Pesquisa de Staphylococcus spp. coagulase negativa em queijo colonial inspecionado: identificação, perfil de genes de enterotoxinas clássicas e de resistência à penicilina e à meticilina

A pesquisa de Staphylococcus spp. coagulase negativa (CNS) em alimentos não é prevista na legislação, entretanto, estas bactérias têm emergido como patógenos oportunistas e sua capacidade enterotoxigênica já foi documentada. O queijo destaca-se entre os principais derivados lácteos associados a intoxicações alimentares e a presença de cepas enterotoxigênicas do gênero Staphylococcus neste alimento representa um risco ao consumidor. O queijo colonial, tradicionalmente consumido pelos gaúchos, não possui regulamento técnico específico e poucos são os estudos relacionados ao risco do consumo desse alimento, bem como, à identificação de micro-organismos presentes nessa matriz. Sendo assim, os objetivos do presente estudo foram: (i) identificar as espécies de Staphylococcus spp. coagulase negativa, presentes em queijo colonial inspecionado; (ii) pesquisar a presença de genes codificadores de enterotoxinas clássicas (SE), bem como de resistência à penicilina e à meticilina nas cepas isoladas desta matriz. Para tanto, no período de novembro de 2014 a maio de 2015, foram analisadas 205 amostras de queijos coloniais inspecionados, sendo 121 adquiridas em Feiras Modelo e 84 em bancas do Mercado Público de Porto Alegre, compreendendo 17 marcas distintas. O isolamento inicial de Staphylococcus spp. foi realizado de acordo com o protocolo ISO 6888-1:1999, adicionado da triagem fenotípica para CNS. A identificação genotípica dos isolados foi realizada pela amplificação da região V1-V2 do gene 16S rRNA, seguida de sequenciamento e comparação das sequências obtidas no GenBank. A pesquisa de genes de enterotoxinas clássicas foi realizada por amplificação dos genes sea, seb, sec, sed e see. A determinação de resistência à penicilina foi avaliada a partir da amplificação do gene blaZ. Para resistência à meticilina, foi realizado teste de triagem frente à cefoxitina, e confirmação pela pesquisa do gene mecA. O armazenamento sob refrigeração foi observado em quase 90% das amostras coletadas. Entre as 179 colônias retiradas do ágar Baird-Parker, 59 apresentaram-se fenotipicamente compatíveis com CNS e foram identificadas genotipicamente. Treze espécies foram identificadas, sendo Macrococcus caseolyticus (40%) a mais frequente. Das 35 cepas confirmadas como CNS, S. equorum e S. vitulinus foram as espécies predominantes seguidas de S. hyicus, S. saprophyticus e S. epidermidis. O gene blaZ foi detectado em cinco cepas de CNS e em uma cepa de M. caseolyticus, sendo relativamente mais frequente em S. hyicus e S. epidermidis. O gene mecA não foi detectado. Oito cepas de CNS amplificaram algum gene para SE, sendo seb o mais frequente, seguido por sed, sea e see. O gene para enterotoxina C não foi detectado. Onze cepas apresentaram pelo menos um dos genes investigados, das quais, seis cepas apresentaram genes para SE e blaZ, concomitantemente. Os perfis seb/blaZ (n=4) e blaZ (n=3) foram os mais frequentes. Foi possível confirmar a diversidade de Staphylococcus spp. coagulase negativa em queijos coloniais inspecionados, além da baixa frequência de cepas carreadoras de genes para enterotoxinas clássicas e resistência à penicilina e à meticilina.The investigation of coagulase-negative Staphylococcus spp. (CNS) is not included in food monitoring; although these bacteria have emerged as significant opportunistic pathogens and their toxigenic capacity has been documented. Among the dairy products, cheese features as one of the most involved in food poisoning outbreaks. The presence of enterotoxigenic strains of Staphylococcus in this food therefore represents a hazard for the consumers. Colonial cheese, which is a traditionally consumed cheese type in Rio Grande do Sul, does not have a specific technical regulation, and there are few studies targeting the risk for consumers, or aiming to identify its typical microbiota. Thus, the objectives of this study were: (i) to identify coagulase-negative Staphylococcus spp. species in inspected colonial cheese (ii) to investigate the presence of genes encoding classical enterotoxins (SE), and resistance to penicillin and methicillin in strains obtained from this food. For this purpose, from November 2014 to May 2015, 205 cheese samples were analyzed, 121 of which were acquired in street fairs and 84 in Central Market. The samples belonged to 17 different brands. The isolation of Staphylococcus spp. was performed according to the ISO 6888-1: 1999 protocol, followed by the phenotypic screening of CNS. The genotype identification of the isolates was performed by amplification of the V1-V2 region of the 16S rRNA gene, followed by sequencing and the sequences comparison with the GenBank database. Classical enterotoxin genes were investigated by amplification of sea, seb, sec, sed and see genes. The determination of penicillin resistance was evaluated by the amplification of blaZ gene. For methicillin resistance, a screening test with cefoxitin was conducted followed by confirmation through the mecA amplification. The majority (89,7%) of the collected samples were stored under refrigeration. Among the 179 atypical colonies obtained from Baird-Parker agar, 59 were phenotypically compatible with CNS and were further subjected to genotyping. Thirteen bacterial species were identified, being Macrococcus caseolyticus the most frequent (40%). Thirty-five strains were confirmed as CNS, being S. equorum and S. vitulinus the most prevalent followed by S. hyicus, S. saprophyticus and S. epidermidis. The gene blaZ was detected in five strains of CNS and in one strain of M. caseolyticus, being relatively more frequent in S. hyicus and S. warneri. The mecA gene was not detected. Eight CNS strains amplified SE gene: SEB was the most frequent, followed by SED, SEA and SEE. There was no enterotoxin C gene detected. Eleven strains carried at least one of the genes investigated; six strains presented genes for SE and blaZ, concomitantly. The profiles SEB/blaZ (n = 4) and blaZ (n = 3) were the most frequent. The diversity of CNS in inspected colonial cheeses was confirmed. In addition, the low frequency of strains carrying genes for enterotoxins and resistance to penicillin and methicillin was observed

