sau170, 302, 1269, and 1364 with identities between 68-73% were found in the MDS dataset. A conserved region found only in the lipoprotein genes of RF122 and N305 was used for primer design. Although the polymerase chain reaction (PCR) on field isolates of S. aureus did not validate the findings for the transporter, the lipoprotein was able to separate the clinical from the subclinical isolates. These results show that sequence variation among bovine S. aureus, and not only the presence/absence of virulence factors, is an important aspect to consider when comparing isolates causing different mastitis outcomes.

Rhodococcus equi pVAPN type causing pneumonia in a dog coinfected with canine morbillivirus (distemper virus) and Toxoplasma gondii.

Canine morbillivirus (previously, canine distemper virus, CDV) is a highly contagious infectious disease-causing agent that produces immunosuppressive infections and multiple clinical signs. Canine toxoplasmosis is an opportunistic disease characterized by enteric, pulmonary, and neuromuscular signs that might be confused with CDV-induced infections. Rhodococcus equi is a Gram-positive intracellular facultative bacterium that is also opportunistic in nature, and causes pyogranulomatous infections in humans and multiple host animals, although canine rhodococcosis is rare or unrecognized. The pathogenicity of R. equi is intimately related to the presence of plasmid-encoded virulence-associated proteins (Vap). Three host-adapted virulence plasmid types of R. equi have been recognized: the circular pVAPA and pVAPB are associated with equine and porcine strains, respectively, and the recently detected linear pVAPN virulence plasmid is related to bovine isolates. Nevertheless, data regarding the detection of host-adapted virulence plasmid types of R. equi isolated from companion animals are scarce. This report describes a case of an uncommon coinfection due to R. equi, T. gondii and CDV, which was diagnosed in a pet dog with respiratory distress. In this case, CDV most likely induced immunosuppression, which facilitated opportunistic infections by R. equi and T. gondii. The analysis of the virulence profile of R. equi revealed the novel pVAPN plasmid type, initially related to bovine strains. This is the second report of the bovine-associated pVAPN type in a pet dog, with an unusual coinfection with T. gondii and CDV. These findings represent a public health concern due to the close contact between pet animals and their owners, particularly because the pVAPN plasmid type was recently detected in people with HIV/AIDS from the same geographical region.

Tetanus in horses: an overview of 70 cases / Tétano em equinos: um panorama de 70 casos

Tetanus is characterized by high case fatality rates in horses. Comprehensive case series studies involving equine tetanus from different geographic areas enable the evaluation of prognosis, efficacy of treatment, and control measures. We retrospectively investigated some selected epidemiological data (breed, age, gender, use of the horses, history of vaccination, seasonality, presence of wound/history of surgical procedures, clinical outcomes) and main clinical aspects (clinical signs, incubation period, length of hospitalization, and period between onset signs and hospitalization) in 70 cases of equine tetanus over 1990-2015, with emphasis in the association between these data and the clinical outcomes. High mortality rate (72.9%) was observed in this study. Forty (57.1%) horses presented history of wounds or surgical procedures related with tetanus, represented mainly by lesions in the hind limbs (42.5%), front limbs (15.0%), umbilical infections (7.1%), castration (4.3%), and face wounds (4.3%). Hyperesthesia, limb spasticity, cervical stiffness, tetanic spasms, and restriction of jaw movement were the main consistent clinical signs. Besides no statistical association, all the horses with umbilical infections, wounds in face, prolonged recumbency, sweating, dysphagia/aphagia died, and together with delay between onset of first clinical signs and prompt veterinary assistance (< 5 days) were considered indicative of poor prognosis; whereas there was a significant association (p=0.001) between survival and length of hospitalization > 7 days, seemed as an evidence of good prognosis. The high mortality rate of tetanus, even in horses under specific treatment, highlight the need for early diagnosis, prompt veterinary assistance, and establishment of prophylatic measures in equine farms.(AU)

