Estimation by flow cytometry of percentages of survival of Listeria monocytogenes cells treated with tetracycline, with or without prior exposure to several biocides.

In certain circumstances, disinfectants are used at sublethal concentrations. The aim of this research work was to determine whether contact of Listeria monocytogenes NCTC 11994 with subinhibitory concentrations of three disinfectants widely used in food processing environments and in the health-care system, benzalkonium chloride (BZK), sodium hypochlorite (SHY) and peracetic acid (PAA), can cause the adaptation of the strain to the biocides and increase its resistance to tetracycline (TE). The minimum inhibitory concentrations (MIC; ppm) were 2.0 (BZK), 3500.0 (SHY) and 1050.0 (PAA). On exposure to increasing subinhibitory concentrations of the biocides, the maximum concentrations (ppm) of the compounds that allowed the strain to grow were (ppm) 8.5 (BZK), 3935.5 (SHY) and 1125.0 (PAA). Both the control cells (non-exposed) and the cells that had been in contact with low doses of biocides were treated with different concentrations of TE (0 ppm, 250 ppm, 500 ppm, 750 ppm, 1000 ppm and 1250 ppm) for 24, 48 and 72 h, and the survival percentages determined using flow cytometry, following dying with SYTO 9 and propidium iodide. The cells previously exposed to PAA presented higher survival percentages (P < 0.05) than the rest of the cells for most of the concentrations of TE and treatment times trialled. These results are worrying because TE is sometimes used to treat listeriosis, highlighting the importance of avoiding the use of disinfectant at subinhibitory doses. Furthermore, the findings suggest that flow cytometry is a fast and simple technique to obtain quantitative data on bacterial resistance to antibiotics.

Biovolume and spatial distribution of foodborne Gram-negative and Gram-positive pathogenic bacteria in mono- and dual-species biofilms.

The objective of this study was to characterize the biofilms formed by Salmonella enterica serotype Agona, Listeria monocytogenes, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) after 12, 48, 72, 120 and 240 h of incubation at 10 °C. Biofilms containing a single species, together with dual-species biofilms in which S. enterica and a Gram-positive bacterium existed in combination, were formed on polystyrene and evaluated by using confocal laser scanning microscopy (CLSM). All strains were able to form biofilm. The greatest biovolume in the observation field of 14,161 µm2 was observed for mono-species biofilms after 72 h, where biovolumes of 94,409.0 µm3 ± 2131.0 µm3 (S. enterica), 58,418.3 µm3 ± 5944.9 µm3 (L. monocytogenes), 68,020.8 µm3 ± 5812.3 µm3 (MRSA) and 59,280.0 µm3 ± 4032.9 µm3 (VRE) were obtained. In comparison with single-species biofilms, the biovolume of S. enterica was higher in the presence of MRSA or VRE after 48, 72 and 120 h. In dual-species biofilms, the bacteria showed a double-layer distribution pattern, with S. enterica in the top layer and Gram-positive bacteria in the bottom layer. This spatial disposition should be taken into account when effective strategies to eliminate biofilms are being developed.

Genomic and Metabolic Characteristics of the Pathogenicity in Pseudomonas aeruginosa.

In recent years, the effectiveness of antimicrobials in the treatment of Pseudomonas aeruginosa infections has gradually decreased. This pathogen can be observed in several clinical cases, such as pneumonia, urinary tract infections, sepsis, in immunocompromised hosts, such as neutropenic cancer, burns, and AIDS patients. Furthermore, Pseudomonas aeruginosa causes diseases in both livestock and pets. The highly flexible and versatile genome of P. aeruginosa allows it to have a high rate of pathogenicity. The numerous secreted virulence factors, resulting from its numerous secretion systems, the multi-resistance to different classes of antibiotics, and the ability to produce biofilms are pathogenicity factors that cause numerous problems in the fight against P. aeruginosa infections and that must be better understood for an effective treatment. Infections by P. aeruginosa represent, therefore, a major health problem and, as resistance genes can be disseminated between the microbiotas associated with humans, animals, and the environment, this issue needs be addressed on the basis of an One Health approach. This review intends to bring together and describe in detail the molecular and metabolic pathways in P. aeruginosa's pathogenesis, to contribute for the development of a more targeted therapy against this pathogen.

