Isolation and characterization of Listeria monocytogenes from tropical seafood of Kerala, India

Abstract

Listeria monocytogenes, which is an intracellular pathogen, causes various illnesses in human as well as in animals. The pathogenicity of this organism depends upon the presence of different virulence genes. A total of 324 tropical seafood and fishery environmental samples were screened for L. monocytogenes. The incidence of the human pathogenic species L. monocytogenes was 1.2 % of the samples. Listeria spp. was detected in 32.3, 27.1, and 5 % of fresh, frozen, and dry fish samples, respectively. Listeria innocua was found to be the most prevalent species of Listeria in the tropical seafood and environmental samples of Kerala. Listeria monocytogenes and L. innocua isolates were confirmed by multiplex PCR. L. monocytogenes isolates from the four positive samples showed phosphatidylinositol-specific phospholipase C reaction on Chromocult® Listeria selective agar. Molecular characterization of L. monocytogenes isolates for virulence genes revealed the presence of β-hemolysin (hly), plcA, actA, metalloprotease (mpl), iap and prfA genes in all the isolates recovered from the positive samples.

Introduction

Listeria monocytogenes is the major human pathogen among eight species of Listeria. This organism causes abortion, encephalitis, gastroenteritis, arthritis, conjunctivitis, etc. in human (Vázquez-Boland et al. 2001). Apart from L. monocytogenes, L. ivanovii has also been reported to cause human infection in some cases (Guillet et al. 2010). Listeria monocytogenes also causes clinical diseases in animals. In ruminants, listeriosis is characterized as encephalitis showing typical ’circling’ symptoms, third trimester abortion, stillbirth, conjunctivitis, etc. (Hoelzer et al. 2012). This pathogen is widely distributed in nature and is generally transmitted to human through contaminated food and water. Being an intracellular pathogen, it can multiply even inside different phagocytic cells like macrophages (Kuhn et al. 1988). Biofilm formation on food-contact surfaces by this pathogen is an indication of severe public health hazards (Zameer et al. 2010). Listeria monocytogenes is one of very few pathogenic organisms which can grow at refrigerated temperatures. Thus, the storage of food at low temperatures cannot prevent the growth of this pathogen (Junttila et al. 1988).

Listeriosis in human is very often associated with very high mortality which may be as high as 30 % (Griffiths 1989). In USA, approximately 1,591 cases of listeriosis in human have been reported in a year and it contributes to about 0.1 % of total food-borne illnesses, but is responsible for 2.6 % of hospitalizations and 18.87 % of the deaths caused by food-borne illnesses (Scallan et al. 2011). Both the outbreaks and sporadic human listeriosis is caused by transmission of this pathogen through contaminated food and the involvement of a wide variety of foods has been reported throughout the world (Linnan et al. 1988; Ericsson et al. 1997). In USA, the recent multistate outbreak of L. monocytogenes apparently through consumption of contaminated cantaloupe has drawn worldwide attention. A total of 146 persons were infected and 33 deaths were reported in this outbreak in which multiple states of USA were affected (CDC 2012). In 2010, there was an incidence of another outbreak of listeriosis in Texas, USA, apparently through consumption of celery (CIDRAP 2010). Considering its immense pathogenic potentiality and high mortality rate, different regulatory agencies including USFDA have imposed zero tolerance for this pathogen in ready-to-eat (RTE) food products (Hitchins 1998).

The virulence of this pathogen is mediated by different virulence genes, i.e. β-hemolysin (hly), phosphatidylinositol-specific phospholipase C (plcA), prfA, iap, actA, and metalloprotease (mpl) (Domann et al. 1991; Rawool et al. 2007). prfA gene is known as a positive regulatory factor, which controls the expression of other virulence genes (Chakraborty et al. 1992). Although India has not experienced any major outbreak of listeriosis, sporadic cases in human have been reported and L. monocytogenes has been previously isolated from different food products including seafood (Malik et al. 2002; Dhanashree et al. 2003). Clinical cases of listeriosis in human caused by L. monocytogenes have also been reported from Delhi and the Karnataka area of India (Bhujwala et al. 1973; Dhanashree et al. 2003). Therefore, regular screening and constant surveillance of food products including seafood for the presence of this pathogen are required.

