Assessment of biofilm formation by Campylobacter spp. isolates mimicking poultry slaughterhouse conditions.

This research aimed to assess the biofilm formation ability of Campylobacter strains under temperature and oxygen stress conditions, similar to those found in the industrial environment, to explain the persistence of this pathogen on the poultry slaughter line. A collection of C. jejuni and C. coli isolates (n = 143) obtained from poultry samples (cecal content and neck skin), collected at slaughterhouse level, from diverse flocks, on different working days, was genotyped by flaA-restriction fragment length polymorphism (RFLP) typing method. A clustering analysis resulted in the assignment of 10 main clusters, from which 15 strains with different flaA-RFLP genotypes were selected for the assessment of biofilm formation ability and antimicrobial susceptibility. Biofilm assays, performed by crystal violet staining method, were conducted with the goal of mimicking some conditions present at the slaughterhouse environment, based on temperature, atmosphere, and contamination levels. Results indicated that many C. jejuni strains with similar flaA-RFLP profiles were present at the slaughterhouse on different processing days. All the strains tested (n = 15) were multidrug-resistant except for one. Biofilm formation ability was strain-dependent, and it appeared to have been affected by inoculum concentration, temperature, and tolerance to oxygen levels. At 10°C, adherence levels were significantly lower than at 42°C. Under microaerobic and aerobic atmospheres, at 42°C, 3 strains (C. jejuni 46E, C. jejuni 61C, and C. coli 65B) stood out, exhibiting significant levels of biofilm formation. C. jejuni strains 46E and 61C were inserted in clusters with evidence of persistence at the slaughterhouse for a long period of time. This study demonstrated that Campylobacter strains from broilers are capable of forming biofilms under conditions resembling the slaughterhouse environment. These results should be seen as a cue to improve the programs of hygiene implemented, particularly in those zones that can promote biofilm formation.

Short communication: Antimicrobial potential of Lactobacillus plantarum strains isolated from Slovak raw sheep milk cheeses.

The objective of this study was to screen Lactobacillus plantarum strains isolated from the traditional Slovak raw sheep milk cheese for their inhibitory potential. Seventy-two strains were obtained from samples of raw sheep milk and raw sheep milk cheeses collected from April 2017 to September 2018, in 23 geographical areas of Eastern Slovakia, by inoculation of de Man, Rogosa, and Sharpe agar plates (Oxoid, Basingstoke, UK). Using both the genus- and species-specific PCR methods, 43 strains were identified as Lactobacillus spp., and 10 strains were confirmed as Lb. plantarum. First, the whole bacterial cultures of Lb. plantarum strains were tested by disc diffusion assay. All showed very good antibacterial activities against 6 selected foodborne pathogens, including Escherichia coli, Salmonella Enteritidis, Pseudomonas aeruginosa, Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus. Then, cell-free neutralized supernatants and partially purified bacteriocins were prepared from the 4 Lb. plantarum strains that exhibited the best antibacterial potential, and they were tested the same way as the whole bacterial cultures. Seven of the 10 Lb. plantarum strains harbored the plnEF gene, 3 strains harbored the plnD gene, and 2 strains possessed both the plnA and plnC genes that encode the production of the respective plantaricins. The presence of both plnR and plnL genes was only detected in a single Lb. plantarum isolate. Based on the results of this study, 4 strains of Lb. plantarum appeared to be suitable candidates for further testing in the dairy manufacturing sector, particularly in the production of raw sheep milk products.

Genetic Characterization of Listeria monocytogenes Isolates from Industrial and Retail Ready-to-Eat Meat-Based Foods and Their Relationship with Clinical Strains from Human Listeriosis in Portugal.

Listeria monocytogenes isolates (n = 81) recovered from ready-to-eat meat-based food products (RTEMP) collected in industrial processing plants and retail establishments were genetically characterized for comparison with those from human clinical cases of listeriosis (n = 49). The aim was to assess RTEMP as a possible food source for human infection. L. monocytogenes was detected in 12.5% of the RTEMP samples, and in some cases, counts were above the European food safety criteria. All isolates were assessed by multiplex PCR for serogroup determination and detection of virulence-associated genes inlA, inlB, inlC, inlJ, plcA, hlyA, actA, and iap. Serogroups IIb and IVb dominated in RTEMP and human isolates, and all were positive for the assessed virulence genes. Antibiotic susceptibility testing by the disk diffusion method revealed a low level of resistance among the isolates. Pulsed-field gel electrophoresis (PFGE) of L. monocytogenes isolates, using restriction enzymes ApaI and AscI, revealed genetic variability and differentiated the isolates in five clusters. Although some pulsed-field gel electrophoresis profiles of particular RTEMP and human isolates seemed to be highly related, exhibiting more than 90% similarity, which suggests a possible common source, in most cases the strains were not genetically or temporally matched. The close genetic relatedness of RTEMP and human listeriosis strains stressed the importance of preventive measure implementation throughout the food chain.

