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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

Aims: In the past eight to 10 years, reports of antibiotic resistance in food-borne isolates in many countries have increased, and this work examined the susceptibility of 1001 food isolates of Listeria species.

Methods and Results: Susceptibility/resistance to eight antibiotics was determined using the Bauer–Kirby disc diffusion assay, and 10·9% of the isolates examined displayed resistance to one or more antibiotics. Resistance to one or more antibiotics was exhibited in 0·6% of Listeria monocytogenes isolates compared with 19·5% of Listeria innocua isolates. Resistance was not observed in Listeria seeligeri or Listeria welshimeri. Resistance to tetracycline (6·7%) and penicillin (3·7%) was the most frequently observed, and while resistance to one antibiotic was most common (9·1%), isolates resistant to two or more antibiotics (1·8%) were also observed.

Conclusions: While resistance to the antibiotics most commonly used to treat human listeriosis was not observed in L. monocytogenes, the presence of such resistance in other Listeria species raises the possibility of future acquisition of resistance by L. monocytogenes.

Significance and Impact of the Study: The higher level of resistance in L. innocua compared with L. monocytogenes suggests that a species-related ability to acquire resistance to antibiotics exists.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

The bacterium Listeria monocytogenes is a Gram-positive, motile organism capable of growth between –0·4 and 50 °C (Farber and Peterkin 1991). The causative agent of listeriosis, it is ubiquitous in the environment. It has been recognized as an animal pathogen since the 1920s but in the past two decades, it has been implicated in several outbreaks of food-borne illness in humans (Schlech et al. 1983; Anon. 1999). While it has been established that food-borne transmission constitutes the main acquisition route of listeriosis (Farber and Peterkin 1991; Pimmer et al. 1992), most healthy humans are not significantly affected by the intake of small numbers of L. monocytogenes in foods. However, certain sections of the population are predisposed to the development of listeriosis due to the presence of existing chronic illness, suppression of the immune system, pregnancy, or extreme youth or age (under 1 year or over 60 years) (Lorber 1990). This presents a significant public health problem because in such sections of the population, listeriosis is fatal in up to 30% of cases (Farber and Peterkin 1991; Jones and MacGowan 1995).

In the past, those individuals who develop listeriosis have usually been treated with penicillin or ampicillin in conjunction with an aminoglycoside (Charpentier and Courvalin 1999), although tetracycline, erythromycin or chloramphenicol, alone or in combination, have also been used (Hof 1991). Current therapy of choice for all forms of listeriosis is a combination of ampicillin and gentamicin (Lorber 1997; Schlech 2000). Listeria spp. have been reported as susceptible to antibiotics active against Gram-positive bacteria (Hawkins et al. 1984) but more recently, reports of resistance in Listeria spp. have been published (Franco Abuín et al. 1994; Roberts et al. 1994; Abrahim et al. 1998). Such increases in antibiotic resistance among Listeria spp. are in line with a general worldwide pattern of an increasing prevalence of antibiotic resistance, including multiple antibiotic resistance among many groups of bacteria. Many pathogens are developing resistance to most currently used antibiotics, and there are increasingly frequent reports of pathogens which are resistant to almost all available antibiotics (Levy 1998). Antibiotic resistance in bacteria has been linked to over-use of antibiotics in animals and humans (Davies 1998; Rao 1998) since these therapeutic compounds were identified nearly 60 years ago. Such resistance may arise from a mutation in an intrinsic chromosomal gene, or by acquisition of exogenous genetic material carrying single or multiple resistance determinants (Levy 1994). It is now clear that such transfer is possible between unrelated bacterial species (Kruse and Sørum 1994), and that these interactions are a frequent and important means of genetic exchanges among micro-organisms.

It is evident that antibiotic resistance is becoming more and more widely reported in all bacteria, not just pathogens, and that the occurrence of antibiotic resistance in non-pathogens poses major, if less direct, risks to human health. While many antibiotic-resistant bacteria in foods are currently saprophytic or commensal in habit, their resistance genes can be transferred to other food-borne bacteria, including pathogenic species within the gastrointestinal tract (Perreten et al. 1997). This process may have undesirable clinical implications for the host, and for the wider population coming into contact with derived antibiotic-resistant pathogens. Thus, more information is urgently required on the patterns of dispersion and transmission of antibiotic resistance among the wider prokaryotic kingdom. This study examined the antibiotic susceptibility of 1001 strains of Listeria spp. (351 L. monocytogenes, 549 L. innocua, 62 L. seeligeri and 39 L. welshimeri) isolated from retail foods purchased in the greater Dublin area.

METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

The Listeria strains used in this study were isolated from 67 retail food samples (Walsh et al. 1998) maintained on nutrient agar slopes at 1 ± 1°C and recovered on Tryptone Soya Agar (Oxoid) at 30°C for 24 h immediately prior to examination.