Antimicrobial Resistance Profile of Bacteria Isolated from Canine and Feline Samples at the Preventive Veterinary Laboratory of the Federal University of Rio Grande do Sul (UFRGS) - Brazil

Background: Antimicrobial resistance (AMR) is a major global health threat. In small animals such as dogs and cats, antimicrobials are most commonly prescribed for skin and genitourinary diseases; therefore, the AMR of bacteria involved in these infections should be monitored. In addition, the results of antimicrobial susceptibility testing (AST) may be interpreted as a local epidemiological history of AMR. The Preventive Veterinary Medicine Laboratory (PVML) received clinical samples from dogs and cats for bacterial isolation and AST. Thus, this study aimed to assess the AMR of bacteria isolated from the samples of dogs and cats received at the Preventive Veterinary Medicine Laboratory (PVML). Materials, Methods & Results: Data from bacteriological examinations performed at the PVML of the Universidade Federal do Rio Grande do Sul (UFRGS) during 5 years were analyzed. Skin and ear canal samples were inoculated in 5% sheep blood agar, and urine samples were streaked on CHROMagar™ orientation. After incubation at 36±1°C for up to 72 h, identification and AST were performed according to routine protocols. Of 1,534 samples submitted to the PVML, 1,086 (70.8%) were collected from dogs and  29.2% from feline patients. Otological swabs (n = 533, 49.1%) were the most frequent samples from dogs, while cat urine samples (n = 384, 84.8%) predominated by far. Considering the canine samples, no bacterial growth (NBG) was observed in 443 (40.8%) samples, while only one colony type was noted in 516 (47.5%) samples. Gram-positive bacteria (n = 298) were more frequent than gram-negative bacteria (n = 77) in the skin. In urine samples, gram-negative bacteria (n = 94) were isolated more frequently than gram-positive bacteria (n = 47). In feline samples, a high number of NBG (n = 308, 68%) was observed. Gram-positive (n = 22) was predominant in comparison to gram-negative bacteria (n = 9) in cultures from the ear and skin swabs. Enterococcus spp. and Escherichia coli were the most frequently identified bacteria in urine samples. Among the Staphylococcus sp. strains of any origin, AMR frequency varied from 4.22% (amikacin) to 50.70% (sulfa/trimethoprim). Enterococcus spp. showed AMR frequencies from 12.5% (amoxicillin/clavulanic acid) to 62.06% (enrofloxacin). Among the gram-negative genera, E. coli presented AMR frequencies from 10.20% (gentamicin) to 60.0% (neomycin). The frequency of AMR was stable over time, and a profile of much higher resistance to fluoroquinolones in comparison to beta-lactams was observed. Discussion: The recurrence of skin and urinary infections implies the need for frequent treatment with antibiotics, which exerts selection pressure for resistance and multidrug resistance. In this study, the frequency of multidrug resistance was low, and the resistance to the tested antimicrobials showed high variation. However, a trend of high resistance to the fluoroquinolone group was observed in contrast to the low resistance to beta-lactams. This trend was consistent among the isolated bacteria, regardless of the type of sample or origin. The overprescription of fluoroquinolones in small animal practices has been widely documented in several countries. However, this class of antimicrobials, is highly prioritized for the treatment of infections in humans. Therefore, the selection of resistant strains has gained special emphasis, especially when considering the possibility of the transmission of resistant bacteria between pets and humans. In summary, the results of bacteriological tests conducted at the PVML-Universidade Federal do Rio Grande do Sul confirmed that ubiquitous bacteria predominate in clinical samples of dogs and cats. The high frequency of resistance to the fluoroquinolone group, while a predominance of susceptible strains in the first-choice drugs such as amoxicillin/clavulanic acid, may indicate excessive and empirical use of the second-choice drugs in clinical practice. Keywords: otitis, dermatitis, urinary infection, dogs, cats, antibiotic resistance, AMR

