Analysis of two sampling treatments of beef for microbiome studies based on metataxonomic.

Microbial enumeration by serial dilution is one of the best resources to estimate cellular density for microbiological analysis. However, for metataxonomic analysis, it is not clear if serially diluted samples may accurately be used for metataxonomic analysis to represent species composition in beef samples. In this study, the effect of sampling preparation of beef samples on the bacterial composition was evaluated by the comparison of dilution and exudate. Based on the obtained results, data obtained from the exudate of the samples were more robust in terms of number of generated reads, but no significant differences in terms of biological diversity were observed (P < .05, Wicoxon Test). Besides, both sample preparation procedures evidenced equivalent results of bacterial composition as well as its relative abundances. In conclusion, the use of exudate allows bacterial enumeration and metataxonomic analysis, which is interesting for the point of view of food microbiologists as cellular loads and microbial composition of culturable and unculturable bacteria could be compared.

Presence and growth prediction of Staphylococcus spp. and Staphylococcus aureus in Minas Frescal cheese, a soft fresh cheese produced in Brazil.

Physical-chemical characteristics of Minas Frescal cheese (MFC) favor the growth of Staphylococcus spp. and allow the production of enterotoxins by specific strains. Here, we aimed to characterize the physical-chemical aspects (pH, storage temperature, and salt content) and the presence of Staphylococcus spp. in MFC samples (n = 50) to support a modeling study for the growth by this microorganism. Coagulase-positive staphylococci isolates were obtained and subjected to PCR assays to identify them as Staphylococcus aureus (nuc) and to detect staphylococcal enterotoxin-related genes (sea, seb, sec, sed, see). Staphylococcus aureus growth kinetics (maximum growth rate, Grmax, and lag time) were predicted based on ComBase model and MFC physical-chemical aspects. Mean counts of Staphylococcus spp. ranged from 3.3 to 6.7 log cfu/g, indicating poor hygiene practices during production. Selected isolates (n = 10) were identified as S. aureus, but none presented classical enterotoxin-related genes. pH, temperature, and salt content ranged from 5.80 to 6.62, 5°C to 12°C, and 0.85% to 1.70%, respectively. The Grmax values ranged from 0.012 to 0.419 log cfu/g per h. Independent of the storage temperature, the lowest Grmax values (0.012 to 0.372 log cfu/h) were obtained at pH 5.80 associated with salt content of 1.7%; independent of the pH and salt content, the best temperature to avoid staphylococcal growth was 7.5°C. Hygienic conditions during MFC production must be adopted to avoid staphylococcal contamination, and storage at temperatures lower than 7.5°C can prevent staphylococcal growth and the potential production of enterotoxins.

Short communication: Potential use of passion fruit (Passiflora cincinnata) as a biopreservative in the production of coalho cheese, a traditional Brazilian cheese.

Passion fruit (Passiflora cincinnata Mast.) is a native fruit from the Caatinga, a typical ecoregion in northeastern Brazil, and it has potential for use by the food and pharmaceutical industries. In this study, we characterized the antimicrobial activity of P. cincinnata and its application in the production of coalho cheese, a traditional Brazilian product. Aqueous extract of P. cincinnata exhibited high inhibitory activity against Listeria spp. (n = 4, reference strains), Staphylococcus aureus (n = 3, reference strains), and multidrug-resistant Staph. aureus (n = 8), and low inhibitory activity against lactic acid bacteria (LAB, n = 3, reference strains). Based on these results, we produced coalho cheese using goat milk with and without (control) passion fruit. Cheeses were stored at 10°C for 14 d and populations of mesophilic aerobes, Staph. aureus, and presumptive LAB were monitored at d 1, 7 and 14. The passion fruit cheese had lower counts of mesophilic aerobes, Staph. aureus (after 7 and 14 d), and presumptive LAB (after 14 d) than the control cheese. Adding ground passion fruit contributed to a reduction of Staph. aureus counts in goat cheese, although these differences were not significant. These results indicated the inhibitory potential of passion fruit and its potential use for controlling microbial populations in a cheese model; further studies are needed to characterize the active molecules that are responsible for such activity.

Technological properties of beneficial bacteria from the dairy environment and development of a fermented milk with the beneficial strain Lactobacillus casei MRUV6.