Tracking Listeria monocytogenes contamination and virulence-associated characteristics in the ready-to-eat meat-based food products industry according to the hygiene level.

Listeria monocytogenes isolates collected from final products and food contact surfaces of 10 ready-to-eat meat-based food products (RTEMP) producing industries were analyzed to relate their virulence-associated characteristics and genetic profiles with the hygiene assessment of those industries. Together with sample collection, an audit was performed to evaluate the implemented food safety management system and to investigate the specific audit requisites more associated to the occurrence of those L. monocytogenes serogroups frequently related with human disease. L. monocytogenes was present in 18% of the samples. The isolates (n=62) were serogrouped and detection of virulence-associated genes inlA, inlB, inlC and inlJ, and also plcA, hlyA, actA and iap was done by multiplex PCR. After this initial characterization, selected isolates (n=31) were submitted to antibiotic resistance testing by the disk diffusion method for the currently most used human and veterinary antibiotics and resistance was low. These isolates were also subtyped by pulsed-field gel electrophoresis. Genotyping and serogrouping of L. monocytogenes isolates revealed a genetically diverse population. Our data indicate that contamination of final products does not seem to be uniquely related to the sampled food surfaces. The occurrence of those L. monocytogenes serogroups more commonly associated with human disease in industries with a high hygienic audit classification could be the result of a previous identification of the pathogen, with an enforcement of the hygiene program without recognizing the real source of contamination. This reinforces the importance of a conjoined diagnosis using audit data and microbiological testing. Food safety management systems of those industries need improvement, particularly in cleaning and sanitizing operations, analytical control, preventive maintenance, personal hygiene and root cause analysis.

Antimicrobial resistance among Campylobacter spp. strains isolated from different poultry production systems at slaughterhouse level.

The aim of the current work was to evaluate the prevalence and antimicrobial susceptibility of Campylobacter spp. isolated from different chicken production systems at the slaughterhouse level. Chicken sampling at slaughterhouse was performed for cecum, carcass, and breast meat from flocks of organic (n = 6), extensive indoor (n = 14), and intensive production (n = 14), totaling 34 ceca pools, 64 neck skin pools, and 132 breasts, representing 96,386 chickens. A collection of 167 strains were identified as Campylobacter coli (n = 85) and Campylobacter jejuni (n = 82) and were tested for susceptibility to 11 antimicrobial agents by the disk diffusion method. The frequency of Campylobacter in chicken samples from different production systems was between 79 and 100%. Campylobacter isolated from all origins were resistant to the fluoroquinolones studied (80-98%). However, for ciprofloxacin and ofloxacin, the Campylobacter isolates from extensive indoor chicken were significantly (P < 0.05) less resistant (77 and 58%) than that from organic (97 and 91%) and intensive production (96 and 95%). A high probability of tetracycline resistance occurrence was also found for the Campylobacter spp. tested (58% for C. jejuni and 76% for C. coli). A more frequent profile of multidrug resistance was noticed for isolates from intensive and organic production than for extensive indoor production. These results reinforce the need of efficient strategy implementation to control and reduce Campylobacter in chickens at production and slaughter levels, and the necessity to reduce the use of antimicrobials in poultry sector.

Biogenic amine formation in turkey meat under modified atmosphere packaging with extended shelf life: Index of freshness.

The aim of this study was to evaluate the effect of modified atmosphere packaging (MAP) on biogenic amine production in turkey meat according to its shelf life period, determining an index of freshness. Sliced meat samples of different meat quality categories (according to color and pH24) were individually packaged under aerobiosis (aerobic package) and in 6 different modified atmospheres containing different gas mixtures: MAP1, 50% N2/50% CO2; MAP2, 0.5% CO/50% CO2/49.5% N2; MAP3, 50% Ar/50% N2; MAP4, 0.5% CO/80% CO2/19.5% N2; MAP5, 100% N2; and MAP6, 50% Ar/50% CO2. All samples were stored at 0 ± 1°C in the dark for between 12 and 25 d. Meat samples packaged in aerobic packaging were analyzed for their microbial and physicochemical characteristics on d 0, 5, and 12 of storage, and then extended to 19 and 25 d when samples were under MAP. The production of biogenic amines analyzed in turkey meat increased over time. The values of putrescine, cadaverine, and tyramine increased significantly (P < 0.05) during storage time in samples packaged under aerobiosis, MAP3, and MAP5. Histamine was not detected in turkey meat packaged under study conditions, or when present, the levels were below the limit of quantification (1.03 mg/kg). Tyramine in turkey meat under MAP was not the best amine indicator of meat deterioration, with cadaverine being suggested instead, or the sum of the amines putrescine, cadaverine, and tyramine, to characterize and quantify meat freshness. After 25 d of storage, the meat packaged under MAP with a mixture containing a higher concentration of CO2 and with CO was the one with a lower index value (11.36 mg/kg), although not significantly different from the indices provided by the meat packaged with MAP1, 2, and 6.