Antimicrobial resistance profile of bacteria isolated from canine and feline samples at the Preventive Veterinary Laboratory of the Federal University of Rio Grande do Sul (UFRGS) – Brazil

Background: Antimicrobial resistance (AMR) is a major global health threat. In small animals such as dogs and cats, antimicrobials are most commonly prescribed for skin and genitourinary diseases; therefore, the AMR of bacteria involved in these infections should be monitored. In addition, the results of antimicrobial susceptibility testing (AST) may be interpreted as a local epidemiological history of AMR. The Preventive Veterinary Medicine Laboratory (PVML) received clinical samples from dogs and cats for bacterial isolation and AST. Thus, this study aimed to assess the AMR of bacteria isolated from the samples of dogs and cats received at the Preventive Veterinary Medicine Laboratory (PVML). Materials, Methods & Results: Data from bacteriological examinations performed at the PVML of the Universidade Federal do Rio Grande do Sul (UFRGS) during 5 years were analyzed. Skin and ear canal samples were inoculated in 5% sheep blood agar, and urine samples were streaked on CHROMagar™ orientation. After incubation at 36±1°C for up to 72 h, identification and AST were performed according to routine protocols. Of 1,534 samples submitted to the PVML, 1,086 (70.8%) were collected from dogs and 29.2% from feline patients. Otological swabs (n = 533, 49.1%) were the most frequent samples from dogs, while cat urine samples (n = 384, 84.8%) predominated by far. Considering the canine samples, no bacterial growth (NBG) was observed in 443 (40.8%) samples, while only one colony type was noted in 516 (47.5%) samples. Gram-positive bacteria (n = 298) were more frequent than gram-negative bacteria (n = 77) in the skin. In urine samples, gram-negative bacteria (n = 94) were isolated more frequently than gram-positive bacteria (n = 47). In feline samples, a high number of NBG (n = 308, 68%) was observed. Gram-positive (n = 22) was predominant in comparison to gram-negative bacteria (n = 9) in cultures from the ear and skin swabs. Enterococcus spp. and Escherichia coli were the most frequently identified bacteria in urine samples. Among the Staphylococcus sp. strains of any origin, AMR frequency varied from 4.22% (amikacin) to 50.70% (sulfa/trimethoprim). Enterococcus spp. showed AMR frequencies from 12.5% (amoxicillin/clavulanic acid) to 62.06% (enrofloxacin). Among the gram-negative genera, E. coli presented AMR frequencies from 10.20% (gentamicin) to 60.0% (neomycin). The frequency of AMR was stable over time, and a profile of much higher resistance to fluoroquinolones in comparison to beta-lactams was observed. Discussion: The recurrence of skin and urinary infections implies the need for frequent treatment with antibiotics, which exerts selection pressure for resistance and multidrug resistance. In this study, the frequency of multidrug resistance was low, and the resistance to the tested antimicrobials showed high variation. However, a trend of high resistance to the fluoroquinolone group was observed in contrast to the low resistance to beta-lactams. This trend was consistent among the isolated bacteria, regardless of the type of sample or origin. The overprescription of fluoroquinolones in small animal practices has been widely documented in several countries. However, this class of antimicrobials, is highly prioritized for the treatment of infections in humans. Therefore, the selection of resistant strains has gained special emphasis, especially when considering the possibility of the transmission of resistant bacteria between pets and humans. In summary, the results of bacteriological tests conducted at the PVML-Universidade Federal do Rio Grande do Sul confirmed that ubiquitous bacteria predominate in clinical samples of dogs and cats. The high frequency of resistance to the fluoroquinolone group, while a predominance of susceptible strains in the first-choice drugs such as amoxicillin/clavulanic acid, may indicate excessive and empirical use of the second-choice drugs in clinical practice

