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

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

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

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

[EN]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.SIThis research was funded by the Junta de Castilla y León (Consejería de Educación, Spain, grant number LE018P20), the Ministerio de Ciencia, Innovación y Universidades (Spain, grant number RTI2018-098267-R-C33), and the Universidad de León (Spain, grant number 18BB282)

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

[EN]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.SIThis research was founded by the Junta de Castilla y León (Consejería de Educación, Spain, grant number LE018P20), and the Ministerio de Ciencia, Innovación y Universidades (Spain, grant number RTI2018-098267-R-C33)

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 healthMinisterio de Ciencia, Innovación y Universidades | Ref. RTI2018-098267-R-C33Junta de Castilla y León | Ref. LE164G1

Relationship with Clonal Lineages and Antimicrobial Resistance

Funding: NORTE-01-0145-FEDER-030101 the projects UIDB/CVT/00772/2020 and LA/P/0059/2020 funded by the Portuguese Foundation for Science and Technology (FCT). The Ministerio de Ciencia, Innovación y Universidades (Spain, grant number RTI2018-098267-R-C33) and the Junta de Castilla y León (Consejería de Educación, Spain, grant number LE018P20). Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.This study aimed to compare the biofilm formation ability of Staphylococcus aureus isolated from a wide range of animals and study the association between biofilm formation and antimicrobial resistance and genetic lineages. A total of 214 S. aureus strains isolated from pets, livestock, and wild animals were evaluated regarding their ability to form biofilms by the microtiter biofilm assay and their structure via confocal scanning laser microscopy. Statistical analysis was used to find an association between biofilm formation and antimicrobial resistance, multidrug resistance, sequence types (STs), spa and agr-types of the isolates. The antimicrobial susceptibility of 24 h-old biofilms was assessed against minimum inhibitory concentrations (MIC) and 10× MIC of amikacin and tetracycline, and the biomass reduction was measured. The metabolic activity of biofilms after antimicrobial treatment was evaluated by the XTT assay. All isolates were had the ability to form biofilms. Yet, significant differences in biofilm biomass production were detected among animal species. Multidrug resistance had a positive association with biofilm formation as well as methicillin-resistance. Significant differences were also detected among the clonal lineages of the isolates. Both tetracycline and amikacin were able to significantly reduce the biofilm mass. However, none of the antimicrobials were able to eradicate the biofilm at the maximum concentration used. Our results provide important information on the biofilm-forming capacity of animal-adapted S. aureus isolates, which may have potential implications for the development of new biofilm-targeted therapeutics.publishersversionpublishe

Exploring the Biofilm Formation Capacity in S. pseudintermedius and Coagulase-Negative Staphylococci Species

NORTE-01-0145-FEDER-030101 UIDB/CVT/00772/2020 LA/P/0059/2020 RTI2018-098267-R-C33 LE018P20The ability of biofilm formation seems to play an important role in the virulence of staphylococci. However, studies reporting biofilm formation of coagulase-negative staphylococci isolated from animals are still very scarce. Thus, we aimed to evaluate the biofilm-forming capacity of CoNS and S. pseudintermedius isolated from several animal species and to investigate the effect of conventional antimicrobials on biofilm reduction. A total of 35 S. pseudintermedius and 192 CoNS were included. Biofilm formation was accessed by the microtiter plate assay and the biofilms were stained by crystal violet. Association between biofilm formation and staphylococci species and antimicrobial resistance was also performed. Biofilm susceptibility testing was performed with tetracycline and amikacin at the minimum inhibitory concentration (MIC) and 10 × MIC. The metabolic activity of the biofilm cells after antimicrobial treatment was accessed by the XTT assay. All isolates formed biofilm, with S. urealyticus producing the most biofilm biomass and S. pseudintermedius producing the least biomass. There was a positive association between biofilm formation and multidrug resistance as well as resistance to individual antimicrobials. Neither tetracycline nor amikacin were able to eradicate the biofilm, not even at the highest concentration used. This study provides new insights into biofilm formation and the effects of antimicrobials on CoNS species.publishersversionpublishe