Lactic Acid Bacteria against Listeria monocytogenes

Background: Listeria monocytogenes is a pathogenic bacterium that can contaminate food and cause public health problems due its ability to form biofilms and resistance to sanitizers, it is responsible for sanitary and economic losses in food producing establishments. The difficulties in controlling biofilms and increasing resistance to traditional antibacterial agents is motivating studies of alternative potential biological agents for the control of pathogenic biofilms, among which lactic acid bacteria (LABs) are included. The objective of this work was to evaluate the activity of LABs against Listeria monocytogenes biofilm formation on polystyrene plates, a surface commonly used in the food industry.Materials, Methods & Results: Lyophilized commercial strains of Bifidobacterium animalis, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus salivaris and Lactobacillus acidophilus were used. The strain of Listeria monocytogenes (L4) was isolated from polystyrene mats from a poultry slaughterhouse cutting room and demonstrated the ability to attach to microplates and resistance to sanitizers (sodium hypochlorite and hydrogen peroxide) at all times, temperatures and tested surfaces. The antimicrobial activity of LABs was evaluated by the agar diffusion method. The LABs that presented action on Listeria monocytogenes were selected for the inhibition and/or removal of biofilms in microplates, and all experiments were carried out in triplicate. Only Bifidobacterium animalis and Lactobacillus plantarum demonstrated action against Listeria. monocytogenes in the agar diffusion assays and were selected for inhibition and competition assays. Furthermore, competition of LABs against Listeria monocytogenes adhesion was evaluated. There was no significant difference between LABs and Listeria monocytogenes, alone or in combination, at temperatures of 30ºC and 37ºC in the Listeria monocytogenes inhibition assays on polystyrene surface. The lactic acid bacteria evaluated did not demonstrate inhibition of Listeria monocytogenes adhesin testes with optical density visualization, however, it was possible to identify a reduction in Listeria monocytogenes counts with the application of Bifidobacterium animals and Lactobacillus plantarum in the testes of competition against biofilm formation. In competition tests Bifidobacterium animalis and Lactobacillus plantarum have an injunction in Listeria monocytogenes, indicating that these lactic acid bacteria can retard Listeria biofilm formation on polystyrene surfaces and thus help control the pathogen in the food industry.Discussion: A potential mechanism to control biofilm adhesion and formation of pathogens for nutrients and fixation on surfaces, multiplication factors and surfaces are a challenge in controlling biofilms of pathogenic microorganisms, alternative measures to traditional methods for inactivating pathogens and biofilm formers bacteria are necessary. In this sense, lactic acid bacteria generate high levels of bacteriocin and are effective in inhibiting the biofilm of pathogenic bacteria, however, our study did not reveal this. We verified that Bifidobacterium animalis and Lactobacillus plantarum have an inhibitory action on Listeria monocytogenes, indicating that these lactic acid bacteria can be used to delay the formation of biofilms by Listeria on polystyrene surfaces, helping to control this pathogen in food industry.Keywords: control of biofilm, pathogenic bacteria, food industry, polystyrene surface, FTDs

SEROVARS AND ANTIMICROBIAL RESISTANCE OF Salmonella spp. ISOLATED FROM TURKEY AND BROILER CARCASSES IN SOUTHERN BRAZIL BETWEEN 2004 AND 2006

