Prevalence, antimicrobial susceptibility and virulotyping of Listeria species and Listeria monocytogenes isolated from open-air fish markets.

BACKGROUND: The aim of this study was to investigate the prevalence and characterization of Listeria species and Listeria monocytogenes isolated from raw fish and open-air fish market environments. Eight hundred and sixty two samples including raw fish and fish market environments (samples from workers' hands, workers' knives, containers and work surface) were collected from the open-air fish markets in the Northern region of Iran.
RESULTS: Listeria spp. was isolated from 104/488 (21.3%) raw fish and 29/374 (7.8%) of samples from open-air fish market environment. The isolates of Listeria spp. included L. innocua (35.3%), L. monocytogenes (32.3%), L. seeligeri (18%), and L. ivanovii (14.3%). Of the 43 L. monocytogenes isolates, 31 (72.1%), 10 (23.3%) and 2 (4.7%) belonged to serovars 1/2a, 4b, and 1/2b, respectively. The inlA, inlB, inlC, inlJ, actA, hlyA, iap, plcA, and prfA virulence-associated genes were detected in almost all of the L. monocytogenes isolates. The Listeria spp. isolates showed high resistance against tetracycline (23.3%), penicillin G, and cephalothin (each 16.5%). Besides, we observed significant resistance level to tetracycline (27.9%), ampicillin (20.9%), cephalothin, penicillin G, and streptomycin (each 16.3%) in the L. monocytogenes isolates. All of the isolates were susceptible to cefotaxime, gentamicin, kanamycin, and pefloxacin. We found that tetM (25.6%), tetA (23.3%), ampC (14%), and penA (11.6%) were the most prevalent antibiotic resistance genes in the L. monocytogenes isolates.
CONCLUSIONS: Recovery of potentially pathogenic L. monocytogenes from raw fish and environment of open-air fish market samples in this study is a convincing evidence for the zoonotic potential of listeriosis.

Background

The genus Listeria includes facultatively anaerobic Gram-positive bacteria. L. ivanovii and L. monocytogenes from this genus are known as pathogenic species and have shown the ability to cause severe diseases in animals and both humans and animals, respectively. Listeriosis is a foodborne infection with high mortality rates which is caused by L. monocytogenes in humans. The pathogen is ubiquitous and has been isolated from animals, different types of foods, and environments worldwide [1-3].

There is a high possibility of L. monocytogenes contamination in the captured fish from contaminated waters and environments. The contamination could also happen during transportation and in the environment of fish markets. Although L. monocytogenes has been isolated from seafood, fish and fishery products, no major listeriosis outbreaks with these products has been reported so far [4]. However, contaminated fish and fish products are considered as the most frequent causes of a number of sporadic listeriosis cases [5].

L. monocytogenes can be divided into 13 serovars which serovars 1/2a, 1/2b and 4b have been mainly reported from foods and human cases of listeriosis [6-8]. The 1/2a is a predominant serovar isolated from food samples [9]. However, serovar 4b is known to be the cause of the largest number of human listeriosis outbreaks [10, 11].

Different Listeria determinants, which are well known as important factors in pathogenicity of L. monocytogenes, include actin (encoded by actA gene), internalin (encoded by inlA, inlB, inlC, and inlJ genes), invasion associated protein (encoded by iap gene), listeriolysin O (encoded by hlyA gene), phosphatidylinositol phospholipase C (encoded by plcA gene), and virulence regulator (encoded by prfA gene) [12-15].

Detection of multi-drug resistant pathogenic bacteria in foods is considered as a public health risk worldwide. Excessive application of antibiotics in veterinary medicine may lead to distribution of antibiotic-resistant pathogens in the environment [16]. Recently, multi-drug resistant isolates of L. monocytogenes have been detected in animals, different types of foods and human cases of listeriosis [17-20]. Since multi-drug resistant isolates could transmit to humans via contaminated food, proper measures should be taken to prevent their environmental spread. Monitoring of the multi-drug resistant pathogens isolated from food and environment could be useful to identify the patterns of resistance to antibiotics [21].

Mazandaran province, which is located in the North of Iran and near Caspian Sea, fish and fish products are consumed more than other places in Iran. Despite the high consumption of fish and fish products in this area, there is no study on the prevalence and characterization of Listeria spp. and L. monocytogenes from fish and environment of fish markets. Hence, this study was done to investigate the prevalence, serotyping, virulotyping and the resistance patterns of Listeria spp. and L. monocytogenes isolates detected from fish and environment of fish markets in Mazandaran province and the Northern region of Iran.

