Antibiotic Susceptibility in Benzalkonium Chloride-Resistant and -Susceptible Listeria monocytogenes Strains

Abstract

This study aimed to investigate whether Listeria monocytogenes strains with resistance to a commonly used biocide display any cross-resistance to antibiotics. Using pulsed-field gel electrophoresis (PFGE), 29 different PFGE types were previously identified in an Iberian pig abattoir and processing plant. Only three PFGE types were resistant to benzalkonium chloride (BAC), but they represented a significant proportion of the PFGE types surviving in the plant after 4 years. In the present study, a subset of 29 strains, representing the 29 different PFGE types, underwent antibiotic susceptibility testing. Antibiotic susceptibility was assessed by Etest, utilizing 12 commonly prescribed antibiotics. All of the 29 strains were susceptible to all of the antibiotics tested. The study revealed that this group of different PFGE types of L. monocytogenes, including those resistant to BAC, possesses uniform sensitivity to antibiotics.

Introduction

Foodborne listeriosis represents a significant concern for the food industry because of the high case fatality rate it causes in humans. Despite the low cure rate for listeriosis, almost all Listeria monocytogenes strains are susceptible to most of the antibiotics active against Gram-positive bacteria (Lungu et al., 2011). There are, however, L. monocytogenes strains of both clinical (Morvan et al., 2010) and food origin (Granier et al., 2011) that are antibiotic resistant, although the resistance rate still does not appear to be very high. These data indicate that it is essential to strengthen microbiological surveillance of resistant strains of L. monocytogenes.

The biocides that are commonly used in the food industries include quaternary ammonium compounds (QACs) such as benzalkonium chloride (BAC). Developed microbial resistance is a drawback of QACs compared with other biocides (Buffet-Bataillon et al., 2012). Concerns have been raised about the possibility that biocide and antibiotic resistance in bacteria might be linked. In vitro studies demonstrating increase in resistance have often been cited as evidence for cross-resistance. However, the scientific evidence of environmental and clinical development of cross-resistance between biocides and antibiotics is limited, and the information from the literature is often difficult to interpret and compare (Buffet-Bataillon et al., 2012).

Investigation of the sources of L. monocytogenes contamination in an Iberian pork-processing plant using pulsed-field gel electrophoresis (PFGE) typing resulted in the identification of 29 PFGE types. Three PFGE types surviving 4 years in that plant were characterized by their low-level resistance to BAC, showing that resistance to an increased sublethal concentration of biocides may lead to the prolonged survival of L. monocytogenes in the food plants (Ortiz et al., 2014).

The aim of this study was to characterize the 29 PFGE types to further investigate their susceptibility to antibiotics. The results may help to increase our knowledge of the actual extent of antibiotic resistance in this significant foodborne pathogen.

Materials and Methods

Food-related strains and standard culture methods

In a previous study, 541 isolates of L. monocytogenes recovered over a period of 3 years in an Iberian pork plant were subtyped by polymerase chain reaction (PCR)–based serogrouping and PFGE. A total of 29 unique PFGE types of four different PCR serogroups were identified among the characterized isolates. One isolate with a unique PFGE type was considered the PFGE type strain of each PFGE type (Ortiz et al., 2014). Three PFGE type strains of PCR serogroup IIa were the only strains found resistant to BAC (Ortiz et al., 2014). The 29 PFGE type strains were used in the present study.

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 Biolife (Milan, Italy).

Testing of susceptibility to antimicrobial agents

Antibiotic susceptibility testing of L. monocytogenes was performed by the Epsilon Test (Etest) method (http://www.biomerieux-diagnostics.com) on MHA, as recommended by the Spanish Society for Infectious Diseases and Clinical Microbiology (Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica SEIMC, http://www.seimc.org) and following the Clinical Laboratory Standards Institute performance standards for antimicrobial susceptibility tests (CLSI, 2012). Staphylococcus aureus ATCC 29213 (American Type Culture Collection, http://www.atcc.org) and Enterococcus faecalis ATCC 29212 were always included as quality controls (CLSI, 2012). A minimum of 2 independent determinations of the susceptibility of each strain of L. monocytogenes to 12 antibiotics was made. The criteria for selecting these 12 agents were based on the recommendations of the Spanish antibiogram committee for the selection of antimicrobials for susceptibility testing (MENSURA, 2000). They represent relevant classes of antibiotics active against Gram-positive bacteria, and include those used for human listeriosis treatment (Table 1).

