Escherichia coli isolates from femoral bone marrow of broilers exhibit diverse pheno- and genotypic characteristics that do not correlate with macroscopic lesions of bacterial chondronecrosis with osteomyelitis.

The pheno- and genotypic relatedness among Escherichia coli isolates from broilers with and without macroscopic lesions of the femoral head were investigated. In total, 219 isolates obtained from the bone marrow were characterized by serotyping, antimicrobial resistance (AMR) profiles, phylogenetic grouping, detection of virulence-associated genes (VAGs) and pulsed-field gel electrophoresis (PFGE). Serotyping revealed that 48.4% of the isolates were assigned to one of the three serotypes (O78:K80: 21.0%, O2:K1: 18.7%, O1:K1: 8.7%). Substantial phenotypic variation was also noticed in AMR testing as most of the birds harboured E. coli isolates with different AMR profiles, which is of high clinical relevance. The majority of isolates could be classified into phylogenetic groups D (54.3%) and B2 (25.6%), followed by A (11.4%) and B1 (8.7%). Virulotyping showed that the highest number of isolates contained genes iucD (86.8%) and iss (84.9%), whereas papC (16.0%) and astA (12.3%) were present in least number of isolates. PFGE resulted in 58 different profiles from 200 typeable isolates. No correlation was found between specific serotypes, AMR profiles, phylogenetic groups, PFGE types or VAG profiles of E. coli and the occurrence of bacterial chondronecrosis with osteomyelitis, contradicting the hypothesis of a specific bacterial pheno- or genotype being involved in the disease.

First case report of non-human primates (Alouatta clamitans) with the hypervirulent Klebsiella pneumoniae serotype K1 strain ST 23: A possible emerging wildlife pathogen.

BACKGROUND: Hypervirulent strain of Klebsiella pneumoniae genotype K1 isolates have recently emerged, causing severe pyogenic liver abscess complicated by devastating metastatic infections in humans. METHODS: We describe a short outbreak of the non-human primate (NHP) research center, associated with a hypervirulent K. pneumoniae. The genetic similarity of the strains was evaluated by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) techniques, and virulence encoding genes were detected by polymerase chain reaction (PCR). RESULTS: The isolates were phenotypically like strains causing community-acquired invasive liver abscess syndrome in humans. All strains exhibited identical PFGE patterns and were found to belong to ST23 and presented a hypermucovisity phenotype and possessed magA and rmpA gene. CONCLUSION: This is the first case report of NHPs caused by K. pneumoniae displaying a hypermucoviscosity phenotype and belonging to capsular serotypes K1 and ST23.

Antimicrobial resistance profiles of Staphylococcus aureus clusters on small dairy farms in southern Brazil / Perfil de resistência a antimicrobianos de grupos clonais de Staphylococcus aureus isolados de pequenas propriedades leiteiras do sul do Brasil

In intensive dairy farming, persistent intramammary infection has been associated with specific Staphylococcus (S.) aureus strains, and these strains may be resistant to antimicrobials. The objective of this study was to evaluate the antimicrobial resistance phenotypes of S. aureus isolates and to assess the distribution and the persistence of clonal groups in small dairy herds of southern Brazil. Milk samples were collected from all lactating cows from 21 dairy farms over a two-year period, totaling 1,060 samples. S. aureus isolates were tested for susceptibility to thirteen antimicrobials using the disk diffusion method. The total DNA of the isolates was subjected to SmaI digestion followed by pulsed-field gel electrophoresis (PFGE). Banding patterns differing by ≤4 bands were considered members of a single PFGE cluster. The frequency of S. aureus isolation ranged from 3.45% to 70.59% among the 17 S. aureus-positive herds. Most S. aureus isolates (87.1%) were susceptible to all antimicrobials; resistance to penicillin (18.2%) was the most frequently observed. The 122 isolates subjected to macrorestriction analysis were classified into 30 PFGE-clusters. Among them, only 10 clusters were intermittent or persistent over the two-year period. The majority (93.6%) of isolates belonging to persistent and intermittent clusters were susceptible to all tested antimicrobials. S. aureus intramammary colonization in small dairy farms of southern Brazil is most frequently caused by sporadic PFGE clusters, although some persistent clusters can arise over time. Both sporadic and persistent isolates were highly susceptible to antimicrobials.(AU)