Methods

Sampling

Between March 2012 and Jun 2014, 488 raw fish samples were purchased at the open-air fish market places in five major cities in Mazandaran Province, North of Iran. The purchased samples included Ctenopharyngodon idella (n = 135), Rutilus kutum (n = 124), Liza auratus (n = 120), and Hypophthalmichthys molitrix (n = 109). In addition, 374 environmental and workers swab samples from workers’ hands (n = 96), workers’ knives (n = 96), work surface (n = 92), and containers (n = 90) were collected from the open-air fish markets. All of the samples were transported in ice boxes to the laboratory within 3 h after sampling. All samples were obtained with the informed consent of the workers and ethics approval for this study was granted by the Islamic Azad University, Iran.

Isolation and detection of Listeria spp.

Listeria spp. were isolated and detected using ISO11290-1 method [22]. Briefly, samples were pre-enriched by half Fraser broth (Oxoid, Basingstoke, UK) and enriched by Fraser broth (Oxoid, Basingstoke, UK) for 48 and 24 h at 37 °C, respectively. Finally, the enriched Fraser broth-culture was streaked onto Palcam agar (Oxoid, Basingstoke, UK) and Oxford agar (Oxoid, Basingstoke, UK) followed by 24 to 48 h incubation at 37 °C. The presumed colonies were verified by biochemical tests and API Listeria (bioMérieux, Marcy l’Étoile, France). The isolates of Listeria spp. were then further confirmed by PCR [23].

Serotyping of L. monocytogenes isolates

The detected L. monocytogenes isolates were serotyped using the commercially prepared Listeria antisera against somatic (O) and flagellar (H) antigens according to the manufacturer (Denka-Seiken Co. Ltd., Tokyo, Japan).

Detection of virulence genes by multiplex-PCR

Two multiplex-PCR were used for detection of nine virulence-associated genes. Detection of inlA, inlB, inlC, and inlJ genes was performed by a multiplex-PCR using primers and cycling conditions as described by D Liu, ML Lawrence, FW Austin and AJ Ainsworth [13], and H Jamali and KL Thong [24]. The PCR assay was done as previously described for the actA, hlyA, iap, plcA, and prfA genes in L. monocytogenes isolates [25].

Phenotypic detection of antimicrobial resistance in Listeria spp. isolates

Antibiotic susceptibility testing was done using the Kirby–Bauer disc diffusion method on Mueller Hinton agar (Oxoid, Basingstoke, UK) supplemented with 5 % defibrinated sheep blood [26]. Ampicillin (30 μg), chloramphenicol (30 μg), cephalothin (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), cefuroxime (30 μg), erythromycin (15 μg), florfenicol (30 μg), gentamycin (10 μg), kanamycin (30 μg), pefloxacin (5 μg), penicillin G (10 unit), rifampicin (5 μg), streptomycin (30 μg), tetracycline (30 μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg), and vancomycin (30 μg) were applied as antibiotic agents. As CLSI breakpoints for Listeria species only include a few antimicrobial agents such as sulfamethoxazole-trimethoprim, ampicillin, and penicillin; therefore CLSI [26] breakpoints for Enterococcus were used for the other antimicrobial agents as recommended by M Conter, D Paludi, E Zanardi, S Ghidini, A Vergara and A Ianieri [27], and Q Li, J Sherwood and C Logue [28].

Antimicrobial resistance genes profiling of L. monocytogenes isolates

The tetracycline resistance genes (tetA, tetB, tetC, tetL, tetM, and tetS), ampicillin resistance gene (ampC), vancomycin resistance gene (vanA and vanB), erythromycin resistance gene (ermB), florfenicol resistance gene (floR), chloramphenicol resistance gene (cmlA), and streptomycin resistance gene (strA, and strB) were detected using PCR as previously described [29-38].

Results and discussion

Although the people in the Northern region of Iran rely on fish as their primary animal protein source, there is still no study done on the prevalence and characterization of L. monocytogenes in fish and environment of fish markets. The prevalence of Listeria spp. in raw fish and fish markets environments in this study is presented in Table 1. Out of the 862 tested samples, 133 (15.4 %) were contaminated with Listeria spp., out of which 104 (78.2 %) and 29 (21.8 %) were isolated from raw fish and fish market environments, respectively. The Listeria spp. isolates included L. monocytogenes (n = 43, 32.3 %), L. innocua (n = 47, 35.3 %), L. seeligeri (n = 24, 18 %) and L. ivanovii (n = 19, 14.3 %). All 43 isolates of L. monocytogenes, identified by biochemical tests, were also confirmed using PCR.

