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Short Communication · Volume 52, Issue 2, 15 February 2020 · Open Access

Isolation and Identification of Listeria monocytogenes from Raw Vegetables and Meat Sold in Quetta, Pakistan

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Affiliations & Notes ⌄
  1. 1Center for Advanced studies in Vaccinology and Biotechnology, University of Balochistan, Quetta, Pakistan 2Bolan Medical College, Quetta 3Department of Zoology, Sardar Bahadur Khan Women University, Quetta , * Corresponding author: tauseefcasvab@gmail.com
Article Info ⌄
Journal:
Pakistan Journal of Zoology
Published:
15 February 2020
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From the article

  • The application of molecular techniques has facilitated the identification and characterization of major virulence-associated genes in L. monocytogenes.
  • The ten isolates of L. monocytogenes isolated from different food sources were analyzed for the presence of virulence associated genes employing the multiplex PCR (Supplementary Fig. 1 and 2).
  • The overall prevalence of L. monocytogenes contamination in food samples observed in the present investigation was 1.25% (10/800).
  • L. monocytogenes carried the virulence genes including hlyA, plcA, prfA, actA, iap, inlA, inlB, inlC and inlJ genes indicating the pathogenic potential of the Listeria isolates.

Selected from the article's own text.

Abstract

Water and soil have been reported as the pivotal reservoirs for Listeria monocytogenes and its subsequent transmission to food chain, plant material and animals. In present study, 800 food samples consisting of beef and chicken meat, milk, salads and fresh vegetables were collected from retail markets of Quetta, Pakistan. Multiplex PCR was employed to identify the prevalent species and genes associated with virulence. Only 10 (1.25%) samples were found contaminated with Listeria monocytogenes. Milk samples were the most contaminated as 4 samples collected from retail shops or dairy farm were found positive for Listeria. While 2 samples of each beef, chicken meat and salad were contaminated with Listeria. The virulent genes inlA, inlB, prfA, hlyA, actA, plcA and iap were detected in all 10 positive samples for Listeria. The positive samples inventories were analyzed and were found that water used to wash the meat and vegetables was contaminated with Listeria. This study concludes that better hygienic measures should be adopted to keep the food material germ free.

Materials and methods

From January to December, 800 food samples were collected randomly from street vendors and retailers in different areas of Quetta, Pakistan. The food items included poultry meat (n=200), beef meat (n=200), vegetables/salad (n=200) and fresh milk (n=200). The collected samples were transported to laboratory in sealed cold box for further processing.

The collected food samples were analyzed to determine the presence of L. monocytogenes. The enrichment, isolation and characterization were performed as been described earlier in detail .

Multiplex PCR was employed to detect the Listeria isolated from different food samples by targeting the virulent genes by specific primers. The details of primers used are given in Table I.

Results and discussion

To ensure food safety, detection of pathogenic bacteria should be a fundamental objective. The application of molecular techniques has facilitated the identification and characterization of major virulence-associated genes in L. monocytogenes. L. monocytogenes is made of multiple strains showing varied virulence potential. To implement effective control and prevention measures against L. monocytogenes infections, it is necessary to identify virulent, disease-causing strains from avirulent, non-pathogenic strain that are relatively harmless.

In current study, 800 samples were collected and screened for L. monocytogenes. Overall, the prevalence of L. monocytogenes was 1.25% (10/800). Out of 10, L. monocytogenes isolates, 1% (1/100) was found in fresh chicken, 1% (1/100) in processed chicken, 2% (2/100) in minced beef, 1% (1/100) in milk collected from dairy farm, 3% (3/100) in milk obtained from retail shops and 2% (2/100) in fresh salad whereas no L. monocytogenes isolate was found in diced beef samples (0/100) and fresh vegetables (0/100) (Table II).

The ten isolates of L. monocytogenes isolated from different food sources were analyzed for the presence of virulence associated genes employing the multiplex PCR (Supplementary Fig. 1 and 2).

All the ten Listeria isolates (100%) were positive for hlyA, plcA, prfA, actA, iap, inlA, and inlB genes while 70% of the isolates were positive for inlC gene and only 50% for inlJ gene (Table III).

The overall prevalence of L. monocytogenes contamination in food samples observed in the present investigation was 1.25% (10/800). However, results of this study indicate lower prevalence as compared to other studies conducted by Nayak et al. (2015) in India (9%), Braga et al. (2017) in Uruguay (11.2%), Ismaiel et al. (2014) in Egypt (13.3%), Chen et al. (2015) in China (22%) and by Montero et al. (2013) in Chile (25%) in foods. Likewise, investigating the raw meat samples in China Wu et al. (2015) found a higher prevalence of L. monocytogenes isolates (23.7%). In Iran Pournajaf et al. (2016) isolated L. monocytogenes in 5.2% of the processed meat samples and 7.4% of dairy products. Difference in prevalence may be due to the types of analyzed food during the investigation in this study.