Implications of antibiotics use during the COVID-19 pandemic: present and future.

COVID-19 is caused by the SARS-CoV-2 virus, which has infected more than 4 million people with 278 892 deaths worldwide as of 11 May 2020. This disease, which can manifest as a severe respiratory infection, has been declared as a public health emergency of international concern and is being treated with a variety of antivirals, antibiotics and antifungals. This article highlights the administration of antimicrobials in COVID-19 patients worldwide, during the 2019-20 pandemic. It is imperative to be aware of the unreported amounts of antibiotics that have been administered worldwide in just a few months and a marked increase in antimicrobial resistance should therefore be expected. Due to the lack of data about antimicrobial use during this pandemic, the global impact on the emergence of new antimicrobial resistance is as yet unknown. This issue must be at the forefront of public health policymaking and planning in order that we are prepared for the potentially severe consequences for human and animal health and the environment.

High Efficacy of Ozonated Oils on the Removal of Biofilms Produced by Methicillin-Resistant Staphylococcus aureus (MRSA) from Infected Diabetic Foot Ulcers.

Ozone has a high wound healing capacity and antibacterial properties and can be used as a complementary treatment in infections. Methicillin-resistant S. aureus (MRSA) is the most common pathogen found in infected diabetic foot ulcers. Most of MRSA are resistant to several classes of antibiotics and, therefore, there is a need for new, effective, and well-tolerated agents. Thus, we aimed evaluate the antimicrobial and antibiofilm potentials of ozonated vegetable oils against MRSA strains isolated from diabetic foot ulcers. Six ozonated oils were produced with concentrations of ozone ranging from 0.53 to 17 mg of ozone/g of oil. The peroxide values were determined for each oil. Ozonated oils content on fatty acid was determined by gas chromatography equipped with a flame ionization detector. The antimicrobial susceptibility testing was performed by the Kirby-Bauer disk diffusion method and the effect of ozonated oils on biofilm formation ability and on established biofilms was investigated. In general, the content in identified unsaturated fatty acid in oils decreased with the increase of ozonation time and, consequently, the peroxide value increased. Most bacterial strains were inhibited by ozonated oil at a concentration of 4.24 mg/g. Ozonated oils had moderate to high ability to remove adhered cells and showed a high capacity to eradicate 24 h old biofilms. Our results show promising use of ozonated oils on the treatment of infections, in particular those caused by multidrug-resistant MRSA strains.

Susceptibility of Listeria monocytogenes planktonic cultures and biofilms to sodium hypochlorite and benzalkonium chloride.

The susceptibility of four L. monocytogenes isolates from pork to sodium hypochlorite (SHY) and benzalkonium chloride (BZK) was tested. Minimum inhibitory concentration (MIC) values of 3500 ppm (SHY), or between 3 ppm and 13 ppm (BZK), were found. Minimum bactericidal concentration (MBC) values ranged from 3500 ppm to 4500 ppm (SHY), and from 3 ppm to 14 ppm (BZK). The effect of SHY and BZK on the architecture and cellular viability of 24-h-old biofilms formed by such strains on polystyrene was determined through confocal laser scanning microscopy (CLSM) in conjunction with fluorescent dyes for live cells (SYTO 9) and dead cells (propidium iodide). Strains were able to form biofilm (biovolume values in the observation field of 14,161 µm2 ranged between 103,928.3 ±â€¯6730.2 µm3 and 276,030.9 ±â€¯42,291.9 µm3). Treatment of biofilms for 10 min with SHY (1MIC or 1.5MIC) or BZK (0.5MIC, 1MIC or 1.5MIC) decreased the biovolume of live (potentially dangerous) cells. SHY reduced the cellular viability of biofilms by more than 90%. On the other hand, BZK was able to remove most biofilm mass (live and dead cells), but decreased cellular viability only to a lesser extent, this suggesting strong biofilm detachment and dissemination of live cells.

Effect of Low Doses of Disinfectants on the Biofilm-Forming Ability of Listeria monocytogenes.

