Pesquisa de indicadores de biofilmes de Escherichia coli, Listeria monocytogenes e Salmonella sp. em abatedouros frigoríficos de bovinos e aves localizados no DF, Entorno e Goiás

Trabalho de Conclusão de Curso (graduação)—Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, 2018.Os biofilmes representam uma preocupação à indústria de alimentos pois resistem a tratamentos antimicrobianos e a sanitizantes, além de causarem perda da qualidade, deteriorarem os alimentos e veicularem patógenos. O objetivo deste trabalho foi verificar a presença de indicadores de biofilmes de Escherichia coli, Listeria monocytogenes e Salmonella spp. em abatedouros frigoríficos de bovinos e aves localizados na região do Distrito Federal, Entorno e Goiás. As amostras de swabs foram coletadas de ambientes e equipamentos/utensílios em diferentes pontos da indústria. Foram realizadas seis visitas aos estabelecimentos, onde foi coletado um total de 144 amostras de swabs. Foram detectadas 89 cepas (61,8%) de E. coli do total de amostras de swabs, sendo 58/89 cepas (65,2%) isoladas de abatedouros de aves e 31/89 cepas (34,8%) de abatedouros de bovinos. As esteiras e as tubulações que direcionam miúdos foram os locais com maior detecção de cepas nos abatedouros de aves, enquanto nos abatedouros de bovinos foram os ralos da sala de abate e da câmara fria. Todas as cepas de Listeria monocytogenes foram detectadas em abatedouros frigoríficos de aves, perfazendo um total de 15/144 (10,4%) das cepas isoladas, sendo as tubulações que direcionam o quarto traseiro das aves, coração, fígado e moela para embalagem os locais mais contaminados. Nenhuma bactéria do gênero Salmonella foi encontrada nas instalações e equipamentos de ambos abatedouros frigoríficos. A presença desses microrganismos no ambiente industrial antes da higienização para o início de suas atividades diárias sugere que há formação de biofilmes nas instalações e equipamentos/utensílios coletados, provavelmente devido a falhas na limpeza e podendo resultar na contaminação dos alimentos produzidos nestes locais.Biofilms represent a concern for the food industry due to the resistance to a antimicrobial and sanitizing treatments. Is also a common causing loss of quality, deterioration of food and transmission of pathogens. The aim of this study was to verify the presence of biofilms indicators of Escherichia coli, Listeria monocytogenes and Salmonella spp. in cattle and poultry slaughterhouses located in Federal District, Entorno and Goiás. The swab samples were collected in the facilities and equipment/ utensils from different points in the industry, six visits were made to the industries and 144 swab samples were obtained. A total of 89/144 strains of E. coli (61.8%) were isolated, 58/89 strains (65.2%) isolated from poultry slaughterhouses and 31/89 strains (34.8%) from cattle slaughterhouses. The tables and tubes that direct viscera were the sites with the largest number of strains in poultry slaughterhouses, while in the cattle slaughterhouses the largest number of strains were in the drains of the slaughter room and the cold room. All strains of Listeria monocytogenes were detected in poultry slaughterhouses, making a total of 15/144 (10.4%) isolated strains, the most contaminated spots were the tubes that direct the hindquarters of poultry, heart, liver and gizzard to packaging. The Salmonella spp. was not found in the facilities and equipments of the visited slaughterhouses. The presence of these microorganisms in the industrial environment, prior to the hygienization process to start their daily activities, suggests a formation of biofilms in the facilities and equipment/utensils collected, probably due to cleaning failures, which can result in the contamination of the food produced in these places

Molecular characterization and biofilm-formation analysis of Listeria monocytogenes, Salmonella spp., and Escherichia coli isolated from Brazilian swine slaughterhouses