In this research paper we describe the technological properties of beneficial lactic acid bacteria (LAB) obtained from a dairy production chain and the development of a fermented milk produced with Lactobacillus casei MRUV6. Fifteen LAB isolates (Lactobacillus sp., Pediococcus sp. and Weissela sp.) presented acidifying abilities (pH ranges from 0.73 to 2.11), were able to produce diacetyl (except by 5 isolates) and exopolysaccharides, and two were proteolytic. L. casei MRUV6 was selected for producing a fermented milk, stored up to 35 d at 4 and 10°C. Counts on MRS agar with added vancomycin (10 mg/l) and MRS agar with added bile salts (1.5% w/v) ranged from 9.7 to 9.9 log CFU/g, independently of the tested conditions, indicating stability and intestinal resistance of L. casei MRUV6, despite some significant differences (P < 0.05). The study demonstrated the technological potential of a potential probiotic candidate strain, L. casei MRUV6, to be used as a starter culture in the dairy industry.

Whole-genome sequencing reveals Listeria monocytogenes diversity and allows identification of long-term persistent strains in Brazil.

Advances in whole-genome sequencing (WGS) technologies have documented genetic diversity and epidemiology of the major foodborne pathogen Listeria monocytogenes (Lm) in Europe and North America, but data concerning South America are scarce. Here, we examined the population structure and genetic diversity of this major foodborne pathogen collected in Brazil. Based on core genome multilocus sequence typing (cgMLST), isolates from lineages I (n = 22; 63%) and II (n = 13; 37%) were distributed into 10 different sublineages (SLs) and represented 31 new cgMLST types (CTs). The most prevalent SLs were SL9 (n = 9; 26%), SL3 (n = 6; 17%) and SL2 and SL218 (n = 5; 14%). Isolates belonging to CTs L2-SL9-ST9-CT4420 and L1-SL315-ST520-CT4429 were collected 3 and 9 years apart, respectively, revealing long-term persistence of Lm in Brazil. Genetic elements associated with stress survival were present in 60% of isolates (57% SSI-1 and 3% SSI-2). Pathogenic islands were present in 100% (LIPI-1), 43% (LIPI-3) and 6% (LIPI-4) of the isolates. Mutations leading to premature stop codons were detected in the prfA and inlA virulence genes. This study is an important contribution to understanding the genomic diversity and epidemiology of Lm in South America. In addition, the results highlight the importance of using WGS to reveal Lm long-term persistence.

Combined effect of bacteriocin produced by Lactobacillus plantarum ST8SH and vancomycin, propolis or EDTA for controlling biofilm development by Listeria monocytogenes.

The Listeria monocytogenes strains selected in the present study exhibited similar behavior in biofilm formation, independently of the tested conditions (bacteriocin from L. plantarum ST8SH, vancomycin, propolis (a natural antimicrobial product) and EDTA (chelating agent)), individual or in associations. The individual application of vancomycin had better inhibitory activity than that of propolis and EDTA; however, the association of the previously mentioned antimicrobial agents with bacteriocins resulted in better performance. However, when we compared the effects of vancomycin, propolis and EDTA, we could clearly observe that the combined application of bacteriocin and vancomycin was more effective than the combination of bacteriocin and propolis, and bacteriocin and EDTA. Considering the current need to reduce the use of antimicrobials and chemical substances in food processing, propolis can represent an alternative to improve the inhibitory effect of bacteriocins against L. monocytogenes biofilm formation, based on the obtained results. In general, high concentrations of bacteriocin produced by L. plantarum ST8SH were more effective in biofilm inhibition, and similar results were observed for vancomycin and propolis; however, all tested EDTA concentrations had similar effect on biofilm formation.

Listeria monocytogenes From Farm to Fork in a Brazilian Pork Production Chain.