Tétano é caracterizado por elevada mortalidade em equinos. Estudos envolvendo séries de casos da doença provenientes de diferentes áreas geográficas possibilitam avaliar o prognóstico, eficácia do tratamento e ações de controle. Foram avaliados, retrospectivamente, os principais achados epidemiológicos (raça, idade, sexo, uso dos equinos, história de vacinação, sazonalidade, presença de lesões/histórico de procedimentos cirúrgicos e evolução dos casos) e aspectos clínicos (sinais clínicos, período de incubação, tempo de hospitalização e período entre início dos sinais e hospitalização) em 70 casos de tétano em equinos atendidos entre 1990 e 2015, com ênfase na associação entre os dados clínico-epidemiológicos e a evolução dos casos (mortalidade). Foi observada alta mortalidade (72,9%) nos equinos. Do total de equinos, 57,1% apresentavam lesões ou histórico de procedimentos cirúrgicos relacionados com tétano, representados principalmente por lesões em membros posteriores (42,5%), membros anteriores (15,0%), infecções umbilicais (7,1%), castração (4,3%) e lesões de face (4,3%). Hiperestesia, espasticidade dos membros, rigidez de pescoço, espasmos tetânicos e restrição aos movimentos de mandíbula foram os principais sinais clínicos observados. Apesar da ausência de significância estatística, todos os animais com presença de infecções umbilicais, lesões na face, decúbito prolongado, sudorese e disfagia/afagia evoluíram para óbito e, associado ao início dos sinais clínicos e a demora no atendimento (< 5 dias), foram considerados indicativos de prognóstico reservado. Em contraste, foi observada associação significante (p=0,001) entre a sobrevivência dos animais e o tempo de hospitalização maior que 7 dias, considerado uma indicação de bom prognóstico. A elevada mortalidade do tétano, mesmo em equinos tratados, enfatiza a necessidade do diagnóstico precoce, do rápido atendimento veterinário e adoção de medidas profiláticas nos criatórios.(AU)

Frequency, serotyping and antimicrobial resistance pattern of Salmonella from feces and lymph nodes of pigs / Isolamento, sorotipagem e padrões de resistência a antimicrobianos de Salmonella em fezes e linfonodos de suínos

Salmonellosis is a foodborne disease caused by bacteria of the genus Salmonella, being pigs and pork-products potentially important for its occurrence. In recent decades, some serovars of Salmonella have shown increase of resistance to conventional antimicrobials used in human and animal therapy, with serious risks for public health. The aim of this study was to evaluate feces (n=50), mediastinal (n=50), mesenteric (n=50) and mandibular (n=50) lymph nodes obtained from slaughter houses for Salmonella spp. Positive samples were serotyped and subjected to an in vitro antimicrobial susceptibility test, including the extended-spectrum beta-lactamase (ESBL) production. Salmonella species were identified in 10% (20/200) of total samples. From these, 20% (10/50) were identified in the submandibular lymph nodes, 18% (9/50) in the mesenteric lymph nodes, 2% (1/50) in feces and 0% (0/50) in the mediastinal lymph nodes. The serotypes found were Salonella Typhimurium (55%), S. enterica subsp. enterica 4,5,12: i: - (35%), S. Brandenburg and S. Derby with 5% (5% each). All strains showed resistance to at least one antimicrobial; 90% were resistant to four or more antimicrobials, and 15% were multidrug-resistant. Resistance to ciprofloxacin, tetracycline and nalidixic acid was particularly prevalent amongst the tested serovars. Here, we highlighted the impact of pigs in the epidemiological chain of salmonellosis in domestic animals and humans, as well as the high antimicrobial resistance rates of Salmonella strains, reinforcing the necessity for responsible use of antimicrobials for animals as an emergent One Health issue, and to keep these drugs for human therapy approaches.(AU)