This study was intended to investigate the effect of contact with concentrations close to the minimum inhibitory concentration (MIC) (0.5, 1, and 1.5 MIC; MIC of planktonic cells was determined using a microdilution broth method) of sodium hypochlorite (SHY) or benzalkonium chloride (BZK) during the process of formation of biofilm (24 h), upon the architecture and viability of the biofilms formed by four L. monocytogenes isolates of molecular serotype 1/2a: S2-1 (BZK-susceptible strain; MICBZK = 3.0 ppm), S2-2 (BZK-resistant strain qacH positive; MICBZK = 13 ppm), CDL 69 (BZK-resistant strain bcrABC positive; MICBZK = 10 ppm), and S2BAC (BZK-resistant laboratory mutant of S2-1, with multidrug resistance phenotype; MICBZK = 9 ppm). Images were examined through confocal laser scanning microscopy after staining with SYTO 9 and Propidium Iodide. Biovolume values in the observation field (14,161 µm2) in the absence of biocides ranged from 103,928.3 ± 6,730.2 µm3 (S2BAC) to 276,030.9 ± 42,291.9 µm3 (S2-1). Exposure to SHY at 0.5 MIC reduced (p < 0.05) the biovolume of biofilms formed by S2-1 and S2BAC and did not modify (p > 0.05) the biovolume of biofilms by S2-2 and CDL 69. Exposure to sub-MICs of BZK decreased (p < 0.05; S2-1) or enhanced (p < 0.05; S2-2, CDL 69 and S2BAC) biofilm development. Exposure to biocides at 1 or 1.5 MIC inhibited biofilm formation. This study provides clear evidence that BZK at sub-MICs can enhance the biofilm-forming ability of BZK-resistant L. monocytogenes strains. Because biofilms contribute to the persistence of bacteria throughout the food chain and represent a major source of food contamination, our findings suggest the importance of avoiding sub-MICs of disinfectants in food-handling environments.

Effect of several packaging conditions on the microbiological, physicochemical and sensory properties of ostrich steaks during refrigerated storage.

A total of 365 ostrich steaks were packaged in air (AIR), vacuum (VAC), MAP1 (70% O2 + 30% CO2), MAP2 (30% O2 + 30% N2 + 40% CO2), MAP3 (20% O2 + 30% N2 + 50% CO2), MAP4 (50% N2 + 50% CO2), MAP5 (20% N2 + 80% CO2) or MAP6 (100% CO2). Microbial counts (10 groups), pH, Aw and sensory properties (nine-point hedonic scale) were determined on days 0, 1, 3, 7 and 15 of storage (4 °C). On day 0, microbial counts (log10 cfu/g) ranged from undetectable levels (Brochothrix thermosphacta, enterococci) to 3.21 ± 0.63 (total aerobic counts -TAC-). The highest and the lowest microbial loads throughout storage were observed in AIR and MAP6, respectively. On day 15 TAC as high as 9.96 ± 0.20 log10 cfu/g were found in AIR. The shelf-life (time until overall acceptability score fell below 5) was 3 days (MAP1, MAP2), 7 days (MAP3, AIR) or 15 days (MAP4, MAP5, MAP6). Only for VAC the shelf-life limit extended beyond 15 days.

Antibiotic susceptibility of methicillin-resistant staphylococci (MRS) of food origin: A comparison of agar disc diffusion method and a commercially available miniaturized test.

Methicillin-resistant staphylococci (MRS) are a major concern to public and animal health. Thirty MRS (Staphylococcus aureus, S. cohnii, S. epidermidis, S. haemolyticus, S. hominis, S. lentus, S. lugdunensis, S. sciuri, and S. xylosus) isolates from meat and poultry preparations were tested for antimicrobial susceptibility to 11 antimicrobials (belonging to seven different categories) of clinical significance using both the standard agar disc diffusion method and a commercially available miniaturized system (Sensi Test Gram-positive). It is worth stressing that 16 isolates (53.33%) exhibited an extensively drug-resistant phenotype (XDR). The average number of resistances per strain was 4.67. These results suggest that retail meat and poultry preparations are a likely vehicle for the transmission of multi-drug resistant MRS. Resistance to erythromycin was the commonest finding (76.67% of strains), followed by tobramycin, ceftazidime (66.67%), ciprofloxacin (56.67%) and fosfomycin (53.33%). An agreement (kappa coefficient) of 0.64 was found between the two testing methods. Using the agar disc diffusion as the reference method, the sensitivity, specificity and accuracy of the miniaturized test were 98.44%, 69.44% and 83.33%, respectively. Most discrepancies between the two methods were due to isolates that were susceptible according to the disc diffusion method but resistant according to the miniaturized test (false positives).