Table 1.

Susceptibility of 29 Strains of Listeria monocytogenes to 12 Antibiotics

Agent	Resistance breakpoints (μg/mL)	MIC range (μg/mL) ^a
Ampicillin	>1.0^c	0.125–0.5
Chloramphenicol	>8.0^d	3.0–8.0
Cotrimoxazole^b	>0.03^c	0.008–0.023
Erythromycin	>1.0^c	0.125–0.38
Gentamicin	>1.0^d	0.19–1.0
Imipenem	>0.25^e	0.064–0.19
Levofloxacin	>2.0^d	0.38–1.0
Linezolid	>4.0^d	1.5–3.0
Penicillin G	>1.0^c	0.094–0.5
Rifampicin	>0.5^d	0.004–0.125
Tetracycline	>2.0^d	0.38–1.0
Vancomycin	>2.0^d	0.75–2.0

Limits of two Listeria strain replicates.

Trimethoprim concentration in a 1:19 ratio with sulfamethoxazole. Breakpoint is expressed as the trimethoprim concentration.

Listeria monocytogenes breakpoints (EUCAST, 2013).

Staphylococcus spp. breakpoints (EUCAST, 2013).

Listeria monocytogenes breakpoint for meropenem (EUCAST, 2013).

MIC, minimum inhibitory concentration.

Results and Discussion

The results for susceptibility to 12 clinically relevant antibiotics, determined using the Etest of the 29 strains of L. monocytogenes, are shown in Table 1. All of the strains analyzed were susceptible to all 12 of these antibiotics.

The Etest correlates with the reference disk diffusion, broth microdilution, and agar dilution tests (Baker et al., 1991). For this reason, the breakpoints for the MICs obtained with standardized reference methods can also be utilized to classify an isolate as susceptible or resistant using the Etest. In this study, the MIC results were interpreted according to the L. monocytogenes breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing for ampicillin, cotrimoxazole, erythromycin, meropenem, and penicillin G, and by using the values for Staphylococcus spp. for antibiotics lacking interpretation criteria for L. monocytogenes (EUCAST, 2013; Table 1).

Tetracycline and ciprofloxacin resistances are the most prevalent types in L. monocytogenes (Morvan et al., 2010; Lungu et al., 2011), which makes the absence of resistance to tetracycline in this study all the more striking. Resistance to ciprofloxacin could be hidden by the fact that we used levofloxacin as the fluoroquinolone representative (Godreuil et al., 2003). Levofloxacin was selected because it is more clinically relevant than ciprofloxacin (i.e., it has a more rapid bactericidal effect against intracellular bacteria [MENSURA, 2000]), and clinical isolates of L. monocytogenes resistant to ciprofloxacin do not show resistance to levofloxacin (Godreuil et al., 2003). Nevertheless, surveillance of ciprofloxacin resistance is important for this kind of epidemiological studies (Morvan et al., 2010; Granier et al., 2011), as well as for in vitro studies of potential development of combined resistance to benzalkonium chloride and ciprofloxacin (Lungu et al., 2011; Ortiz et al., 2014).

This study demonstrated that BAC-resistant L. monocytogenes might not show cross-resistance to antibiotics. The results also showed the natural uniform sensitivity to antibiotics, which can still be found in certain highly related populations of L. monocytogenes from specific food-processing environments. PFGE typing results indicated that the 29 PFGE types were clonally related (Ortiz et al., 2014), and thus, this uniform sensitivity might be affected by the common origin of the strains and their low genetic diversity.

Footnotes

Acknowledgment

This work was supported by Research Project grant RTA2011-00098-C02 (INIA FEDER) from the Spanish Ministry of Economy and Competitiveness.

Disclosure Statement

No competing financial interests exist.

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