A infecção intramamária persistente em bovinos leiteiros tem sido associada com estirpes de Staphylococcus (S.) aureus específicos, os quais podem ser resistentes a antimicrobianos. Os objetivos deste estudo foram avaliar os fenótipos de resistência aos antimicrobianos de isolados de S. aureus e a distribuição e persistência de grupos clonais em pequenos rebanhos leiteiros do sul do Brasil. As amostras de leite foram coletadas de todas as vacas em lactação de 21 propriedades leiteiras, ao longo de um período de dois anos, perfazendo um total de 1.060 amostras. Isolados de S. aureus foram testados quanto à resistência frente a treze antimicrobianos, pelo método de disco-difusão. O DNA total dos isolados foi clivado com a enzima Smal e submetido a eletroforese em gel de campo pulsado (PFGE). Padrões de bandas diferentes por ≤4 bandas foram considerados como pertencentes ao mesmo grupo clonal. A freqüência de S. aureus variou de 3,45% até 70,59%, entre os 17 rebanhos com isolamento positivo de S. aureus. A maioria dos isolados de S. aureus (87,1%) foi suscetível a todos os antimicrobianos; resistência à penicilina (18,2%) foi a mais freqüentemente observada. Os 122 isolados submetidos à análise de macrorestrição foram classificados em 30 grupos clonais de PFGE. Entre eles, apenas dez grupos clonais foram intermitentes ou persistentes ao longo do período de dois anos. A maioria (93,6%) dos isolados pertencentes a grupos clonais persistentes e intermitentes foram suscetíveis a todos os antimicrobianos testados. Concluiu-se que a colonização intramamária em bovinos de pequenas propriedades leiteiras do Sul do Brasil é mais frequentemente causada por grupos clonais esporádicos de S. aureus, embora alguns grupos clonais persistentes possam ocorrer ao longo do tempo. Em ambos os grupos clonais os isolados foram majoritariamente suscetíveis a antimicrobianos.(AU)

Caracterização molecular de Listeria monocytogenes oriundas de cortes cárneos bovinos e de abatedouros frigoríficos de bovinos localizados no Distrito Federal, Brasil / Molecular characterization of Listeria monocytogenes from beef samples and cattle slaughterhouses located in the Federal District, Brazil

Este trabalho teve como objetivo realizar a detecção de cepas de Listeria monocytogenes de cortes cárneos bovinos bem como no ambiente de abatedouros frigoríficos localizados no Distrito Federal, promover a sorotipificação pela reação em cadeia da polimerase (PCR), realizar antibiograma e submeter às cepas à eletroforese de campo pulsado (Pulsed-field gel electrophoresis - PFGE). Foram analisados um total de 125 cortes cárneos bovinos, 45 amostras de swabs de carcaças e 43 amostras de swabs em que foram detectados 13 cepas de Listeria monocytogenes, sendo 11 em cortes cárneos bovinos e 2 swabs de ambiente em um abatedouro frigorifico. Não foram isoladas cepas de swabs de carcaça. Dentre as 13 cepas de Listeria monocytogenes foram encontradas seis cepas do sorotipo 4b, cinco do sorotipo 1/2c e duas cepas do sorotipo 1/2a. Dentre as 11 cepas de L. monocytogenes encontradas em cortes cárneos bovino, uma (9,1%) cepa apresentou resistência a eritromicina, outra (9,1%) cepa a gentamicina e outra a ciprofloxacina (9,1%) e todas as cepas (100%) apresentaram resistência ao Ác. Nalidíxico. Das duas (2) cepas oriundas de ralos de abatedouro frigorífico, todas (100%) apresentaram resistência ao Ác. Nalidíxico e a sulfonamidas. A análise por eletroforese de campo pulsante (PFGE) demonstrou 13 diferentes pulsotipos, em que foram agrupados em 3 diferentes grupos clonais, que coincidentemente se correlacionavam com os 3 diferentes sorotipos encontrados sugerindo uma ampla disseminação desses perfis no Distrito Federal.(AU)

The aim of the study was the analysis of Listeria monocytogenes strains in beef samples as well as slaughterhouse environment, located in the Federal District, promote serotyping by polymerase chain reaction (PCR), perform antibiotic susceptibility and submit the strains to Pulsed-field gel electrophoresis (PFGE). A total of 125 beef samples were analyzed, 45 samples of carcasses swabs and 43 swab samples. It detected 13 strains of Listeria monocytogenes, 11 in beef samples. and 2 in slaughterhouse environment. No carcass swabs strains were isolated. Among the 13 strains of L. monocytogenes six strains of serotype 4b were found, five serotype 1/2c and two strains of serotype 1/2a. Among the 11 strains of L. monocytogenes found in beef, one (9.1%) strain showed resistance to erythromycin, one (9.1%) strain to gentamicin, one to ciprofloxacin (9.1%) and all strains (100%) were resistant to nalidixic acid. The two strains coming from the slaughterhouse drains, all (100%) were resistant to nalidixic acid and Sulfonamides. The analysis by pulsed field gel electrophoresis (PFGE) showed 13 different pulsotypes; they were grouped into three different clonal groups, coincidentally correlated with the three different serotypes found, what suggests a widespread dissemination of these profiles in the Federal District, Brazil.(AU)

Acute and Chronic Erysipelothrix rhusiopathiae Infection in Lambs.