Structure and viability of 24- and 72-h-old biofilms formed by four pathogenic bacteria on polystyrene and glass contact surfaces.

The biofilms formed by Salmonella Hadar (SH174), Listeria monocytogenes (LM6), methicillin-resistant Staphylococcus aureus (MRSA125) and vancomycin-resistant Enterococcus faecium (VRE61s) on polystyrene and glass after 24 h and 72 h of incubation at 37 °C were examined by confocal laser scanning microscopy (CLSM) after staining with SYTO9 and propidium iodide (PI). A lower average biovolume (P < 0.05) was observed for SH174 biofilms (73,073.61 ±â€¯52,365.90 µm3 in the observation field of 14,161 µm2) than for LM6 (180,804.50 ±â€¯32,554.62 µm3), MRSA125 (208,605.34 ±â€¯57,534.93 µm3) and VRE61s (212,543.91 ±â€¯39,718.62 µm3) biofilms. SH174 showed the greatest (P < 0.05) biovolume on glass, as compared with polystyrene. Biofilms of LM6, MRSA125 and VRE61s were produced at comparable levels on both contact surfaces. After 24 h, SH174 formed small scattered cell clusters, and biovolume of biofilms increased (P < 0.05) after 72 h. By contrast, LM6, MRSA125 and VRE61s formed compact biofilms quickly (24 h) on both contact surfaces. Seventy-two-hour-old biofilms showed the largest biovolumes of dead or damaged (PI-stained) cells, except for MRSA125 (polystyrene) and VRE61s (polystyrene and glass). Appropriate procedures for the disinfection of food processing surfaces immediately after use are required to prevent the formation of biofilm by pathogenic bacteria.

Effect of sub-inhibitory concentrations of biocides on the architecture and viability of MRSA biofilms.

The effect of sub-minimum inhibitory concentrations (sub-MICs) of three biocides (benzalkonium chloride [BZK], trisodium phosphate [TSP] and sodium hypochlorite [SHY]) upon the architecture and viability of the biofilms formed by a methicillin-resistant Staphylococcus aureus strain of food origin (MRSA 48a) was investigated. Images were examined through confocal laser scanning microscopy (CLSM) after staining with SYTO9 and propidium iodide. Sub-MICs of BZK or TSP reduced the ability of MRSA to produce biofilm. In contrast, the presence of sub-MICs of SHY enhanced the biofilm-forming ability of MRSA when cells had undergone previous adaptation to this compound (biovolume in the observation field was 137,785.31 ± 47,682.79 µm3 for biofilms formed in the presence of SHY, and 70,204.13 ± 31,603.98 µm3 in the absence of biocides; P < 0.05). The largest amount of live (green stained) cells (P < 0.05) was observed in biofilms grown in the presence of SHY relative to the other conditions tested (58,999.75 ± 55,312.37 µm3vs 31,976.29 ± 38,594.98 µm3). Findings from the present work constitute the first report of biofilm production by MRSA being induced by sub-inhibitory concentrations of SHY. The data suggest that repeated use of SHY at low concentrations could represent a public health risk.

Effect of Sub-Lethal Concentrations of Biocides on the Structural Parameters and Viability of the Biofilms Formed by Salmonella Typhimurium.