Salmonella spp. causes diseases in fowls, when species-specific serovars (Salmonella Pullorum and S.Gallinarum) are present in flocks, and public health problems, when non-typhoid serovars are isolated, as well as possible bacterial resistance induced by the preventive and therapeutic use of antimicrobials in animal production. This study describes the serovars and bacterial resistance of 280Salmonella spp. strains isolated from turkey and broiler carcasses in Southern Brazil between 2004 and 2006. SalmonellaEnteritidis was the most prevalent serovar (55.7%), followed by Heidelberg (5.0%), Agona (4.3%), Bredeney (3.9%), Hadar (3.2%), and Typhimurium (2.9%). Tennessee and S. Enterica subspecies enterica(O: 4.5) were isolated only in turkeys, and Hadar (18.6%) was the most prevalent serovar in this species. Antimicrobial susceptibility tests were performed in 178 isolates (43 from turkeys and 135 from broilers). All isolates were sensitive to amoxicillin + clavulanic acid, polymyxin B, ciprofloxacin, and norfloxacin, and were resistant to bacitracin and penicillin. Broiler carcass isolates showed resistance to nalidixic acid (48.9%), nitrofurantoin (34.3%), neomycin (9.6%), tetracycline (5.2%), and kanamycin (8.9%); and turkey carcass isolates were resistant to nalidixic acid (62.8%), tetracycline (34.9%), and neomycin (30.2%), with a significant difference in turkeys when compared to broiler carcass isolates. These results indicate the need for judicious use of antimicrobials in livestock production, given that the serovars identified are potential causes of food poisoning

Campylobacter jejuni e Campylobacter coli EM CARCAÇAS DE FRANGO RESFRIADAS E CONGELADAS

Espécies de Campylobacter spp. termotolerantes são agentes de surtos de campilobacteriose em humanos e os produtos de origem avícola são considerados uma importante fonte de infecção. Foram identificados Campylobacter jejuni e Campylobacter coli em carcaças de frango resfriadas e congeladas coletadas em três abatedouros entre 2014 e 2015. A detecção de Campylobacter spp. foi realizada por microbiologia convencional e a identificação de C. jejuni e C. coli por multiplex-PCR. Dentre as amostras avaliadas verificou-se Campylobacter spp. termotolerante em 63,8%, sendo 72,2% em carcaças resfriadas e 55,5% em carcaças congeladas. Destas, 83,3% foram positivas para C. jejuni e 66,6% para C. coli, enquanto 50% foram positivas para ambas as espécies. A presença de Campylobacter spp. termotolerante em carcaças de frangos de corte prontas para consumo representa uma importante fonte de transmissão destes patógenos para humanos.Palavras-chave: abatedouros avícolas; campilobacteriose; termotolerantes

Campylobacter jejuni AND Campylobacter coli IN CHILLED AND FROZEN CHICKEN CARCASSES

Species of thermophilic Campylobacter spp. are agents of campylobacteriosis outbreaks in humans, and poultry products are implicated as major sources of infection. The detection of Campylobacter spp. was performed by conventional microbiology in poultry carcasses collected in slaughterhouses and species were identifiied by multiplex-PCR. Thermophilic Campylobacter spp. was verified in 63.8% of the samples, being 72.2% in chilled carcasses and 55.5% in frozen carcasses. Of these, 83.3% were positive for C. jejuni and 66.6% to C. coli, while 50% were positive for both. The presence of thermophilic Campylobacter spp. in ready-to-eat poultry products represents a potential source of human campylobacteriosis. Keywords: campylobacteriosis; poultry slaughterhouses; thermophilic bacteria

Salmonella spp. Isolated by Miniaturized Most Probable Number and Conventional Microbiology in Poultry Slaughterhouses