Polyarthritis caused by Erysipelothrix rhusiopathiae is a relatively common infection in lambs characterized by low mortality and high morbidity. E. rhusiopathiae is a ubiquitous Gram-positive bacterium that is both a commensal and a pathogen of vertebrates. The disease was studied during an outbreak in a Norwegian Spæl sheep flock. In the acute phase, 48 of 230 (20%) lambs developed clinical signs and 4 died (1.7%). One acute case was necropsied and E. rhusiopathiae was cultured from all major organs investigated and from joints. There was a fibrinous polyarthritis, increased presence of monocytes in vessels, and necrosis of Purkinje cells. Sixteen of the diseased animals (33%) developed a chronic polyarthritis. Eight of these lambs were necropsied; all had lesions in major limb joints, and 3 of 8 also had lesions in the atlanto-occipital joint. At this stage, E. rhusiopathiae was cultured only from the joints in 7 of 8 (87.5%) lambs, but by real-time polymerase chain reaction, we showed persistence of the bacterium in several organs. Pulsed-field gel electrophoresis typing of the bacterial isolates indicated that the same strain caused the acute and chronic disease. Five of 6 (83%) chronically affected animals had amyloidosis of the spleen, and 6 of 8 (75%) had amyloidosis of the liver. All chronically affected animals had a glomerulonephritis, and 6 of 8 (75%) had sparse degeneration in the brain. Ceruloplasmin and haptoglobin were significantly increased in the chronically diseased lambs. These results show that chronic ovine erysipelas is not restricted to joints but is a multisystemic disease.

Diagnóstico de un brote de Salmonella Typhimurium en Chinchillas (Chinchilla lanigera) mediante la electroforesis en gel de campo pulsado / Diagnosis of an autbreak of Salmonella Typhimurium in chinchillas (Chinchilla lanigera) by pulsed-fiel gel electrophoresis

Empleando estudios anatomopatológicos y microbiológicos se examinó a un grupo de chinchillas (Chinchilla lanigera) adultas que murieron súbitamente en 2012 en una granja de la ciudad de La Plata (Buenos Aires, Argentina). Se aisló Salmonella enterica serovar Typhimurium (S. Typhimurium) del hígado, el bazo, el corazón, los pulmones, los riñones y los intestinos de los cinco animales evaluados. Los cinco aislamientos estudiados (uno por animal) fueron sensibles a ampicilina, cefalotina, cefotaxima, ácido nalidíxico, gentamicina, estreptomicina, cloranfenicol, fosfomicina, nitrofurantoína y trimetoprima-sulfametoxazol, y resistentes a tetraciclina. El análisis de dichos aislamientos por electroforesis en gel de campo pulsado [pulsed-field gel electrophoresis (PFGE)] con XbaI mostró un perfil electroforético idéntico con 15 bandas, idéntico a su vez al patrón ARJPXX01.0220 del banco nacional argentino de datos de PulseNet, que cuenta con patrones de PFGE de Salmonella. El presente trabajo describe por primera vez el diagnóstico postmortem de un brote de salmonelosis en chinchillas usando un método molecular, como la electroforesis en gel en campo pulsado

Adult chinchillas (Chinchilla lanigera) that had suddenly died in a commercial farm located in La Plata City, Buenos Aires Province, Argentina, in July 2012 were macroscopically, histopathologically, and microbiologically examined. Salmonella enterica serovar Typhimurium (S. Typhimurium) was isolated from the liver, spleen, heart, lungs, kidneys and intestines from each of the five animals evaluated. The five strains were susceptible to ampicillin, cephalotin, cefotaxime, nalidixic acid, gentamicin, streptomycin, chloramphenicol, fosfomycin, nitrofurantoin and trimethoprimsulfamethoxazole, and resistant to tetracycline. Each of the five S. Typhimurium isolates was analyzed by XbaI- pulsed-field gel electrophoresis (PFGE), showing an identical electrophoretic profile with 15 defined bands, which was found to be identical to pattern ARJPXX01.0220 of the PulseNet Argentine National database of Salmonella PFGE patterns. This is the first work describing the postmortem diagnosis of an outbreak of salmonellosis in chinchillas by using molecular methods such as PFGE

Molecular epidemiology of environmental MRSA at an equine teaching hospital: introduction, circulation and maintenance.

The role that environmental contamination might play as a reservoir and a possible source of Methicillin-resistant Staphylococcus aureus (MRSA) for patients and personnel at equine veterinary hospitals remains undefined, as the environment has only been monitored during outbreaks or for short periods. Therefore, the objectives of this study were to determine the monthly presence, distribution, and characteristics of environmental MRSA at an equine hospital, and to establish patterns of contamination over time using molecular epidemiological analyses. For this purpose, a yearlong active MRSA surveillance was performed targeting the environment and incoming patients. Antimicrobial susceptibility testing, SCCmec typing, PFGE typing, and dendrographic analysis were used to characterize and analyze these isolates. Overall, 8.6% of the surfaces and 5.8% of the horses sampled were positive for MRSA. The most common contaminated surfaces were: computers, feed-water buckets, and surgery tables-mats. Ninety percent of the isolates carried SCCmec type IV, and 62.0% were classified as USA500. Molecular analysis showed that new pulsotypes were constantly introduced into the hospital throughout the year. However, maintenance of strains in the environment was also observed when unique clones were detected for 2 consecutive months on the same surfaces. Additionally, pulsotypes were circulating throughout several areas and different contact surfaces of the hospital. Based on these results, it is evident that MRSA is constantly introduced and frequently found in the equine hospital environment, and that some contact surfaces could act as "hot-spots". These contaminated surfaces should be actively targeted for strict cleaning and disinfection as well as regular monitoring.

In vivo spread of macrolide-lincosamide-streptogramin B (MLSB) resistance--a model study in chickens.

