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)

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)

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

Prevalence and characteristics of Shiga toxin-producing Escherichia coli (STEC) from cattle in Korea between 2010 and 2011

A total of 156 Shiga-like toxin producing Escherichia coli (STEC) were isolated from fecal samples of Korean native (100/568, 18%) and Holstein dairy cattle (56/524, 11%) in Korea between September 2010 and July 2011. Fifty-two STEC isolates (33%) harbored both of shiga toxin1 (stx1) and shiga toxin2 (stx2) genes encoding enterohemolysin (EhxA) and autoagglutinating adhesion (Saa) were detected by PCR in 83 (53%) and 65 (42%) isolates, respectively. By serotyping, six STEC from native cattle and four STEC from dairy cattle were identified as O-serotypes (O26, O111, O104, and O157) that can cause human disease. Multilocus sequence typing and pulsed-field gel electrophoresis patterns highlighted the genetic diversity of the STEC strains and difference between strains collected during different years. Antimicrobial susceptibility tests showed that the multidrug resistance rate increased from 12% in 2010 to 42% in 2011. Differences between isolates collected in 2010 and 2011 may have resulted from seasonal variations or large-scale slaughtering in Korea performed to control a foot and mouth disease outbreak that occurred in early 2011. However, continuous epidemiologic studies will be needed to understand mechanisms. More public health efforts are required to minimize STEC infection transmitted via dairy products and the prevalence of these bacteria in dairy cattle.

Distribution, quantitative load and characterization of Salmonella associated with swine farms in upper-northern Thailand

This study was conducted to analyze the prevalence and quantitative loads of Salmonella spp. on pig farms in Chiang Mai, Lamphun, Thailand to assess loading levels before slaughtering. The serotype diversity, antimicrobial-resistance pattern and pulse-field type of Salmonella spp. were also characterized to assess the dynamic propagation of the pathogen. The Salmonella-positive prevalence was 246/805 (30.56%), and the quantitative loads varied from 1.48~4.04 Log10MPN/g, with a mean +/- standard deviation of 2.11 +/- 0.57. AMP/S/TE (ampicillin/streptomycin/tetracycline) was the highest frequency antimicrobial resistance pattern found in this study. In addition, Salmonella Rissen was the primary serotype in this region. PFGE results indicated the occurrence of infection by cross contamination among pig farms. Our study showed that pork is easily contaminated with this pathogen. Farm control programs must be based on strict biosecurity and hygienic measures, which could further reduce the contamination pressure at slaughterhouses or retail shops.

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.

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.

Determination of staphylococcal exotoxins, SCCmec types, and genetic relatedness of Staphylococcus intermedius group isolates from veterinary staff, companion animals, and hospital environments in Korea

The Staphylococcus (S.) intermedius group (SIG) has been a main research subject in recent years. S. pseudintermedius causes pyoderma and otitis in companion animals as well as foodborne diseases. To prevent SIG-associated infection and disease outbreaks, identification of both staphylococcal exotoxins and staphylococcal cassette chromosome mec (SCCmec) types among SIG isolates may be helpful. In this study, it was found that a single isolate (one out of 178 SIG isolates examined) harbored the canine enterotoxin SEC gene. However, the S. intermedius exfoliative toxin gene was found in 166 SIG isolates although the S. aureus-derived exfoliative toxin genes, such as eta, etb and etd, were not detected. SCCmec typing resulted in classifying one isolate as SCCmec type IV, 41 isolates as type V (including three S. intermedius isolates), and 10 isolates as non-classifiable. Genetic relatedness of all S. pseudintermedius isolates recovered from veterinary staff, companion animals, and hospital environments was determined by pulsed-field gel electrophoresis. Strains having the same band patterns were detected in S. pseudintermedius isolates collected at 13 and 18 months, suggesting possible colonization and/or expansion of a specific S. pseudintermedius strain in a veterinary hospital.

Dissemination and tracking of Salmonella spp. in integrated broiler operation

Controlling Salmonella in integrated broiler operation is complicated because there are numerous potential sources of Salmonella contamination, including chicks, feed, rodents, wild poultry operations, and the processing plant. The objective of this study was to investigate the distribution of Salmonella through all phases of two integrated broiler operations and to determine the key areas related to the control of all known sources of infection. Two different Salmonella serotypes were observed at integrated broiler chicken company A. S. enteritidis, the predominant company A isolate, was consistently found in the breeder farm, hatcheries, broiler farms, and chicken slaughterhouse. At company B, a total of six different serotypes, S. heidelberg, S. senftenberg, S. enteritidis, S. blockley, S. gallinarum, and S. virchow, were detected. Although S. heidelberg was not found in the broiler farms, it was consistently found in the breeder farm, hatcheries, and chicken slaughterhouse. In addition, S. enteritidis was found in the hatcheries, broiler farm, and chicken slaughterhouse. In order to obtain the genetic clonality, 22 S. enteritidis isolates were digested with XbaI and analyzed by pulsed-field gel electrohporesis (PFGE). A difference in the PFGE pattern was found to be related to the origin of the integrated broiler operation. These data support the critical need to control Salmonella in breeder farms and hatcheries, and demonstrate important points related to the control of infection in large-scale poultry operations of Korea.

Persistent occurrence of a single Streptococcus equi subsp. zooepidemicus clone in the pig and monkey population in Indonesia

In the present study 41 mucoid growing Streptococcus equi subsp. zooepidemicus strains (37 strains isolated from healthy two from diseased pigs, two strains isolated from healthy monkeys) appeared to be phenotypically and genotypically identical to mucoid growing S. equi subsp. zooepidemicus strains isolated from a previously described outbreak among the pig and monkey population on the island of Bali, Indonesia. These findings indicate that the mucoid growing S. equi subsp. zooepidemicus clone was still present in the pig and monkey population in Indonesia.