Background: Salmonella spp. are frequently isolated from fowls, and their detection in poultry products varies according to the breeding system and the slaughtering process, bringing risks to the consumer and compromising the marketability. The control of Salmonella in poultry slaughterhouses is based on the detection of bacteria, but the quantification of the agent would be important in assessing risk, as well as in obtaining data to determine the capacity of each step of the process to decrease or increase bacterial contamination. The aims of this study were to propose a method for the quantification of Salmonella in poultry slaughterhouses, frequency of isolation and serovars identified.Materials, Methods & Results: Twenty-one broiler flocks from seven federally inspected slaughterhouses in southern Brazil, totaling 1,071 samples, were assessed by miniaturized most probable number (mMPN) and conventional microbiology. The samples were collected in triplicate at 17 points, which included cloacae, transportation cages before and after sanitization, water (scald tank, supply, pre-chiller and chiller), and carcasses (before and after scalding, defeathering, rinsing, evisceration, final rinsing, chilling at 4ºC, and freezing at -12°C for 24 h, 30 and 60 days). Typical Salmonella colonies were submitted to TSI, LIA, SIM, urea, and polyvalent anti-O antiserum tests, and to final identification by Microarray by Check&Trace. Nine of the 1,071 (0.83%) samples analyzed by mMPN and by conventional microbiology were positive for Salmonella and the following serovars were identified: Anatum, Brandenburg, Agona, Tennessee, Bredeney, Schwarzengrund and Infantis.Discussion: This positive rate was lower than that described by other authors, whose rates ranged from 3% and 39% for the isolation of Salmonella spp. from different sources, such as slaughterhouses and retail sales in samples collected in Brazil. The low frequency of isolation of Salmonella in this study can be attributed to the efficiency of control systems used from the field to the slaughterhouse, such as Good Manufacturing Practices (GMP) and Sanitation Standard Operating Procedures (SSOP), which are HACCP requirements. Also, when slaughtering technology actions are properly managed, such as water replacement and temperatures lower than 4ºC in the chiller, the initial contamination by Salmonella spp. can be reduced, with a decline in contamination from 70% to 20%, and with a reduction in the contamination of broiler carcasses after chilling from 15.8% to 3.3%. On the other hand the contamination of carcasses by Salmonella before pre-chilling and in post-chilling might be due to the automated system, inadequate temperatures during chilling, and inappropriate water chlorination in the assessed meat-packing plant. Of the 17 points evaluated, seven were positive for Salmonella, especially the cages after sanitization and frozen carcasses. The contamination by Salmonella spp. in transportation cages after sanitization indicates inefficiency of the automated system as well as possible bacterial resistance to the sanitizers used in SSOP while the isolation in carcasses frozen for 24 h and 60 days demonstrates the thermal resistance of the bacterium to a conservation method widely used in the food industry. In this work, just one of the nine positive samples for Salmonella was identified by conventional methods (CM) and mMPN. The discrepancy between methods can be explained by the heterogeneous distribution of Salmonella and other bacteria in naturally contaminated samples. Samples that were positive in the qualitative test but negative in the mMPN protocol could have had a number of Salmonella below the detection amount

Número mais provável (NMP) de Salmonella sp. em cecos de frangos de corte e correlação com a população linfocitária bursal

A contaminação de produtos avícolas por Salmonella tem sido um real desafio à avicultura desde o reconhecimento da carne de frango e ovos como importantes fontes de transmissão de salmonelas para humanos. O estado imune das aves tem um papel crítico na defesa contra diferentes patógenos. Lotes de aves com imunossupressão sofrem aumento da incidência de infecções secundárias e têm seu desempenho afetado negativamente. Entre os patógenos virais aviários que têm capacidade imunossupressora encontra-se o vírus da doença infecciosa da bursa ou doença de Gumboro. Neste estudo, avaliou-se a população linfocitária bursal através de exame histopatológico e a contagem de Salmonella em cecos de frango de corte através do método do número mais provável (NMP) buscando-se correlacionar estes fatores. Foram coletados os cecos e as bolsas de Fabricius (BF) de 347 frangos de corte com 28 dias de idade. Os cecos foram congelados e analisados pela microbiologia convencional para determinar a presença ou ausência de Salmonella sp e as amostras positivas submetidas à técnica de contagem pelo NMP. Foram identificadas duas amostras positivas (0,58%) para Salmonella Enteritidis e estas apresentaram 0 e 21 NMP/g, enquanto os cecos correspondentes apresentaram entre 60-70% de depleção linfocitária. Outros gêneros bacterianos identificados foram Providencia rettgeri, Proteus mirabilis e E. coli. Os escores histopatológicos evidenciaram depleção linfocitária moderada a severa nas bursas, porém o isolamento de Salmonella Enteritidis em duas amostras não possibilitou maiores inferências a respeito do envolvimento da Doença de Gumboro quanto à persistência de salmonelas no ceco das aves analisadas, sob a presente metodologia

