Characterisation of Listeria monocytogenes from Food and Human Clinical Samples at Duhok, Kurdistan Region of Iraq

Listeria monocytogenes is one of the most important foodborne pathogens in human worldwide. In present study, this bacterium was isolated from different animal products and human clinical samples. The isolates were characterized by antibiotic susceptibility tests, serotyping, virulence genes and 16SrRNA sequencing. Out of 1362 investigated samples, Listeria monocytogenes were identified in 48(3.5%) of samples. Seven samples 1.1% were from human, while 41(5.7%) were from food samples. The majority of food isolates were resistant to penicillin, cephalexin, doxycycline, ampicillin and vancomycin; while variable resistance to the other antibiotics was observed. Serotyping of food and human isolates found that 7 of human isolates and 28 of food isolates belonged to serogroup 1/2a (3a). While, 8 isolates from food samples belonged to the serogroup 4b. Five fresh red meat isolates belonged to the serogroup 1/2b. All food and human isolates contained virulence genes actA, hlyA, plcA and iap genes. Phylogenetic analysis based on 16SrRNA sequencing showed that the L. monocytogenes isolated from milk were not closely related to the meat and human isolates. This data suggests that the antibacterial resistant Listeria monocytogenes are widely spread within the animal products rather than the clinical samples. The most common serogroup within the isolated strains was 1/2a (3a). Surprisingly, all isolates found to be virulent strains depending on the virulence genes detection. Therefore, it is highly recommended to apply strict biosecurity measurements on food and food processing environment to avoid or to maintain the spread of the bacterial infection within the area.


INTRODUCTION
Listeria monocytogenes is a facultative anaerobic gram-positive intracellular pathogen and mesophilic. This microorganism is considered as a fatal foodborne bacterium with a great impact on a public health; the bacterial infection in human is associated with a high mortality rate (20-30%) 1,2 . The disease is a major risk among immunocompromised persons due to the suppression of T cell 3 ; and among old people, pregnant woman, neonates, transplant and AIDS patients 4 . Listeria causes septicemia, meningitis, encephalitis, stillbirth and abortion, in addition to gastrointestinal 5,6 . Transmission of Listeria to the human could be through ingestion of undercooked and contaminated food 7,8 .
This organism is distributed widely in food products such as meat, poultry and seafood. Studies found that approximately 99% of listeriosis cases occur through consumption of contaminated food 8 . Different serogroups are responsible for the different epidemiological features such as outbreak, sporadic, and epidemic, therefore, the proper serotyping of human isolates is important in order to identify the proper serogroups and the source of infection of this microorganisms 2,16 . However, little information on the prevalence of clinical listeriosis and L. monocytogenes prevalence among food products in Duhok province of Iraq is available. To our best knowledge insufficient studies took place to identify this bacterium in food products 17,18 and no study, as yet, has been done to detect the prevalence rate of L. monocytogenes among human in Duhok province. Therefore, the present study was set to determine the prevalence of L. monocytogenes from human clinical samples in Duhok province, and to isolate and determine their serotypes and virulence potential. Besides, antibiotics susceptibility profile and genetic diversity between isolates were investigated.

MATERIAL AND METHODS Collection of samples
A total of 1362 samples were obtained from various sources during July 2016 to May 2017 in Duhok province, Iraqi Kurdistan Region. Three hundred and nine (309) frozen chicken samples were taken from the directorate of prevention affairs, 167 raw goat and mutton meat samples were collected from local butcher shops. Furthermore, 239 samples from milk and milk products (118 of raw milk from local sheep, and 121 samples of local soft cheese) were collected from local shops. In addition to food samples, a total of 400 human samples were collected from third trimester pregnant women (Urine =200 and High vaginal swab=200). The rest were 247 blood samples which were taken from different groups of immunocompromised patients such as renal failures patients (N=101), septicemia cases (N=83), premature babies (N=44), meningitis cases (N=15) and Heart failure patients (N=4). The urine and high vaginal swabs samples were collected from the patients who visited Duhok Obstetrics and Gynecology Teaching Hospital, while the blood samples were collected from Azadi Teaching Hospital, Hevi Pediatric Teaching Hospital, and Duhok kidney and diseases transplantation center. The samples were collected under the aseptic condition and were delivered in cold box to the directorate of prevention affairs where processed shortly within 24 hrs of collection. L. monocytogenes isolation and identification L. monocytogenes were isolated from food samples according to the standard double enrichment method recommended by ISO 11290:1 with some changes 19 . A ratio of 1:9 of all samples were collected, 1 ml of milk was add to 9 ml of half fraser broth (pre-enrichment medium) and 25 g of meat and soft cheese were inoculated into 225 ml of half fraser broth (LabM, UK), and then incubated for 24h at 30°C. From overnight incubate broth, 100ul (0.1ml) was transferred into 10ml of fully concentrated fraser broth (LabM, UK) as a second enrichment then incubated at 37°C for 48h. Loop full of inoculum from second enrichment was subsequently, platted on PALCAM agar (LabM, UK) and re-incubated for 48h at 37°C. The Gray-greenish colonies with black center were picked and streaked on Harlequin™ Listeria Chromogenic Agar (LabM, UK), the blue/green colonies surrounded by an opaque halo were then chosen and confirmed using 13 biochemical reactions such as aesculin hydrolysis, acid production from rhamnose, xylose (Microgen, UK) 20 .

