Abstract

INTRODUCTION:

This study aimed to determine the prevalence, and virulence factors of Listeria monocytogenes isolated from various samples by multiplex polymerase chain reaction (MPCR).

METHODS:

A total of 617 isolates were obtained and MPCR was employed for detection of the inlA, inlC, and inlJ genes.

RESULTS:

L. monocytogenes was detected in 46 (7.45%) of the 617 specimens. inlA, inlC, and inlJ were detected in 100%, 76.26%, and 71% isolates, respectively.

CONCLUSIONS:

This study validated MPCR in the analysis and rapid detection of L. monocytogenes. The role of the genes in pathogenesis of the strains can also be affirmed.

Keywords:
Listeria monocytogenes; Virulence genes; Multiplex-PCR

Listeria monocytogenes is a gram positive, non- sporulating and facultative intracellular bacteria that caused infection in both humans and animals. L. monocytogenes infection in humans, listeriosis, is a serious illness that affects mostly immunosuppressed individuals, newborns, and the elderly, with symptoms ranging from septicemia, meningitis, encephalitis, abortions, to occasional death¹1. Ingianni A, Floris M, Palomba P, Madeddu M, Quartuccio M, Pompei R. Rapid detection of Listeria monocytogenes in foods, by a combination of PCR and DNA probe. Mol Cell Probes 2001; 15:275-280.. In recent years, many outbreaks of listeriosis have been implicated in the contamination of trading nutriments such as vegetables, milk, and meat foodstuffs²2. Lotfollahi L, Nowrouzi J, Irajian G, Masjedian F. Prevalence and antimicrobial resistance profiles of Listeria monocytogenes in spontaneous abortions in humans. Afr J Microbiol Res 2011; 5:1990-1993.. Listeria monocytogenes detection from specimens based on selective enrichment media followed by biochemical studies is arduous and requires at least 5 days for a positive diagnosis. Polymerase chain reaction (PCR) is a rapid method with high sensitivity and specificity for specific deoxyribonucleic acid (DNA) sequences and permits direct detection of the pathogens²2. Lotfollahi L, Nowrouzi J, Irajian G, Masjedian F. Prevalence and antimicrobial resistance profiles of Listeria monocytogenes in spontaneous abortions in humans. Afr J Microbiol Res 2011; 5:1990-1993.. A PCR employing amplification of the iap, prfA, hly, inl, and plcA gene sequences was recently reported for L. monocytogenes detection¹1. Ingianni A, Floris M, Palomba P, Madeddu M, Quartuccio M, Pompei R. Rapid detection of Listeria monocytogenes in foods, by a combination of PCR and DNA probe. Mol Cell Probes 2001; 15:275-280.. Liu et al. showed that the inlA gene is species-specific and the inlC and inlJ genes are virulence-specific for L. monocytogenes³3. Liu D, Lawrence ML, Austin FW, Ainsworth AJ. A multiplex PCR for species- and virulence-specific determination of Listeria monocytogenesJ Microbiol Methods 2007; 71:133-140.. The inlA gene is present in all L. monocytogenes isolates irrespective of the source and serovar, but not in other Listeria spp. and other bacteria³3. Liu D, Lawrence ML, Austin FW, Ainsworth AJ. A multiplex PCR for species- and virulence-specific determination of Listeria monocytogenesJ Microbiol Methods 2007; 71:133-140.. The InlA protein facilitates interaction between L. monocytogenes and the host cell receptor E-cadherin and leads to L. monocytogenes adhesion and invasion of epithelial cells in the human intestine³3. Liu D, Lawrence ML, Austin FW, Ainsworth AJ. A multiplex PCR for species- and virulence-specific determination of Listeria monocytogenesJ Microbiol Methods 2007; 71:133-140.. InlC (also known as IrpA) is a virulence marker responsible for the post-intestinal dissemination of L. monocytogenes infection⁴4. Bierne H, Sabet C, Personnic N, Cossart P. Internalins: a complex family of leucine-rich repeat-containing proteins in Listeria monocytogenes. Microbes Infect 2007; 9:1156-1166.. Only four proteins of the internalin family are part of the repertoire of secreted proteins, and among these, inlC is the most important. The gene encoding the inlC protein is present in pathogenic L. monocytogenes and L. ivanovii species, but not in other Listeria species¹1. Ingianni A, Floris M, Palomba P, Madeddu M, Quartuccio M, Pompei R. Rapid detection of Listeria monocytogenes in foods, by a combination of PCR and DNA probe. Mol Cell Probes 2001; 15:275-280.. The InlJ (or lmo2821) gene is responsible for passage of L. monocytogenes through the intestinal barrier and can be used for evaluating virulence of L. monocytogenes⁵5. Jalali M, Abedi D. Prevalence of Listeria species in food products in Isfahan, Iran. Int J Food Microbiol 2008; 122:336-340.. Detection of virulent genes is necessary as it decreases the time and labor required for diagnosis and will be useful in a large-scale investigation for detecting virulent strain of Listeria monocytogenes species includes a range of strains with varying virulence and pathogenicity, and while numerous L. monocytogenes strains are naturally virulent and capable of produce major illness and death, others are avirulent²2. Lotfollahi L, Nowrouzi J, Irajian G, Masjedian F. Prevalence and antimicrobial resistance profiles of Listeria monocytogenes in spontaneous abortions in humans. Afr J Microbiol Res 2011; 5:1990-1993.. Key virulence-related proteins and the genes encoding them can betargeted to better evaluate virulent strains of L. monocytogenes⁵5. Jalali M, Abedi D. Prevalence of Listeria species in food products in Isfahan, Iran. Int J Food Microbiol 2008; 122:336-340.^{) (6}6. Sattari M, Forouzandeh M. Isolation and identification of Listeria monocytogenes in vaginal samples by PCR. Pathobiol Res 2009; 12: 51-58.. Therefore, it is imperative to develop a rapid and precise laboratory method that can easily discriminate between virulent and avirulent strains. A rapid method that provides information about epidemiological feature of microorganism can helpful in the control and prevention of listeriosis. In addition, it will also provide insights regarding the outbreak and prevalence of naturally both virulent and avirulent strains. This study aimed to determine the prevalence, and virulence factors of L. monocytogenes isolated from clinical and non-clinical samples by using multiplex PCR.