This study aimed to verify the presence of Listeria monocytogenes, Salmonella spp., and Escherichia coli in two Brazilian swine slaughterhouses, as well as to perform antibiograms, detect virulence and antimicrobial resistance genes, and evaluate the in vitro biofilm-forming capability of bacterial isolates from these environments. One Salmonella Typhi isolate and 21 E. coli isolates were detected, while L. monocytogenes was not detected. S. Typhi was isolated from the carcass cooling chamber’s floor, resistant to several antimicrobials, including nalidixic acid, cefazolin, chloramphenicol, doxycycline, streptomycin, gentamicin, tetracycline, and sulfonamide, and contained resistance genes, such as tet(B), tet(C), tet(M), and ampC. It also showed moderate biofilm-forming capacity at 37°C after incubating for 72 h. The prevalence of the 21 E. coli isolates was also the highest on the carcass cooling chamber floor (three of the four samplings [75%]). The E. coli isolates were resistant to 12 of the 13 tested antimicrobials, and none showed sensitivity to chloramphenicol, an antimicrobial prohibited in animal feed since 2003 in Brazil. The resistance genes MCR-1, MCR-3, sul1, ampC, clmA, cat1, tet(A), tet(B), and blaSHV, as well as the virulence genes stx-1, hlyA, eae, tir α, tir β, tir γ, and saa were detected in the E. coli isolates. Moreover, 5 (23.8%) and 15 (71.4%) E. coli isolates presented strong and moderate biofilm-forming capacity, respectively. In general, the biofilm-forming capacity increased after incubating for 72 h at 10°C. The biofilm-forming capacity was the lowest after incubating for 24 h at 37°C. Due to the presence of resistance and virulence genes, multi-antimicrobial resistance, and biofilm-forming capacity, the results of this study suggest a risk to the public health as these pathogens are associated with foodborne diseases, which emphasizes the hazard of resistance gene propagation in the environment

Pesquisa e avaliação de formação de biofilmes de Listeria monocytogenes, Salmonella spp. e Escherichia coli em ambiente de abatedouro frigorífico de suínos

Dissertação (mestrado) — Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, Programa de Pós-Graduação em Saúde Animal, 2021.O objetivo deste estudo foi verificar a presença de Listeria monocytogenes, Salmonella e Escherichia coli, bem como realizar antibiograma, detectar genes de resistência antimicrobiana e virulência, e avaliar a capacidade de formação in vitro de biofilmes dos isolados de ambientes, equipamentos e utensílios de abatedouros frigoríficos de suínos localizados no Distrito Federal. Foram detectados 21 isolados de Escherichia coli, 01 isolado de Salmonella Typhi e não houve detecção de Listeria monocytogenes. A Salmonella Typhi foi isolada do piso da câmara de resfriamento de carcaças, e apresentou multirresistência a antimicrobianos, sendo eles o ácido nalidíxico, cefazolina, cloranfenicol, doxiciclina, estreptomicina, gentamicina, tetraciclina e sulfonamida, também foram detectados os genes de resistência tet(B), tet(C), tet(M) e ampC. Apresentou ainda capacidade moderada de formação de biofilme a 37°C após incubação de 72h. Em relação aos 21 isolados de E. coli, o local com o maior número de detecções foi o piso da câmara de resfriamento de carcaças, onde o microrganismo foi isolado em 3 (75%) das 4 coletas. Os isolados apresentaram resistência a 12 dos 13 antimicrobianos testados, sendo que nenhum isolado apresentou sensibilidade ao cloranfenicol, antimicrobiano de uso proibido na alimentação animal desde 2003 no Brasil. Houve presença dos genes de resistência MCR-1, MCR-3, sul1, ampC, clmA, cat1, tet(A), tet(B) e blaSHV, também foram detectados os genes de virulência stx-1, hlyA, eae, tir α, tir β, tir γ e saa nos isolados de E. coli. No que se refere à capacidade de formação de biofilme, verificou-se forte capacidade de formação de biofilmes em 5 (23,8%) dos isolados de E. coli e capacidade moderada em 15 (71,4%) isolados deste mesmo microrganismo. Em relação às diferentes temperaturas, tempo de incubação e seus locais de origem, o isolado oriundo da mesa de toalete do abatedouro frigorífico apresentou a maior capacidade de formação de biofilme, enquanto o oriundo da parede da câmara de resfriamento não formou biofilme em nenhuma das condições tempo e temperatura verificados neste estudo. No geral, os isolados apresentaram maior capacidade de formação de biofilmes a 10°C após 72h de incubação. A menor capacidade de formação de biofilmes foi verificada na temperatura de 37°C após 24h de incubação. Visto que os isolados apresentaram genes de resistência e virulência, multirresistência a antimicrobianos e capacidade de formação de biofilmes, os resultados deste estudo sugerem risco à saúde de pública devido a associação destes patógenos a doenças transmitidas por alimentos, sendo pertinente ressaltar o risco de propagação de genes de resistência no ambiente.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).The aim of this work was to verify the presence of Listeria monocytogenes, Salmonella and Escherichia coli, as well as perform antibiogram, detect genes of antimicrobial resistance and virulence, and evaluate the in vitro capacity of biofilm formation of strains isolated from swine slaughterhouse environments, equipment and utensils located at the Distrito Federal area, Brazil. Twenty-one strains of Escherichia coli and one strain of Salmonella Typhi were detected, there was no detection of Listeria monocytogenes. Salmonella Typhi was isolated from the cold storage chamber floor and showed multiresistance to antimicrobials (nalidixic acid, cefazolin, chloramphenicol, doxycycline, streptomycin, gentamicin, tetracycline and sulfonamide), were also detected the resistance genes tet(B), tet(C), tet(M) and ampC. The Salmonella strain showed a moderate capacity for biofilm formation at 37°C after 72h incubation. Regarding the 21 E. coli strains, the place with the highest number of detections was the cold storage chamber floor, where the microorganism was isolated in 3 (75%) of the 4 collected swab samples. The strains were resistant to 12 of the 13 antimicrobials tested, none of the isolates were sensitive to chloramphenicol, an antimicrobial that has been banned in animal feed since 2003 in Brazil. The resistance genes MCR-1, MCR-3, sul1, ampC, clmA, cat1, tet(A), tet(B), blaSHV, and the virulence genes stx-1, hlyA, eae, tir α, tir β, tir γ were detected in E. coli strains. Concerning the capacity of biofilm formation, a strong biofilm formation was found in 5 (23.8%) of the E. coli strains and a moderate biofilm formation in 15 (71.4%) strains of this same microorganism. Regarding the different temperatures, incubation period and their places of origin, the strain isolated from the abattoir evisceration table had the greatest capacity to form a biofilm, while the one from the cold storage chamber walls did not form biofilm under any of the time vs. temperature conditions in this study. In general, the strains showed higher capacity to form biofilms at 10°C after 72h incubation. The lowest capacity for biofilm formation was verified at 37°C after 24h of incubation. Since the strains presented resistance and virulence genes, multiresistance to antimicrobials and the capacity to form biofilms, the results of this study suggest a risk to public health due to the association of these pathogens with foodborne diseases, it is also pertinent to emphasize the risk of spreading resistance genes in the environment