Table 1

Prevalence of Listeria spp. in raw fish and environmental samples

	No. of samples	Listeria spp.	L. monocytogenes	L. innocua	L. seeligeri	L. ivanovii
Ctenopharyngodon idella	135	32 (23.7 %)	9 (6.7 %)	14 (10.4 %)	4 (3 %)	5 (3.7 %)
Rutilus kutum	124	27 (21.8 %)	14 (11.3 %)	10 (8.1 %)	1 (0.8 %)	2 (1.6 %)
Liza auratus	120	21 (17.5 %)	7 (5.8 %)	6 (5 %)	5 (4.2 %)	3 (2.5 %)
Hypophthalmichthys molitrix	109	24 (22 %)	7 (6.4 %)	4 (3.7 %)	11 (10.1 %)	2 (1.9 %)
workers’ hands	96	11 (11.5 %)	2 (2.1 %)	5 (5.2 %)	1 (1 %)	3 (3.1 %)
workers’ knives	96	3 (3.1 %)	1 (1 %)	2 (2.1 %)	0	0
Work surface	92	12 (13 %)	2 (2.2 %)	6 (6.5 %)	2 (2.2 %)	2 (2.2 %)
Containers	90	3 (3.3 %)	1 (1.1 %)	0	0	2 (2.2 %)
Total	862	133 (15.4 %)	43 (5 %)	47 (5.5 %)	24 (2.8 %)	19 (2.2 %)

Among the 862 samples, 32 Ctenopharyngodon idella (23.7 %), 27 Rutilus kutum (21.8 %), 24 Hypophthalmichthys molitrix (22 %), and 21 Liza auratus (17.5 %) were naturally contaminated with Listeria spp. In addition, 12 work surface (13 %), 11 workers’ hands (11.5 %), and 3 containers (3.3 %), and 3 workers’ knives (3.1 %) harboured Listeria spp. Several studies on the prevalence of Listeria spp. in fish, fish products and environments have been performed worldwide [39, 40]. The prevalence of Listeria spp. isolated from raw fish and fish markets in this study concurred with the earlier findings in Iran and other countries [41, 42]. However, our findings showed higher prevalence of Listeria spp. compared with the previous studies [43, 44].

In the present study, L. monocytogenes was isolated from 37/488 raw fish (7.6 %) and 6/374 environments of fish markets (1.6 %). Previous reports by H Momtaz and S Yadollahi [43] and VS Parihar, S Barbuddhe, M-L Danielsson-Tham and W Tham [42], indicated contamination of 7.7 and 9 % of fish samples with the pathogen. L. monocytogenes has been isolated from fish and fish products in earlier studies in different provinces of Iran, including Khuzestan (4 %) [41], West Azarbaijan (2.6 %) [45], and Gilan (2.2 %) [46]. Furthermore, other reports has shown contamination of fish market environment samples with the pathogen in central part of Iran (16.5 %) and Northern Greece (5 %) [40, 47]. It is known that L. monocytogenes is ubiquitous and can be found in soil, vegetation, sewage, surface water as well as foods [11]. Hence, it is clear that a large source of the pathogen could survive inside fish and also in its surroundings.

Of the 43 L. monocytogenes isolates from raw fish and environment of fish market samples, 31 (72.1 %), 10 (23.3 %), and 2 (4.7 %) were serovar 1/2a, 4b, and 1/2b, respectively (Table 2). We found that the 1/2a was the predominant L. monocytogenes serovar in the samples tested in the present study. The high percentage rate of serovar 1/2a in raw fish and environment of fish markets in this study is in agreement with the previous reports from Iran, Finland and Estonia [46-49]. Serovar 4b was isolated from raw fish only. The 4b was predominant serovar in fish and seafood products in Iran, China, and Israel [43, 50, 51]. On the other hand, B Siriken, ND Ayaz and I Erol [52] recently reported that serovar 1/2b was common serovar in Turkish raw and processed seafood products. Our finding on the presence of serovar 4b indicates that fish could be a potential source of human listeriosis, in the Northern region of Iran.