The present study has been undertaken to assess the occurrence of this pathogen in the tropical seafood and fisheries environment of Kerala, India, and also to screen the presence of different virulence genes in L. monocytogenes isolates recovered from the samples.

Materials and methods

Samples

A total of 324 samples of seafood comprising of 255 seafood and 69 fisheries environment samples were screened for the presence of L. monocytogenes during the period from March 2005 to March 2006 and from November 2007 to December 2011 (Table 1). The samples were procured from fish hatcheries, fish landing centers, and retail outlets located at Ernakulum, Alappuzha, and Kottayam and Kollam districts of Kerala, India.

Table 1 Listeria spp. in the fish and fisheries environment of tropical area of Kerala, India, based on types of samples

Standard cultures used in this study

Listeria monocytogenes ATCC 19115 and L. innocua ATCC 330390 were procured from the American type culture collection, USA. Listeria monocytogenes MTCC 657 was procured from the Microbial type culture collection, Institute of Microbial Technology, Chandigarh. Listeria monocytogenes NCTC 11994 was maintained in the Microbiology, Fermentation and Biotechnology Division. Staphylococcus aureus NCIM 5022 was procured from the National Collection of Industrial Microorganisms, National Chemical laboratory, Pune. All the type cultures were maintained on Tryptic soya agar slants at 4 °C with intermittent subculture.

Isolation and identification of the organism

The screening of the samples for the presence of Listeria spp. was carried out using the ISO 11290-I method mentioned in Scotter et al. (2001). Twenty five grams of sample was taken in 225 ml of half-Fraser’s broth, macerated in a stomacher blender (Seward), and incubated at 30 °C for 24 h. Then, 0.1 ml was transferred to Fraser’s broth and was incubated at 37 °C for 48 h. Plating from both half-Fraser’s broth and Fraser’s broth was done on Listeria selective agar (Oxford formulation) and PALCAM agar plates. Oxford and PALCAM plates were incubated at 30 and 37 °C, respectively, for 48 h. The typical colonies from Listeria selective agar (Oxford and PALCAM) were isolated, purified again on corresponding agar media (Oxford or PALCAM), and stored on Tryptic soya agar slants at 4 °C. Identification of the isolates was done by standard biochemical tests, restricted β-hemolysis on sheep blood agar, and the CAMP test as mentioned in Hitchins (1998). Listeria monocytogenes isolates were further confirmed by spot inoculation on Chromocult® Listeria selective agar (Merck, Germany) for phosphatidylinositol-specific phospholipase C activity. The plates were incubated at 37 °C for 48 h and were examined for typical blue-green colonies of L. monocytogenes with an opaque halo surrounding the colony.

Serogrouping of the L. monocytogenes strains and isolates

Listeria monocytogenes isolates recovered from seafoods in this study, two freshwater fish isolates from Goa, India, L. monocytogenes ATCC 19115 and L. monocytogenes MTCC 657, were employed in this study. They were serogrouped using Bacto Listeria Type 1 and Type 4 antisera (Difco, USA) by the slide agglutination test as per the manufacturer’s instruction. Two L. monocytogenes isolates from Goa were kindly donated by Dr. S.B. Barbuddhe, Senior Scientist, ICAR Research Complex, Goa, India.

Preparation of bacterial lysate for multiplex PCR

All the cultures were grown in BHI broth for the preparation of lysate. Lysate from the bacteria was prepared by boiling method as done by Fitter et al. (1992) with slight modifications. The bacterial culture was centrifuged at 7,000 g for 10 min at 4 °C and the resultant pellet was washed once in normal saline. The pellet was suspended in 150 μl sterile distilled water and kept in boiling water bath for 10 min and frozen immediately at −30 °C. Before use as the PCR template, it was thawed and centrifuged at 4,000 g to pellet the debris. Five μl of the supernatant was used as template in the PCR reaction.