This study aimed to investigate the effect of sub-minimum inhibitory concentrations (sub-MICs) of three food-grade biocides (benzalkonium chloride -BZK-, trisodium phosphate -TSP-, and sodium hypochlorite -SHY-) on Salmonella biofilms. The structural parameters and bacterial viability of the biofilms formed by a S. Typhimurium isolate from poultry was investigated by means of confocal laser scanning microscopy after staining with SYTO9 and propidium iodide. The MIC values for Salmonella cells before exposure to subinhibitory concentrations of biocides were 8.0 µg/mL (BZK), 18.0 mg/mL (TSP), and 6.0 mg/mL (SHY). The cultures exhibited a stable acquired tolerance to BZK and SHY. The maximum concentrations of biocides that allowed bacterial growth after several passages through gradually higher concentrations of such compounds were 30.4 µg/mL (BZK) and 10.1 mg/mL (SHY). The architecture and viability of S. Typhimurium biofilms varied in response to sub-MICs of different biocides. Previous adaptation to SHY enhanced (p < 0.001) biofilm formation (average biovolume in the observed field -14,161 µm2-: 139,856.15 ± 155,213.27 µm3) relative to unexposed cells (53,779.05 ± 55,535.62 µm3) and cells previously exposed to BZK (58,216.97 ± 58,644.45 µm3) or TSP (30,052.13 ± 28,290.56 µm3). This was particularly marked when biofilm was grown in the absence of biocides or in the presence of sub-MICs of SHY. The highest percentage of dead cells was shown by biofilms formed by cultures previously exposed to TSP relative to the other conditions tested (34.08% ± 13.74% vs. 23.70% ± 16.16%; p < 0.001). The importance of maintaining higher than MICs of SHY during sanitizing procedures to fight foodborne infections by Salmonella biofilms is highlighted.

Antimicrobial resistance and virulence genes in enterococci from wild game meat in Spain.

A total of 55 enterococci (45 Enterococcus faecium, 7 Enterococcus faecalis, and three Enterococcus durans) isolated from the meat of wild game animals (roe deer, boar, rabbit, pheasant, and pigeon) in North-Western Spain were tested for susceptibility to 14 antimicrobials by the disc diffusion method. All strains showed a multi-resistant phenotype (resistance to between three and 10 antimicrobials). The strains exhibited high percentages of resistance to erythromycin (89.1%), tetracycline (67.3%), ciprofloxacin (92.7%), nitrofurantoin (67.3%), and quinupristin-dalfopristin (81.8%). The lowest values (9.1%) were observed for high-level resistance to gentamicin, kanamycin, and streptomycin. The average number of resistances per strain was 5.8 for E. faecium isolates, 7.9 for E. faecalis, and 5.7 for E. durans. Genes encoding antimicrobial resistance and virulence were studied by polymerase chain reaction. A total of 15 (57.7%) of the 26 vancomycin-resistant isolates harboured the vanA gene. Other resistance genes detected included vanB, erm(B) and/or erm(C), tet(L) and/or tet(M), acc(6')-aph(2″), and aph(3')-IIIa in strains resistant to vancomycin, erythromycin, tetracycline, gentamicin, and kanamycin, respectively. Specific genes of the Tn5397 transposon were detected in 54.8% of the tet(M)-positive enterococci. Nine virulence factors (gelE, agg, ace, cpd, frs, esp, hyl, efaAfs and efaAfm) were studied. All virulence genes, with the exception of the frs gene, were found to be present in the enterococcal isolates. At least one virulence gene was detected in 20.0% of E. faecium, 71.4% of E. faecalis and 33.3% of E. durans isolates, with ace and cpd being the most frequently detected genes (6 isolates each). This suggests that wild game meat might play a role in the spreading through the food chain of enterococci with antimicrobial resistance and virulence determinants to humans.

Exposure of Escherichia coli ATCC 12806 to sublethal concentrations of food-grade biocides influences its ability to form biofilm, resistance to antimicrobials, and ultrastructure.

Escherichia coli ATCC 12806 was exposed to increasing subinhibitory concentrations of three biocides widely used in food industry facilities: trisodium phosphate (TSP), sodium nitrite (SNI), and sodium hypochlorite (SHY). The cultures exhibited an acquired tolerance to biocides (especially to SNI and SHY) after exposure to such compounds. E. coli produced biofilms (as observed by confocal laser scanning microscopy) on polystyrene microtiter plates. Previous adaptation to SNI or SHY enhanced the formation of biofilms (with an increase in biovolume and surface coverage) both in the absence and in the presence (MIC/2) of such compounds. TSP reduced the ability of E. coli to produce biofilms. The concentration of suspended cells in the culture broth in contact with the polystyrene surfaces did not influence the biofilm structure. The increase in cell surface hydrophobicity (assessed by a test of microbial adhesion to solvents) after contact with SNI or SHY appeared to be associated with a strong capacity to form biofilms. Cultures exposed to biocides displayed a stable reduced susceptibility to a range of antibiotics (mainly aminoglycosides, cephalosporins, and quinolones) compared with cultures that were not exposed. SNI caused the greatest increase in resistances (14 antibiotics [48.3% of the total tested]) compared with TSP (1 antibiotic [3.4%]) and SHY (3 antibiotics [10.3%]). Adaptation to SHY involved changes in cell morphology (as observed by scanning electron microscopy) and ultrastructure (as observed by transmission electron microscopy) which allowed this bacterium to persist in the presence of severe SHY challenges. The findings of the present study suggest that the use of biocides at subinhibitory concentrations could represent a public health risk.

