Molecular Characterization and Phylogenetic Analysis of Listeria monocytogenes Isolated from Milk and Milk Products in Kaduna, Nigeria

In this study, Listeria (L.) monocytogenes isolated from milk and milk products in Kaduna, Nigeria, were subjected to a multiplex PCR assay to identify virulence-associated genes (such as prf A, inl A, hly A, act A, and iap). Of the 36 isolates, 9 (25%) were positive for one or two virulence-associated genes. Based on the sample type, 6 (16.9%) of the isolates that possessed virulence-associated genes were obtained from raw milk, 2 (3.2%) from “Manshanu,” and 1 (2.8%) from “Kindrimo.” Sequence and phylogenetic analysis based on the 16S rRNA revealed that Nigerian L. monocytogenes isolates (NGA 34A, NGA 35A, NGA 41A, and NGA 38A), when compared with reference L. monocytogenes, were grouped into two distinct clusters, A and B, with sequence (NGA 34A, NGA 35A, and NGA 41A) phylogenetically closer to J1776; N1-011A; R2-502; J1816; and J2-031, whereas L. monocytogenes isolate (NGA 38A) clustered with EDG; J1-220; J1926; J1817; and J2-1091. The separation of the Nigerian L. monocytogenes isolates into linage A (responsible for epidemic listeriosis) and lineage B (responsible for sporadic cases of listeriosis) is of public health concern and that local isolates might have potentials for human food borne listeriosis based on the virulence factors so far identified.


Introduction
Listeriosis is a food borne infection caused by opportunistic bacterial pathogen Listeria (L.) monocytogenes that is abundant in the environment [1]. The bacteria have been isolated from the soil, water, plants, faeces, decaying vegetables, meat, sea food, dairy products, and asymptomatic human and animal carriers [2]. It has been isolated from sheep, goat, and cow milk [3]. L. monocytogenes is a Gram-positive facultative intracellular food borne pathogen causing listeriosis, a rare but severe infection in humans and animals with a mortality rate of 25-30% [4]. Listeriosis is either noninvasive, self-limiting, and gastrointestinal, occurring in healthy individuals, or invasive and systemic, occurring in immunocompromised individuals such as pregnant women and newborns, resulting in meningitis, encephalitis, septicaemia, mother-to-foetus infection, and abortion [5]. There are several virulence genes so far identified in L. monocytogenes. These include the internalins (encoded by inl A, inl C, and inl J), listeriolysin O (LLO encoded by hly A), actin (act A), phosphatidylinositol-phospholipase C (PI-PLC encoded by plc A), iap (invasion associated protein encoded by iap), 2 Canadian Journal of Infectious Diseases and Medical Microbiology and virulence regulator (encoded by prf A). These virulence factors play significant role in the bacterial pathogenicity and infection outcome [6,7].
It has been observed that Listeria monocytogenes employ these virulence factors that act synergistically in the intracellular pathogenicity of the bacteria [8]. For example, expressions of virulence genes, such as listeriolysin O (LLO), phospholipases (plc A and plc B), and internalins A and B (inl A and inl B), are regulated by prf A, and these genes facilitate the intracellular growth and spread of the bacterium within the mammalian host [9]. Point mutation is responsible for low virulence of Listeria monocytogenes strains in a number of virulence genes and as a result of spontaneous mutations, some L. monocytogenes strains may lose one or more virulence determinants [10,11]. A virulence factor of phospholipase, called PI-PLC, is expressed by pathogenic species of L. monocytogenes and Listeria ivanovii only, and this virulence factor is an important marker for the discrimination between pathogenic and nonpathogenic Listeria species [12]. There is growing evidence that virulence is not a stable property but can be influenced by environmental conditions. For example, it has been shown that acid and salt stress increase the expression of virulence genes and in vitro pathogenicity of the bacteria [13]. Virulence potentials are also influenced by temperature, the presence or absence of oxygen, osmotic stress, and pH [14]. L. monocytogenes has 13 serotypes, of these only four serotypes (1/2a, 1/2b, 1/2c, and 4b) are known to cause human listeriosis [15]. Based on the data generated from molecular subtyping methods L. monocytogenes isolates have been grouped into two major genetic divisions or lineages, termed as lineage I and lineage II [15]. A third lineage, predominantly associated with animals, has been described by some studies as well [16]. Serotypes 1/2a, 3b, 3c, and 4b belong to lineage I, while serotypes 1/2a, 1/2c, and 3a belong to lineage II [17]. Studies have shown that lineage I strains are significantly higher among human clinical listeriosis cases and contaminated foods [1], while lineage II strains show a significantly higher prevalence among food isolates and animal clinical cases than among human clinical listeriosis [16]. Pathogenic potentials of lineage I isolates are greater than those of lineage II, as determined by their ability to spread to neighbouring host cells in a cell culture plaque assay [1]. It will be observed that there is a consensus that lineage I strains may represent a human host-adaptive lineage while lineage II strains may represent an environmental adapted lineage [15]. The application of multiplex PCR for the detection of more than one virulence gene in a single tube is desirable because it reduces cost and labour and will be useful in a large scale detection of virulent strains of Listeria [18].
The prevalence and molecular characteristics of Listeria monocytogenes are generally unknown in Nigeria and thus this present study was carried out with the aim of characterizing L. monocytogenes isolates from raw milk and milk products using PCR and to determine the virulence and the phylogenetic characteristics of the isolates.