The influence of specific and non-specific antibiotic pressure on in vivo spread of macrolide-lincosamide-streptogramin B (MLSB) resistance was evaluated in this study. Chickens repeatedly inoculated with Enterococcus faecalis harbouring the plasmid pAMß1 carrying the erm(B) gene were perorally treated for one week with tylosin, lincomycin (both specific antibiotic pressure) and chlortetracycline (non-specific antibiotic pressure). Antibiotic non-treated but E. faecalis inoculated chickens served as a control. To quantify the erm(B) gene and characterise intestinal microflora, faecal DNA was analysed by qPCR and 454-pyrosequencing. Under the pressure of antibiotics, a significant increase in erm(B) was observed by qPCR. However, at the final stage of the experiment, an increase in erm(B) was also observed in two out of five non-treated chickens. In chickens treated with tylosin and chlortetracycline, the increase in erm(B) was accompanied by an increase in enterococci. However, E. faecalis was at the limit of detection in all animals. This suggests that the erm(B) gene spread among the gut microbiota other than E. faecalis. Pyrosequencing results indicated that, depending on the particular antibiotic pressure, different bacteria could be responsible for the spread of MLSB resistance. Different species of MLSB-resistant enterococci and streptococci were isolated from cloacal swabs during and after the treatment. PFGE analysis of MLSB-resistant enterococci revealed four clones, all differing from the challenge strain. All of the MLSB-resistant isolates harboured a plasmid of the same size as pAMß1. This study has shown that MLSB resistance may spread within the gut microbiota under specific and non-specific pressure and even in the absence of any antimicrobial pressure. Finally, depending on the particular antibiotic pressure, different bacterial species seems to be involved in the spread of MLSB resistance.

Yersiniosis in Atlantic cod, Gadus morhua (L.), characterization of the infective strain and host reactions.

A disease outbreak in farmed Atlantic cod caused by Yersinia ruckeri is reported. Mortality started following vaccination of cod reared in two tanks (A and B). The accumulated mortality reached 1.9% in A and 4.8% in B in the following 30 days when treatment with oxytetracycline was applied. Biochemical and molecular analysis of Y. ruckeri isolates from the cod and other fish species from fresh and marine waters in Iceland revealed a high salinity-tolerant subgroup of Y. ruckeri serotype O1. Infected fish showed clinical signs comparable with those of Y. ruckeri -infected salmonids, with the exception of granuloma formations in infected cod tissues, which is a known response of cod to bacterial infections. Immunohistological examination showed Y. ruckeri antigens in the core of granulomas and the involvement of immune parameters that indicates a strong association between complement and lysozyme killing of bacteria. Experimental infection of cod with a cod isolate induced disease, and the calculated LD50 was 1.7 × 10(4) CFU per fish. The results suggest that yersiniosis can be spread between populations of freshwater and marine fish. Treatment of infected cod with antibiotic did not eliminate the infection, which can be explained by the immune response of cod producing prolonged granulomatous infection.

Molecular epidemiology and extended-spectrum Beta-lactamases production of Klebsiella pneumoniae isolated from three dairy herds / Epidemiologia molecular e produção de Beta- lac-tamases de espectro estendido de Klebsiella pneumoniae isoladas de três propriedades leiteiras

The objectives of this study were to isolate Klebsiella pneumoniae from different sources in three dairy cattle herds, to use the pulsed-field gel electrophoresis (PFGE) to measure genotypic similarities between isolates within a dairy herd, to verify the production of extended-spectrum β-lactamases (ESBLs) by the double-disk synergy test (DDST), and to use the PCR to detect the main ESBLs subgroups genes. Three dairy farms were selected based on previous mastitis outbreaks caused by K. pneumoniae. Milk samples were collected from lactating cows and from the bulk tank. Swabs were performed in different locations, including milking parlors, waiting room, soil, animal's hind limbs and rectum. K. pneumoniae was isolated from 27 cases of intramammary infections (IMI) and from 41 swabs. For farm A isolates from IMI and bulk tank were considered of the same PGFE subtype. One isolate from a bulk tank, three from IMI cases and four from environmental samples were positive in the DDST test. All eight DDST positive isolates harbored the bla shv gene, one harbored the bla tem gene, and three harbored the bla ctx-m gene, including the bulk tank isolate. Our study confirms that ESBL producing bacteria is present in different locations in dairy farms, and may be responsible for IMI. The detection of ESBLs on dairy herds could be a major concern for both public and animal health.

Os objetivos deste estudo foram isolar Klebsiella pneumoniae de diferentes localidades em três propriedades leiteiras, utilizar a eletroforese em campo pulsátil para averiguar similaridades genotípicas entre os isolados de uma mesma propriedade, verificar a produção de beta-lactamases de espectro estendido (ESBLs) pela prova da disco-difusão dupla associada (DDST) e utilizar a PCR para detecção dos principais subgrupos genéticos de ESBLs. Três propriedades leiteiras foram selecionadas baseando-se em surtos prévios de mastites causadas por K. pneumoniae. Amostras de leite foram coletadas de vacas em lactação e do tanque de expansão. Swabs foram realizados em diferentes localidades, incluindo salas de lactação, salas de espera, solo, reto e membros posteriores de animais. K. pneumoniae foi isolada de 27 casos de infecções intramamária (IMI) e de 41 swabs. Para a propriedade A os isolados de IMI e do tanque de expansão foram considerados do mesmo subtipo molecular. Um isolado do tanque de expansão, três de casos de IMI e quatro de amostras ambientais foram considerados positivos no teste da DDST. Todos os oito isolados DDST positivos portavam o gene bla shv, um portava o gene bla tem, e três portavam o gene bla ctx-m, incluindo um isolado de tanque de expansão. Nosso estudo confirma que bactérias produtoras de ESBLs estão presentes em diferentes localidades em propriedades leiteiras, e podem ser responsáveis por quadros de IMI. A detecção de ESBLs em propriedades leiteiras pode representar uma grande preocupação para saúde pública e para a saúde animal.