Antibiotic-resistant bacteria: a challenge for the food industry.

Antibiotic-resistant bacteria were first described in the 1940s, but whereas new antibiotics were being discovered at a steady rate, the consequences of this phenomenon were slow to be appreciated. At present, the paucity of new antimicrobials coming into the market has led to the problem of antibiotic resistance fast escalating into a global health crisis. Although the selective pressure exerted by the use of antibiotics (particularly overuse or misuse) has been deemed the major factor in the emergence of bacterial resistance to these antimicrobials, concerns about the role of the food industry have been growing in recent years and have been raised at both national and international levels. The selective pressure exerted by the use of antibiotics (primary production) and biocides (e.g., disinfectants, food and feed preservatives, or decontaminants) is the main driving force behind the selection and spread of antimicrobial resistance throughout the food chain. Genetically modified (GM) crops with antibiotic resistance marker genes, microorganisms added intentionally to the food chain (probiotic or technological) with potentially transferable antimicrobial resistance genes, and food processing technologies used at sub-lethal doses (e.g., alternative non-thermal treatments) are also issues for concern. This paper presents the main trends in antibiotic resistance and antibiotic development in recent decades, as well as their economic and health consequences, current knowledge concerning the generation, dissemination, and mechanisms of antibacterial resistance, progress to date on the possible routes for emergence of resistance throughout the food chain and the role of foods as a vehicle for antibiotic-resistant bacteria. The main approaches to prevention and control of the development, selection, and spread of antibacterial resistance in the food industry are also addressed.

Decontamination treatments can increase the prevalence of resistance to antibiotics of Escherichia coli naturally present on poultry.

The main objective of this study was to determine the ability of various decontaminants to increase the prevalence of resistance to antibiotics in Escherichia coli populations on poultry. Chicken legs were dipped for 15 min into aqueous solutions (wt/vol) of trisodium phosphate (TSP; 12%), acidified sodium chlorite (ASC; 1200 ppm), ascorbic acid (AA; 2%) or citric acid (CA; 2%), or tap water (control). Samples were analyzed immediately after treatment (day 0) and after five days of storage at 7 ± 1 °C. A total of 250 E. coli isolates (50 from each group of samples; 25 on day 0 and 25 on day 5) were tested against twelve antibiotics of clinical significance by means of a standard disc-diffusion technique. A high prevalence of resistance to antibiotics was observed for E. coli strains from control samples, with three (6.0%) isolates sensitive, three (6.0%) resistant to one antibiotic and 44 (88.0%) isolates resistant to two or more antibiotics. Isolates from control samples had a lower prevalence of resistance than those from treated samples to ampicillin-sulbactam (P < 0.01, samples treated with TSP), amoxicillin-clavulanic acid (P < 0.001, ASC, AA and CA), cephotaxime (P < 0.05, TSP), trimethoprim-sulphamethoxazole (P < 0.05, AA; P < 0.01, CA), tetracycline (P < 0.01, CA), ciprofloxacin (P < 0.001, ASC; P < 0.05, AA; P < 0.01, CA) and nitrofurantoin (P < 0.01, TSP). These results suggest that the chemical decontaminants tested could favor the emergence, selection and/or proliferation of antibiotic-resistant strains in microbial populations on poultry meat.

Effect of Sodium Nitrite, Nisin and Lactic Acid on the Prevalence and Antibiotic Resistance Patterns of Listeria monocytogenes Naturally Present in Poultry.

