Multidrug-Resistance Cases of Listeria monocytogenes Isolated from Fresh Meats ^†

Abstract

The aim of this study is to provide an overview of multidrug-resistance cases of Listeria monocytogenes isolated from fresh meat and meat products from the north of Portugal. Samples of fresh meat preparations and meat products from hypermarkets and small traditional local shops were subjected to microbiological analysis and antimicrobial resistance tests. The strains were identified using morphological and molecular methods. Antibiotic resistance was determined using the Kirby–Bauer disk diffusion method. The overall prevalence of L. monocytogenes among screened samples was 32%. A total of nine isolates were obtained from minced meat, displaying a multidrug-resistance profile.

1. Introduction

In the last few decades, the selective pressure resulting from the use of antibiotics has led to the emergence of bacterial strains that exhibit a growing resistance to these agents with an increase in bacteria showing multi-resistance profiles. Listeria monocytogenes is a major bacteria that can contaminate meat and meat products, having an important impact on public health, being a potential cause of foodborne diseases.

The aim of this study is to provide an overview of multidrug-resistance cases of L. monocytogenes isolated from fresh meat and meat products from the north of Portugal.

2. Materials and Methods

2.1. Sample Collection and Bacterial Isolates

From April to September 2022, 75 samples of meat preparations were collected from hypermarkets and small traditional local shops, including fresh meats and meat-based products. Of the total samples, 20% were meat-based products (“alheira” and “moura”), 12% were meatballs and hamburgers, 12% were meat skewers, 8% were breaded meat, 40% were minced meat and 8% were fresh sausage. Samples were transported to the laboratory under refrigeration conditions in 10 min, which was followed by microbiological analysis.

2.2. Multi-Drug Resistance in Bacterial Isolates

The antimicrobial susceptibility testing was performed by the Kirby–Bauer disk diffusion method, which followed the recommendations given in the European Committee on Antimicrobial Susceptibility Testing (EUCAST) 2023 guidelines with the exception of kanamycin that followed the Clinical and Laboratory Standards Institute (CLSI) 2017 standards. The following antibiotic discs were used: ampicillin (10 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), erythromycin (15 µg), gentamicin (10 µg), kanamycin (30 µg), linezolid (30 µg), meropenem (10 µg), rifampicin (5 µg), tetracycline (30 µg), trimethoprim/sulfamethoxazole (1.25/23.75 µg) and vancomycin (30 µg). A reference strain L. monocytogenes (ATCC 7973) was used as a quality control strain.

3. Results

The overall prevalence of L. monocytogenes among screened samples was 28% with highest percentages in meat-based products and meat skewers. The percentage of resistance to each antibiotic is shown in Figure 1.

There were three strains of L. monocytogenes that displayed a multidrug-resistance profile (25%) since they showed resistance to at least three different classes of antimicrobials. Their multidrug-resistance pattern was as follows: one isolate was resistant to four classes of antimicrobials (corresponding to a sample of turkey minced meat), and two isolates were resistant to six classes of antimicrobials (corresponding to a sample of bovine minced meat and turkey minced meat).

Table 1. Antimicrobial-resistant phenotype from multidrug-resistant L. monocytogenes isolates.

Identification	Multidrug-Resistant Phenotype
1	RD, CIP, KAN, ERY, MRP, SXT
2	RD, CIP, ERY, TET, MRP, SXT
3	CIP, ERY, MRP, SXT

Abbreviations. RD: rifampicin; CIP: ciprofloxacin, K: kanamycin; ERY: erythromycin; TET: tetracycline, MRP: meropenem; SXT: trimethoprim–sulfamethoxazole.

shows the phenotype of L. monocytogenes isolated, regarding their multidrug antibiotic resistance.

All three strains have resistance to ciprofloxacin, erythromycin, meropenem and trimethoprim–sulfamethoxazole. Only one strain is resistant to tetracycline and one was resistant to kanamycin. Strain number 3 is susceptible to rifampicin.

4. Discussion

In this study, a prevalence of 28% of L. monocytogenes in meat and meat products was obtained in retail markets. Research conducted under similar conditions, in meat obtained from retail markets in Brazil and China, show a maximum prevalence of L. monocytogenes of 19% [1,2].

Regarding antimicrobial resistance, all samples were susceptible to ampicillin, chloramphenicol, gentamicin, linezolid and vancomycin with no resistance reported. In 2020, Matle et al. [3] published a review about antimicrobial resistance in L. monocytogenes from meat and meat products where they found resistance against most of the antimicrobials that we tested [4]. However, the percentage of resistance is considerably higher in our study, as shown by other authors. Gómez et al. (2014) [2] registered no resistance towards chloramphenicol nor ciprofloxacin and only 0.5% of resistance with tetracycline [4]. Additionally, Maung et al. (2019) [5] encountered no resistance to ampicillin, gentamycin, erythromycin, vancomycin or sulfamethoxazole–trimethoprim and just one case of resistance to linezolid [5]. In our study, we found three isolates that showed a multidrug-resistance profile, being resistant to more than three classes of antimicrobials. This percentage is in accordance with a systematic review from Tayeb et al. (2023) [6] that had 22.97% of multidrug resistance in meat and meat products.

5. Conclusions

A moderate frequency of L. monocytogenes (29%) was found among meat and meat products for human consumption. Six strains were multidrug resistant with a diversity of antimicrobial resistance. These results show us that livestock animals may be an important reservoir of antimicrobial-resistance genes. This is of great concern for public health, since most of the antimicrobial resistances detected were antimicrobials considered to be essential and frequently used in human medicine. Furthermore, the frequent monitoring of strains from livestock is essential to understand the spread and the changes of the genetic repertoire as well as the zoonotic potential of these strains. Some measures to overcome antimicrobial resistance in meat in Portugal should be taken into consideration, such as the education of livestock producers, limiting the availability of antibiotics and the implementation of more restrictive legislation concerning antimicrobial prescription.