Strain-specific pathogenicity of putative host-adapted and nonadapted strains of Streptococcus uberis in dairy cattle.

Streptococcus uberis is an important cause of intramammary infection in dairy cattle. Strains of Strep. uberis appear to differ in their ability to cause disease based on previous epidemiological studies. We explored the pathogenicity of 2 strains of Strep. uberis, where one strain represented a putatively host-adapted type based on its ability to cause persistent infection and to spread from cow to cow in a lactating herd. This type was part of a clonal complex that is commonly associated with bovine mastitis. The other strain, which was isolated from a transient infection in a single animal in the same herd and did not belong to any known clonal complex, was selected as putatively nonadapted type. Cows (6 per strain) were experimentally challenged in a single hind quarter and the adjacent hind quarter was used as mock challenged control quarter. Both strains showed an equal ability to grow in the milk of challenge animals in vitro. All cows that were challenged with the putatively host-adapted strain developed clinical signs of mastitis, including fever and milk yield depression as well as elevated somatic cell count due to influx of polymorphonuclear leucocytes and lymphocytes. The cytokine response followed a specific order, with an increase in IL-1ß, IL-6, and IL-8 levels at the time of first SCC elevation, followed by an increase in IL-10, IL-12p40, and tumor necrosis factor-α levels approximately 6h later. In 4 of 6 animals, IL-17A was detected in milk between 57 and 168 h postchallenge. The increase in IL-17A levels coincided with inversion of the prechallenge CD4(+)-to-CD8(+) T lymphocyte ratio, which was observed from 96 h postchallenge. This was followed by normalization of the CD4(+)-to-CD8(+) ratio due to continued increase of the CD8(+) concentration up to 312 h postchallenge. Spontaneous resolution of infection was observed in 5 animals and coincided with a measurable IL-17A response in 4 animals, suggesting that IL-17 may be involved in the resolution of intramammary infection. With the exception of minor elevation of IL-8 levels, no clinical, cytological, or immunological response was detected in quarters challenged with the nonadapted strain. The observed strain-specific pathogenicity was consistent across animals, implying that it is determined by pathogen factors rather than host factors.

In vivo screening of five phytochemicals/extracts and a fungal immunomodulatory protein against colibacillosis in broilers.

Five phytochemicals/extracts (an extract from Echinacea purpurea, a ß-glucan-rich extract from Shiitake, betaine [Betain™], curcumin from Curcuma longa [turmeric] powder, carvacrol and also a recombinant fungal immunomodulatory protein [FIP] from Ganoderma lucidum) cloned and expressed in Escherichia coli were investigated for their anticolibacillosis potential in three chicken experiments, which were conducted in floor pens. Birds that were inoculated with E. coli intratracheally were treated with the phytochemicals/extracts or the FIP and compared with doxycycline-medicated and non-medicated infected broilers. Non-medicated and non-infected birds were used as negative controls. Mortality, colibacillosis lesions and body weight gains were used as parameters. Considering the sum of dead birds and chickens with generalized colibacillosis per group, there was no significant difference between the positive control groups and birds treated with phytochemicals/extracts or the FIP. In contrast, doxycycline-treated birds showed significantly lower mortality and generalized colibacillosis. Moreover, none of the phytochemicals/extracts and the FIP improved recovery from colibacillosis lesions, while all doxycycline-treated broilers recovered completely. The negative control birds and doxycycline-treated groups consistently showed the highest weight gains. Pulsed-field gel electrophoresis of reisolates showed that they were genetically indistinguishable from the inoculation strain. In conclusion, none of the tested phytochemicals/extracts and the FIP significantly reduced the E. coli-induced mortality and generalized colibacillosis, and nor did they improve recovery from colibacillosis lesions.

Molecular typing of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) isolated from animals and retail meat in North Dakota, United States.