Table 2

Prevalence of L. monocytogenes serovars in raw fish and environmental samples

	Serovars of L. monocytogenes
	1/2a	4b	1/2c
Ctenopharyngodon idella	6 (60 %)	3 (30 %)	0
Rutilus kutum	9 (64.3 %)	4 (28.6 %)	1 (7.1 %)
Liza auratus	6 (85.7 %)	1 (14.3 %)	0
Hypophthalmichthys molitrix	4 (57.1 %)	2 (28.6 %)	1 (7.1 %)
Workers’ hands	2 (100 %)	0	0
Workers’ knives	1 (100 %)	0	0
Work surface	2 (100 %)	0	0
Containers	1 (100 %)	0	0
Total	31 (72.1 %)	10 (23.3 %)	2 (4.7 %)

All 43 isolates of L. monocytogenes were tested for the presence/absence of virulence genes inlA, inlB, inlC, and inlJ. Although, inlA, inlB, and inlC genes were observed in all of the L. monocytogenes, inlJ gene was detected in 42/43 (97.7 %) of the L. monocytogenes isolates. The surface-associated internalin is alleged to play a role in the pathogenesis of listeriosis [53]. This is the first study, to the best of our knowledge, to examine L. monocytogenes isolates from raw fish and environment of fish markets for the presence of four main internalin genes (inlA, inlB, inlC, and inlJ). Similar results were obtained in the previous studies, where the tested internalin genes were present in almost all of the examined L. monocytogenes isolates from animals [17], human listeriosis [54], different kinds of foods [15, 18, 24] and environmental samples [55].

Although, the actA, hlyA, and iap genes were detected in all of the L. monocytogenes isolates, plcA and prfA genes were observed in 41 (95.3 %) and 42 (97.7 %) of the isolates, respectively. The prevalence rate of these five virulence genes in this study is in concurrence with the earlier studies in Iran [43] and India [56] where the actA, hlyA, iap, plcA, and prfA genes were observed in all the L. monocytogenes isolates recovered from seafood samples. The presence of nine virulence-associated genes in almost all of the L. monocytogenes isolates suggests that these isolates could be potentially virulent.

In total, 53 and 57 Listeria spp. isolates (39.8 and 42.9 %) were resistant to one and two antibiotics, respectively (Table 3). In addition, nine isolates of Listeria spp. (6.8 %) were multi-drug resistant. The most frequent antibiotic-resistance was resistance to tetracycline (23.3 %), followed by penicillin G, cephalothin (each 16.5 %), streptomycin (15.8 %), florfenicol (15 %), erythromycin (14.3 %), ampicillin (12 %), trimethoprim-sulfamethoxazole (12.5 %), ceftazidime (8.3 %), vancomycin (6 %), rifampicin (3.8 %), chloramphenicol (1.5 %), and cefuroxime (1.2 %). Out of 43 L. monocytogenes isolates, 15 (34.9 %), 20 (46.5 %) and 6 (6.4 %) were resistant to one, two and more than two antibiotics. The L. monocytogenes isolates indicated high resistance to tetracycline (27.9 %), ampicillin (20.9 %), cephalothin, penicillin G, and streptomycin (each 16.3 %). All of the Listeria spp. isolates were sensitive to cefotaxime, gentamicin, kanamycin, and pefloxacin.

Table 3

Resistance profiles of Listeria spp. isolated from raw fish and environmental samples

	Listeria spp. (n = 133)	L. monocytogenes (n = 43)	L. innocua (n = 47)	L. seeligeri (n = 24)	L. ivanovii (n = 19)
Ampicillin	16 (12 %)	9 (20.9 %)	5 (10.6 %)	2 (8.3 %)	0
Chloramphenicol	2 (1.5 %)	1 (2.3 %)	1 (2.1 %)	0	0
Cefotaxime	0	0	0	0	0
Ceftazidime	11 (8.3 %)	6 (14 %)	4 (8.5 %)	0	1 (5.3 %)
Cefuroxime	1 (1.2 %)	0	1 (3.6 %)	0	0
Cephalothin	22 (16.5 %)	7 (16.3 %)	11 (23.4 %)	3 (12.5 %)	1 (5.3 %)
Erythromycin	19 (14.3 %)	6 (14 %)	9 (19.1 %)	3 (12.5 %)	1 (5.3 %)
Florfenicol	20 (15 %)	6 (14 %)	11 (23.4 %)	2 (8.3 %)	1 (5.3 %)
Gentamycin	0	0	0	0	0
Kanamycin	0	0	0	0	0
Pefloxacin	0	0	0	0	0
Penicillin G	22 (16.5 %)	7 (16.3 %)	9 (19.1 %)	2 (8.3 %)	4 (21.1 %)
Rifampicin	5 (3.8 %)	1 (2.3 %)	2 (4.3 %)	2 (8.3 %)	0
Streptomycin	21 (15.8 %)	7 (16.3 %)	8 (17 %)	3 (12.5 %)	3 (15.8 %)
Tetracycline	31 (23.3 %)	12 (27.9 %)	11 (23.4 %)	5 (20.8 %)	3 (15.8 %)
Trimethoprim-sulfamethoxazole	14 (10.5 %)	5 (11.6 %)	5 (10.6 %)	3 (12.5 %)	1 (5.3 %)
Vancomycin	8 (6 %)	3 (7 %)	4 (8.5 %)	1 (4.2 %)	0
Resistant to 1 antibiotic	53 (39.8 %)	15 (34.9 %)	14 (29.8 %)	10 (41.7 %)	14 (73.7 %)
Resistant to 2 antibiotics	57 (42.9 %)	20 (46.5 %)	27 (57.4 %)	8 (33.3 %)	2 (10.5 %)
Resistant to > 2 antibiotics	9 (6.8 %)	6 (14 %)	3 (6.4 %)	0	0