Gas mixtures approach to improve turkey meat shelf life under modified atmosphere packaging: the effect of carbon monoxide.

Gas mixtures with CO have been applied to beef and pork meat, but no data have been reported regarding their application to poultry meat. The aim of this study was to evaluate the effect of an anaerobic gas mixture with CO on the growth of spoilage flora, color, and lipid oxidation stability of turkey meat under modified atmosphere packaging (MAP) stored at 0°C. Sliced meat samples were individually packaged under aerobiosis (aerobic packaging) and in 4 different modified atmospheres containing different gas mixtures: MAP 1, 50% N(2) and 50% CO(2); MAP 2, 0.5% CO, 50% CO(2), and 49.5% N(2); MAP 3, 0.5% CO, 80% CO(2), and 19.5% N(2); and MAP 4, 100% N(2). All the samples were stored at 0 ± 1°C in the dark for 12 to 25 d. Meat samples packaged in aerobic packaging were analyzed for their microbial and physicochemical characteristics on d 0, 5, and 12 of storage, which was extended to 19 and 25 d when samples were under MAP. For meat packaged with MAP 3, the total mesophilic and psychrotrophic counts were significantly lower (P < 0.001) than those observed in condition MAP 1. The introduction of CO, added to a higher concentration of CO(2), inhibited microbial flora in general, with particular action on Brochothrix thermosphacta. In terms of microbial quality, the shelf life of turkey meat under the MAP study conditions was longer than that of meat in aerobic packaging (5 d): 12 d for mixture MAP 4, 19 d for MAP 1 and MAP 2, and 25 d for MAP 3. Only MAP 4 without CO(2) or CO prevented lipid oxidation of the meat. The presence of CO in anoxic gas mixtures with CO(2) for turkey meat under MAP was useful, giving the bright pink color preferred by consumers without leading to the appearance of undercooked meat.

Antibiotic resistance and virulence factors among Enterococci isolated from chouriço, a traditional Portuguese dry fermented sausage.

Enterococci are ubiquitous microorganisms, found as part of the normal intestinal microbiota of many animals. They can be present in food products, for example, the Portuguese dry fermented sausage chouriço. Twenty enterococci were isolated from chouriço in two processing units; after identification and typification by conventional-molecular methods, the isolates were screened for virulence factors and antibiotic resistance. Identification allocated all enterococci to the species Enterococcus faecalis, and PCR fingerprinting demonstrated that each isolate was specific to the processing unit and chouriço from which it was recovered. Regarding the screening for virulence factors, 1 strain produced cytolysin and 4 were gelatinase positive, but none produced lipase. The ace gene was detected in 1 enterococci, ebpABC and efaA(fs) in 16 isolates each, esp in 3, fsrB in 5, gelE in 7, and cylA in 1. A multiresistant phenotype was observed in 8 isolates, 6 belonging to factory A. The antibiotic resistance gene ere(B) was detected in 9 enterococci, whereas the genes tet(M), aac(6')-Ie-aph(2''), and vanA were detected in 8 isolates each. As some of the E. faecalis chouriço isolates present a multiresistant profile and harbor virulence and/or resistance genes, to assess further the safety of Portuguese dry sausages, a larger number of products and processing units must by analyzed.

Distribution of aminogenic activity among potential autochthonous starter cultures for dry fermented sausages.

Any bacterial strain to be used as starter culture should have suitable characteristics, including a lack of amino acid decarboxylase activity. In this study, the decarboxylase activity of 76 bacterial strains, including lactic acid bacteria and gram-positive, catalase-positive cocci, was investigated. These strains were previously isolated from European traditional fermented sausages to develop autochthonous starter cultures. Of all the strains tested, 48% of the lactic acid bacteria strains and 13% of gram-positive, catalase-positive cocci decarboxylated one or more amino acids. Aminogenic potential was strain dependent, although some species had a higher proportion of aminogenic strains than did others. Thus, all Lactobacillus curvatus strains and 70% of Lactobacillus brevis strains had the capacity to produce tyramine and beta-phenylethylamine. Some strains also produced other aromatic amines, such as tryptamine and the diamines putrescine and cadaverine. All the enterococcal strains tested were decarboxylase positive, producing high amounts of tyramine and considerable amounts of beta-phenylethylamine. None of the staphylococcal strains had tyrosine-decarboxylase activity, but some produced other amines. From the aminogenic point of view, Lactobacillus plantarum, Lactobacillus sakei, and Staphylococcus xylosus strains would be the most suitable for use as autochthonous starter cultures for traditional fermented sausages.