Table II. The prevalence of Listeria monocytogenes isolates in different food items in Quetta.

Food items

No. of samples analyzed

Samples contaminated with Listeria (n/%)

Fresh chicken

100

1 (1%)

Processed chicken

100

1(1%)

Beef diced

100

0(0%)

Beef minced

100

2(2%)

Milk dairy farm

100

1(1%)

Milk retail shops

100

3(3%)

Fresh vegetables

100

0(0%)

Fresh salad

100

2(2%)

Total

800

10 (1.25%)

Table III. Occurrence of virulence-associated genes present in L. monocytogenes isolated from various foods.

Source

No. of isolates

Virulence-associated genes

hlyA

plcA

prfA

actA

iap

inlA

inlB

inlC

inlJ

Food

100%

+

+

+

+

+

+

+

-

-

70%

-

-

-

-

-

-

-

+

-

50%

-

-

-

-

-

-

-

-

+

In this study the highest prevalence of L. monocytogenes contamination was observed in milk samples (3%) obtained from retail shops followed by fresh salad (2%) and minced beef samples (2%). These findings highlight the potential role of these food types in the spread of L. monocytogenes and alert the health personnel about proper handling and cooking . Wu et al. (2015) observed higher contamination frequency (5.7%) in vegetables in China. In another study, Cordano and Jacquet (2009) analyzed frozen and fresh vegetables from 2000 to 2005, reporting a L. monocytogenes prevalence of 26 and 10%, respectively.

L. monocytogenes comprises multiple strains that exhibit varying virulence potential. Although the differences in the virulence potential is not completely due to the presence of these genes, but these virulence makers play an important role in the pathogenicity of L. monocytogenes. Thus, detection of these markers may represent a quick approach for preliminary discrimination of potentially virulent strains from avirulent strains of L. monocytogenes. It has been suggested that diagnosis of pathogenic Listeria spp. and listerial infections should ideally be based on virulence markers . Moreover, the importance of PCR has been investigated for detection of L. monocytogenes from foods .

In the present study a multiplex PCR was standardized for the simultaneous detection of inlA gene and the hlyA, plcA, prfA, actA, iap, inlB, inlC and inlJ gene for the virulence determination of the pathogen.

Genotypic characterization of L. monocytogenes isolates from the different food samples in this study revealed the presence of inlA gene in all isolates (100%); these results are similar to those obtained by Liu et al. (2007) in USA and Almeida et al. (2000) in Brazil. Study conducted by Jung et al. (2003) showed that the inlA gene is species-specific, thereby suggesting their species-wide sequence conservation. Multiple virulence factors, including phosphatidylinositol phospholipase C (plcA), haemolysin (hlyA), actin polymerization protein (actA), invasive associated protein (iap), and internalin A (inlA), are necessary for the pathogenesis of L. monocytogenes. Therefore, detection of just one virulence factor by PCR is not always sufficient to identify L. monocytogenes strains. In addition, it is plausible that spontaneous mutations enable the removal of one or more virulence determinants in some L. monocytogenes strains. Thus, simultaneous detection of multiple virulent genes in a single assay is desirable as it reduces the time and labor involved and will be useful for large-scale investigations for detection of pathogenic strains of Listeria . According to this study, the prevalence of inlC and inlJ virulence genes was 70% and 50% respectively which is comparable with the findings by Pounajaf et al. (2016) reporting the prevalence of inlC and inlJ genes in 64% and 57% of clinical samples and 84% and 78% of food specimens, respectively. These findings implicate a role of the virulence genes inlA, inlC and inlJ in the pathogenesis of L. monocytogenes isolates.

In this study the multiplex PCR detected hlyA gene in 100% of L. monocytogenes isolates. Similar findings were reported by Jallewar et al. (2007). However, the result of this study is not in agreement with the result of Usman et al. (2016), who reported the carriage of hlyA gene by 25%. It may be possible that some L. monocytogenes strains may lack one or more virulence determinants because of spontaneous mutations .

Conclusion

The overall prevalence of L. monocytogenes in food was found to be 1.25%. L. monocytogenes carried the virulence genes including hlyA, plcA, prfA, actA, iap, inlA, inlB, inlC and inlJ genes indicating the pathogenic potential of the Listeria isolates. The highest prevalence in milk, fresh salad, minced beef samples in this study highlight the potential that these foods may play an important role in the spread of foodborne pathogen and alert to health personnel about the need to proper handling and cooking in individuals.

Supplementary material

There is supplementary material associated with this article. Access the material online at: https://dx.doi.org/10.17582/journal.pjz/20190402110434

Statement of conflict of interest

The author declare no conflict of interest.

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