Serum neuron-specific enolase as early predictor of outcome after in-hospital cardiac arrest: a cohort study

INTRODUCTION: Outcome after cardiac arrest is mostly determined by the degree of hypoxic brain damage. Patients recovering from cardiopulmonary resuscitation are at great risk of subsequent death or severe neurological damage, including persistent vegetative state. The early definition of prognosis for these patients has ethical and economic implications. The main purpose of this study was to investigate the prognostic value of serum neuron-specific enolase (NSE) in predicting outcomes in patients early after in-hospital cardiac arrest. METHODS: Forty-five patients resuscitated from in-hospital cardiac arrest were prospectively studied from June 2003 to January 2005. Blood samples were collected, at any time between 12 and 36 hours after the arrest, for NSE measurement. Outcome was evaluated 6 months later with the Glasgow outcome scale (GOS). Patients were divided into two groups: group 1 (unfavorable outcome) included GOS 1 and 2 patients; group 2 (favorable outcome) included GOS 3, 4 and 5 patients. The Mann–Whitney U test, Student's t test and Fisher's exact test were used to compare the groups. RESULTS: The Glasgow coma scale scores were 6.1 ± 3 in group 1 and 12.1 ± 3 in group 2 (means ± SD; p < 0.001). The mean time to NSE sampling was 20.2 ± 8.3 hours in group 1 and 28.4 ± 8.7 hours in group 2 (p = 0.013). Two patients were excluded from the analysis because of sample hemolysis. At 6 months, favorable outcome was observed in nine patients (19.6%). Thirty patients (69.8%) died and four (9.3%) remained in a persistent vegetative state. The 34 patients (81.4%) in group 1 had significantly higher NSE levels (median 44.24 ng/ml, range 8.1 to 370) than those in group 2 (25.26 ng/ml, range 9.28 to 55.41; p = 0.034). CONCLUSION: Early determination of serum NSE levels is a valuable ancillary method for assessing outcome after in-hospital cardiac arrest

Risk factors for and protective factors against breastfeeding interruption before 2 years : a birth cohort study

Background: Little is known about the factors associated with the World Health Organization (WHO) recommendation of breastfeeding for at least 2 years. The objective of this study was to identify risk factors for and protective factors against breastfeeding interruption before 2 years of age. Methods: In this live birth cohort, mother and infant dyads were followed for 2 years. Data collection was performed at the maternity ward and subsequently at the children’s homes, monthly during the first 6months of life and then at 9, 12, 18, and 24 months. The outcome of interest was breastfeeding interruption before 2years of age. Median duration of breastfeeding was estimated using Kaplan-Meier’s survival analysis, and the associations were tested using Cox’s hierarchical multivariate model. Significance was set at 5%. Results: Data from a total of 1344 dyads were assessed. Median breastfeeding duration was 385 days. The following risk factors for breastfeeding interruption were identified: white skin color (adjusted hazard ratio [HRa]: 1.31; 95% confidence interval [95%CI]: 1.10–1.56), primiparity (HRa: 1.21; 95%CI: 1.05–1.40), working outside the home (HRa: 1.52; 95%CI: 1.30– 1.77), child sex male (HRa: 1.18; 95%CI: 1.03–1.35) and use of a pacifier (HRa: 3.46; 95%CI: 2.98–4.01). Conversely, the following protective factors were identified: lower family income (HRa: 0.81; 95%CI: 0.71–0.94), mother-infant bed-sharing (HRa:0.61, 95%CI: 0.52–0.73), on-demand breastfeeding in the first month (HRa: 0.64; 95%CI: 0.47–0.89) and exclusive breastfeeding at 4 months (HRa: 0.58, 95%CI: 0.48–0.70). Conclusions: The findings allowed to identify both risk factors for and protective factors against breastfeeding interruption before 2 years of age. Knowledge of these factors may help prevent this event and aid in the development of programs that help women maintain breastfeeding for at least 2 years, as recommended by the WHO