The effect on turkey meat shelf life of modified-atmosphere packaging with an argon mixture.

There is a lack of knowledge related to the action of Ar on microbial development and prevention of oxidation when applied to raw meat under modified-atmosphere package (MAP). The aim of this study was to evaluate the effect of an anaerobic gas mixture with Ar on spoilage flora growth, color, and lipid oxidation stability of turkey meat under MAP stored at 0 degrees C. Breast muscles samples were collected on different working days from turkey carcasses (BUT9 and BIG6), fast-cooled in a tunnel (-2 degrees C, 2 m.s(-1), 90% RH) for 2 h and selected to be deboned according current practices in industrial slaughterhouses. The breasts were cut into slices that were individually packaged under aerobiosis (P0) and in 4 different modified atmospheres containing different gas mixtures as (P1) 100% N2, (P2) 50% Ar-50% N2, (P3) 50% Ar-50% CO2, and (P4) 50% N2-50% CO2. All samples were stored at 0+/-1 degrees C in the dark for between 12 and 25 d. Meat samples packaged in P0 were analyzed for their microbial and physicochemical characteristics on d 0, 5, and 12 of storage and then extended to 19 and 25 d when samples were under MAP. The microbial shelf life period extension of MAP sliced turkey meat compared with aerobic packaging (5-d shelf life) is 1 wk more for P1 and P2 mixtures, 2 wk for P4, and 3 wk for P3. The Ar-CO2 mixture was more efficient in delaying flora development than CO2-N2 with 1 log difference on the 25th day of storage, for total psychrotrophic counts, total anaerobic counts, and Brochothrix thermosphacta. The presence of Ar on gas mixtures did not seem to have any additional protective effect on lipid turkey meat oxidation.

Spoilage of light (PSE-like) and dark turkey meat under aerobic or modified atmosphere package: microbial indicators and their relationship with total volatile basic nitrogen.

1. The aim of this work was to evaluate the shelf life of turkey meat from different colour categories (Pale, Soft and Exudative (PSE)-like), intermediate and dark), packaged under aerobic or modified atmosphere (MAP) conditions; also to establish a relationship between microbial quality and total volatile basic nitrogen (TVB-N), evaluating its capacity for shelf life determination. 2. Breasts were selected according to luminance (L*) and pH(24): L >/= 51 and pH < 5.8 for light colour, 43 < L < 51 for intermediate colour, L 5.8 for dark colour. Sliced meat was packaged under aerobic or MAP conditions with 50% N(2) and 50% CO(2), then stored in the dark at 0 +/- 1 degrees C for periods of 12 or 25 d. Meat under aerobic conditions was evaluated for microbiological characteristics and TVB-N on d 0, 5 and 12. This evaluation was extended to include d 19 and 25 when samples were under MAP conditions. 3. The dark meat group after 12 d of storage in aerobiosis presented significantly higher plate counts of aerobic mesophilic, psychrotrophic micro-organisms and higher TVB-N than other meat colour categories. The shelf life of turkey meat under MAP was one week longer for intermediate and light colour meat (20 d) than for dark meat. TVB-N values of 20 to 30 mg NH(3)/100 g turkey meat correspond to advanced spoilage stages. We proposed 14 mg NH(3)/100 g as the limit of freshness acceptability for turkey meat. 4. TVB-N was an indicator of turkey meat microbial spoilage but was not a suitable early predictor for microbial spoilage and in particular for turkey meat stored under MAP conditions because counts of micro-organisms were moderately correlated (Pseudomonas spp. and Enterobacteriaceae) with this index, as they were inhibited by MAP gas mixture and storage temperature used in the present study.

Traditional dry fermented sausages produced in small-scale processing units in Mediterranean countries and Slovakia. 1: Microbial ecosystems of processing environments.