Nas últimas décadas, o aumento de cepas circulante de Salmonella concomitantemente a resistência microbiana tem despertado a preocupação dos órgãos de Saúde Pública. Deste modo, o objetivo do presente trabalho foi pesquisar a presença de Salmonella a partir de fezes (n=50), linfonodos mediastinos (n=50), mesentéricos (n=50) e submandibular (n=50) oriundos de um abatedouro suíno. As cepas isoladas foram sorotipadas e testadas quanto a resistência antimicrobiana. A presença de Salmonella isolada foram em 10% (20/200) do total de amostras, sendo 20% dos linfonodos submandibulares, 18% dos linfonodos mesentéricos e 2% das fezes. Os sorotipos encontrados foram S. Typhimurium (55%), S. enterica subsp. enterica 4,5,12: i: - (35%), S. Brandenburg (5%) e S. Derby (5%). Todas a cepas apresentaram resistência a pelo menos um antimicrobiano testado, sendo 90% resistente pelo menos quatro antimicrobianos. Destes, 15% foram classificadas como multidrogas resistentes. Os antimicrobianos mais resistentes entre os sorovares isolados foram a ciprofloxacina, tetraciclina e o ácido nalidixico. A presença de cepas de Salmonella resistente a antimicrobianos na espécie suína tem gerado um grande impacto epidemiológico entre homem e animal, reforçando cada vez mais a necessidade do uso adequado de drogas principalmente relacionado com o tema "One Health".(AU)

Diversity of class 1 and 2 integrons detected in Escherichia coli isolates from diseased and apparently healthy dogs.

Escherichia coli is one of the major pathogens causing urinary tract infections (UTIs) and pyometra in dogs. The aims of this study were to investigate canine E. coli isolates for the presence of class 1 and 2 integrons by PCR/sequencing and to characterize these isolates and their integron-carrying plasmids. Isolates were characterized by phylotyping, XbaI-macrorestriction analysis and plasmid transfer experiments. Plasmids were analyzed by S1 nuclease-PFGE, replicon typing, conjugation and restriction analysis. Antimicrobial resistance was investigated by antimicrobial susceptibility testing and PCR/sequencing. From 158 E. coli of dogs suffering from UTIs (n=51) and pyometra (n=52) or being apparently healthy (n=55), 13 isolates harboured class 1 (n=10) or class 2 integrons (n=3). They were distributed among the phylogenetic groups A (3/13), B1 (6/13), B2 (3/13) and D (1/13). Two isolates showed indistinguishable XbaI-patterns, but differed in the remaining characteristics. Another two isolates (UTI or apparently healthy) displayed different XbaI-patterns, but harboured similar plasmids. Integrons were found on plasmids of incompatibility groups IncF, IncF-IncFIC, IncFIB-IncHI2, IncFIB-IncN, IncFIC or IncHI2 and three of them were conjugative. Resistances to aminoglycosides, sulphonamides and trimethoprim were commonly detected. Class 1 integrons carried the gene cassette arrays dfrA12-orfF-aadA28, ΔdfrA17-aadA5, dfrA29, aadA7, aadA29 or dfrA12-orfF-aadA2-cmlA1-aadA1. Class 2 integrons carried the array dfrA1-sat2-aadA30. Two extended-spectrum ß-lactamase genes (blaCTX-M-2) and one AmpC ß-lactamase gene (blaCMY-2) were also detected on plasmids. These findings indicate the potential risk of the dissemination and persistence of E. coli and/or integron-carrying plasmids in companion animals.

Evaluation of three formulations of culture media for isolation of Brucella spp. regarding their ability to inhibit the growth of contaminating organisms.

Three culture media (Brucella agar, Farrell medium, and CITA) were compared for their effectiveness in inhibiting contamination and for isolating Brucella spp. One hundred lymph nodes from pigs (n = 50) and wild boars (n = 50) with lymphadenitis were collected in slaughterhouses in the State of São Paulo and were assessed on these three selective media for Brucella spp. All of the samples were negative for Brucella spp. on the three culture media. On the agar medium, fungal (70 plates) and Gram-positive bacterial (59 plates) contaminants were observed; in the CITA medium, the absence of fungal and Gram-positive bacteria on 15 plates was observed; no bacterial or fungal growth was observed on the Farrell media. The results demonstrated that the CITA and Farrell media inhibited the growth of contaminants better than the Brucella agar.

Evaluation of ERIC-PCR as genotyping method for Corynebacterium pseudotuberculosis isolates.

