Abstract
Listeria monocytogenes is still recognized as being commonly susceptible to antibiotics; however, there have been reports of reduced susceptibility in recent years. The significance of this resistance is not clear, in part due to the disparity in the antimicrobial susceptibility testing methods used. EUCAST (European Committee on Antimicrobial Susceptibility Testing) has recently proposed a standardized method for antibiotic susceptibility testing of L. monocytogenes. The aim of this work was to evaluate the susceptibility to 11 antibiotics in clinical use of 50 pulsed-field gel electrophoresis types of L. monocytogenes representing 347 isolates from a poultry industry setting using the EUCAST method and to compare the results with those obtained 10 years before. All poultry strains were sensitive to all the antibiotics tested but one strain was resistant to benzylpenicillin according to the EUCAST criteria. The current findings supported the previous study and confirmed that in certain food-associated L. monocytogenes populations, antibiotic sensitivity has remained stable.
Introduction
Listeria monocytogenes is a bacterium widely distributed in the environment of agri-food industries (Orsi et al, 2024). It causes listeriosis, an infectious disease that recorded the highest hospitalization and mortality rates (96.0 and 18.1%, respectively) in the European Union in 2022 among foodborne diseases (EFSA [European Food Safety Authority]; ECDC [European Centre for Disease Prevention and Control], 2024). One of the major public health challenges of the 21st century is the emergence and spread of antibiotic-resistant bacteria. L. monocytogenes appears to show low resistance to the antibiotics used to treat human cases of listeriosis (Baquero et al., 2020; Moura et al., 2024), although there are conflicting reports on the significance of this resistance, partly due to the disparity in the antibiotic susceptibility testing methods used. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) has recently proposed a standardized method for antibiotic susceptibility testing by disk diffusion with clinical cut-off points for minimum inhibitory concentrations (MICs) based on the epidemiological cut-off (ECOFF) of L. monocytogenes (EUCAST, 2024).
The aim of this work was to evaluate the susceptibility to 11 antibiotics in clinical use of 50 pulsed-field gel electrophoresis (PFGE) types of L. monocytogenes representing 347 isolates from a poultry industry (López-Alonso et al., 2020) using the EUCAST standardized disk diffusion method (EUCAST, 2024) and to compare the results with those obtained 10 years before (Ortiz et al., 2014).
Materials and Methods
Food-related strains and standard culture methods
A total of 50 L. monocytogenes strains of 50 different subtypes obtained by PFGE, representing 347 isolates from the poultry industry, were selected (López-Alonso et al., 2020). Clustering of the 50 initial PFGE types according to the protocol standardized and optimized by PulseNet for L. monocytogenes revealed 39 different PFGE types (López-Alonso et al., 2020). Although the number of strains is low (n = 50), they represented 347 isolates. Thus, the different PFGE types must be considered an added value for a study of susceptibility testing of 39 genetically different strains. The origin of all L. monocytogenes isolates was a poultry production company in Spain. L. monocytogenes was detected at three production stages: a broiler abattoir, a processing plant, and retail stores marketing fresh poultry products from the same company. These three stages spanned three locations in three provinces of Spain (López-Alonso et al., 2020).
Strains were stored in tryptic soy yeast extract broth (TSYEB) containing 15% glycerol at −20°C prior to use. Before each test, bacteria were subcultured on two consecutive days on Mueller-Hinton agar (MHA). TSYEB and MHA were obtained from Sigma–Aldrich (Spain) and Scharlab (Spain), respectively.
Antimicrobial susceptibility testing
Mueller-Hinton agar with 5% defibrinated horse blood and 20 mg/L β-NAD (Mueller-Hinton Fastidious, MH-F) obtained from Becton Dickinson (Spain) was used. Antibiotic disks obtained from Oxoid (Spain) included the five antibiotics contained in the standardized method (ampicillin [AMP 2 µg], benzylpenicillin [PEN 1 U], erythromycin [ERY 15 µg], meropenem [MEM 10 µg], and trimethoprim-sulfamethoxazole [SXT 1.25–23.75 µg]), and six other antibiotics not included in the standardized method [ciprofloxacin (CIP 5 µg), chloramphenicol (C 30 µg), gentamicin (CN 10 µg), rifampicin (RD 5 µg), tetracycline (TET 30 µg), and vancomycin (VAN 30 µg)]. The inhibition zones were interpreted according to EUCAST criteria for L. monocytogenes or, alternatively, for Staphylococcus spp. L. monocytogenes ATCC BAA-679 and Staphylococcus aureus ATCC 29213 were used as controls.