ABSTRACT: Listeria monocytogenes contamination was assessed in different steps of a pork production chain. Ten lots of pigs were sampled at termination barns, at slaughter (after bleeding, after buckling, after evisceration, and after final washing), at processing (knives, deboning tables, and employees' hands), and of end products (ribs, shoulder, ham, and sausage). All samples (n = 670) were subjected to L. monocytogenes detection, and the obtained isolates (n = 18, identified as Listeria spp.) were characterized by their biochemical characteristics, serogroups, virulence genes, pulsed-field gel electrophoresis profiles, antibiotic resistances (ampicillin, penicillin, gentamicin, and sulfamethoxazole-trimethoprim), and adhesion abilities. The results revealed the low occurrence of Listeria spp. in the evaluated pork production chain. However, four tested sausage samples (40%) were positive for Listeria spp., with L. monocytogenes identified in two (20%) of these samples. Ten isolates were identified as L. monocytogenes (eight from serogroup 1/2a or 3a and two from serogroup 4b, 4d, or 4e): all isolates were also positive for the virulence-related genes hlyA, iap, plcA, actA, inlA, inlB, inlC, and inlJ and susceptible to the tested antibiotics. One sausage sample was contaminated by both serogroups 1/2a or 3a and 4b, 4d, or 4e. Isolates from serogroup 1/2a or 3a obtained during visits 5 and 6 presented distinct genetic profiles by pulsed-field gel electrophoresis, indicating that contamination may come from different sources. The adhesion potential exhibited by Listeria spp. isolates (n = 18) ranged from weak (serogroup 4b, 4d, or 4e) to moderate (L. innocua and L. monocytogenes serogroup 1/2a or 3a). Despite the low occurrence of L. monocytogenes, pathogenic serogroups were detected in sausages, demanding control measures by the industry.

Enumeration of starter cultures during yogurt production using Petrifilm AC plates associated with acidified MRS and M17 broths.

The efficiency of Petrifilm AC (3M Microbiology, St. Paul, MN, USA) associated with the broths M17 and de Mann-Rogosa-Sharpe (MRS) at pH 5.4 was evaluated to enumerate Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus during the yogurt production. Commercial and reference strains of these microorganisms were experimentally inoculated in nonfat milk and incubated at 42 degrees C for 4 h for yogurt production. At the moment of inoculation and after incubation, aliquots were collected, submitted to dilution using the broths M17 and MRS at pH 5.4, and plated for Strep. salivarius and Lb. bulgaricus enumeration according ISO 9232 and at Petrifilm AC plates, respectively. M17 plates were incubated at 42 degrees C, and MRS plates were incubated at 35 degrees C under anaerobiosis. After 48 h, the formed colonies were enumerated and the counts were compared by correlation and analysis of variance (P<0.05). In addition, colonies were randomly selected from all plates and characterized according to Gram staining and morphology. The obtained results indicated that Petrifilm AC plates associated to M17 and MRS at pH 5.4 can be considered as a suitable alternative for Strep. salivarius and Lb. bulgaricus enumeration during yogurt production, with slight interferences due to the acidity of MRS at the moment of inoculation, and due to the acidity of yogurt at the end of fermentation process. It was also observed that the MRS at pH 5.4 was not sufficiently selective for Lb. delbrueckii enumeration, despite it is indicated by the official protocol from ISO 9232.

Effect of selected feed additives to improve growth and health of dairy calves.

The aim was to evaluate the effect of different feed additives on intake, performance, and fecal consistency index (FCI) of dairy calves from 6-60 d of age and its residual effect 15 d after weaning. Fifty Holstein calves (38 ± 1.0 kg BW) were fed 5 L/d of milk plus starter feed until weaning, and corn silage and concentrate after weaning. The treatments were: control (CON), monensin (MON; 30 mg/kg of starter), probiotic E. faecium (PROB; 70 mg/kg of starter), essential oils (EO; 300 mg/kg of starter), or PROB + EO (EOPROB). Fecal score and dry matter intake (DMI) were measured daily, and animals were weighed every 15 d. A DNA extraction from feces was performed to identify the presence of microorganisms (E. coli, Hafnia, Shiguella, Lactobacillus spp, Enterococcus spp, and Enterococcus faecium NCIMB 10415) by PCR. Two 72-h digestibility trials were performed at days 20-28 and 50-56, by total fecal collection. The DMI before weaning was greater for EO (903.0 g/d) compared with MON (794.3 g/d) and EOPROB (783.1 g/d). The FCI decreased during pre-weaning for EO and MON. Average daily gain (ADG) and feed efficiency (FE) did not differ among treatments before weaning. After weaning, DMI and FCI did not differ among treatments. The EO had greater ADG (917.5 g/d) compared with CON (615.8 g/d) and PROB (592.6 g/d). The FE improved with EO (0.72 g/g) over CON (0.36 g/g), MON (0.49 g/g), and PROB (0.36 g/g). The PCR results showed absence of E. faecium NCIMB 10415 in animals fed PROB and CON. Animals fed PROB had greater intake of CP and NDF than animals fed EOPROB. The EO can be added to the dairy calf ration to improve fecal score and increase DMI. The pre-weaning FCI decrease with MON and increase with PROB.

Lactic Acid Bacteria (LAB) and Their Bacteriocins as Alternative Biotechnological Tools to Control Listeria monocytogenes Biofilms in Food Processing Facilities.