Bacterial Strains and Biochemical.
The isolation of Listeria monocytogenes was carried out according to the procedure of Roberts et al. [19]. Briefly, about 10 mL of the incubated homogenate was added to 90 mL Listeria enrichment broth (Oxoid, CM 0862), which contains selective Listeria enrichment supplements (Oxoid, SR 0141E) and incubated at 30 ∘ C for 48 hrs. A loop full of the 48 hrs broth was cultured onto Chromogenic Listeria agar (Oxoid, CM 1080) plates, which contains brilliance6 Listeria differential supplement (Oxoid, SR 0228E) and brilliance6 Listeria selective supplement (Oxoid, SR 00227) and the plates was incubated at 37 ∘ C for 24-48 hrs. Colonies of Listeria monocytogenes having a greenish-blue coloration were picked and streaked on nutrient agar (Oxoid, CM 0003) slants and incubated at 37 ∘ C for 24 hrs, before storage at 4-5 ∘ C. The suspected isolates were further subjected to conventional biochemical test [Gram staining, oxidase, catalase, -hemolysis, bile esculin, and carbohydrate fermentation test (xylose, Mannitol, Rhamnose, Maltose, Inositol, and Sucrose)] as described previously [20]. Isolates found to ferment mannitol and xylose and positive to rhamnose were considered as L. monocytogenes. Further characterization using Microbact6 12L Listeria identification kit (Oxoid, MB 1128) was carried out.
Following cultural identification and biochemical assays stated above 36 Listeria monocytogenes isolates were identified (Table 1).

Polymerase Chain Reaction and Gel Electrophoresis.
Bacterial isolates obtained locally as well as reference strains were subjected to PCR assay. The reference strain designated ATCC 19155 used as positive control was kindly provided by Professor Radu of the Center of Excellence for Food Safety Research, Faculty of Food Science and Technology, Universiti Putra Malaysia, where the molecular studies were carried out. Bacterial genomic DNA was extracted from both local and reference isolates using the Quick-g DNA6 miniprep kit, (Zymo Research, SA) following the manufacturer's instructions.
Multiplex PCR assay was used to detect L. monocytogenes harbouring hly A gene and 16S rRNA of L. monocytogenes [21]. The primer pairs designated as LM1 and LM2 (LM1 5 -CCT AAG ACG CCA ATC GAA-3 and LM2 -5 -AAG CGC TTG CAA CTG CTC-3 ) were used for the detection of L. monocytogenes harbouring hly A gene. On the other hand, primer pairs designated as U1 and LI1 (LI1 5 -CTC CAT AAA GGT GAC CCT-3 and U1 5 -CAG CMG CCG CGG TAATWC-3 ) were used to amplify 938 bp region in the 16S rRNA gene specific for the detection of Listeria genus [21]. These primers were synthesized by Invitrogen, USA, and were 18-20 bp in length. L. monocytogenes ATCC 19155 was used as a positive control.
The PCR amplification was carried out in a 25 L reaction mixture that consisted of 5 L of 5x PCR buffer, dNTPs (0.5 L), MgCl 2 (2 L), Taq DNA polymerase (0.5 L), 0.5 L of each 20 pM primer [LM1, LM2, U1, and LI1], 13 L of distilled water, and 2 L of DNA template. PCR amplification was carried out in a programmed thermocycler with the following thermal conditions: hot start PCR plate at 95 ∘ C for 3 mins, followed by 35 cycles each of 30 sec denaturation at 94 ∘ C, 15 sec annealing at 53 ∘ C, 90 sec extension for 72 ∘ C, and final extension at 72 ∘ C for 7 mins.
Following PCR amplification, about 5 L of the PCR product was mixed with DNA loading dye (6x) and electrophoresed in 1.0% Agarose gel in TAE buffer using a minitank at 80 V, 400 amp, and 45 min. The electrophoresed product on the gel was stained with ethidium bromide for 30 min, destained for 20 min, and visualized under UV illuminator (SYNGENE, Biosystems, UK). A 100 bp DNA ladder (Promega Corporation, USA) was included to estimate the size of the amplified products.