The Bauer–Kirby technique (Anon. 1997) was used to determine susceptibility to antibiotics. Three to five well-isolated colonies were transferred into 10 ml Brain Heart Infusion Broth (Oxoid), incubated at 37°C for 24 h, diluted 1:10 in 9 ml 0·1% Peptone Water (Oxoid) and inoculated onto the entire surface of a dried Mueller–Hinton Agar (MHA)(Oxoid) plate containing 5% Horse Blood (BLE) using a cotton swab. The MHA plates were held at room temperature for 10 min to allow evaporation/absorption of free surface liquid.

Antibiotic discs containing 10 μg ampicillin (A10), 30 μg chloramphenicol (C30), 15 μg erythromycin (E15), 10 μg gentamicin (CN10), 10 units penicillin G (P10), 10 μg streptomycin (S10), 30 μg tetracycline (TE30) or 30 μg vancomycin (VA30) (all Oxoid) were placed on the surface of each inoculated plate (four discs per plate) using a multi-disc dispenser (Oxoid). After incubation for 24 ± 4 h at 37°C, the diameter (in mm) of the zone around each disc was measured, and interpreted in accordance with the National Committee for Clinical Laboratory Standards (NCCLS) guidelines, to classify the antibiotic sensitivity of each isolate as ‘susceptible’, “intermediate’ or ‘resistant’. A number of quality control strains obtained from The National Collection of Industrial and Marine Bacteria (NCIMB), Scotland, were included within the analyses, i.e., Escherichia coli NCIMB 12210 (ATCC 25922), Pseudomonas aeruginosa NCIMB 12469 (ATCC 27853) and Staphylococcus aureus NCIMB 12702 (ATCC 25923).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

Of the 1001 isolates tested, 109 (10·9%) displayed resistance, as classified by NCCLS, to at least one antibiotic (Table 1). The level of resistance in L. monocytogenes was low (0·6%) compared with L. innocua (19·5%), and no resistance was observed in L. seeligeri or L. welshimeri. Resistance to one antibiotic was more common than multiple resistance, i.e., 91 (9·0%) isolates were resistant to one antibiotic, 13 to two antibiotics (1·3%), three to three antibiotics (0·3%), one to four antibiotics (0·01%) and one isolate, L. innocua, from a frozen burger, was resistant to five antibiotics (0·01%) (Table 2). No multiple resistance was observed in L. monocytogenes.

Table 1.   Resistance and multi-resistance patterns in Listeria species tested by the Bauer–Kirby technique Thumbnail image of
Table 2.   List of foods from which isolates displaying resistance to more than one antibiotic were isolated. All of the isolates were Listeria innocuaThumbnail image of

Resistance to tetracycline was most common (Table 3), with 64 isolates showing resistance to this antibiotic. Penicillin G was the next most common, with 37 isolates showing resistance, then ampicillin (20 isolates), streptomycin (six isolates), erythromycin (four isolates), vancomycin (two isolates), chloramphenicol (one isolate) and gentamicin (one isolate).

Table 3.   Number of Listeria monocytogenes and L. innocua isolates resistant to antibiotics Thumbnail image of

Table 4 shows the food types examined. The highest numbers of antibiotic-resistant Listeria spp. were detected in frozen burgers. A number of samples of ready-to-eat (RTE) products, including cooked sliced meats, salads, lettuce and ice cream, were found to contain antibiotic-resistant Listeria spp. The bulk of the isolates tested were from frozen burgers and sliced cooked meats, though most of the antibiotic-resistant Listeria spp. were found in the burgers.

Table 4.   Occurrence and antibiotic resistance of Listeria spp. in food samples Thumbnail image of

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

The results of this study suggest that the overall incidence of antibiotic resistance in Listeria spp. is still relatively low, i.e., less that 7% in the case of tetracycline, the most frequently observed resistance. However, the study does confirm that since the first report of antibiotic-resistant strains of Listeria spp. and L. monocytogenes (Poyart-Salmeron 1990), there has been a continuing pattern of the emergence of strains of Listeria spp. isolated from food, the environment or from clinical cases of listeriosis, which are resistant to one or more antibiotics (Arpin et al. 1992; Charpentier et al. 1995). Although the incidence of antibiotic resistance is currently low, the range of antibiotics to which resistance has been acquired is wide. It is of concern that this expanding range now includes a number of antibiotics used to treat listeriosis, e.g. penicillin, ampicillin, tetracycline and gentamicin.

Thus, while the relative proportions of antibiotic-resistant L. monocytogenes were low, it is clear that a number of mechanisms are operating to facilitate the introduction of undesirable antibiotic-resistant genes into the genus in general, and into L. monocytogenes in particular. Previous experiments demonstrated a number of these mechanisms. In vitro and in vivo studies have shown conjugative transfer of antibiotic resistance, i.e., receipt of enterococcal and streptococcal plasmids into the genus Listeria spp. and re-transfer of such plasmids within the genus, including L. monocytogenes (Perez-Diaz et al. 1982; Flamm et al. 1984; Doucet-Populaire et al. 1991). Such transfers have been reported to confer a number of the resistances noted in this study, including resistance to streptomycin, erythromycin and chloramphenicol (Vicente et al. 1988). Alternatively, transfer of non-plasmid-borne erythromycin resistance genes from L. innocua to L. monocytogenes has also been reported (Roberts et al. 1994).