The impact of treating minced chicken meat with sodium nitrite (SN, 100 ppm), nisin (Ni, 10 ppm) and lactic acid (LA, 3000 ppm) on the levels of some microbial groups indicating hygiene quality were investigated. Specifically, aerobic plate counts and culture-based counts of psychrotrophic microorganisms and enterobacteria were obtained. Additionally, the prevalence of Listeria monocytogenes and the resistance of 245 isolates from this bacterium to 15 antibiotics were documented. L. monocytogenes was isolated using the ISO 11290-1:2017 method and confirmed with polymerase chain reaction using the lmo1030 gene. Antibiotic resistance was established using the disc diffusion technique (EUCAST and CLSI criteria). Twenty-four hours after treatment, the microbial load (log10 cfu/g) was reduced (p < 0.05) relative to controls in those samples treated with LA, with counts of 5.51 ± 1.05 (LA-treated samples) vs. 7.53 ± 1.02 (control) for APC, 5.59 ± 1.14 (LA) vs. 7.13 ± 1.07 (control) for psychrotrophic microorganisms and 2.33 ± 0.51 (LA) vs. 4.23 ± 0.88 (control) for enterobacteria. L. monocytogenes was detected in 70% (control samples), 60% (samples receiving SN), 65% (Ni) and 50% (LA) (p > 0.05) of samples. All strains showed resistance to multiple antimicrobials (between 3 and 12). In all, 225 isolates (91.8%) showed a multi-drug resistant (MDR) phenotype, and one isolate (0.4%) showed an extensively drug-resistant (XDR) phenotype. The mean number of resistances per strain was lower (p < 0.01) in the control samples, at 5.77 ± 1.22, than in those receiving treatment, at 6.39 ± 1.51. It is suggested that the use of food additives might increase the prevalence of resistance to antibiotics in L. monocytogenes, although additional studies would be necessary to verify this finding by analyzing a higher number of samples and different foodstuffs and by increasing the number of antimicrobial compounds and concentrations to be tested.

Viability and Virulence of Listeria monocytogenes in Poultry.

The prevalence of Listeria monocytogenes in 30 samples of poultry was determined using culture-dependent (isolation on OCLA and confirmation by conventional polymerase chain reaction -PCR-, OCLA&PCR) and culture-independent (real-time polymerase chain reaction, q-PCR) methods. L. monocytogenes was detected in 15 samples (50.0%) by OCLA&PCR and in 20 (66.7%) by q-PCR. The concentrations (log10 cfu/g) of L. monocytogenes (q-PCR) ranged from 2.40 to 5.22 (total cells) and from <2.15 to 3.93 (viable cells). The two methods, q-PCR using a viability marker (v-PCR) and OCLA&PCR (gold standard), were compared for their capacity to detect viable cells of L. monocytogenes, with the potential to cause human disease. The values for sensitivity, specificity and efficiency of the v-PCR were 100%, 66.7% and 83.3%, respectively. The agreement between the two methods (kappa coefficient) was 0.67. The presence of nine virulence genes (hlyA, actA, inlB, inlA, inlC, inlJ, prfA, plcA and iap) was studied in 45 L. monocytogenes isolates (three from each positive sample) using PCR. All the strains harbored between six and nine virulence genes. Fifteen isolates (33.3% of the total) did not show the potential to form biofilm on a polystyrene surface, as determined by a crystal violet assay. The remaining strains were classified as weak (23 isolates, 51.1% of the total), moderate (one isolate, 2.2%) or strong (six isolates, 13.3%) biofilm producers. The strains were tested for susceptibility to a panel of 15 antibiotics. An average of 5.11 ± 1.30 resistances per isolate was observed. When the values for resistance and for reduced susceptibility were taken jointly, this figure rose to 6.91 ± 1.59. There was a prevalence of resistance or reduced susceptibility of more than 50.0% for oxacillin, cefoxitin, cefotaxime, cefepime ciprofloxacin, enrofloxacin and nitrofurantoin. For the remaining antibiotics tested, the corresponding values ranged from 0.0% for chloramphenicol to 48.9% for rifampicin. The high prevalence and level of L. monocytogenes with numerous virulence factors in poultry underline how crucial it is to follow correct hygiene procedures during the processing of this foodstuff in order to reduce the risk of human listeriosis.