Bacteriocins are antimicrobial peptides produced by bacteria Gram-negative and Gram-positive, including lactic acid bacteria (LAB), organisms that are traditionally used in food preservation practices. Bacteriocins have been shown to have an aptitude as biofilm controlling agents in Listeria monocytogenes biofilms, a major risk for consumers and the food industry. Biofilms protect pathogens from sanitization procedures, allowing them to survive and persist in processing facilities, resulting in the cross-contamination of the end products. Studies have been undertaken on bacteriocinogenic LAB, their bacteriocins, and bioengineered bacteriocin derivatives for controlling L. monocytogenes biofilms on different surfaces through inhibition, competition, exclusion, and displacement. These alternative strategies can be considered promising in preventing the development of resistance to conventional sanitizers and disinfectants. Bacteriocins are "friendly" antimicrobial agents, and with high prevalence in nature, they do not have any known associated public health risk. Most trials have been carried out in vitro, on food contact materials such as polystyrene and stainless steel, while there have been few studies performed in situ to consolidate the results observed in vitro. There are strategies that can be employed for prevention and eradication of L. monocytogenes biofilms (such as the establishment of standard cleaning procedures using the available agents at proper concentrations). However, commercial cocktails using alternatives compounds recognized as safe and environmental friendly can be an alternative approach to be applied by the industries in the future.

Listeria monocytogenes: Occurrence in beef and identification of the main contamination points in processing plants.

This study aimed to establish the occurrence of Listeria spp., especially L. monocytogenes and its main serotypes, in beef and processing plants. A total of 443 samples were obtained from equipment, installations and products from 11 meat processing establishments from Paraná state, Brazil. All samples were analyzed using USDA methodology for Listeria spp. detection, followed by species identification. The occurrence of Listeria spp. in the samples was 38.1% of which 51.4% were from equipment, 35.4% from installations and 30.2% from products. The identified species were: L. monocytogenes (12.6%), L. innocua (78.4%), L. seeligeri (1.2%), L. welshimeri (7.2%) and L. grayi (0.6%). The identified serotypes of L. monocytogenes were 1/2a and 4b. The results demonstrate the significance of equipment and installations as sources of contamination by Listeria spp. and L. monocytogenes in the processing of beef and meat products.

Genotypic and antimicrobial characterization of pathogenic bacteria at different stages of cattle slaughtering in southern Brazil.

Meat can be contaminated in different stages of the slaughtering process and the identification of these stages is the starting point to implement adequate control measures. The objectives of this study were to assess the presence of pathogenic microorganisms in cattle carcasses, to identify the most important contamination points of the slaughtering process, and to evaluate the possible risk factors related to them in a cattle slaughterhouse. To this aim, 108 cattle carcasses were sampled at three stages of the slaughtering process: Point 1 (hides after bleeding); Point 2 (carcasses after hide removal); and Point 3 (carcasses immediately after division). Escherichia coli O157:H7, Listeria monocytogenes and Salmonella Livingstone were isolated from the carcasses. Phenotypic and genotypic characterization indicated that there was cross-contamination among animals, since bacteria with identical genotypic and phenotypic profiles were isolated from different animals at the same sampling day. Furthermore, this is the first report about the isolation of E. coli O157:H7 in a bovine slaughterhouse from southern Brazil.

Combined effect of bacteriocin produced by Lactobacillus plantarum ST8SH and vancomycin, propolis or EDTA for controlling biofilm development by Listeria monocytogenes / Efecto combinado de la bacteriocina producida por Lactobacillus plantarum ST8SH y la vancomicina, el própolis o el EDTA para controlar el desarrollo del biofilm producido por Listeria monocytogenes

The Listeria monocytogenes strains selected in the present study exhibited similar behavior in biofilm formation, independently of the tested conditions (bacteriocin from L. plantarum ST8SH, vancomycin, propolis (a natural antimicrobial product) and EDTA (chelating agent)), individual or in associations. The individual application of vancomycin had better inhibitory activity than that of propolis and EDTA; however, the association of the previously mentioned antimicrobial agents with bacteriocins resulted in better performance. However, when we compared the effects of vancomycin, propolis and EDTA, we could clearly observe that the combined application of bacteriocin and vancomycin was more effective than the combination of bacteriocin and propolis, and bacteriocin and EDTA. Considering the current need to reduce the use of antimicrobials and chemical substances in food processing, propolis can represent an alternative to improve the inhibitory effect of bacteriocins against L. monocytogenes biofilm formation, based on the obtained results. In general, high concentrations of bacteriocin produced by L. plantarum ST8SH were more effective in biofilm inhibition, and similar results were observed for vancomycin and propolis; however, all tested EDTA concentrations had similar effect on biofilm formation.

