Papel de la industria alimentaria en el control de la resistencia bacteriana a los antibióticos

Discurso que estudia un problema de relevancia creciente en el ámbito de la Salud Pública, tal y como ha sido reconocido por numerosos organismos nacionales e internacionales mereciendo la pena destacar el importante papel del Veterinario en el control de la resistencia a los antibióticos, papel que muchas veces se ha infravalorado y que ira desgranando a lo largo de la intervención

Prevalence and types of methicillin-resistant Staphylococcus aureus (MRSA) in meat and meat products from retail outlets and in samples of animal origin collected in farms, slaughterhouses and meat processing facilities. A review

[EN]Methicillin-resistant Staphylococcus aureus (MRSA) is a frequent cause of nosocomial and community infections, in some cases severe and difficult to treat. In addition, there are strains of MRSA that are specifically associated with food-producing animals. For this reason, in recent years special attention has been paid to the role played by foodstuffs of animal origin in infections by this microorganism. With the aim of gaining knowledge on the prevalence and types of MRSA in meat and meat products, a review was undertaken of work published on this topic since 2001, a total of 259 publications, 185 relating to meat samples from retail outlets and 74 to samples of animal origin collected in farms, slaughterhouses and meat processing facilities. Strains of MRSA were detected in 84.3% reports (156 out of 185) from retail outlets and 86.5% reports (64 out of 74) from farms, slaughterhouses and meat processing facilities, although in most of the research this microorganism was detected in under 20% of samples from retail outlets, and under 10% in those from farms, slaughterhouses and meat processing facilities. The meat and meat products most often contaminated with MRSA were pork and chicken. In addition to the mecA gene, it is crucial to take into consideration the mecB and mecC genes, so as to avoid misidentification of strains as MSSA (methicillin-susceptible Staphylococcus aureus). The great variety of methods used for the determination of MRSA highlights the need to develop a standardized protocol for the study of this microorganism in foods.SIES16RFOP009 FEDER 2014-2020 DE CASTILLA Y LEÓN, Actuación:20007-CL - Apoyo Consorcio BUCL

The One Health approach in food safety: Challenges and opportunities

[EN] According to theWorldHealth Organization, contaminated food causes approximately 600 million cases of illness and 420,000 associated deaths worldwide each year. In this scenario, food safety is a crucial task, which is based in the application of correct hygiene practices throughout the entire food production chain: from farm to table. Ensuring food safety requires the implementation of practical, comprehensive, affordable, and effective approaches such as Hazard Analysis and Critical Control Points, Good Agricultural Practices, Good Manufacturing Practices, use of low cost technologies for traceability, regular inspection and auditing, as well as education and training, among other factors. In addition, it is essential to establish more comprehensive strategies based on the One Health approach. Since human health and animal health are interdependent and linked to the health of the ecosystems inwhich they coexist, collaboration between multiple disciplines is necessary to achieve optimal health for people, animals, and the environment.Within the food system, the One Health approach can be useful, for example, in the fight against zoonoses or antibiotic resistance. This paper describes various aspects related to the main biotic food-borne diseases, the problem of biofilms and the impact of different antimicrobials (whether biocides or antibiotics) on antibiotic resistance, listing a range of alternatives to the use of classic sanitizing compounds. All of this has the aim of giving an overview of the different factors, including those connected to the environment or animal health, which can influence food safety and thus public health.SIConsejería de Educación, Junta de Castilla y León, Grant/Award Number: LE018P2

Effect of low doses of biocides on the susceptibility of Listeria monocytogenes and Salmonella enterica to various antibiotics of clinical importance

[EN] The use of subinhibitory concentrations of biocides in food processing environments requires special attention because it is related to potential increases in antibiotic resistance. In this study, we determined the effect of exposure to low doses of four biocides (sodium hypochlorite, SHY; benzalkonium chloride, BZK; peracetic acid, PAA; trisodium phosphate, TSP) on the resistance to 10 antibiotics and on the hydrophobicity of the cellular surface of a strain of Listeria monocytogenes serotype 1/2a (LM) and a strain of Salmonella enterica serotype Agona (SA), both of meat origin. The cultures were exposed at 37 °C in Mueller Hinton II cation-adjusted broth with 0.6% yeast extract (with 0.2% of laky horse blood added in the case of LM) to increasing concentrations of the biocides, starting with half the minimum inhibitory concentration (MIC/2) and incrementing by 1.5 times the concentration until growth was no longer observed, calculating the MIC of the antibiotics before (control cultures) and after exposure. After exposure to TSP, LM was able to grow in the presence of a concentration of the biocide 2.53 times higher than the MIC of unexposed cultures. No adaptation was observed for SHY, BZK or PAA. SA demonstrated adaptation to BZK (it tolerated a concentration 1.13 times higher than the MIC for the unadapted strain) and PAA (2.53 times). LM cultures presented increased resistance (from susceptibility to reduced susceptibility, from susceptibility to resistance, or from reduced susceptibility to resistance) to erythromycin (strains exposed to BZK, PAA and TSP) and fosfomycin (all compounds). Regarding SA, after exposure its resistance to cefoxitin (all compounds), gentamicin (all compounds), tetracycline (TSP), fosfomycin (SHY, BZK and TSP) and enrofloxacin (BZK, PAA and TSP) increased. The cell surface hydrophobicity (determined through the microbial adhesion to solvents -MATS- test) increased (LM exposed to PAA and TSP; SA exposed to BZK) or decreased (SA exposed to PAA) after contact with the biocides. These findings suggest that the use of biocides at subinhibitory concentrations can contribute to the increase in bacterial resistance to antibiotics, in addition to modifying the hydrophobicity of the cellular surface, which is related to the capacity of bacteria to form biofilm.S