Biofilm formation by Salmonella enteritidis at different incubation temperatures

Background: The genus Salmonella, associated with poultry products, is considered the leading cause of foodborne outbreaks in humans in many countries. In Brazil, Salmonella Enteritidis (SE) is the serovar remains as one most frequently isolated from humans, and it is also a major serovar found in animals, food, animal feed, and environmental samples, despite all the efforts to control this pathogen. Also, the bacterium is able to form biofilms on different surfaces, protecting cells from both cleaning and sanitizing procedures in the food industries. This study aimed to verify the ability of Salmonella Enteritidis isolates to form biofilm on polystyrene at different incubation temperatures. Materials, Methods & Results: A total of 171 SE samples were isolated from foodborne outbreaks (foods and stool cultures) and poultry products between 2003 and 2010. The biofilm-forming ability of samples was measured at four different temperatures (3°C, 9ºC, 25ºC, and 36ºC), for 24 h, simulating temperatures usually found in poultry slaughterhouses. Later, 200 μL of each bacterial suspension was inoculated, in triplicate, onto 96-well, flat-bottomed sterile polystyrene microtiter plates, washed, after that, the biofilm was fixed with methanol. The plates were dried at ambient temperature, stained with 2% Hucker’s crystal violet. Afterwards, absorbance was read using an ELISA plate reader and the optical density (OD) of each isolate was obtained by the arithmetic mean of the absorbance of three wells and this value was compared with the mean absorbance of negative controls (ODnc). The following classification was used for the determination of biofilm formation: no biofilm production, weak biofilm production, moderate biofilm production and strong biofilm production. Results demonstrated all isolates from stool cultures and foods involved in foodborne outbreaks, at least one of the four temperatures tested, were able to form biofilm, even at 3°C, undescribed as possible for the growth of SE. SE strains from poultry products also formed biofilm at least at one of the temperatures. Discussion: The prevention of biofilms formation is very important, once they can be difficult to remove from utensils and food equipment surfaces, becoming a chronic source of microbial contamination of foods, possible dissemination of diseases, and increase of resistance to cleaning and sanitization procedures. A high ability for biofilm formation on plastic surfaces was observed. We may consider that Salmonella has the capacity to bind to surfaces, with relevant impacts on public health. Although biofilm formation could be affected by temperature, most of the SE isolates analyzed in our study were strong biofilm producers at all temperatures, including at 3°C, a temperature used for food preservation and until then not acknowledged as worrisome regarding the development of Salmonella spp. There is a common sense that maintenance of food at low temperatures, particularly below 5°C, is safer to consumers as low temperatures reduce microbial multiplication. However, our results show that the growth of SE in its sessile form is possible under refrigeration. These findings lead to the assumption that the ability of SE to form biofilms, especially at low temperatures, is related to its endurance in inhospitable environments, eventually infecting humans, and that may be one of the factors associated with the high prevalence of this serovar in outbreaks of foodborne diseases. To our knowledge, this is the first publication about biofilm formation by Salmonella Enteritidis at 3ºC

Detection and quantification of Campylobacter spp. in Brazilian poultry processing plants

Introduction: Campylobacteriosis is considered the most common bacteria-caused human gastroenteritis in the world. Poultry is a major reservoir of Campylobacter. Human infection may occur by consumption of raw and undercooked poultry or by contamination of other foods by these items. The aim of this study was to assess the prevalence of Campylobacter spp. in poultry processing plants with conventional culture method and real-time PCR. Methodology: A total of 108 poultry processing plant samples were collected to test with conventional microbiology and qPCR. Sampling included cloacal swabs, swabs of transport crates (before and after the cleaning and disinfection process) and carcasses (after the chiller, cooled at 4°C and frozen at −12°C). Results: Positivity in cloacal swabs indicated that poultry arrived contaminated at the slaughterhouse. Contamination in transport cages was substantially increased after the cleaning process, indicating that the process was ineffective. The detection of Campylobacter on carcasses was higher than that on cloacal swabs, which could indicate cross-contamination during the slaughtering process. Conventional microbiology and molecular methods revealed a prevalence of 69.4% and 43.5%, respectively. Lower detection by qPCR can be attributed to the high specificity of the kit and to biological components that could inhibit PCR reactions. Conclusions: Our results indicate that poultry arrive contaminated at the slaughterhouse and that contamination can increase during the slaughtering process due to cross-contamination. The isolation of Campylobacter in cooled and frozen carcasses corroborates the bacterial survival even at temperatures considered limiting to bacterial growth which are routinely used for food preservation