The aim of this study was to evaluate the Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) as a tool for molecular typing of C. pseudotuberculosis isolates from eight different hosts in twelve countries. Ninety-nine C. pseudotuberculosis field strains, one type strain (ATCC 19410T) and one vaccine strain (1002) were fingerprinted using the ERIC-1R and ERIC-2 primers, and the ERIC-1R+ERIC-2 primer pair. Twenty-nine different genotypes were generated by ERIC 1-PCR, 28 by ERIC 2-PCR and 35 by ERIC 1+2-PCR. The discriminatory index calculated for ERIC 1, ERIC 2, and ERIC 1+2-PCR was 0.89, 0.86, and 0.92, respectively. Epidemiological concordance was established for all ERIC-PCR assays. ERIC 1+2-PCR was defined as the best method based on suitability of the amplification patterns and discriminatory index. Minimal spanning tree for ERIC 1+2-PCR revealed three major clonal complexes and clustering around nitrate-positive (biovar Equi) and nitrate-negative (biovar Ovis) strains. Therefore, ERIC 1+2-PCR proved to be the best technique evaluated in this study for genotyping C. pseudotuberculosis strains, due to its usefulness for molecular epidemiology investigations.

Qualidade do leite de vacas criadas no sistema silvipastoril no Vale do Cauca, Colômbia / Milk quality indicators from cows bred on silvopastoral system in Cauca Valley, Colombia

O sistema silvipastoril caracteriza-se por aumentar a produção de leite, com maior número de vacas por hectare devido ao maior aporte de proteína na dieta. Neste sistema as vacas são alimentadas, além do pasto, de pequenas árvores e arbustos. O objetivo do presente estudo foi determinar os principais indicadores de qualidade do leite e agentes causais de mastite em vacas criadas em sistema silvipastoril. Foram avaliadas a composição (teor de gordura, proteína total, lactose, extrato seco, extrato seco desengordurado e nitrogênio uréico), contagem de células somáticas (CCS), contagem bacteriana total (CBT), ocorrência de mastite clínica e subclínica, isolamento microbiológico, perfil de sensibilidade bacteriana "in vitro" e detecção de resíduos antimicrobianos no leite produzido por 100 vacas, bem como do tanque de expansão e latões em propriedades do Vale do Cauca, Colômbia. Os teores médios dos principais constituintes do leite foram 3,24% de gordura, 3,27% de proteína total, 4,40% de lactose, 10,62% de extrato seco, 8,57% de extrato seco desengordurado e 15,82mg/dL de nitrogênio uréico, enquanto do tanque de expansão e latões foi 3,51% de gordura, 3,20% de proteína total, 4,34% de lactose, 11,72% de extrato seco, 8,47% de extrato seco desengordurado e 14,57mg/dL de nitrogênio uréico. A celularidade média dos quartos mamários e do tanque de expansão foi 141.252,75 CS/mL e 363.078,05 CS/mL respectivamente. A CBT média dos quartos mamários e do tanque de expansão foi 4.466,84 UFC/mL e 24.547,01 UFC/mL. Os principais micro-organismos isolados dos quartos mamários foram Corynebacterium bovis, Staphylococcus aureus, Staphylococcus hyicus, Staphylococcus spp., Streptococcus dysgalatiae, enquanto do tanque de expansão foram identificados Streptococcus spp., Enterobacter cloacae, Hafnia alveii, Streptococcus hemolítico e Streptococcus spp., com maior frequência. A presença de resíduos de antimicrobianos em leite de vacas e do tanque ou latão foi detectada em 30% e 86% das amostras, respectivamente. O sistema silvipastoril mostrou ser uma boa alternativa para produção de leite em vacas. No entanto, são necessários cuidados no tratamento mamário para evitar resíduos no leite e a análise de todos os parâmetros de qualidade para garantir um produto diferenciado.