Las cepas de Listeria monocytogenes seleccionadas en el presente estudio presentaron comportamientos similares en la formación de biofilms, independientemente de los tratamientos a las que fueron sometidas (bacteriocina de Lactobacillus plantarum ST8SH, vancomicina, própolis (produto natural antimicrobiano) y EDTA (agentes quelante)), individual o en combinaciones. La aplicación individual de vancomicina presentó una mejor actividad inhibitoria frente a las aplicaciones individuales de própolis y de EDTA; sin embargo, la combinación de estos agentes antimicrobianos con las bacteriocinas resultó en un mejor desempeño. Se observó claramente que la aplicación combinada de bacteriocina y vancomicina fue más efectiva para controlar el desarrollo de biofilm en comparación con la combinación de la bacteriocina y el própolis o de la combinación de la bacteriocina y el EDTA. Considerando la necesidad actual de reducir el uso de sustancias antimicrobianas y químicas en el procesamiento de alimentos y sobre la base de los resultados obtenidos, se puede afirmar que el própolis representa una alternativa para mejorar el efecto inhibitorio de bacteriocinas contra la formación de biofilm de L. monocytogenes. En general, altas concentraciones de la bacteriocina producida por L. plantarum ST8SH fueron más eficaces en la inhibición del biofilm, y se observaron resultados similares para la vancomicina y el própolis; sin embargo, todas las concentraciones de EDTA evaluadas tuvieron un efecto similar en la formación de biofilm.

Screening and enumeration of lactic acid bacteria in milk using three different culture media in Petrifilm Aerobic Count plates and conventional pour plate methodology.

This study aimed to compare Petrifilm Aerobic Count (AC) plates and the conventional pour plate methodology using de Mann-Rogosa-Sharpe (MRS), Kang-Fung (KF) and Kang-Fung-Sol (KFS) culture media for screening and enumeration of lactic acid bacteria (LAB) in milk. Suspensions of 10 LAB species in reconstituted powder skim milk and 30 raw milk samples, without experimental inoculation, were tested. For selective enumeration, all samples were previously diluted in MRS, KF and KFS broths and then plated in Petrifilm AC and conventional pour plate methodology, using the same culture media with added agar. All plates were incubated at 37 degrees C for 48 h in anaerobic conditions. Differences in the counts were observed only for raw milk samples using KFS in conventional methodology, when compared with the counts obtained from MRS and KF (P0.05). The results showed excellent correlation indexes between both methodologies using the three culture media for LAB suspensions (r=0.97 for MRS, KF and KFS). For raw milk samples, the correlation indexes were excellent (r=0.97, for MRS) and good (r=0.84 for KF, and r=0.82 for KFS), showing some interference in Petrifilm AC when supplements were added, especially lactic acid. These results indicate the possibility of using Petrifilm AC plates for enumeration of LAB in milk, even with the use of selective supplements.

Frequency of 2, 3, 5-triphenyltetrazolium chloride (TTC) non-reducing bacteria in pasteurized milk

2, 3, 5-triphenyltetrazolium chloride (TTC) is a dye largely used for enumeration of microbial colonies in solid culture media, being a key component of the dry rehydratable film system used for microbiological analysis of food. This dye is colorless in the oxidized form and red when reduced by microorganisms, due to formation of formazan. In this study, TTC was added to Plate Count Agar (PCA) for enumeration of microorganisms in thirty four pasteurized milk samples, with the aim to verify the frequency of microorganisms that are unable to reduce TTC. Milk samples were decimally diluted in saline and pour-plated in PCA plus 0.015(per cent) TTC. Colonies were counted after 24h and 48 h of incubation at 35(degree)C. From a total of 50,574 colonies 19,665(38.88 per cent) did not reduce TTC in 48h. It was observed that 571(6.36 per cent) colonies that were colorless in 24h became red in 48h. From those that didn't reduce TTC in 48h, 233 were purified and Gram stained. 229(98.71 per cent) of them were Gram positive cocci and bacilli. The results show that there is a high percentage of microorganisms unable to reduce TTC in pasteurized milk, which cannot be detected by laboratory procedures based on the formation of red colonies.