Effect of exposure to sub-inhibitory concentrations of biocides on the susceptibility to antibiotics of sessile cells of "Listeria monocytogenes" and "Salmonella enterica"

Journal Pre-proof. Publicación en abierto financiada por el Consorcio de Bibliotecas Universitarias de Castilla y León (BUCLE), con cargo al Programa Operativo 2014ES16RFOP009 FEDER 2014-2020 DE CASTILLA Y LEÓN, Actuación:20007-CL - Apoyo Consorcio BUCLE[EN] The effect of sub-inhibitory concentrations of several disinfectants on the antibiotic resistance of sessile cells of Listeria monocytogenes and Salmonella enterica was investigated. Biofilms were formed in the presence of biocides at MIC/2. Five compounds were used: peracetic acid (PAA), benzalkonium chloride (BZK), sodium hypochlorite (SHY), polyvinyl pyrrolidone iodine (PVI) and bis(3-aminopropyl)amine (BAPA). Two strains of L. monocytogenes (LM30 and LM28) and S. enterica (S. Typhimurium -ST- and S. Enteritidis -SE-) were used. Mono-species biofilms (MSB) and mixed biofilms (MB, LM30 + ST and LM28 + SE) were tested. LM30 went from susceptible to resistant to tetracycline and erythromycin after exposure to BAPA in MB. LM28 went from susceptible to resistant to tetracycline in the presence of PVI in MSB, from susceptible to intermediate to chloramphenicol after exposure to PAA in both MSB and MB, and from susceptible to resistant to ampicillin after exposure to SHY and BAPA in MB. ST went from intermediate to resistant to gentamicin after exposure to SHY, PVI and BAPA (MSB), or to PVI and BAPA (MB). This strain went from susceptible to intermediate to cefoxitin after exposure to BZK and PVI (MSB) or to BZK, PVI and BAPA (MB). For fosfomycin, ST went from susceptible to intermediate after exposure to PAA, PVI and PAPA (MSB) or to SHY, BZK, PVI and BAPA (MB). In the case of cephalothin, ST moved from intermediate to resistant category in MSB exposed to BAPA. SE moved from susceptible to intermediate to fosfomycin after contact with PAA in both MSB and MB. By contrast, in several occasions, strains increased their susceptibility to antibiotics after exposure to biocides. In some additional cases, contact with disinfectants caused significative modifications in the MIC of various antibiotics without the strains changing from one category to another. These findings show that contact with low concentrations of biocides can modify the antibiotic resistance of sessile cells of L. monocytogenes and S. enterica. The need to use adequate doses of disinfectants in food facilities in order to avoid changes in bacterial resistance is highlighted.SIJunta de Castilla y León (Consejería de Educación, Spain, grant number LE018P20

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

[EN] 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.S

Prevalence, quantification and antibiotic resistance of Listeria monocytogenes in poultry preparations

.A total of 100 samples of fresh poultry preparations were obtained from 10 retail outlets in North-Western Spain. Listeria spp. were found in 73 samples. Isolates were identified through polymerase chain reaction (PCR) as Listeria monocytogenes (56 samples), Listeria innocua (32), Listeria grayi (3), Listeria seeligeri (1) and Listeria spp. (6). In 24 samples, several different Listeria species were found. The loads of L. monocytogenes detected by quantitative polymerase chain reaction (q-PCR) in the 56 positive samples ranged from 0.05) on concentrations of L. monocytogenes. A total of 163 L. monocytogenes isolates were tested (disc diffusion) against 15 antimicrobials of clinical significance. The average number of resistances per isolate was 5.83 ± 1.64. All strains showed resistance to multiple antimicrobials (between 4 and 11). In all, 80 isolates (49.1%) showed a multi-drug resistant (MDR) phenotype, and two isolates (1.2%) showed an extensively drugresistant (XDR) phenotype. More than 50.0% of isolates showed resistance or reduced susceptibility to oxacillin, cefoxitin, cefotaxime, cefepime, rifampicin, ciprofloxacin, enrofloxacin or nitrofurantoin. This is a cause for concern because these substances are among the antibiotics used to treat human listeriosis, with rifampicin and fluoroquinolones frequently being used. The results from this research work show that poultry preparations are a potential major source of resistant L. monocytogenes strains, since these are present in some samples at high concentrations. This highlights the pressing need to handle poultry preparations correctly, so as to ensure they are sufficiently cooked and to avoid cross-contamination events.S

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) 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.Junta de Castilla y León | Ref. LE018P20Agencia Estatal de Investigación | Ref. RTI2018-098267-R-C33Agencia Estatal de Investigación | Ref. PID2022-142329OB-C3

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

[EN] 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) 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 testedSIThis research was funded by the Junta de Castilla y León (Consejería de Educación, Spain, grant number LE018P20) and the Ministerio de Ciencia e Innovación (Spain, grant numbers RTI2018-098267-R-C33 and PID2022-142329OB-C31

Viability and Virulence of Listeria monocytogenes in Poultry

Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/)[EN] 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.SIThis research was funded by the Junta de Castilla y León (Consejería de Educación, Spain, grant number LE018P20) and the Ministerio de Ciencia e Innovación (Spain, grant numbers RTI2018- 098267-R-C33 and PID2022-142329OB-C31). Sarah Panera-Martínez is the recipient of a predoctoral research fellowship from the Junta de Castilla y León (Consejería de Educación, Spain) co-financed by the European Social Fund