Results and Discussion
All poultry industry-related strains were sensitive to four of the five antibiotics included in the EUCAST standardized method for L. monocytogenes (EUCAST, 2024) and also to the six antibiotics that are not included in the standardized method for L. monocytogenes, with which the Staphylococcus cut-off points were used (Fig. 1). Only one strain out of the 50 poultry industry-related strains was resistant to benzylpenicillin according to the EUCAST criteria (EUCAST, 2024) (Fig. 1).
Distribution of inhibition diameters for 50 strains of L. monocytogenes against 11 antibiotics (ampicillin [AMP], benzylpenicillin [PEN], erythromycin [ERY], meropenem [MEM], trimethoprim-sulfamethoxazole [SXT], ciprofloxacin [CIP], chloramphenicol [C], gentamicin [CN], rifampicin [RD], tetracycline [TET], and vancomycin [VAN]). Dotted lines correspond to EUCAST zone diameter breakpoints of the five antibiotics included in the standardized method for L. monocytogenes (AMP, PEN, ERY, MEM, and SXT) and the six antibiotics that are not included in the standardized method for L. monocytogenes, with which the Staphylococcus cut-off points were used (CIP, C, CN, RD, TET and VAN) (EUCAST, 2024).
In contrast to other published work on antibiotic-resistant L. monocytogenes, but not confirmed by further study of resistant strains, acquired antibiotic resistance in this population representing 347 L. monocytogenes isolates from a poultry industry is very low. For example, Hailu et al. (2021) reported that 89.5% of 67 L. monocytogenes isolates from dairy cattle and poultry manure were found to have ampicillin resistance, while in our case the 100% sensitivity of the isolates to ampicillin and gentamicin, the later being the antibiotics of choice for the treatment of disseminated listeriosis, as well as the 100% sensitivity to the trimethoprim-sulfamethoxazole combination used in cases of aminopenicillin allergy or nephrotoxicity with gentamicin, are especially noteworthy. On the contrary, in Spain Markovich et al. (2024) using EUCAST disk diffusion and minimum inhibitory concentration (MIC) methods, reported that L. monocytogenes (n = 132) from food or human sources showed no antibiotic resistance. In a large-scale study in France, Moura et al. (2024) analyzed 5339 L. monocytogenes isolates (2908 clinical and 2431 food isolates), and using disk diffusion method according to the guidelines of EUCAST, they reported that acquired antimicrobial resistance was rare (2.23% isolates). Our results are similar to those obtained in France (Moura et al., 2024) and suggest that the incidence of antibiotic resistance in L. monocytogenes also remains low in these poultry associated strains, similarly to the pork associated strains reported previously (Ortiz et al., 2014).
Thus, in the studied poultry-associated L. monocytogenes population, antibiotic sensitivity has remained stable. Ecological factors could explain this common susceptibility, as L. monocytogenes is a ubiquitous bacterial pathogen most frequently found in reservoirs unexposed to antibiotics (Baquero et al., 2020; Orsi et al., 2024).
Nevertheless, multidrug-resistant Listeria species strains have been isolated from numerous sources including clinical isolates (Luque-Sastre et al., 2018) and L. monocytogenes can acquire antibiotic resistance genes from other organisms (Baquero et al., 2020). In fact, according to Moura et al. (2024), the most common acquired resistance phenotype in both clinical and food isolates of L. monocytogenes was against tetracyclines, mainly due to the presence of Tn916-carrying tetM genes. Resistance surveillance is therefore mandatory in certain environments (e.g., food-processing, hospital), where antibiotic selective pressure is higher than other environments, due to the often widespread use of antibiotics in those earlier environments (Baquero et al., 2020; Orsi et al., 2024).
Footnotes
Authors’ Contributions
R.M. and J.V.M.-S.: Conceptualization. Writing—original draft preparation. R.M., D.F., P.L., S.O., D.P.-B., and J.V.M.-S.: Methodology, data curation. R.M., J.L.A., and J.V.M.-S.: Supervision. Writing—reviewing and editing.
Disclosure Statement
No competing financial interests exist.
Funding Information
This work was in part supported by the Spanish Ministry of Science, Innovation and Universities (Research Project grant PID2022-142329OB-C32).