Antibiotic Resistance of Listeria monocytogenes Isolated from Meat-Processing Environments,Beef Products,and Clinical Cases in Brazil

Introduction

L isteria monocytogenes is a gram-positive intracellular foodborne pathogen that is widely distributed in the environment. This pathogen can survive on a diversity of surfaces, resulting in persistence in food-processing environments and cross contamination with end products. ¹⁵ Listeriosis is the disease caused by L. monocytogenes and it is considered an emergent illness since the 1980s, when a number of cases and outbreaks were reported and associated with the consumption of foods contaminated with the pathogen.^8,20,23 This disease is characterized by severe infections in high-risk groups requiring rapid treatment with antibiotics.^31,32

Serogrouping of L. monocytogenes isolates obtained from clinical and food samples is important to predict the possible risks to consumers. Strains from serotypes 1/2a, 1/2b, and 4b are considered the most virulent and are frequently associated with human listeriosis cases and outbreaks.^5,31

A diversity of proteins are associated with the virulence activity of L. monocytogenes, such as internalins, listeriolysins, and phospholipases. ¹³ The internalin-related genes (inlA, inlC, and inlJ) are involved with the passage through the intestinal barrier, cell adhesion, and invasion, and their presence in L. monocytogenes isolates suggests a potential pathogenicity for consumers.

Most L. monocytogenes isolates are susceptible to the antibiotics that are usually employed to fight gram-positive bacteria, and the first report of a multiresistant strain was described in 1990 by Poyart-Salmeron et al. ²⁷ Nowadays, several studies have shown resistance among isolates obtained from food-processing environments and foods.^{17,18,21,25,34} The emergence of antimicrobial resistance has serious consequences for public health, such as failures in the treatment of diseases and limitations in therapeutic choice, and may require the use of more modern drugs. ²

The present study aimed to assess the antimicrobial resistance and the presence of virulence markers among L. monocytogenes isolates obtained from different sources (environment, food, clinical cases) and different Brazilian regions.

Materials and Methods

Results

Table 1 shows the different origins of L. monocytogenes isolates and also the serogrouping results. Among 69 isolates from meat-processing environments, a predominance of isolates from serogroups 4b, 4d, 4a, or 4c, and 1/2c or 3c was observed, and among 43 isolates from beef products, most were identified as belonging to serogroups 1/2b or 3b, followed by 4b, 4d, 4a, or 4c, and 1/2c or 3c. Finally, among 25 clinical strains, the most common serogroups were 4b, 4d, 4a, or 4c.

All L. monocytogenes isolates presented positive PCR results for the internalin genes (inlA, inlC, and inlJ) and were susceptible to ampicillin (10 μg), penicillin G (10 units), erythromycin (15 μg), gentamicin (10 μg), imipenem (10 μg), rifampin (5 μg), chloramphenicol (30 μg), tetracycline (30 μg), trimethoprim/sulfamethoxazole (25 μg), and vancomycin (30 μg). However, resistance to clindamycin (2 μg) and oxacillin (1 μg) was found in most of the L. monocytogenes isolates (Table 2). The detailed results for each isolate and related to each antibiotic are presented in the Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/mdr).

Discussion

The conventional agglutination method is the reference protocol for serotyping L. monocytogenes isolates obtained from clinical and food samples. However, molecular methodologies have been proposed for identification of the main L. monocytogenes serogroups associated with listeriosis. These methods provide rapid and low-cost results, but the exact identification of the serotype is not possible since these protocols propose a serogroup categorization that includes different serotypes, that is, usually the most prevalent serotype and other nonfrequent serotypes.^5,14

In this study, we showed that most isolates recovered from different Brazilian states from food-processing environments and foods belonged to serogroups 4b, 4d, 4a, or 4c (Table 1), which is in agreement with previous studies in Brazil.^3,6,22 Moreover, serotypes 1/2a or 3a, 1/2b or 3b, and 1/2c or 3c also have been identified in food-processing environments and foods in different states, similar to previous studies.^6,7,24 All isolates from clinical cases belonged to serogroups 4b, 4d, 4a, or 4c, 1/2a or 3a, and 1/2b or 3b (Table 1), which are usually associated with the majority of outbreaks and sporadic cases of listeriosis. ³¹

The internalin-related genes play important roles in the virulence mechanisms of L. monocytogenes and their presence suggests potential pathogenicity. The inlJ gene is directly related to the L. monocytogenes passage through the intestinal barrier; the InlC gene contributes to the postintestinal steps of infection; and the inlA gene plays an important role to entry into host cells.^19,32 In this study, all L. monocytogenes strains were positive for virulence markers inlA, inlC, and inlJ, as observed in a number of other studies with strains from serotypes 1/2a, 1/2c, 1/2b, and 4b.^19,30,34 However, additional characterization, such as sequencing the entire inlA gene, is important to properly assess the virulence potential of L. monocytogenes strains since some mutations are associated with the expression of truncated proteins, which can result in low virulence potential.^28,30

Treatment of listeriosis is done using β-lactam antibiotics (ampicillin or amoxicillin) in association, or not, with an aminoglycoside (gentamicin). However, other drugs can also be used, such as erythromycin, tetracycline, chloramphenicol, rifampicin, trimethoprim/sulfamethoxazole, and linezolid.^31,33 In our study, all isolates were susceptible to antibiotics used in listeriosis treatment, but a high level of resistance and intermediate resistance was observed against clindamycin and oxacillin.

Resistance to lincosamides and penicillins has been described previously.^{12,16–18,29,34} Antimicrobial resistance in microorganisms can occur due to endogenous and exogenous factors, and the environment plays an important role in allowing interactions with other bacteria and consequent gene or plasmid transfer.^9,11,18,21 Oxacillin and clindamycin resistance has been attributed to efflux pumps or 23S ribosomal RNA modifications,^1,18 as well as excessive use of both drugs in veterinary medicine. ¹ However, Bertsch et al. ⁴ suggested that Listeria fleischmannii, which is resistant to clindamycin, may possess a transferable transposon that remains to be identified. While these drugs are not used to treat listeriosis, the emergence of resistance demands attention due to the possibility of horizontal transfer to other bacteria. ²⁵

This study showed the prevalence of pathogenic serogroups among isolates from food-processing environments, foods, and clinical cases in Brazil. Despite having susceptibility to most of the antibiotics used to treat listeriosis, the presence of high antimicrobial resistance to oxacillin and clindamycin is a serious concern for public health, and more in-depth research is needed to better understand the mechanisms of antimicrobial resistance. In addition, all isolates harbored virulence marker genes, demanding further analysis to properly characterize their pathogenic potential.