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.

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.

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.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) for Twelve Antimicrobials (Biocides and Antibiotics) in Eight Strains of Listeria monocytogenes.

When selecting effective doses of antimicrobials, be they biocides or antibiotics, it is essential to know the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of these substances. The present research determined the MICs and MBCs for three biocides, sodium hypochlorite (SH), benzalkonium chloride (BC), and peracetic acid (PAA), and nine antibiotics in eight strains of Listeria monocytogenes of varying serotypes. Marked intra-species differences were observed in the resistance of L. monocytogenes to the biocides and antibiotics. The MICs (ppm) for the biocides ranged between 1750 and 4500 for SH, 0.25 and 20.00 for BC, and 1050 and 1700 for PAA. Their MBCs (ppm) ranged from 2250 to 4500 for SH, 0.50 to 20.00 for BC, and 1150 to 1800 for PAA. The MICs (ppm) for antibiotics lay between 1 and 15 for ampicillin, 8 and 150 for cephalothin, 20 and 170 for cefoxitin, 0.05 and 0.20 for erythromycin, 4 and 50 for chloramphenicol, 3 and 100 for gentamicin, 2 and 15 for tetracycline, 2 and 80 for vancomycin, and 160 and 430 for fosfomycin. The corresponding MBCs (ppm) were from 5 to 20 for ampicillin, 9 to 160 for cephalothin, 70 to 200 for cefoxitin, 4 to 5 for erythromycin, 9 to 70 for chloramphenicol, 5 to 100 for gentamicin, 3 to 30 for tetracycline, 3 to 90 for vancomycin, and 160 to 450 for fosfomycin. Notably, erythromycin showed considerable efficacy, demonstrated by the low values for both MIC and MBC. Based on EUCAST and the CLSI criteria, all strains were susceptible to erythromycin. All strains were resistant to cephalothin, cefoxitin, gentamicin, and fosfomycin. Further values for resistance were 87.50% for ampicillin and vancomycin, 75.00% for tetracycline, and 62.50% for chloramphenicol. The high prevalence of antibiotic resistance is a matter for concern. A positive correlation was found between MIC and MBC values for most of the biocides and antibiotics. The higher the hydrophobicity of the cell surface, the higher the susceptibility to biocides, suggesting that surface characteristics of bacterial cells influence resistance to these compounds.

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.

Diversity, Antibiotic Resistance, and Biofilm-Forming Ability of Enterobacteria Isolated from Red Meat and Poultry Preparations.

A total of 44 samples of beef, pork, and poultry preparations were tested. Average counts (log cfu/g) of enterobacteria were 1.99 ± 0.99 (beef preparations), 1.96 ± 1.44 (pork), 2.09 ± 0.92 (chicken), and 2.17 ± 1.06 (turkey) (p > 0.05). Two hundred enterobacterial strains were identified and 13 genera (21 species) were distinguished, including species that are a significant cause of infection. The most common genera were Escherichia (32.5% of strains), Serratia (17.0%), Hafnia (12.5%), and Salmonella (12.0%). Isolates were screened by disc diffusion for susceptibility to 15 antibiotics. A total of 126 strains (63% of the isolates) were multirresistant (having resistance to two or more antibiotics), 46 (23%) were resistant to one antibiotic, and 28 (14%) were sensitive to all antibiotics. The average number of resistances per strain was 2.53 ± 2.05. A higher (p < 0.05) average number of resistances was observed in strains from turkey (3.14 ± 2.55) than in strains from beef (2.15 ± 1.22), pork (2.16 ± 1.39), or chicken (2.44 ± 2.22). At least 50% of strains showed resistance or reduced susceptibility to ampicillin, cefotaxime, ceftazidime, or streptomycin, considered to be "critically important" antimicrobial agents in human medicine. Seventy-nine strains (39.5%), 60 strains (30.0%), and 46 strains (23.0%) were weak, moderate, and strong biofilm producers (crystal violet assay), respectively. This investigation provides evidence that bacteria from red meat and poultry preparations pose major potential risk to consumers.

Effect of low doses of biocides on the antimicrobial resistance and the biofilms of Cronobacter sakazakii and Yersinia enterocolitica.