The silvopastoral system is characterized by increasing the production of milk with a greater number of cows per hectare due to the higher amount of protein in the diet. In silvopastoral system cows are fed in addition to pasture, small trees and shrubs. The aim of this study was determinate the main indicators of milk quality and mastitis causal agents in cows bred on silvopastoral system. We evaluated the composition (fat, total protein, lactose, solids, dry extract, nonfat dry and urea nitrogen), somatic cell count (SCC), total bacterial count (TBC), occurrence of clinical and subclinical mastitis, microbiological isolation, in vitro antimicrobial sensitivity profile and detection of antimicrobial residues in milk produced by 100 cows raised in silvopastoral systems, as well as the bulk tank and churns in farms of Cauca Valley, Colombia. The concentration of the major constituents of milk were 3.24% fat, 3.27% total protein, 4.40% lactose, 10.62% dry extract, 8.57% nonfat dry and 15.82mg/dL urea nitrogen, while the bulk tank and churns was 3.51% fat, 3.20% total protein, 4.34% lactose, 11.72% dry extract, 8.47% nonfat dry and 14.57mg/dL urea nitrogen. The cell count of the cows and the bulk tank was 141,252.75 CS/mL and 363,078.05 CS/mL respectively. The TBC mean in cows and the bulk tank was 4,466.84 CFU/mL and 24,547.01 CFU/mL respectively. The main microorganisms isolated from the udder cows were Corynebacterium bovis, Staphylococcus aureus, Staphylococcus hyicus, Staphylococcus spp., Streptococcus dysgalatiae, while the bulk tank were identified more often Streptococcus spp., Enterobacter cloacae, Hafnia alveii, hemolytic Streptococcus and Streptococcus spp. Antimicrobial residues in cow milk and bulk or churn were detected in 30% and 86% respectively. The silvopastoral system showed to be good alternative to milk production in cow. However is important the care with antimicrobial residues in milk and the analysis of all quality parameters to ensure a differentiated product.

Molecular identification and antimicrobial susceptibility of Nocardia spp. isolated from bovine mastitis in Brazil.

Nocardia spp. infections can cause severe damage to the mammary gland due to suppurative pyogranulomatous lesions and lack of clinical cure in response to conventional antimicrobial therapy. Although Nocardia infections are considered relatively uncommon in cows, there has been an apparent worldwide increase in the incidence of bovine mastitis caused by Nocardia spp, perhaps due to environmental transmission of this ubiquitous pathogen. The objectives of present study were to determine: (i) species distribution of 80 Nocardia isolates involved in bovine mastitis (based on molecular methods); and (ii) antimicrobial susceptibility pattern of all isolates from three geographical areas in Brazil. In this study, Nocardia nova (80%) was the most frequently isolated species, followed by Nocardia farcinica (9%). Additionally, Nocardia puris, Nocardia cyriacigeorgica, Nocardia veterana, Nocardia africana, and Nocardia arthritidis were detected using 16S rRNA sequencing. This is apparently the first report of N. puris, N. veterana, N. cyriacigeorgica, N. arthritidis and N. africana in association with bovine mastitis. Based on the disk diffusion test, isolates were most frequently resistant to cloxacillin (75%), ampicillin (55%) and cefoperazone (47%), whereas few Nocardia spp. were resistant to amikacin, cefuroxime or gentamicin.

Citologia aspirativa no diagnóstico da linfadenite em ovinos / Fine needle aspiration in diagnosis of lymphadenitis in sheep

O presente estudo investigou o uso da cito-(73,0 por cento), Arcanobacterium pyogenes (6,0 por cento), Streptococlogia aspirativa com agulha fina no diagnóstico da lin-cus spp. β hemolítico (5,0 por cento) e Escherichia coli (4,0 por cento) fadenite em ovinos e a ocorrência de microrganismos foram os microrganismos mais frequentes nos animais nos linfonodos com lesões, com ênfase no isolamento de com linfadenite. Streptococcus spp. (21,0 por cento) e Staphylo-Corynebacterium pseudotuberculosis. Foram utilizados coccus spp. (7,0 por cento) foram as bactérias isoladas com 100 linfonodos de ovinos com aumento de volume su-maior frequência nos linfonodos sem lesões colhidos em gestivos de linfadenite e 100 linfonodos de ovinos sem abatedouro. A punção aspirativa com agulha fina perlesões, colhidos em abatedouro. C. pseudotuberculosis mitiu identificar microrganismos "corineformes" em 79 (79,0 por cento) animais com linfadenite e, destes, 73 (73,0 por cento) foram identificados como C. pseudotuberculosis. Nenhuma linhagem de C. pseudotuberculosis foi isolada dos linfonodos dos animais sem lesões. Concluiu-se que C. pseudotuberculosis foi o microrganismo mais freqüente nos ovinos com linfadenite, e que a citologia aspirativa pode ser utilizada como método de triagem no diagnóstico da linfadenite caseosa ovina.