Between December 2012 and November 2015, 617 samples, including both clinical and non-clinical specimens, were collected from Tehran hospitals, Iran. Five milliliters of fluid specimens (including blood and urine), fecal and vaginal swabs, and 25g of the placental tissue were inoculated into 50, 10, and 225ml of TSYBE broth (tryptic soy broth positive 0.6% yeast extract) (Merck Co., Germany), respectively. Solid specimens (including dairy products and processed meat) were inoculated into 225ml of TSYBE. After inoculation in TSYBE, the samples were incubated for 7-16 days. In addition, samples stored at 4°C for 6 months were inoculated into PALKAM agar (Merck Co., Germany) supplemented with 5mg polymixin B, 2.50mg acriflavin, and 10mg ceftazidime and incubated at 35°C for 48h. Approximately 5-6 suspend grown colonies from both culture media were inoculated into Brain Heart Infusion (BHI) agar (Merck, Germany) and analyzed using standard microbiological and biochemical tests such as catalase, CAMP test, beta hemolysis on blood agar, bile esculin, hydrolysis of sodium hippurate, and motility at 4ºC. Bacterial DNA was extracted from colonies grown overnight in BHI broth at 37°C by using a DNA extraction Kit (Roche Co, New York, USA) according to the manufacturer's protocol. Primers used in the M-PCR assay were from a previously published study by Liu et al.³3. Liu D, Lawrence ML, Austin FW, Ainsworth AJ. A multiplex PCR for species- and virulence-specific determination of Listeria monocytogenesJ Microbiol Methods 2007; 71:133-140.. The reaction mixture consisted of 2µl extracted DNA, 2.5µl 10× PCR buffer, 1.5µl MgCl₂ (50mM), 0.5µl dNTP (10mM), 1.25µl each primer (10pmol/uL), 0.4µl Taq DNA polymerase (5U/µl) and deionized water to a final volume of 25µl. The reaction mixture was amplified in a thermo cycler (Eppendorf, Germany) with the following PCR conditions: denaturation at 94°C for 5 min, 31 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s and extension at 72°C for 45 s, and final extension at 72°C for 5 min. The PCR products were further analyzed by electrophoresis in 1.50% agarose gel for 40 min in Tris-acetate buffer, visualized by ethidium bromide staining, illuminated by a UV-Trans illuminator, and the images were captured by a gel documentation apparatus (UVP Gel Seq Software, England). L. monocytogenes ATCC-764 was used as a positive control and L. ivanovii ATCC 19119, Staphylococcus aureus ATCC-25923, Streptococcus pyogenic ATCC-19615, Enterococcus feacalis ATCC-29212, Escherichia coli ATCC 25922, and Acinetobacter baumannii ATCC 19606 were used as negative controls. Data analysis was conducted by employing descriptive statistics, t-test, and chi square test, using SPSS-21 software and a 5% significance level was set in all analyses.