The susceptibility of Cronobacter sakazakii ATCC 29544 (CS) and Yersinia enterocolitica ATCC 9610 (YE) to sodium hypochlorite (10% of active chlorine; SHY), peracetic acid (39% solution of peracetic acid in acetic acid; PAA) and benzalkonium chloride (BZK) was tested. Minimum inhibitory concentration (MIC) values (planktonic cells; microdilution broth method) of 3,800 ppm (SHY), 1,200 ppm (PAA) and 15 ppm (BZK) for CS, and 2,500 ppm (SHY), 1,275 ppm (PAA) and 20 ppm (BZK) for YE, were found. In some instances, an increase in growth rate was observed in presence of sub-MICs (0.25MIC, 0.50MIC or 0.75MIC) of biocides relative to the samples without biocides. The cultures exhibited an acquired tolerance to biocides and an increase in antibiotic resistance after exposure to sub-MICs of such disinfectants. Strains were able to form strong biofilms on polystyrene after 48 hours (confocal laser scanning microscopy), with average biovolumes in the observation field (14,161 µm2) of 242,201.0 ± 86,570.9 µm3 (CS) and 190,184.5 ± 40,860.3 µm3 (YE). Treatment of biofilms for 10 minutes with disinfectants at 1MIC or 2MIC reduced the biovolume of live cells. PAA (YE) and BZK (CS and YE) at 1MIC did not alter the percentage of dead cells relative to non-exposed biofilms, and their effect of countering biofilm was due principally to the detachment of cells. These results suggest that doses of PAA and BZK close to MICs might lead to the dissemination of live bacteria from biofilms with consequent hazards for public health.

Persistent Listeria monocytogenes Isolates from a Poultry-Processing Facility Form more Biofilm but Do Not Have a Greater Resistance to Disinfectants Than Sporadic Strains.

Some strains of Listeria monocytogenes can persist in food-processing environments, increasing the likelihood of the contamination of foodstuffs. To identify traits that contribute to bacterial persistence, a selection of persistent and sporadic L. monocytogenes isolates from a poultry-processing facility was investigated for biofilm-forming ability (crystal violet assay). The susceptibility of sessile cells to treatments (five minutes) with sodium hypochlorite having 10% active chlorine (SHY: 10,000 ppm, 25,000 ppm, and 50,000 ppm) and benzalkonium chloride (BZK: 2500 ppm, 10,000 ppm, and 25,000 ppm) was also studied. All isolates exhibited biofilm formation on polystyrene. Persistent strains showed larger (p < 0.001) biofilm formation (OD580 = 0.301 ± 0.097) than sporadic strains (OD580 = 0.188 ± 0.082). A greater susceptibility to disinfectants was observed for biofilms of persistent strains than for those of sporadic strains. The application of SHY reduced biofilms only for persistent strains. BZK increased OD580 in persistent strains (2500 ppm) and in sporadic strains (all concentrations). These results indicate that the use of BZK at the concentrations tested could represent a public health risk. Findings in this work suggest a link between persistence and biofilm formation, but do not support a relationship between persistence and the resistance of sessile cells to disinfectants.

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.

Effects of Bacteriophage P100 at Different Concentrations on the Structural Parameters of Listeria monocytogenes Biofilms.

Because listeriosis is one of the deadliest foodborne diseases, controlling and eradicating Listeria monocytogenes biofilms is a serious challenge for food safety. Biofilms (24 h old) formed on polystyrene by a L. monocytogenes strain of food origin were exposed for a further 24 h to 12 different concentrations (from 100 to 1011 PFU/mL) of the bacteriophage P100 (Listex P100). The structural parameters of biofilms were studied by using confocal laser scanning microscopy and digital image analysis. The biovolume in the observation field (14,121 µm2) of control (untreated) biofilms was 237,333.1 ± 2,692.6 µm3. The biomass of treated biofilms ranged from 164.7 ± 89.0 µm3 (biofilms exposed to 1010 PFU/mL) to 231,170.5 ± 15,142.0 µm3 (100 PFU/mL). The lowest biomass was achieved after treatment with 108 PFU/mL, with no further decrease in biovolume when higher phage concentrations were used. A strong ( P < 0.001) correlation was found between phage concentration (log units) and biovolume (-0.965), surface coverage (-0.939), roughness (0.976), maximum thickness (-0.853), and average thickness (-0.965). Findings from this research suggest that bacteriophage P100 at concentrations equal to or greater than 8 log PFU/mL successfully removes L. monocytogenes biofilms from polystyrene surfaces.

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.

Lactic acid concentrations that reduce microbial load yet minimally impact colour and sensory characteristics of beef.

Lactic acid (LA) has recently been approved in the EU as beef decontaminant. In order to identify the most appropriate concentration, beef samples were spray-treated with LA (2%, 3%, 4% or 5%) or left untreated (control). Microbial load (aerobic plate counts, psychrotrophs and Enterobacteriaceae), pH, instrumental colour and sensory properties were investigated at 0, 24, 72 and 120h of refrigerated storage. The reductions in bacteria after spraying ranged from 0.57 to 0.95 log units. A residual antimicrobial effect was observed so that at 120h LA reduced microbial load by up to 2 log units compared with the control samples. Samples treated with 5% LA showed the lowest redness value (a*) and hedonic scores at all sampling times. Only for samples treated with 4% LA did the sensorial shelf-life limit extend beyond 120h. It is suggested that treatment of beef with 4% LA not only may improve microbiological quality, but also may enhance sensory properties and shelf-life.