The fine needle aspiration in diagnosis of lymphadenitis in sheep, and occurrence of microorganisms in the lymph nodes, with emphasis to identification of Corynebacterium pseudotuberculosis were investigated. One hundred sheep with lymphadenitis and 100 lymph nodes without lesions collected from sheep in slaughterhouses were evaluated. C. pseudotuberculosis (73.0 percent), Arcanobacterium pyogenes (6.0 percent), Streptococcus spp. β hemolytic (5.0) and Escherichia coli (4.0 percent) were the more frequent causal agents in sheep with lymphadenitis. Streptococcus spp. (21.0 percent) and Staphylococcus spp. (7.0 percent) were the more common microorganisms identified in lymph nodes without lesions sampled in slaughterhouse. Fine needle aspiration enable identify 79 (79.0 percent) microorganisms with "corynebacteria" aspect in sheep with lymphadenitis. From these, 73 (73.0 percent) were identified as C. pseudotuberculosis. None strain of C. pseudotuberculosis was isolated from lymph nodes of animals without lesions. The results showed the high frequency of C. pseudotuberculosis in sheep with lymphadenitis, and possibility the use of fine needle aspiration in initial diagnosis of lymphadenitis in sheep.

Caracterização de sorotipos em linhagens do gênero Salmonella isoladas de diferentes afecções em animais domésticos / Serotype characterization of Salmonella strains isolated from different affections in domestic animals

Foram caracterizados os sorotipos, o perfil de sensibilidade microbiana e os achados clínico-epidemiológicos em 53 linhagens do gênero Salmonella isoladas de 41 cães, nove equinos e três bovinos, acometidos por diferentes manifestações clínicas entre 1997 e 2007. Salmonella Typhimurium (45,3 por cento), Salmonella enterica (22,6 por cento), Salmonella Enteritidis (7,5 por cento), Salmonella enterica subsp enterica 4,5,12i (5,7 por cento), Salmonella Newport (5,7 por cento), Salmonella Dublin (3,8 por cento), Salmonella Agona (3,8 por cento), Salmonella Glostrup (3,8 por cento), Salmonella Saintpaul (1,8 por cento) foram os sorotipos encontrados. Ciprofloxacina (100,0 por cento), norfloxacina (100,0 por cento) e gentamicina (100,0 por cento) foram os antimicrobianos mais efetivos, enquanto a maior resistência das linhagens foi observada para ceftiofur (28,5 por cento) e florfenicol (7,0 por cento). As linhagens foram isoladas de animais com enterite, infecção do trato urinário, septicemia, piometra, pneumonia e conjuntivite. Ressalta-se para o predomínio do sorovar Typhimurium nas diferentes manifestações da salmonelose nos animais. Destaca-se, também, a identificação de sorotipos nos animais que também são observados em casos de salmonelose em humanos

The serotype characterization, antimicrobial susceptibility profile, and clinical-epidemiological findings were evaluated in 53 Salmonella spp. strains isolated from 41 dogs, nine horses and three cattle presenting different clinical manifestations between 1997 at 2007. Salmonella Typhimurium (45.3 percent), Salmonella enterica (22.6 percent), Salmonella Enteritidis (7.5 percent), Salmonella enterica subsp. enterica 4,5,12i (5.7 percent), Salmonella Newport (5.7 percent), Salmonella Dublin (3.8 percent), Salmonella Agona (3.8 percent), Salmonella Glostrup (3.8 percent), Salmonella Saintpaul (1.8 percent) were the more common serotypes. Ciprofloxacin (100.0 percent), norfloxacin (100.0 percent) and gentamicin (100.0 percent) were more effective drugs while resistance of isolates was observed to ceftiofur (28.5 percent) and florfenicol (7.0 percent). The strains were isolated from animals with enteritis, urinary tract infections, septicaemia, pyometra, pneumonia and conjuntivits. The results showed the high frequency of Salmonella Typhimurium serotype in different animals studied. The study highlighted also the presence of serotypes in our animals that also have been identified in humans with salmonellosis.