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Applied and Environmental Microbiology, November 2006, p. 7410-7412, Vol. 72, No. 11
0099-2240/06/$08.00+0     doi:10.1128/AEM.00956-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Occurrence and Antibiotic Sensitivity of Listeria monocytogenes Strains Isolated from Oysters, Fish, and Estuarine Water

O. R. Rodas-Suárez,² J. F. Flores-Pedroche,² J. M. Betancourt-Rule,² E. I. Quiñones-Ramírez,¹ and C. Vázquez-Salinas^2*

Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Carpio y Plan de Ayala S/N, Col. Casco de Santo Tomás, C.P. 11340, México,¹ Departamento de Biotecnología, División de Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana, Michoacán y Purísima S/N, Col. Vicentina, C.P. 09340, México²

Received 22 April 2006/ Accepted 4 September 2006

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We analyzed the presence of Listeria spp. in oyster, fish, and seawater samples and tested isolates for antibiotic sensitivity. Listeria monocytogenes was found in 4.5% of fish samples and 8.3% of seawater samples and was not recovered from oysters. Multiresistant environmental strains were found, representing a potential threat to human health.

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Human listeriosis is a public health problem of low incidence but high mortality, requiring prompt diagnosis and adequate antibiotic therapy (1). Over the last 2 decades a high number of food-borne listeriosis outbreaks have occurred, some with high mortality rates (2, 13, 19). Antibiotic resistance and inefficient empirical treatment of Listeria infections could be responsible for this increased mortality (4). Since the first multiresistant Listeria monocytogenes strain was observed in France (14), different antibiotic resistance patterns in environmental, food, and clinical sources have been reported (7, 12, 20). Information on the presence of Listeria monocytogenes in Mexico is scarce, and the frequency of listeriosis is unknown. The purpose of this study was to determine the presence of Listeria spp. in fish, oysters, and saline waters in an area where fish are caught for local and regional consumption and to determine the sensitivities of the L. monocytogenes isolates to different antimicrobial agents.

A total of 66 oyster, 66 fish, and 144 estuarine water samples were collected over a 12-month period (June 2001 to May 2002) from 12 sites of the Pueblo Viejo lagoon, Veracruz, Mexico (Fig. 1). Fish and oyster samples were transported on dry ice in separate thermal containers, and estuarine water samples were collected in sterile plastic bottles (Nalgene) and transported to the laboratory on ice. Oyster and fish samples were processed as previously described (9). Seawater samples were filtered through a 14-cm-diameter and 0.45-µm-pore membrane (Millipore). Twenty-five milliliters of seawater or 25 g oyster or fish samples was added to 225 ml enrichment broth (EB; Merck) and incubated at 30°C for 24 to 48 h. The filter used for the water samples was washed with 100 ml peptone solution (0.1%), added to 225 ml EB, and incubated at 4°C for 7 days. Afterwards, a 0.1-ml sample was streaked in Oxford agar (Oxoid) and incubated at 30°C for 24 to 48 h. L. monocytogenes isolates were identified and serotyped as previously described (8). Antibiotic sensitivity was assessed using the Kirby-Bauer disk diffusion assay. The test and control strains were seeded in Mueller-Hinton agar supplemented with 0.5% defibrinated sheep blood and 0.1% esculin (17). Commercially available disks (Bio-Rad) with the following antibiotics were used: ampicillin, cephalothin, cefotaxime, ceftazidime, cefuroxime, dicloxacillin, erythromycin, gentamicin, pefloxacin, penicillin, tetracycline, and trimethoprim-sulfamethoxazole. MICs at which 50% of the isolates were inhibited (MIC₅₀s) and MIC₉₀s were calculated by following the CLSI (formerly NCCLS) guidelines (11). L. monocytogenes ATCC 19114, Escherichia coli ATCC 29922, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 25923 strains were used as controls in all assays.

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FIG. 1. Map of Pueblo Viejo lagoon in northeastern Mexico (reprinted from Hidrobiológica [3a] with the permission of the publisher).