Influence of serotype on the growth kinetics and the ability to form biofilms of Salmonella isolates from poultry.

The influence of the serotype on the growth behaviour and the ability to form biofilms of Salmonella enterica strains was investigated. The relationships between biofilm formation and growth kinetic parameters were also determined. A total of 69 strains (61 isolates from poultry and 8 reference strains from culture collections) belonging to 10 serotypes (S. enterica serotype Typhimurium, S. Newport, S. Paratyphi B, S. Poona, S. Derby, S. Infantis, S. Enteritidis, S. Virchow, S. Agona and S. Typhi) were tested. All Salmonella strains produced biofilms on polystyrene micro-well plates (crystal violet assay). Isolates were classified as weak (35 strains), moderate (22), or strong (12) biofilm producers. S. Agona and S. Typhi produced the most substantial (P < 0.001) biofilms. Growth curves were performed at 37 °C in tryptone soy broth by means of optical density (OD(420-580)) measurements from 0 to 48 h. Growth kinetic parameters (Gompertz model) varied between serotypes. The maximum growth rate (ΔOD(420-580)/h) ranged from 0.030 ± 0.002 (S. Typhi) to 0.114 ± 0.011 (S. Agona). The ability of Salmonella strains to form biofilms was not related to their growth kinetic parameters. The formation of biofilms by Salmonella on polystyrene constitutes an issue of concern because plastic materials are frequently used in food facilities. The findings suggest that special efforts must be made for the effective control of Salmonella in food-processing environments when S. Agona or S. Typhi strains are present.

Quantification of Total and Viable Cells and Determination of Serogroups and Antibiotic Resistance Patterns of Listeria monocytogenes in Chicken Meat from the North-Western Iberian Peninsula.

Twenty samples of minced chicken meat procured from butcher's shops in León (Spain; 10 samples) and Vila Real (Portugal; 10 samples) were analyzed. Microbial concentrations (log10 cfu/g) of 7.53 ± 1.02 (viable aerobic microbiota), 7.13 ± 1.07 (psychrotrophic microorganisms), and 4.23 ± 0.88 (enterobacteria) were found. The detection method described in the UNE-EN ISO 11290-1 standard (based on isolation from the chromogenic medium OCLA) with confirmation by the polymerase chain reaction (PCR; lmo1030) (OCLA−PCR), revealed Listeria monocytogenes in 14 samples (70.0% of the total), nine of Spanish origin and five of Portuguese (p > 0.05). The levels of viable and inactivated L. monocytogenes in the samples were determined with a q-PCR using propidium monoazide (PMAxx) as a viability marker. Seven samples tested positive both with the OCLA−PCR and with the q-PCR, with estimated concentrations of viable cells varying between 2.15 log10 cfu/g (detection limit) and 2.94 log10 cfu/g. Three samples tested negative both with the OCLA−PCR and with the q-PCR. Seven samples were positive with the OCLA−PCR, but negative with the q-PCR, and three samples tested negative with the OCLA−PCR and positive with the q-PCR. The percentage of viable cells relative to the total ranged between 2.4% and 86.0%. Seventy isolates of L. monocytogenes (five from each positive sample) were classified in PCR serogroups with a multiplex PCR assay. L. monocytogenes isolates belonged to serogroups IIa (52 isolates; 74.3%), IIc (7; 10.0%), IVa (2; 2.9%), and IVb (9; 12.9%). The susceptibility of the 70 isolates to 15 antibiotics of clinical interest was tested. The strains presented resistance to between three and eight antibiotics. The average number of resistances was greater (p < 0.001) among strains isolated from Spanish samples (6.20 ± 1.08), than in those from Portugal (5.00 ± 1.08). In both groups of strains, a prevalence of resistance higher than 95% was observed for oxacillin, cefoxitin, cefotaxime, and cefepime. The need to handle minced chicken meat correctly, taking care to cook it sufficiently and to avoid cross-contamination, so as to reduce the danger of listeriosis, is emphasized. A combination of culture-dependent and culture-independent methods offers complementary routes for the detection in food of the cells of L. monocytogenes in various different physiological states.