The objective of this study was to determine the prevalence and molecular typing of methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in food-producing animals and retail meat in Fargo, North Dakota. A two-step enrichment followed by culture methods were used to isolate S. aureus from 167 nasal swabs from animals, 145 samples of retail raw meat, and 46 samples of deli meat. Positive isolates were subjected to multiplex polymerase chain reaction in order to identify the genes 16S rRNA, mecA, and Panton-Valentine Leukocidin. Pulsed-field gel electrophoresis and multilocus sequence typing were used for molecular typing of S. aureus strains. Antimicrobial susceptibility testing was carried out using the broth microdilution method. The overall prevalence of S. aureus was 37.2% (n=133), with 34.7% (n=58) of the animals positive for the organism, and the highest prevalence observed in pigs (50.0%) and sheep (40.6%) (p<0.05); 47.6% (n=69) of raw meat samples were positive, with the highest prevalence in chicken (67.6%) and pork (49.3%) (p<0.05); and 13.0% (n=6) of deli meat was positive. Five pork samples (7.0%) were positive for MRSA, of which three were ST398 and two were ST5. All exhibited penicillin resistance and four were multidrug resistant (MDR). The Panton-Valentine Leukocidin gene was not detected in any sample by multiplex polymerase chain reaction. The most common clones in sheep were ST398 and ST133, in pigs and pork both ST398 and ST9, and in chicken ST5. Most susceptible S. aureus strains were ST5 isolated from chicken. The MDR isolates were found in pigs, pork, and sheep. The presence of MRSA, MDR, and the subtype ST398 in the meat production chain and the genetic similarity between strains of porcine origin (meat and animals) suggest the possible contamination of meat during slaughtering and its potential transmission to humans.

Reproduction of the Escherichia coli peritonitis syndrome in laying hens.

In five experiments, each consisting of four or six groups with seven or 14 brown laying hens per group, birds were inoculated with an Escherichia coli strain, isolated from a layer with the E. coli peritonitis syndrome (EPS) by different routes between 23 and 33 weeks of age. Aerosol-exposed hens inhaled 10(5.1) to 10(6.2) colony-forming units per hen; hens inoculated by other routes received 10(7.6) to 10(9.1) colony-forming units per hen. In one experiment, one-half of the birds of each group were injected intraperitoneally with sterile egg yolk simultaneously with E. coli. Dead and surviving birds were necropsied and bacteriological examination of the bone marrow was performed. The percentage of birds with EPS that died was 89 (159/179). Nearly all dead birds showed septicaemia (155/159 = 97%), while most had septicaemia and peritonitis (126/159 = 79%). Surviving hens with EPS (20/179 = 11%) showed chronic peritonitis and inactive ovaries. Taking all experiments together, exposure of hens by the intravenous, intratracheal and intraperitoneal routes induced EPS in 84% (41/49), 80% (55/69) and 76% (16/21), respectively, while aerosol and intravaginal exposure resulted in EPS percentages of 57% (32/56) and 49% (28/57), respectively. Except for orally inoculated groups (7/56 = 13% EPS), in all other groups the EPS rates differed significantly (P <0.01) from those of the placebo-exposed groups (0/42). Neither the age of hens nor the presence of free yolk in the abdomen influenced the EPS rate. The results of the present study are suggestive of the respiratory and vaginal origin of EPS in the field.

Streptococcus equi subsp. zooepidemicus isolates from equine infectious endometritis belong to a distinct genetic group.

Streptococcus equi subsp. zooepidemicus is the pathogen most commonly isolated from the uterus of mares. S. zooepidemicus is an opportunistic pathogen and part of the resident flora in the caudal reproductive tract. The aim of this study was to investigate whether a genotypically distinct subpopulation of S. zooepidemicus is associated with endometritis in the mare, by genotyping and comparing uterine S. zooepidemicus strains with isolates from the vagina and clitoral fossa. Mares with (n=18) or without (n=11) clinical symptoms of endometritis were included. Uterine samples were obtained using a guarded endometrial biopsy punch, whereas a swab was used to recover samples from the cranial vagina and the clitoral fossa. If S. zooepidemicus was present, up to three colonies were selected from each anatomical location (max. 9 isolates per mare). Bacterial isolates were characterized by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). S. zooepidemicus was isolated from the endometrium of 12 mares. A total of 88 isolates were analyzed by PFGE: 31 from the endometrium, 26 from the cranial vagina and 31 isolates from the clitoral fossa. For MLST 21 isolates were chosen. Results demonstrated a higher genetic similarity of the isolates obtained from infectious endometritis compared to isolates obtained from the caudal reproductive tract. In conclusion, we demonstrate for the first time that a genetically distinct group of S. zooepidemicus is associated with infectious endometritis in the mare.

Análise fenotípica e genotípica da virulência de Staphylococcus spp. e de sua dispersão clonal como contribuição ao estudo da mastite bovina / Phenotypic and genotypic analysis of virulence in Staphylococcus spp. and its clonal dispersion as a contribution to the study of bovine mastitis