The 43 L. monocytogenes were examined for the presence of resistance genes. Six of 43 L. monocytogenes isolates (14 %) harbored more than one antimicrobial resistance gene. Among the evaluated serovars of L. monocytogenes isolates, a higher prevalence of antimicrobial resistance genes was detected in serovar 1/2a (81.5 %), followed by serovar 4b (18.5 %). However, the resistance genes were not found in serovar 1/2c isolates.

For tetracycline resistance, the tetM and tetA genes were present in 91.7 and 83.3 % of the tetracycline-resistant isolates, respectively and 71.4 % of the penicillin-resistant isolates harboured penA gene. Out of 7 streptomycin-resistant isolates, 42.9 and 14.3 % isolates contained strA, and strB, respectively. Furthermore, the ampC and vanA resistance genes were found in 66.7 and 33.3 % of the ampicillin- and vancomycin-resistant isolates, respectively. However, the tetB, tetC, tetL, tetS folR, cmlA, and vanB were not detected in the examined L. monocytogenes isolates from raw fish and open-air fish market environments. The prevalence rate of the tetM, tetA, penA and strA genes in the present study is in agreement with earlier investigations, in which a high frequency of these resistance genes in L. monocytogenes isolates was reported by C Poyart-Salmeron, P Trieu-Cuot, C Carlier, A MacGowan, J McLauchlin and P Courvalin [57], and V Srinivasan, H Nam, L Nguyen, B Tamilselvam, S Murinda and S Oliver [29].

In the current study, the phenotypic resistance profiles of the L. monocytogenes isolates were not confirmed by detection of resistance genes. For instance, 2 of 6 vancomycin-resistant isolates which showed phenotypic resistance to vancomycin, harbored vanA gene and none of them contained vanB. Likewise, out of 43 L. monocytogenes isolates, 7 (16.3 %) were phenotypically resistant to penicillin G, however, only 6 of the isolates (11.6 %) carried penA resistance gene. The same results were reported in earlier studies [29]. This inconsistency suggests that mutation in ribosomal protein gene or decreased outer membrane permeability can contribute to antimicrobial resistance phenotypes [58, 59].

A high resistance of L. monocytogenes to tetracycline and penicillin G was observed in the present study. Our findings were in agreement with a previous investigation, in which a high resistance of L. monocytogenes to tetracycline and penicillin G was also reported by AA Fallah, SS Saei-Dehkordi and M Mahzounieh [47], and O Rodas-Suárez, J Flores-Pedroche, J Betancourt-Rule, EI Quiñones-Ramírez and C Vázquez-Salinas [39]. Tetracycline and fluoroquinolones are widely applied as growth supplement and therapeutic agents in Iranian fish farms, respectively. The presence of antibiotic-resistant L. monocytogenes as well as multi-drug resistant isolates in fish on the one hand and transmission of the pathogen through contaminated fish on the other hand, clarify major public health concerns associated with this pathogen.

Conclusions

In conclusion, recovery of potentially pathogenic L. monocytogenes from raw fish and environment of open-air fish market samples evidences the zoonotic potential of listeriosis. Hence, further surveillance of the prevalence of L. monocytogenes and also of emerging antibiotic resistance is required to enable the recognition of the contaminated foods, as well as ensure the effective antibiotic treatment.