Avaliação da sobrevivência de Salmonella enterica inoculada em salame de carne suína

Pork salami is an embedded, cured and ripened product commonly consumed in Brazil, and the presence of Salmonella enterica has already been reported in this product. During its preparation, the microbiological safety depends on the meat quality, addition of ingredients with antimicrobial activity, hygiene during processing, pH and water activity (Aw) reduction during maturation. In Brazil, the maturation protocol has not been determined in food regulation; therefore, the objectives of this study were (a) to identify the fermentation and drying phases during salami maturation; (b) to test the survival of S. enterica during salami processing; and (c) to compare xylose lysine deoxycholate (XLD) and thin agar layer (TAL) agar for recovering Salmonella. The salami samples were prepared with a cocktail of S. enterica strains, fermented at 30°C and dried at 20°C with controlled relative humidity (RH). Periodic sampling for S. enterica quantification and Aw and pH analyses were performed during maturation, and curves were constructed. Fermentation occurred during the first 66 hours, and the pH decreased while the population of S. enterica increased over the first 21 hours. The drying step was able to reduce the bacterial population by approximately 5 log CFU after 875 hours, reaching an Aw of less than 0.78. However, elimination of S. enterica was not achieved. For Salmonella recovery, TAL agar was more efficient than XLD agar.O salame de carne suína é um produto embutido, curado e maturado comumente consumido no Brasil no qual a presença de Salmonella enterica tem sido relatada. Durante a sua elaboração, a segurança microbiológica depende da qualidade da carne, adição de ingredientes com atividade antimicrobiana, higiene durante a produção, redução de pH e atividade de água (Aw) durante a sua maturação. O protocolo de maturação ainda não está determinado na legislação brasileira; portanto o estudo objetivou: (a) identificar as fases de fermentação e dessecação durante a maturação de salame; (b) testar a sobrevivência de S. enterica durante o processamento de salame e (c) comparar os meios de cultura xilose lisina dextrose (XLD) e thin agar layer (TAL) para recuperação de células do referido micro-organismo. Os salames foram elaborados com um coquetel de S. enterica e submetidos à fermentação em 30ºC e secagem a 20ºC com umidade relativa (UR) controlada. Amostragens periódicas para quantificação de S. enterica, análises de Aw e pH foram realizadas durante a maturação e as curvas foram construídas. A fermentação ocorreu nas primeiras 66 horas, quando houve queda do pH do salame; entretanto S. enterica aumentou sua população nas primeiras 21 horas. A etapa de dessecação foi capaz de reduzir aproximadamente 5 log UFC da população bacteriana em 875 horas, alcançando Aw menor que 0,78, mas não foi capaz de eliminar o micro-organismo do alimento. Para enumeração do micro-organismo, o meio sólido TAL foi mais eficiente na recuperação das células submetidas à maturação quando comparado ao ágar XLD comumente utilizado

Detection of Salmonella Heidelberg resistant to colistin in the intestinal content of pigs at slaughter