Raw gel image of Fig 1 - PCR confirmation of <i>Salmonella</i> Typhi.

Row 1) 100-bp marker (Invitrogen®), row 2) negative control, row 3) positive control for Salmonella spp., 204-bp fragment (ompC primer), row 4) 204-bp fragment (ompC primer) for Salmonella spp., and 738-bp fragment (viaB primer) for Typhi serotype. Visualization on a 2% agarose gel stained with 0.5 μg/mL ethidium bromide in an ultraviolet transilluminator (Major Science®). (PDF)</p

<i>Salmonella</i> Typhi.

PCR confirmation of S. Typhi isolated from slaughterhouse A located in the Federal District of Brazil. 1) 100-bp marker (Invitrogen®), 2) negative control, 3) positive control for Salmonella spp., 204-bp fragment (ompC primer), 4) 204-bp fragment (ompC primer) for Salmonella spp. and 738-bp fragment (viaB primer) for Typhi serotype. Visualization on a 2% agarose gel stained with 0.5 μg/mL ethidium bromide in an ultraviolet transilluminator (Major Science®).</p

Resistance genes.

Oligonucleotides used for the antimicrobial resistance gene detection.</p

<i>E</i>. <i>coli</i> biofilm formation.

Biofilm-forming capacity in 21 E. coli isolates incubated for 24 h and 72 h at three different temperatures (37, 24 and 10°C). The classification is based on the parameters described by Stepanović et al. [62], where ODf is the final optical density of the isolates, and ODn is the negative control optical density. ODn = 0.064 and 0.086 in isolates incubated for 24 h and 0.086 in isolates incubated for 72 h, respecti-vely. The isolates were classified into non-biofilm-forming (NF) when ODf ≤ ODn, weak biofilm-forming (ODn (PDF)</p

<i>E</i>. <i>coli</i> genes’ detection and antibiograms.

Results of 21 E. coli antibiograms, detection of resistance and virulence genes, and detection points in slaughterhouses A and B.</p

<i>Salmonella</i> spp. research detection primers.

Oligonucleotides used for Salmonella spp. confirmation and serovar detection of Salmonella spp.</p

Virulence genes and <i>E</i>. <i>coli</i> serotypes.

Oligonucleotides used for virulence gene and serotype detection in E. coli strains.</p