A mastite é uma inflamação da glândula mamária causada principalmente por bactérias, dentre as quais o gênero Staphylococcus ocupa um papel importante. Bactérias pertencentes a este gênero são caracterizadas por expressar fatores de virulência que permitem sua persistência e disseminação no hospedeiro. O presente trabalho teve por objetivo avaliar fenogenotipicamente os fatores de virulência de isolados de Staphylococcus spp. a partir de casos de mastite bovina. Foram analisadas 272 amostras de leite provenientes de oito propriedades da região Sul-Fluminense do Estado do Rio de Janeiro. Após identificação, obteve-se um total de 250 isolados de Staphylococcus spp. Estes foram submetidos às provas fenotípicas de detecção da produção de "slime" em microplaca e em ágar vermelho congo; produção de hemolisinas e sinergismo hemolítico; produção de caseinase e DNase. Posteriormente foram submetidos à técnica de PCR para detecção dos genes de produção de cápsula (cap5 e cap8), fibronectina (fnbA,e fnbB), "slime" (icaA e icaD) e hemolisinas (hla e hlb). Do total avaliado, 58% (145/250) foi identificado como Staphylococcus spp. coagulase-negativos e 42% (105/250) como Staphylococcus spp. coagulase-positivos, destes 36,2% (38/105) foram identificados como S. aureus, 11,4% (12/105) como S. intermedius e 3,8% (4/105) como pertencentes ao grupo SIG. Apenas 6,4% (16/250) dos isolados foram produtores de α-hemólise, 4,8% (12/250) de β-hemólise e, 1,6% (4/250) de α e β-hemólise. A produção de caseinase foi observada em 66,4% (166/250), e a produção de "slime" avaliada pela técnica da microplaca em 76,8% (192/250) dos isolados, respectivamente. A DNase foi detectada em ECNs (38/145) e S. aureus (14/38). Os marcadores genéticos avaliados para a produção de slime, icaA e icaD apresentaram nenhuma ou leve concordância com a produção fenotípica, respectivamente, utilizando o coeficiente Kappa. Tal dado parece indicar que outros marcadores genéticos podem estar envolvidos com a expressão desta característica. Os demais genes detectados com frequência de 4% (10/250) para cap5 e para cap8, 32,8% (82/250) para fnbA, 4,4% (11/250) para fnbB, 19,2% (48/250) para hla e 18% (45/250) para hlb. O perfil circulante nas propriedades foi o 1: isolado produtor de "slime" e caseinase. O gene spaA foi positivo em todos os S. aureus, apresentando amplicons de tamanhos variados, sendo o tamanho prevalente o de 300pb. A amplificação do gene coa apresentou nove tipos polimórficos distintos, sendo prevalente o amplicon de 600pb. O gene agr foi detectado em todos os S. aureus, com amplicon de 200pb. Foi observado que os genes de virulência estudados estavam distribuídos de modo aleatório entreos 6 distintos perfis eletroforéticos obtidos através da Eletroforese em Gel de Campo Pulsado (PFGE).

Mastitis is an inflammation of one or more mammary glands caused mainly by bacteria, among which the genus Staphylococcus plays an important role. Bacteria belonging to this genus are known to express virulence factors which allow their persistence and spread in the host. This study aimed to evaluate the phenotypic and genotypic aspects of virulence factors in Staphylococci spp. isolates from bovine mastitis clinical cases. A total of 272 milk samples from 8 farms in the South-Fluminense region of Rio de Janeiro were analyzed. The samples underwent conventional bacterial identification, yielding 250 Staphylococci spp. isolates. These were tested for the phenotypic detection of slime production by the microplate and Congo Red Agar methods. The hemolysins production, hemolytic synergism, caseinase and DNase production were also evaluated. The isolates were then assayed through the Polymerase Chain Reaction method to detect genes associated with virulence factors such as: capsule (cap5, cap8), fibronectin (fnbA, fnbB), slime (icaA, icaD) and hemolysins (hla e hlb). Regarding the number of isolates assessed, 58% (145/250) were identified as coagulase-negative Staphylococcus spp. and 42% (105/250) as coagulase-positive Staphylococcus spp. The latter comprised 36.2% (38/105) of isolates identified as S. aureus, 11.4% (12/105) as S. intermedius and 3.8% (4/105) belonging to the SIG group. The hemolisin production was not significant, whereas only 6,4% (16/250) produced alfa hemolysis, 4,8% (12/250) produced beta hemolysis and 1,6% (4/250) was able to produce both. Caseinase production was observed in 66.4% (166/250) and slime production assayed through the microplate method was positive in 76,8% (192/250). DNAse was detected in coagulase-negative Staphylococcus spp. (38/145) and in S. aureus (14/38). Low association between genetic detection of icaA (38/250) and icaD (54/250) and slime phenotypic expression (192/250) suggest that others genetic markers can be involved in this expression. Regarding gene amplification, the isolates did not show significant correlation between the genetic detection of icaA (38/250) and icaD (54/250) and slime production (192/250), indicating that other genetic markers may be involved in this trait expression. The frequency of the occurrence of the others studied genes was of 4% (10/250) for cap5 and cap8, 32,8% (82/250) for fnbA, 4,4% (11/250) for fnbB, 19,2% (48/250) for hla and 18% (45/250) for hlb. The major circulating strain profile on the farms encompassed slime and caseinase producer strains. The spaA gene was found in all of the S. aureus isolates, presenting varying amplicons sizes, with 300bp being the prevalent size. The amplification of the coa gene showed nine polymorphic variants, with 600bp being the prevalent amplicon. The agr gene was also detected in every S. aureus isolate, with an amplicon of 200bp. It was noticed that the presence or absence of the virulence genes assayed in this study were not correlated with the 6 distinct electrophoretic profiles obtained by PFGE.

Prevalence and antimicrobial resistance of Enterococcus spp and Staphylococcus spp isolated from surfaces in a veterinary teaching hospital.