Multiplex PCR for differentiation of L. monocytogenes and L. innocua

Three sets of primers were used for this multiplex PCR reaction. Primers pairs UI/LII, LL5/LL6, and lin0464F/l in0464R are specific to Listeria genus, L. monocytogenes, and L. innocua, respectively (Table 2). Primers pairs UI/LII and LL5/LL6 are based on the 16S rRNA gene and the hly gene, respectively, whereas the primer pair lin0464F/lin0464R is based on a gene with unknown function in L. innocua. This PCR assay was carried out using the protocol of Das et al. (2010). PCR assays were started with an initial denaturation at 95 °C for 3 min followed by 30 cycles of 94 °C for 45 s (denaturation), 55˚ for 45 s (annealing), and primer extension at 72˚ for 45 s. Final extension was carried out at 72 °C for 5 min. PCR products were resolved on electrophoresis on 1.5 % agarose gel containing 0.3 μg/ml ethidium bromide in 0.5× TBE buffer at 7 V/cm for 1 h.

Table 2 Primers used in this study

Screening for virulence genes in L. monocytogenes

Listeria monocytogenes isolates from the four positive samples of this study, as well as the standard strains, were screened separately for the presence of different virulence genes, e.g., β-hemolysin (hly), plcA, actA, metalloprotease (mpl), iap and prfA genes by PCR. Genomic DNA from all the standard strains and isolates were purified as per the method of Liu et al. 2003. The concentration and purity of the genomic DNA were estimated by taking the OD at 260 and 280 nm. For screening of hly, plcA, actA, iap and prfA genes, the primers as mentioned in Rawool et al. (2007) were used. The primer pair targeting the mpl gene has been designed based on the published sequences of mpl genes of L. monocytogenes using Primer 3 software, which is freely available online at http://frodo.wi.mit.edu/. The sequences of all the primers used in this study are shown in Table 2. For amplification of the hly, plcA, actA, iap and prfA genes, PCR was carried out in a 50 μl reaction mixture consisting of 10× PCR buffer (Fermentas), 2 mM MgCl₂, 0.2 mM dNTPs mix, 1U Taq DNA polymerase and 10 μM of the respective primer pair. The PCR cycling condition consisted of initial denaturation at 95 °C for 5 min followed by 35 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 1 min 30 s. Final extension was done at 72 °C for 5 min. The PCR reaction mixture for detection of mpl was same as in case of other virulence genes except the MgCl₂ concentration, which was 1.5 mM. For the amplification of the mpl gene, the same cycling condition was used except for the annealing temperature, which was 59 °C. PCR products were resolved as described previously.

Results

Isolation of Listeria spp.

Among 324 samples, Listeria spp. were recovered from 95 samples (occurrence rate 29.3 %) and L. monocytogenes was detected in 4 samples, i.e. 2 marine fish samples, 1 freshwater fish and 1 ice sample (Table 1). However, L. innocua was found to be the most prevalent species of Listeria present in the tropical water of Kerala with an incidence rate of 28.7 %. The incidence of L. monocytogenes was found to be very low, i.e. only 1.2 % of total samples tested. Among fresh, dry, and frozen samples, the lowest occurrence of Listeria spp. was found in the dry fish (5 %). In fresh and frozen fish samples, the occurrence of Listeria spp. was found to be 32.3 and 27.1 %, respectively. Among fisheries environmental samples, 29 % of the samples (20 out of 69) were found to harbor Listeria spp. and L. monocytogenes was detected in one sample (ice).

Listeria monocytogenes isolates were identified by standard biochemical tests and were confirmed in Chromcult® Listeria selective agar. All the L. monocytogenes type cultures and isolates produced blue-green colonies with opaque halos, whereas L. innocua colonies yielded blue-green colonies but without an opaque halo.

Serogrouping of L. monocytogenes

Listeria monocytogenes isolates from two marine fish and freshwater fish were found to be belong to serogroup 1, whereas ice isolates belong to serogroup 4. On the other hand, both the freshwater fish isolates from Goa belong to serogroup 4. Listeria monocytogenes ATCC 19115 and MTCC 657 also belong to serogroup 4.