Detection of L. monocytogenes Virulence-Associated
Genes. The obtained DNA product was used in performing another multiplex PCR assay with the aim of detecting the presence of five virulence-associated genes [hemolysin A gene (hly A), regulatory gene (prf A), actin gene (act A), invasion associated protein p60 gene (iap), and surface protein (inl A)] of L. monocytogenes, as described by Rawool et al. [18]. The primers used for the detection of these virulence genes were synthesized by Sigma Aldrich, USA. The details of the primer sequences are shown in Table 2. The DNA obtained using the Quick-g DNA miniprep-uncapped column kit (Zymo Research) was used for the detection of virulence-associated genes.
The PCR was standardized for the detection of virulenceassociated genes of L. monocytogenes by optimizing the annealing temperatures between 50 and 60 ∘ C. Based on optimization trials, the standardized PCR protocol for a 25 L reaction mixture included 5 L PCR buffer (100 mMTris-HCl buffer, pH 8.3 containing 500 mM KCl, 15 mM MgCl 2 , and 0.01% gelatine), 1 L dNTP mix, 3 L MgCl 2 and 0.5 L of forward and reverse primers, 1 L of 5 units of Taq DNA polymerase, 5 L DNA template, and 5 L nuclease free water to make up the reaction volume. The DNA amplification reaction was performed in a Master Cycler gradient thermocycler (Eppendorf, Hamburg, Germany) with a preheated lid in PCR tubes (0.2 mL). The cycling conditions for PCR included an initial denaturation of DNA at 95 ∘ C for 2 min followed by 35 cycles each of 15 sec denaturation at 95 ∘ C, 30 sec annealing at 53 ∘ C, and 90 sec extension at 72 ∘ C. Final was carried out in 10 min at 72 ∘ C and then PCR product held at 4 ∘ C.
The resultant PCR products were separated by electrophoresis in 1.5% Agarose gel for 45 min at 100 V in Tris-acetate EDTA buffer, stained with ethidium bromide (0.5 g/mL), and visualized with a UV transilluminator (Syngene Frederick, MD). The gel image was documented by a gel documentation apparatus, and DNA size was determined by a 100 bp DNA ladder (Promega).

2.4.
Purification of PCR Products. 5 L volume PCR product for each of the 9 hly A and 16S rRNA harbouring L. monocytogenes positive isolates was analysed on a 1.5% Agarose gel to confirm successful amplification of the 16S rRNA. The remaining 20 L of each of the PCR product was later run and each corresponding 16S rRNA band was cut and purified by using the MEGA quick-spin6 Total Fragment DNA purification kit (iNtRON Biotechnology, Gyeonggi-do, South Korea).

DNA Sequencing and Phylogenetic Analysis.
Purified gel was used for DNA sequencing. All sequencing reactions were commercially done by iNtRON (iNtRON Biotechnology Gyeonggi-do, South Korea) using the Sangers sequencing method. Both forward and reverse primers were used to ensure adequate sequencing coverage. For sequencing and phylogenetic tree, only 15 samples were randomly selected and tested.  TAA TAT CAT TTG CTG TTT TAT CTG TC-3  The obtained partial 16S rRNA sequences were proofread and assembled in Chromas Lite version 2.1.1. Multiple sequence alignment of the DNA sequences was performed using ClustalW in Bioedit software version 7.2.5 (Biological Sequence Alignment Editor for Win 95/98NT/2 K/XP/7), as described by Hall, 1999 [22]. To compare the sequence of local L. monocytogenes with reference isolates available in the GenBank database, sequence similarity search was performed using the NCBI nucleotide BLAST search to determine the similarity of the local L. monocytogenes sequence (http://blast.ncbi.nlm.nih.gov/).
Phylogenetic analysis was carried out based on partial sequence of 16S rRNA gene of L. monocytogenes. In this regard, the aligned sequence was subjected to a Maximum Likelihood (ML) method, in MEGA 6.0 software [23]. However, prior to performing ML analysis, best substitution model that described the sequence data set was obtained taking into considerations the lowest Bayesian inferences. Equally, 1000 bootstraps values were used to determine the confidence interval of the resultant tree. In order to root the tree, a 16S rRNA sequence from Mycobacterium tuberculosis was used as an out-group.
All local L. monocytogenes sequences used in the phylogenetic study were submitted to GenBank for archiving. The accession numbers of these sequences together with the reference isolates are summarised in Table 3.