Of the 617 specimens, 170 and 447 samples were collected from clinical and non-clinical sources, respectively. Clinical samples including blood, urine, placenta tissue, rectal swabs, and vaginal swabs were obtained from patients with spontaneous abortions hospitalized in Shariati hospital, Tehran, Iran. Non-clinical samples were obtained from dairy products, processed meat, and animals. Dairy products and processed meat samples were obtained from supermarkets and retail market from Tehran, Iran. Animal specimens were obtained from the Tehran university veterinary clinic (Table 1). The multiplex PCR method was performed using the inlA, inlC, and inlJ primers that amplify regions of 800, 517, and 237 bp, respectively. As indicated in Figure 1, the distribution of species and virulence-specific genes in the clinical samples (n; 170, 27.4%) showed that 14 (8.2%), 9 (5.3%), and 8 (4.7%) strains harbored the inlA, inlC, and inlJ genes, respectively. Of the 447 (72.6%) non-clinical samples, 32 (7.11%), 27 (6%), and 25 (5.60 isolates were positive for the inlA, inlC and inlJ genes, respectively. As shown in Table 2, there were no significant association between the bacterial isolation and non-clinical sources (p = 0.65). In addition, the prevalence of the nlA, inlC, and InlJ genes was not statistically significant between the clinical group and non-clinical group (p > 0.05).

FIGURE 1
Multiplex-PCR amplification of the InlA, InlC and InlJ genes in the eight selected isolates of Listeria monocytogenes. Lane 1: DNA Ladder 1kb; Lane 2: positive control (Listeria monocytogenes ATCC7644); Lane 3-4: strains isolated from clinical samples; Lane 5-6: strains isolated from dairy samples; Lane 7-8: strains isolated from meat samples; Lane 9: strain isolated from animal samples; Lane 10: Listeria ivanovii ATCC 19119; Lane 11: Staphylococcus aureus ATCC 25923; Lane 12: Streptococcus pyogenes ATCC 19615; Lane 13: Enterococcus faecalis ATCC 29212; Lane 14: Escherichia coli ATCC 25922. (band 800 bp: inlA, band 517 bp: inlC, and band 237bp: inlJ). PCR: polymerase chain reaction; DNA: deoxyribonucleic acid.

In total, forty-six (7.5%) L. monocytogenes isolates were recovered from the 617 specimens tested in the present study, which is comparable with the results of the study conducted by Lotfollahi et al²2. Lotfollahi L, Nowrouzi J, Irajian G, Masjedian F. Prevalence and antimicrobial resistance profiles of Listeria monocytogenes in spontaneous abortions in humans. Afr J Microbiol Res 2011; 5:1990-1993.. Four (7.5%), 2 (5.7%), 5 (14.2%), 3 (8.5%), and 0 (0%) L. monocytogenes isolates were obtained from the placental tissue, urine, vaginal swabs, rectal swabs, and blood, respectively. In the present study, vaginal swabs and blood exhibited the highest and lowest prevalence, respectively, amongst the clinical samples. In the current study, L. monocytogenes was isolated from the urine and rectal-vaginal swabs of two women. Of the two cases, L. monocytogenes was isolated from the vaginal and rectal swabs of one patient and in the other case, L. monocytogenes was isolated from placental tissue and rectal and vaginal swabs of the same patient. These results highlight the role of L. monocytogenes in spontaneous abortions in these women. The results of this study are in agreement with previous reports in which the bacterium was isolated 7% of 100 vaginal samples⁵5. Jalali M, Abedi D. Prevalence of Listeria species in food products in Isfahan, Iran. Int J Food Microbiol 2008; 122:336-340., 22 of 428 women with a poor obstetric history⁶6. Sattari M, Forouzandeh M. Isolation and identification of Listeria monocytogenes in vaginal samples by PCR. Pathobiol Res 2009; 12: 51-58., 3 of 100 women⁷7. Kargar M, Ghasemi A. Role of Listeria monocytogenes hlyA gene isolated from fresh cheese in human habitual abortion in Marvdasht. Iranian J Clin Infect Dis 2009; 4:214-218._, and 9 of 670 women⁸8. Ismaiel AA, Ali AE, Enan G. Incidence of Listeria in Egyptian meat and sairy samples. Food Sci Biotechnol 2014; 23:179-185. and therefore, implicate L. monocytogenes as a causative agent of human abortions. Of the 107 different dairy products, 9 (7.4%) L. monocytogenes were isolated from 5 (7.1%) cheese samples, 2 (7%) cream samples (10%) and 2 kashk samples. Kargar and Ghasemi reported a higher prevalence of L. monocytogenes (13.1%) than that reported in our study⁷7. Kargar M, Ghasemi A. Role of Listeria monocytogenes hlyA gene isolated from fresh cheese in human habitual abortion in Marvdasht. Iranian J Clin Infect Dis 2009; 4:214-218.. This is probably due to differences in geographical regions in which the two studies were conducted. L. monocytogenes was recovered from 5.2% of processed meat product samples tested in the present study. Our results showed a much lower prevalence in contrast to those observed in studies conducted by Hudson et al. (New Zealand), Simon et al. (Spain ) and Ismaiel et al. (Egypt), where the prevalence of L. monocytogenes was 12.5%, 17.3%, and 13.3% (frozen beef), respectively⁸8. Ismaiel AA, Ali AE, Enan G. Incidence of Listeria in Egyptian meat and sairy samples. Food Sci Biotechnol 2014; 23:179-185.^{) (9}9. Hudson JA. Efficacy of high sodium chloride concentrations for the destruction of Listeria monocytogenes Lett Appl Microbiol 1992; 14:178-180.^{) (10}10. Simon MC, Gray DI, Cook N. DNA extraction and PCR methods for the detection of Listeria monocytogenes in cold-smoked salmon. Appl Environ Microbiol 1996; 62:822-824.. This discrepancy may be due to the lower number of meat specimens that investigated in our study. However, our findings are consistent with those of studies conducted by Wang et al. and Ismaiel et al., where the prevalence of the pathogen was 4.7% (in meat samples) and 5.3% (in frozen chicken samples)⁸8. Ismaiel AA, Ali AE, Enan G. Incidence of Listeria in Egyptian meat and sairy samples. Food Sci Biotechnol 2014; 23:179-185.^{) (9}9. Hudson JA. Efficacy of high sodium chloride concentrations for the destruction of Listeria monocytogenes Lett Appl Microbiol 1992; 14:178-180..