Microbial ecosystems were surveyed in 314 environmental samples from 54 Southern and Eastern European small-scale processing units (PUs) manufacturing traditional dry fermented sausages. The residual microflora contaminating the surfaces and the equipment were analysed after cleaning and disinfection procedures. All the PU environments were colonised at various levels by spoilage and technological microflora with excessive contamination levels in some of the PUs. Sporadic contamination by pathogenic microflora was recorded. Salmonella and Listeria monocytogenes were detected in 4.8% and 6.7% of the samples, respectively, and Staphylococcus aureus was enumerated in 6.1% of the samples. Several critical points were identified, such as the machines for S. aureus and the tables and the knives for L. monocytogenes; this knowledge is crucial for the improvement of hygiene control systems in small and traditional meat processing industries. The variability of the residual contamination emphasized the different cleaning, disinfecting and manufacturing practices routinely followed by these small-scale processing units.

Incidence of pectoralis major turkey muscles with light and dark color in a Portuguese slaughterhouse.

Turkey meat and processed products are very popular in Portugal. However, no studies have been made to assess turkey meat quality. The main objective of this study was to evaluate the quality of turkey breast meat in a Portuguese slaughterhouse, differentiating it to obtain better industrial management, performance, and consumer contentment. Nine hundred and seventy-seven male turkeys (from 16 to 20 wk old) from different flocks (BUT 9 and BIG 6) were evaluated to assess meat quality. Turkeys were slaughtered on different days, electrically stunned (225 V/3 s), and scalded in a vertical water bath at 81 degrees C/5 min. On the slaughter line, the pH and temperature were measured on the pectoralis muscle 15 min postmortem. The carcasses were fast-cooled in a tunnel (-2 degrees C/2 m.s(-1)/90% RH) for 2 h and kept in a refrigeration chamber (0 degree C/85% RH) until deboning (approximately 24 h postmortem). Color and pH 24 h postmortem (pH(24)) were measured on the pectoralis major muscle after carcass deboning. Pectoralis major muscles were selected according to criteria used by Barbut (1996) and drip loss, cooking loss, and total pigments analysis were performed on 67 different sliced meat samples. Muscles classified by pH decline rate, called rapid glycolytic, did not present final quality characteristics that could relate them with pale, soft, and exudative- (PSE) like meat, because there was no relationship between pH 15 h postmortem and lightness (L*), drip loss, or cooking loss. The differences, founded on physicochemical characteristics within pectoralis major muscles, allowed us to establish a criteria of turkey meat quality for dark and PSE-like meat, with L* < or = 44 and pH(24) > or = 5.8 and L* > or = 50 and pH(24) < 5.8, respectively. Based on criteria, the studied population presented 8.1% of carcasses with PSE-like muscles and 12.1% with dark muscles. The association of pH(24) and L* as criteria classification can be useful to classify turkey meat quality.

Influence of lairage environmental conditions and resting time on meat quality in pigs.

This study investigated the influence of lairage environmental conditions and resting time on pig carcasses and meat quality. The experimental material consisted of 1001 cross Pietrain-Duroc-Hampshire × Belgium-LR-LW pigs, held in lairage for either ≈30 min (direct slaughter) or between 2-3 h under 12 °C/90% relative humidity (RH), 20 °C/80% or 90% RH and 35 °C/50% or 85% RH. Prior to arrival at the lairage plant they were transported for about 45-60 min and subjected to a fasting period of 36 h before loading. Unloading operation and the driving of pigs to the point of stunning were carried out according to the practices used in the plant (sticks and electrical goads were used). Batches of 20-30 mixed pigs were used in each trial, held at a stocking density of approximately 0.55 m(2)/pig (≈100 Kg live weight). Lairage environmental conditions (LC), significantly affected almost all measurements, but not pH(1), in Semi-membranosus (SM) and Longissimus dorsi (LD) muscles and the carcass damage score. The influence of resting time (RT) was basically exerted on pH(u), deep ham temperature and in pH(1), of SM, the internal muscle reflectance being mostly unaffected. There were also significant batch (B) effects in a large range of parameters. Factors greatly interacted their influence on carcass and meat quality, denoting LC × B, LC × RT × B and LC × RT the most significant effects. RT × B only showed two low significant interactions for rigor value and pH(1), in SM, suggesting that, conversely to the lairage environmental conditions the influence of resting time is practically unaffected by the day of slaughter. The increase of lairage temperature decreased the frequency of normal carcasses, followed by an expressive higher incidence of PSE status. The influence of lairage relative humidity on the PSE/DFD muscle incidence depended on the associated temperature, but the most important detrimental effects were noticed in experiments carried out at 35 °C. In respect to lairage resting time, the influence on meat quality is strictly related to environmental conditions, mainly the temperature. Nevertheless, and excepting the assays at 35 °C/85% RH, direct slaughter of pigs (= 30 min in pens) generally produced less carcasses of normal quality than resting periods up to 2-3 h.