Multiplex PCR

In multiplex PCR assay, all the L. monocytogenes type cultures and isolates from four positive samples yielded 267 and 938 bp PCR products, whereas L. innocua isolates yielded 938 and 749 bp PCR products (Fig. 1). Staphylococcus aureus NCIM 5022 (used as negative control) and the isolates, which were not confirmed as Listeria spp. did not yield any amplified product (not shown in the image).

Fig. 1

Identification of isolates by multiplex PCR. Lane M Generuler™ DNA ladder plus, lane 1 L. monocytogenes NCTC 11994, lanes 2–5 L. innocua isolates, lane 6 L. monocytogenes isolates from fish, lanes 7–8 L. innocua isolates

Virulence genes in L. monocytogenes

Listeria monocytogenes standard strains and isolates were screened for the presence of different virulence genes. All the isolates tested showed the presence of 456, 1,484, 131, 1,060 and 1,376 bp amplified products in hlyA, plcA, iap, prfA and mpl gene-specific PCR, respectively. actA gene-specific PCR using primers yielded 839 bp PCR products in all the strains tested except L. monocytogenes MTCC 657.

Discussion

The ability to grow in different food products at low temperatures and cause different types of human illnesses render L. monocytogenes a potential public health hazard in food industry. This pathogen is ubiquitous in nature and is more resistant to different sanitizing agents including quaternary ammonium compounds (Mereghetti et al. 2000). The presence of L. monocytogenes in food is sourced from raw materials as well as food processing environments, which include different food contact surfaces on which there is the possibility of the formation of biofilm by this pathogen. Being an important and emerging food-borne pathogen, it requires special attention with special reference to its prompt detection and control of growth in food. In this study, it has been observed that, in the tropical water of India, the incidence of L. monocytogenes was 1.2 % in fish and environmental samples. Listeria monocytogenes isolates of both serogroups 1 and 4 have been detected in the present study. The most prevalent species of Listeria was L. innocua, as the incidence has been found to be 28.7 %. These findings are in agreement with those of Fuchs and Surendran (1989), who observed that 33.3 % of the seafood samples were positive to L. innocua. In a study on the incidence of Listeria spp. in fishes of Mysore, India, it was found that 37.8 % of the samples were positive to Listeria spp., whereas L. monocytogenes was detected in 1.83 % of the samples (Moharem et al. 2007). However, Jeyasekaran et al. (2003) observed that 7.6 % of the fisheries environmental samples of Mangalore, India, were positive for both Listeria spp. and L. monocytogenes. Listeria innocua and L. monocytogenes share a common ecological niche and L. innocua is sometimes considered as an indicator organism for L. monocytogenes (Bubert et al. 1999). Thus, the frequent screening of seafood samples for the presence of L. monocytogenes is of utmost importance.

Different virulence genes, i.e. β-hemolysin, phosphatidylinnositol-specific phospholipase C, iap, actA, mettaloprotease, and prf A gene play important roles in the virulence of L. monocytogenes. All the virulence factors act synergistically in intracellular survival and pathogenicity of L. monocytogenes (Vázquez-Boland et al. 2001). Listeria monocytogenes isolates from all the four positive samples as well as two freshwater fish isolates from Goa showed the presence of mpl, hly, plcA, actA, prfA and iap genes, which are responsible for key steps of L. monocytogenes cellular proliferation. The presence of these virulence genes is indicative of probable virulence.

The present study indicates that L. monocytogenes harboring key virulence genes is present in seafood and the fisheries environment of Kerala, India. Thus, this psychrotrophic pathogen requires constant monitoring and surveillance in fish and fisheries products for effective control.

Conclusion

In this study, L. monocytogenes was isolated in 4 out of 324 samples showing the low occurrence rate (1.2 %). But a comparatively high occurrence rate was found in the case of L. innocua (28.7 %). All the virulence genes screened were detected in the L. monocytogenes isolates recovered from the positive samples. Isolates of both serogroups 1 and 4 were detected in the tropical water of Kerala. Thus, it can be concluded that constant monitoring and surveillance for the presence of this psychrotrophic pathogen in Indian seafood are required.