OBJECTIVE: To determine the prevalence and antimicrobial resistance of enterococci and staphylococci collected from environmental surfaces at a veterinary teaching hospital (VTH). DESIGN: Longitudinal study. SAMPLE: Samples collected from surfaces in 5 areas (emergency and critical care, soft tissue and internal medicine, and orthopedic wards; surgery preparation and recovery rooms; and surgery office and operating rooms) of a VTH. PROCEDURES: Selected surfaces were swabbed every 3 months during the 3-year study period (2007 to 2009). Isolates of enterococci and staphylococci were identified via biochemical tests, and antimicrobial susceptibility was evaluated with a microbroth dilution technique. A subset of isolates was analyzed to assess clonality by use of pulsed-field gel electrophoresis. RESULTS: 430 samples were collected, and isolates of enterococci (n = 75) and staphylococci (110) were identified. Surfaces significantly associated with isolation of Enterococcus spp and Staphylococcus spp included cages and a weight scale. Fourteen Enterococcus spp isolates and 17 Staphylococcus spp isolates were resistant to ≥ 5 antimicrobials. Samples collected from the scale throughout the study suggested an overall increase in antimicrobial resistance of Enterococcus faecium over time. Clonality was detected for E faecium isolates collected from 2 different surfaces on the same day. CONCLUSIONS AND CLINICAL RELEVANCE: Although not surprising, the apparent increase in antimicrobial resistance of E faecium was of concern because of the organism's ability to transmit antimicrobial resistance genes to other pathogens. Results reported here may aid in identification of critical control points to help prevent the spread of pathogens in VTHs.

Molecular epidemiology of Mannheimia haemolytica and Mannheimia glucosida associated with ovine mastitis.

While Mannheimia haemolytica and Mannheimia glucosida have been recognized as causes of intramammary infection in sheep, there has been no investigation of the epidemiology of the strains involved. Pulsed field gel electrophoresis was used to study the molecular epidemiology of isolates of these 2 species associated with ovine mastitis. Ten distinct strains were recognized among 12 M. haemolytica isolates, and 7 distinct strains among 13 M. glucosida isolates. The results demonstrate a high diversity of isolates with the ability to cause ovine mastitis. However, the presence of some identical isolates may suggest the possibility of horizontal transmission of these species in some flocks, possibly through lamb sucking, and/or differences in the capacity of some isolates to cause mastitis in sheep.

The first nosocomial outbreak of methicillin-resistant Staphylococcus aureus in horses in Sweden.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) in animals is a rare finding in Sweden. In horses, MRSA was first detected in a screening survey in 2007. In 2008, six clinical cases occurred in an equine hospital, indicating an outbreak. METHOD: All MRSA isolates detected, 11 spa-type t011 and one t064 (n = 12), in infected horses (n = 10) and screening of horses (n = 2) in Sweden from December 2007 to March 2010 were retrospectively analysed with pulsed-field gel electrophoresis (PFGE) using Cfr9I and ApaI restriction enzymes, to study relationship between the isolates. Medical records of infected horses and outbreak investigation notes were scrutinised to monitor the clinical outcome and other aspects of the outbreak. RESULTS: Eight of the 10 infected horses were linked to one equine hospital and two to another hospital in the same region. The six horses infected with MRSA in 2008 underwent surgery during the period 22 May-7 July in one of the hospitals. Four more infections linked to the two hospitals were notified between 2009 and March 2010.Nine of the 11 spa-type t011 isolates had identical Cfr9I and ApaI PFGE pattern. All six infected horses from 2008 presented with this MRSA. Two t011 isolates differed in one and two bands, respectively, in PFGE.Nine horses suffered from surgical site infections (SSI). No antimicrobials were used following the MRSA diagnosis and the infections cleared. The time from surgery to MRSA diagnosis differed greatly between the horses (range 15-52 days). CONCLUSIONS: Association in time and space of six horses infected with an identical MRSA strain of spa-type t011 confirmed an outbreak. Two isolates found in 2009 and 2010 in the outbreak hospital were closely related to the outbreak strain, indicating one circulating strain. Both spa-type t011 and t064 have been reported in horses in Europe prior to these findings. The observation that the infections cleared although antimicrobials were not used is encouraging for future prudent use of antimicrobials. The time from surgery to bacteriological diagnosis was not acceptable in most cases, as contagious spread was a risk. Sampling when symptoms of infection are noticed and accurate analysis are thus important.

Beware of the pet dog: a case of Staphylococcus intermedius infection.

Staphylococcus intermedius is a common commensal and pathogen in dogs and cats and only rarely has been identified as causing human infection. We report a human case of postoperative sinus infection caused by methicillin-resistant S. intermedius. A 28-year-old woman with a history of endoscopic pituitary adenoma resection presented with 3 weeks of foul smelling nasal discharge. Nasal endoscopy revealed purulent sinus drainage. Cultures, initially misidentified as coagulase-negative Staphylococcus and then as Staphylococcus aureus, revealed the presence of S. intermedius. Cultures from the patient's pet dog also grew S. intermedius strains that were confirmed to be identical to those of the patient's by pulse field gel electrophoresis analysis. The patient was successfully treated with endoscopic debridement and a prolonged antibiotic regimen with vancomycin and linezolid. Our case illustrates the possibility of transmission of antibiotic-resistant bacteria causing infection from pets to humans.