Detection of Virulence-Associated Genes of Listeria monocytogenes Isolates.
Analysis of multiplex PCR used for the detection of virulence-associated genes revealed that, of the 36 L. monocytogenes isolates, 9 (25.0%) possessed at least one or more virulence-associated genes (Figure 2). Of the 5 virulence-associated genes screened for only 3 virulenceassociated genes (hly A, iap, and prf A) genes were detected; Listeria monocytogenes FW040025 CP011345 (15) Mycobacterium bovis AY360331 each of 5 (13.8%) isolates possessed the iap and prf A genes. Based on the sample types, 6 (16.9%) of the L. monocytogenes isolates that possessed virulence-associated genes were from raw milk, 2 (3.2%) "Manshanu," and 1 (2.8%) "Kindrimo". respectively. The PCR protocol used in this study was based on the amplification of hly A gene by using a set of primers, LM1 and LM2. Border et al. [21] recommended LM1 and LM2 primers as the best primer pair to be used for the detection of L. monocytogenes, because they are designed to amplify specific fragments in L. monocytogenes strains that are genetically and biochemically assessed as belonging to this specie, on the basis of the presence of the 702 bp and 938 bp amplicons [21].

Phylogenetic Tree
Kuan et al. [25] were also able to amplify the DNA at their expected amplification site. The result of this study is not in agreement with the result of Jallewar et al. [26], who reported the carriage of hly A gene by all the isolates of L. monocytogenes tested. The inability of some of the L. monocytogenes isolates in this study to harbour the hly A gene may be due to the nonvirulent characteristics of some L. monocytogenes isolates because some may be environmental isolates or it may be possible that some L. monocytogenes strains may lack one or more virulence determinants because of spontaneous mutations [11].
The ability to survive and multiply in the macrophages and other host cells is key to the pathogenicity of L. monocytogenes [27]. Rapid methods for the isolation and confirmation of L. monocytogenes in foods are still being explored [28]. Because of the importance of L. monocytogenes epidemiology to human health and the notable diversity in the pathogenicity among strains, subtyping and virulence characterization are of importance [7]. Due to the diverse strains of L. monocytogenes, of varying pathogenicity, the ability to precisely track the strains involved in listeriosis outbreaks and rapidly determine their pathogenic potentials is critical for the control and prevention of further occurrences of this deadly disease [24]. The phenotypic subtyping methods are generally less sensitive, have low differentiation ability, and are not easy to reproduce, whereas the genotypic approaches are more sensitive and reliable [29]. Virulenceassociated genes of L. monocytogenes, namely, prf A, hly A, and iap, were amplified according to their respective fragment sizes of 1060 bp, 702 bp, and 131 bp, respectively. This study revealed that of the 36 L. monocytogenes isolates screened for virulence-associated genes, only 9 (25.0%) isolates harboured one or more of such genes and none of them possessed all the five genes. It is plausible that some L. monocytogenes strains may lack one or more virulence determinants because of spontaneous mutations [11].
Henriques et al. [30] detected hly A genes from L. monocytogenes isolates from Portuguese ready-to-eat meat based food industry, which is similar to the findings of this study where hly A was detected in all the nine L. monocytogenes isolates. However, the detection of act A gene in the work of Henriques et al. [30] differs from this study which failed to detect act A. Listeriolysin O and act A are associated with the bacterium's capability of passing the intestinal barrier, cell to cell spread and motility, cell invasion, and intracellular parasitism [7,18]. Therefore, simultaneous detection of virulence genes in a single step will be desirable as it reduces time and labour, and it will be useful in large scale survey for detecting virulent strains of Listeria [18].
L. monocytogenes consist of at least 4 evolutionary lineages (I, II, III, and IV). Even though only cluster A is statistically supported with a bootstrap value of 100%, cluster B is not statistically supported.
Most L. monocytogenes isolates seem to belong to lineages I and II, which include the serotypes more commonly associated with human clinical cases, including serotypes 1/2b and 4b (lineage I) and serotype 1/2a (lineage II). Since it is a rooted tree, we can infer evolutionary relationship, information regarding the common ancestors, and the path of evolution from the rooted phylogenetic tree.
Since lineage I L. monocytogenes strains are more prevalent in human clinical listeriosis cases and contaminated food [31], it can be said that Nigerian L. monocytogenes isolates (NGA 34A, NGA 35A, and NGA41A) are from contaminated foods (i.e., milk and milk products). From the phylogenic tree, Nigerian L. monocytogenes isolates (NGA 34A, NGA 35A, and NGA 41A) were classified as lineage I, which are known to be of higher pathogenic potentials due to intrinsic virulence features, as compared to lineage II strains of L. monocytogenes [32]. Morobe et al. [33] in their study observed that most of the L. monocytogenes isolates belong to lineage III (4a and 4c), which are known to be more prevalent in animals with clinical listeriosis [31], as opposed to this study where none of the L. monocytogenes isolate were found to belong to lineage III. Isolate NGA 35a was found to belong to lineage II, which is opposed to the findings of Morobe et al. [33], where they observed that none of the L. monocytogenes isolates belong to lineage II.
The implication of the present findings is that L. monocytogenes of lineage 1 originating from animal milk and milk product could be associated with increased human risk to L. monocytogenes infection in Nigeria and thus effort to control the disease should be targeted at improving the food safety and quality.