First Report of Listeria monocytogenes Serotypes Detected from Milk and Milk Products in Sri Lanka

Received: Revised: Accepted: 2014–06–06 2014–07–06 2014–07–09 Listeria monocytogenes is the causative organism of listeriosis, which primarily affects immune– compromised individuals, including pregnant women. Contamination of milk and milk products with L. monocytogenes is a serious problem to the world and to the developing countries like Sri Lanka. Even the presence of low numbers of L. monocytogenes is a potential risk since this organism is capable of multiplying at ambient and under refrigerated conditions. The main aim of the study is to detect the circulating serotypes in dairy industries using molecular methods and primarily to trace the lineage of these serotypes with a focus on food safety and disease prevention. Altogether total of 266 samples from raw milk, pasteurized milk, ice cream, curd, yogurt and cheese were collected from many parts of the country, tested for the presence of L. monocytogenes and studied the serotype. L. monocytogenes was detected in all types of the above–mentioned milk and milk products. Out of total L. monocytogenes strains detected by PCR, 61.51%, 11.53% and 4% belong to serotypes 1/2a (or 3a) [lineage II], 1/2b (or 3b) [lineage I] and, 1/2c (or 3c) [lineage II] respectively. It has been observed previously that serotypes (1/2a), 1/2b) isolated from raw milk samples been linked to multiple human listeriosis outbreaks. This study revealed the serotypes of L. monocytogenes circulating in Sri Lanka, thus makes an alarm to the health authorities to be more vigilant about the probable outbreaks of listeriosis in Sri Lanka. This is the first report on circulating L. monocytogenes serotypes in Sri Lanka. All copyrights reserved to Nexus® academic publishers


INTRODUCTION
Listeria monocytogenes is a pathogenic bacterium that can cause Listeriosis in humans and various animal species.In humans, foodborne L. monocytogenes causes large outbreaks of Listeriosis, with a mortality rate of 9% to 44% (Clark et al., 2010).The primary mode of infection is through the ingestion of contaminated food.Therefore, contamination of milk and milk products with L. monocytogenes is a serious problem to the world (Kozak et al., 1996;Donnelly, 2004;Rudolf and Siegfried 2001;Conly and Johnston, 2008;Pan et al. 2006;Molla et al., 2004).
Only two studies have been reported in Sri Lanka to ascertain the presence of L. monocytogenes in our dairy products.According to their results the percentage of L. monocytogenes contaminated milk samples in Sri Lanka is much higher compared to developed countries (Gunasena et al., 1995 andJayamanne andSamarajeewa, 2001).Only four suspected cases of listeriosis have been reported in Sri Lanka so far (Wijesundera et al., 1992, Withana andMirando, 1967).There is no information about the circulating serotypes in Sri Lanka so far.
The main aim of our study is to detect the circulating serotypes in dairy industries using molecular methods and primarily to trace the lineage of these serotypes, as these serotypes are responsible for outbreaks and epidemics.

Bacterial Strains and Growth Conditions
L. monocytogenes ATCC 51776 and L. innocua ATCC 33090 was used in the sensitivity & specificity studies respectively.All the bacterial strains were grown on blood agar (Oxoid-UK) at 37 0 C for 24 hrs.and sub cultured in Brain Heart Infusion (BHI) broth.Listeria Enrichment broth (LEB) from Oxoid-UK was used for enrichment and the Listeria selective agar medium, (Oxoid-UK) was used for selective plating.Stock cultures were made using skimmed milk (William et al., 2008).

Sample Collection
Samples were collected randomly from different parts of the country (Figure I).A total of 154 samples of raw and pasteurized milk samples were collected from farms and milk collecting centers and 112 samples of milk products such as cheese, curd, yogurt and ice cream were obtained from retail outlets including supermarkets, farm shops, groceries, milk booths and public markets (Table I).

Enrichment and DNA Extraction
A total of 25 g or 25 ml of dairy samples were incubated in 225 ml of LEB at 30 ± 1 °C for 24 and 48 hrs.After 24 hrs.500µL of suspension was extracted according to Gerrit et al (2005) with modifications include three washing steps using PBS.Extracted DNA was kept at -20 o C for further analysis.

PCR Amplifications:
Nested PCR detection was performed according to Lieve et al., 1995.As a modification, to achieve the optimal sensitivity 5 µL, 2 µL and 1 µL of the first PCR mixture were used as template in the second PCR.Thermal cycler 9600 (Perkin-Elmer Corp.) was used to run the PCR reactions.

Serotyping by PCR
Serotyping was done according to Borucki and Call, (2003).

Statistical Analysis
Chi-square test analysis was performed and differences were considered significant at values of P<0.05.

Sensitivity of the Nested PCR
The specificity of the PCR primers has previously been validated (Chiba et al (1998), Border et al) but the sensitivity was reassessed in our laboratory by amplification of 10-fold serial dilutions in milk by spiking axenically grown L. monocytogenes cells.The minimal amount of L. monocytogenes that could be reliably detected was 1cfu.To remove the presence of potential PCR inhibitor(s) in the milk samples, washing steps were added in the DNA extraction as a modification.One micro liter of first PCR gave the optimal sensitivity in the nested PCR.In the present study, a total of 266 raw milk and dairy product samples from all over the country were examined.The study revealed 45 samples of (41.19%) raw milk, 10 samples of (35.71%) ice cream, 10 samples of (18.36%) pasteurized milk, 8 samples of (28.57%) curd, 4 samples of (14.2%) cheese and 3 samples of (10.71%) yoghurt were contaminated with L. monocytogenes (Figure II).The number of samples contaminated by L. monocytogenes found to be significant with the type of dairy products.Although Listeria contamination was generally found in all types of dairy products in all districts, numbers of raw milk samples contaminated at district levels were found to be significant.However, numbers of other dairy products contaminated at district level were not significant (Figure III).With regard to the distribution of raw milk contamination it was observed that the highest contamination was found in districts of Kandy and Gampaha (26%).Moderate amount of raw milk contaminants were observed in Anuradhapura (17%), Polonnaruwa (9%), Hambantota (9%) & Colombo (7%) districts.In contrast lowest contamination occurred in districts of Galle (4%) (Figure IV).