Fagotipos de Salmonella Enteritidis isoladas de amostras clínicas, alimentos e carcassas de frangos no sul do Brasil

Neste trabalho utilizou-se 272 isolados de Salmonella Enteritidis, dos quais 111 foram isolados de carcaças de frango congeladas, 126 de alimentos e material biológico de humanos envolvidos em episódios de toxinfecções alimentares e 35 de diferentes materiais de origem avícola Destas amostras, 111 foram fagotipadas, sendo 57,65% classificadas como fagotipo 4, 32,43% como fagotipo 4a, 3,60% como fagotipo 6a e 0,90% como fagotipo 7, enquanto 5,40% das amostras não foram fagotipáveis A predominância do fagotipo 4 está em concordância com os resultados publicados ao redor do mundo, e reforça a necessidade de estudos relacionados ao significado epidemiológico destes achados.272 isolates of Salmonella Enteritidis (111 isolated from frozen broiler chicken carcasses, 126 from human food and other biological materials involved in food poisoning outbreaks and 35 from different poultry materials) were selected for phage typing. From these, 111 were phage typed, 57.65% being classified as phage type 4, 32.43% as phage type 4a, 3.60% as phage type 6a and 0.90% as phage type 7, whereas 5.40% samples were not phage typeable. The predominance of phage type 4 is in agreement with the results published worldwide, and reinforces the need for studies related to the epidemiological meaning of these findings

Salmonella Enteritidis forma biofilme sob baixas temperaturas em diferentes superfícies da indústria de alimentos

We evaluated the influence of temperature on the ability of Salmonella Enteritidis (SE) to form biofilms on stainless steel, polyethylene, and polyurethane surfaces under different hygiene procedures. These materials were placed on SE culture and incubated at 42±1 ºC, 36±1 ºC, 25±1 ºC, 9±1 ºC, and 3±1 ºC for 4, 8, 12, and 24 h. Hot water at 45 ºC and 85 ºC, 0.5% peracetic acid solution, and 1% quaternary ammonia were used for hygienization. Biofilm formation occurred at all temperatures evaluated, highlighting at 3 ºC which has not been reported as an ideal temperature for the adhesion of SE to these materials. The SE adhered more often to polyethylene surfaces than to polyurethane and stainless steel surfaces (P<0.05). Peracetic acid and water at 85 ºC had similar hygienization efficiency (P<0.05) followed by quaternary ammonia whereas water at 45 ºC was not effective. SE adhered to these materials under low temperatures which to date have been deemed safe for food preservation.Avaliou-se o efeito da temperatura na capacidade de Salmonella Enteritidis (SE) formar biofilme em superfícies de aço inoxidável, polietileno e poliuretano e diferentes processos de higienização. Corpos de prova destes materiais foram postos frente a culturas de SE e incubados a 42±1 ºC, 36±1 ºC, 25±1 ºC, 9±1 ºC e 3±1 ºC por 4, 8, 12 e 24 horas. Para a higienização foram testados água aquecida a 45ºC e 85 ºC e soluções de ácido peracético 0,5% e amônia quaternária 1%. Verificou-se a formação de biofilmes em todas as temperaturas avaliadas, ressaltando-se a 3 ºC, ainda não citada como propícia para adesão de SE. Houve maior adesão ao polietileno do que ao poliuretano e ao aço inoxidável (P<0.05). Para higienização, o ácido peracético e a água a 85 ºC tiveram ação semelhante (P<0.05), seguidos por amônia quaternária, enquanto que a água a 45 ºC não foi eficaz. Todos os materiais avaliados propiciaram a aderência de SE, mesmo sob temperaturas baixas, consideradas até então seguras para a conservação dos alimentos