Listeria spp. were not recovered from oyster samples, in accordance with the results of other studies (6). However, Listeria spp. were found in 22.7% and 30.5% of fish and estuarine water samples, respectively, while L. monocytogenes was identified in 3/66 (4.5%) fish samples and in 12/144 (8.3%) water samples (Table 1). Although Listeria spp. were initially reported as rarely found in water (3), isolation rates as high as 62% in estuarine freshwater samples have been found (6). Listeria sp. isolation frequencies were higher during the rainy seasons, because the increased water volume of the rivers draining into the lagoon reduce lagoon salinity, as occurs with some Vibrio species (10). Our results are comparable to those of Colburn et al. (6), who suggested a consistent input of Listeria spp. from freshwater tributaries draining into estuaries. The percent distribution of Listeria species isolated from seawater (Table 1) was greater than that of species isolated from fish products, although no species or serogroups were predominant at any specific sampling point, showing no evidence of direct influence of human activities or domestic animals near the lagoon.

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TABLE 1. Isolation of Listeria spp. from fish and seawater samples

Variance analysis showed no significant differences in isolation rates among the different sampling points (P < 0.05). Nonlinear regression analysis was used to determine the statistical significance of seasonal, salinity, temperature, and antibiotic sensitivity variations for Listeria isolates. Salinity clearly showed a negative correlation with Listeria recovery rates (r = –0.88; P < 0.0001), which dropped to zero at a salinity of 15 g/liter (Fig. 2). Temperature did not significantly influence recovery rates.

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FIG. 2. Effect of salt concentration on the isolation rate of L. monocytogenes in estuarine water samples.

Because human listeriosis can be food borne, sensitivity and resistance of food isolates to antibiotics should be assessed. No Listeria isolates showed resistance to the ß-lactam antibiotic cefotaxime, and only 5.9% showed resistance to gentamicin. Overall resistance frequencies for other antibiotics varied: 9.7% for dicloxacillin, 13.2% for cefuroxime and cephalothin, 16.7% for tetracycline, 30.9% for erythromycin, 37.4% for trimethoprim-sulfamethoxazole, 57.4% for penicillin, 60.3% for ampicillin, 67.6% for ceftazidime, and 73.5% for pefloxacin. Antibiotic resistance frequencies of individual Listeria species are shown in Table 2, and MICs for antibiotics are shown in Table 3. Importantly, 6% of L. monocytogenes strains isolated showed multiresistance to ampicillin, erythromycin, tetracycline, dicloxacillin, and trimethoprim-sulfamethoxazole. Multiresistant L. monocytogenes strains have been isolated from clinical (14) and other (5) sources. Although it was previously considered that multiresistant strains of Listeria spp. are not commonly found in nature, evidence of the emergence of multiresistant L. monocytogenes strains from various sources has been reported (15, 16, 18). Our results provide further evidence of the emergence of multiresistant strains in nature, representing a potential threat to human health.

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TABLE 2. Frequency of Listeriasp. isolates showing resistance to antibiotics as assessed by the Bauer-Kirby assay

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TABLE 3. MIC50s and MIC90s of 68 L. monocytogenes strains isolated from estuarine water and fish samples

In order to deal with potential Listeria-related public health problems, at-risk sites should be analyzed and control measures should be implemented. Pueblo Viejo lagoon is surrounded by small rural populations, some of which use latrines discharging feces into the lagoon. Furthermore, the waters of this lagoon are very shallow (only 1.5 m deep during the rain season), so that fisherman and dogs and other domestic animals can easily walk through it. The presence of antibiotic-resistant L. monocytogenes strains in these waters suggests that the bacterium is likely to be found in other ecosystems in Mexico with similar sanitary and environmental conditions, posing a health risk for these populations.

 
* Corresponding author. Mailing address: Departamento de Biotecnología, División de Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana, Michoacán y Purísima S/N, Col. Vicentina, C.P. 09340, México D.F., México. Phone: (52 55) 5804-4724. Fax: (52 55) 5804-4712. E-mail: cvs{at}xanum.uam.mx.

Published ahead of print on 15 September 2006.

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Applied and Environmental Microbiology, November 2006, p. 7410-7412, Vol. 72, No. 11
0099-2240/06/$08.00+0     doi:10.1128/AEM.00956-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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