Dairy Type
No

DISCUSSIONS
Dairy products are known to be one of the vehicles through which L. monocytogenes is transferred to human.Therefore many countries have initiated a zero tolerance policy prohibiting the sale of processed ready-to-eat (RTE) food products contaminated with L. monocytogenes.This policy designates L. monocytogenes as a contaminant.Previous studies have shown that L. monocytogenes has a very low occurrence in dairy products (Jalali andAbedi, 2007, Moshtaghi andMohamadpour, 2007).Anyhow in our study as we used molecular detection method with high sensitivity (1 cfu detected) very low level of L. monocytogenes contaminants were able to be detected in the variety of dairy products.
Our results revealed higher number of L. monocytogenes (42%) detected from raw milk samples in comparison to other countries like 0% in center of Iran (Jalali and Abedi, 2008) 0.018% in United States (Frye and Donnelly, 2005) 1.1% in England and Wales (Greenwood et al., 1991) and 1.6% in west of Iran (Moshtaghi and Mohamadpour, 2007).In contrast Pintado et al., (2005) reported higher rate of incidence (up to 46%) in Portugal.These differences may be due to varying environmental condition between different locations as well as method of detection.These figures cautious those hygienic conditions of milking and the subsequent manipulations in our country are substandard.
Isolation of L. monocytogenes from ice cream (35.7%) in this study is another proof that this microorganism can survive freezing temperatures (Cordano and Rocourt, 2001).Increased contamination of ice cream by this bacterium is explained by suitable conditions for its growth, such as pH value, water activity, availability of nutrients and storage temperature (Molla et al., 2004).
Significant amount (20.4%) of contamination of L. monocytogenes of pasteurized milk due to the presence of abnormally high pathogen load in the raw milk may have overwhelmed the pasteurization process (Jayamanne and Samarajeewa, 2010.) or that localization of some L. monocytogenes within bovine cells may have protected them from heat inactivation (Swaminathan, 2001).In Massachusetts, the listeriosis outbreak in 1983 was associated with consumption of pasteurized milk (Fleming et al., 1985).
In fermented products such as curd, yogurt, L. monocytogenes has the ability to survive during production process and storing.Further, low amount of Listeria contaminants were found in yogurt (10.71%) and cheese (14.28%) in this study.The survival of L. monocytogenes in yogurt depends on the sample acidity and this bacterium disappears when the pH falls to 3.5 (Vermeulen et al., 2007).
When consider about the distribution pattern of Listeria contamination of raw milk the highest contamination occur in selected areas of Kandy and Gampaha district (wet zone).It may be due to the high moisture content of the soil.Previous studies also indicated that the favourable places for Listeria growth is high moisture content in the environments where the organism has had opportunity to incubate (Bunning et al., 1992).Apart from this, in coastal areas such as Galle, Hambantota and dry zone areas such as Anuradhapura, Polonnaruwa encountered moderately low amount of raw milk contaminants due to the high salinity and low moisture content of the environment respectively.Previous studies indicated that the growth of L. monocytogenes; the pH values ranged from 5.96 ~ 5.10, with a mean of 5.48; the salt concentrations ranged from 6.30% ~ 3.20%, and the moisture levels averaged 37.79% (Kongo et al., 2006).
In this study serogrouping by PCR suggests serotypes belonging to phylogenetic lineage of division I comprises serogroups 1/2b (or 3b) and division II comprises 1/2a (or 3a), 1/2c (or 3c).Isolates within this group appeared in all dairy types.It was found that serotype 1/2b (or 3b) was rare with only one positive in raw milk and two positives being picked up in pasteurized milk.This is in line with most studies that have found serotypes 1/2a and 1/2b was to be the most common serotypes in food (Aarnisalo et al., 2003: Wallace et al., 2003: Cetinkaya et al. 2014).
One major finding in this study is that the majority of the isolates found in dairy samples belong to lineage II [1/2a (or 3a), 1/2c (or 3c)] this is in line with the previous studies (Gray et al., 2004;Ward et al., 2004).
This study alarms us the possible out breaks of Listeriosis.The presence of serotype of 1/2a & 1/2b further supports this view.From a public health standpoint, it is extremely important to identify the contaminated food vehicle and remove it from food distribution channels as rapidly as possible.This is the first report in Sri Lanka on serotypes circulating in the country, which was identified from the dairy products representing different parts of the country.These findings open the avenue for more research on circulating serotypes and their lineages.The identification and characterization of L. monocytogenes lineages with unique epidemiological, ecological, genetic, and phenotypic characteristics also has important practical implications.For example, it is increasingly apparent that representatives of all lineages need to be included in strain sets that are used to evaluate new detection and sub typing methods.The identified serotypes could be used in genetic analysis and lineage studies, which in turn shed new light on Sri Lankan, isolate characteristics.
Further this study will help in formulating National Listeria Risk Assessment guidelines for dairy industry in Sri Lanka.In future similar study will be done in human samples thus different serotypes of L. monocytogenes prevailing in the Sri Lankan population can be identified which in turn will help us to investigate the molecular epidemiology of the disease listeriosis in the future.

Figure I :FigureFigure
Figure I: Locations of the sample collection Figure VI: Distribution of serotypes in each dairy type