In the present study, the inlA gene was detected in all L. monocytogenes isolates (100%); these results were similar to those obtained by Liu et al. (USA) and Almeida et al. (Brazil)³3. Liu D, Lawrence ML, Austin FW, Ainsworth AJ. A multiplex PCR for species- and virulence-specific determination of Listeria monocytogenesJ Microbiol Methods 2007; 71:133-140.^{) (11}11. Almeida PF, Almeida RCC. A PCR protocol using inl gene as a target for specific detection of Listeria monocytogenes. Food Control 2000; 11:97-101.. Studies conducted by Poyart et al. and Jung et al. showed that the inlA gene is species-specific, thereby suggesting their species-wide sequence conservation¹²12. Poyart C, Trieu-Cuot P, Berche P. The inlA gene required for cell invasion is conserved and specific to Listeria monocytogenes. Microbiol 1996; 142:173-180.^{) (13}13. Jung YS, Frank JF, Brackett RE, Chen J. Polymerase chain reaction detection of Listeria monocytogenes on frankfurters using oligonucleotide primers targeting the genes encoding internalin AB. J Food Prot 2003; 66:237-241.. Rawool et al. showed that multiple virulence factors, including phosphatidylinositol phospholipase C (plcA), hemolysin (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 Listeria¹⁴14. Rawool DB, Malik SVS, Barbuddhe SB, Shakuntala I, Aurora RA. A multiplex PCR for detection of virulence associated genes in Listeria monocytogenes. Int J Food Saf 2007; 9:56-62.. According to our study, the prevalence of inlC and inlJ virulence genes was 9 (64%) and 8 (57%) in clinical samples and 27 (84%) and 25 (78%) in non-clinical specimens, respectively. Similarly, Liu et al. reported that the inlC and inlJ genes are present in 80.5% and 77.81% of L. monocytogenes strains, respectively¹⁰10. Simon MC, Gray DI, Cook N. DNA extraction and PCR methods for the detection of Listeria monocytogenes in cold-smoked salmon. Appl Environ Microbiol 1996; 62:822-824.. These findings implicate a role of the virulence genes inlA, inlC and inlJ in the pathogenesis of L. monocytogenes isolates. Accordingly, the present report describes the development of a multiplex PCR that incorporates the inlA gene primers for confirming species-specificity and the inlC and inlJ gene primers for virulence determination of L. monocytogenes.

In conclusion, a multiplex PCR incorporating the inlA gene primers for L. monocytogenes species-specific recognition and the inlC and inlJ gene primers for virulence determination was developed that enables the rapid and simultaneous confirmation of the L. monocytogenes species and its virulence.

Acknowledgments

The authors would like to thank the Department of Microbiology, Iran University of Medical Science (IUMS), for their technical assistance.

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