Salmonella Heidelberg has increasingly been reported as cause of human salmonellosis worldwide. In Brazil, S. Heidelberg has been reported in poultry but it is infrequently isolated from pigs. Here, we describe the isolation of S. Heidelberg resistant to colistin from slaughter pigs. Five pigs and their carcasses belonging to a same slaughter batch in ten consecutive days were sampled for fragment of intestine in the ileocecal region and sponges rubbed on the carcass surface (400 cm2) before chilling. Salmonella detection was performed according to the ISO 6579:2002. Intestinal content was also subjected to Salmonella enumeration by a miniaturized Most Probable Number (MPN) protocol. Salmonella isolates were characterized by antimicrobial resistance by the disk diffusion test, the minimum inhibitory concentration to colistin determination and to gene mcr-1 investigation by PCR. Salmonella was isolated from the intestinal content of 64% (32/50) of the pigs, in amounts that varied from 2.7 to \u3e1,400 MPN/g. Salmonella Heidelberg was the most frequent serovar identified in the intestinal content samples (20/50; 40%), and this serovar was present in eight of the ten pig batches sampled. At the prechill, Salmonella was isolated from 8% of carcasses, and S. Heidelberg was not detected. Salmonella Heidelberg strains were resistant against ampicillin (n=9), tetracycline (n=8), sulfonamide (n=8) and gentamicin (n=5). Nine multi-drug resistant strains were detected; among them four strains were positive for the gene mcr-1. In these strains the MIC value was 8 μg.mL-1, while in the strains without the mcr-1 gene it ranged from 2 μg.mL-1 to 4 μg.mL-1. Therefore, humans in contact with carrier pigs or their environment may be exposed to S. Heidelberg, including strains harboring the gene mcr-1

Perception of poultry veterinarians on the use of antimicrobials and antimicrobial resistance in egg production

This study aimed to describe the perception of veterinarians who work with commercial laying hens in the state of Rio Grande do Sul, Brazil, regarding the use of antibiotics and their possible impacts on animal, human, and environmental health. A descriptive epidemiological study was carried out through face-to-face or web conferencing interviews with the veterinarians that provide technical assistance at commercial laying hen operations. A standardized and structured questionnaire was developed based on the literature and expert opinion, which contained 1 opened and 40 closed questions. Conventional non-probabilistic sampling was used, based on an initial list of 15 veterinarians registered in the Poultry Production Association of Rio Grande do Sul, followed by the snowball technique. The acquisition of 26 contacts of veterinarians was accomplished, and 16 were interviewed. Through the answers obtained it was possible to verify that the interviewees' understanding regarding both the antibiotic resistance impact and the decision-making about the use of antibiotics seem to be linked to their practical experiences. Besides that, according to the veterinarians, farmers can acquire and administer the antimicrobials on their farms. Moreover, both farm storage and administration of lower doses of antibiotics than the recommended one could be contributing factors to resistant bacteria selection. Furthermore, controversially, the professionals believed that resistant bacteria can be transmitted to humans from eggs, but they said that there are no bacteria in eggs. Therefore, the veterinarians practices can be improved considering national and international guidelines on antimicrobial resistance to minimize the development of resistance. Finally, it is expected that the present results will contribute to a more complex discussion about antimicrobial resistance, helping to formulate public policies in the egg production industry

Escherichia coli in chicken carcasses in southern Brazil : absence of shigatoxigenic (STEC) and isolation of atypical enteropathogenic (aEPEC)

The aim of this study was to investigate the presence of shiga toxin-producing Escherichia coli (STEC) and atypical enteropathogenic Escherichia coli (aEPEC) in frozen chicken carcasses sold at stores in southern Brazil. Typical E. coli colonies were enumerated in 246 chicken carcasses, and the presence of stx1, stx2, eae genes was investigated in their rinse liquid and in E. coli strains isolated from those carcasses. Strains of E. coli were also investigated for the presence of bfp gene. A median of 0.6 cfu.g-1(ranging from <0.1 to 242.7 cfu.g-1) of typical E. coli colonies was found in the carcasses. Shiga toxin-encoding genes (stx1 and stx2) were not detected, indicating that the chicken carcasses were negative for STEC. The intimin protein gene (eae) was detected in E.coli isolated from 4.88% of the carcasses; all tested strains were negative for the bfp gene and were classified as aEPEC. Twenty-two aEPEC strains were tested for resistance to ten antimicrobials and subjected to macrorestriction (PFGE). All the tested aEPEC strains were fully susceptible to cephalosporins, ciprofloxacin and colistin. Resistance to sulfonamide (65%), ampicillin (55%), tetracycline (50%) and gentamicin (45%) were the most frequent. The PFGE profile demonstrated a low level of similarity among the resistant strains, indicating that they were epidemiologically unrelated. The results indicate that aEPEC strains can contaminate chicken meat, and their association with strains implicated in human diarrhea needs to be further investigated