The higher levels of antibiotic resistance in L. innocua than in the other Listeria spp. raises the possibility of species differences in relation to the means and/or rates of acquisition of antibiotic resistance. Rota et al. (1996) found that a higher percentage of L. innocua were resistant to antibiotics than L. monocytogenes. More specifically, Franco Abuín et al. (1994) found that while isolates of L. monocytogenes were susceptible to tetracycline, L. innocua isolates were not. Rota et al. (1996) also noted that a higher percentage of L. innocua was multi-resistant than in the case of L. monocytogenes. The genetic basis for these differences is unclear, although the presence of plasmids encoding antibiotic resistance in L. innocua but not in L. monocytogenes has been reported (Slade and Collins-Thompson 1990). Other workers (Poyart-Salmeron et al. 1990; Charpentier et al. 1995) have reported the presence of plasmid-mediated antibiotic resistance in L. monocytogenes.

The results of this study confirm that major changes in the nature and incidence of antibiotic resistance among Listeria spp. have occurred within the last decade. In 1988, Espaze and Reynaud found little antibiotic resistance among Listeria spp., and no significant differences among the antibiotic sensitivities of the different Listeria spp., except for greater fosfomycin sensitivity in L. ivanovii. These workers correctly predicted the emergence of resistant strains of Listeria spp. as food production techniques changed and the numbers of Listeria spp. in the food environment increased, thereby increasing the likelihood of genetic transfer of resistance determinants. This increased prevalence of Listeria spp. in food-processing environments is now well recognized (Jay 1996), and was reconfirmed by the results of the current study in which Listeria spp. were recovered from almost 30% of samples examined. Within this overall increase in incidence of this genus, L. innocua is reported as occurring more frequently in foods than L. monocytogenes (Walsh et al. 1998). This may be an artefact related to differential selection during enrichment and recovery procedures, or because L. innocua is simply more common in the environment than L. monocytogenes (Sheridan et al. 1994; Walsh et al. 1998). Increasing recognition of the mechanisms by which antibiotic resistance, including multiple antibiotic resistance of the types observed in this study, can move into and among Listeria spp. suggests that such resistance(s) will become an increasingly frequently observed characteristic of L. monocytogenes.

This study has demonstrated that at least some of the range of antibiotic transfer and exchange mechanisms, demonstrated as possible in experimental studies, are active among Listeria occurring in foods or food environments. It is not yet clear which of these mechanisms will prove most significant in clinical terms. However, undesirable transfers are occurring. Although a recent review of antibiotic resistance in Listeria spp. (Charpentier and Courvalin 1999) reported no cases of resistance to penicillin and gentamicin, in strains of Listeria spp. from human, food and environmental sources, the present study detected resistance to these antibiotics among food isolates. While only one gentamicin-resistant isolate was noted, the acquisition of this trait is of particular concern because, as noted earlier, gentamicin (in combination with ampicillin) is the current therapy of choice for all forms of listeriosis. This study adds gentamicin to the list of antibiotics to which Listeria spp. have acquired resistance.

It is possible to postulate the reasons for the frequencies of some of the resistances observed in the present study. The high frequency of tetracycline resistance noted in this and other studies may have developed due to widespread use of this antibiotic in therapy, or as a supplement in animal feeds, with subsequent dissemination through known multiple mechanisms for the transmission of resistance to and among Listeria spp. (Poyart-Salmeron et al. 1992). In other cases, the factors underlying the acquisition and persistence of antibiotic resistance are much less clear. Demonstration of the efficiency of transfer across genus boundaries, e.g. to and from Enterococcus/Streptococcus and even Staphylococcus, particularly associated with the acquisition of multiple drug resistance, suggests that the established view of the antibiotic resistance profile of Listeria spp., including L. monocytogenes, may require significant review. It has been suggested that the privileged site for such acquisitions, and acquisitions from a range of Gram-negative bacteria, is the digestive tract of humans and animals (Doucet-Populaire et al. 1991). Considering the range of antibiotic resistances on offer from the microflora of such environments, it is hardly surprising that Listeria spp., previously thought to be susceptible to all Gram-positive antibiotic agents (Hawkins et al. 1984), has undergone marked changes within the last 20 years.

Although antibiotic resistance in Listeria spp. was noted in relatively recent times (Gellin and Broome 1989) as ‘rare’, the number of reports of antibiotic resistance in this genus has been increasing (Poyart-Salmeron et al. 1990; Barbuti et al. 1994; Roberts et al. 1994; Papa et al. 1996; Rota et al. 1996) to a point where, as with many other pathogens, the list of effective clinical options is becoming disconcertingly small. This study has demonstrated that a wide and rapidly expanding range of undesirable and, in some cases, multi-resistant determinants is currently present in members of the Listeria genus with significant potential for transfer to the currently pathogenic species, i.e., L. monocytogenes. More comprehensive and continuous monitoring of the course and nature of the acquisition and dissemination of antibiotic resistance by this pathogen and other members of the genus is warranted.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. References
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