DNA extraction and Confirmation of L. monocytogenes isolates by PCR
Genomic DNA was recovered from L. monocytogenes isolates using the direct boiling method 21 . The purity and the concentration of the DNA was evaluated using Nanodrop (Thermofisher, UK) through the calculation of optical densities ratio at 260/280 nm. L. monocytogenes isolates were confirmed using PCR primers complementary to the highly conserved 16S rRNA sequence as stated by 22 . The reaction conditions consisted of initial denaturation of DNA template (94°C for 3 min), then 35 cycles of denaturation (94°C for 1 min), annealing (60°C for 2 min) and extension (72°C for 1min). This primer amplified 938 bp of 16S rRNA which is considered the species specific primers depending on the conserved region for L. monocytoges detection. The positive control was obtained from College of Veterinary Medicine-Duhok Research Center 23 .The primers used are listed in Table 1.

Virulence genes detection of L. monocytogenes
Virulence genes amplification (hylA, plcA, actA and iap) was carried out through Multiplex PCR using similar amplification conditions and primer sets as used earlier 27 . Briefly, PCR reaction was carried out in 25µl volume containing 12, 5µl ready to use master mix (1X) (Genet-Bio, South korea), primers (10ng/µl) each and DNA template (50 ng/ µl) under the following conditions: initial denaturation for 2 mints at 94°C, then 30 cycles for (20 s at 94°C, 20 s at 55°C and 50 s at 72°C) and final extension for 2 mints at 72°C. The details of oligonucleotides used in this study are shown in (Table 1).

Serogroup identification of L. monocytogenes
Serogrouping of L. monocytogenes (1/2a, 1/2b, 1/2c, and 4b) was performed using Multiplex PCR using primers Imo0737, Imo1118, ORF2819 and ORF2110 and the amplification conditions as reported before 11 . Amplification was carried out in a 25ul reaction volume containing 12.5µl ready to use master mix (1X) (Genet-Bio, South Korea), Primers were used at 0.1µM and about 50 ng/ul of DNA was added as template. Reaction was carried out with an initial denaturation for 5 min at 94°C, 35 cycles for (30 s at 94°C, 75 s at 54°C and 75 s at 72°C) and final extension for 10 min at 72°C in a thermal cycler (Applied Biosystems 9700, USA).

16S rRNA sequencing and phylogenetic construction
The sequencing of universal 16SrRNA 29 was implemented using primer listed in (Table  1) at Macrogen Company, Korea. The qualities sequences were tested and aligned using BioEdit sequence alignment editor 7.0.0 (Isis Pharmaceuticals, Inc., Carlsbad, USA). The sequences of different isolates were submitted to GenBank to get the GenBank accession Numbers (Fresh red meat isolate MK968361, Frozen chicken meat isolate MK968368, Raw milk isolate MK968369, white soft cheese isolate MK968371, human-vaginal swab MK968366, human-blood MK968364 and human-urine MK968365). The sequence identity comparing with corresponding sequences submitted in GenBank was estimated using the "BLAST" tool on NCBI website. The sequences were aligned using clustalW. The Neighbor-Joining method with Jukes-Cantor model in MEGA7 with 1000 bootstrap replicates was used for construction of phylogenetic tree.

RESULTS AND DISCUSSION Prevalence of L. monocytogenes in different samples
In the present study, 715 food samples were tested, out of these, a total of 309 frozen chicken samples and 167 fresh red meat samples were tested. L. monocytogenes was isolated at a rate of 8.73% from frozen chicken samples and 5.98% from fresh red meat. Our findings are in agreement with the prevalence rate of 8% and 7.1% in Iraq 17,18 both in chicken samples. Similarly, a study conducted in Egypt reported 8.1% isolation which is very close to our findings 30 . However, other studies reported much higher values compared to our results, particularly 94.7% recorded in Turkey 31 and 14.1% in Iran 32 . The lowest prevalence to our knowledge was 0.8% from a study conducted in South Korea33. Regarding to the raw red meat, the prevalence rate of L. monocytogenes in this study was close to what was found in France (5.0%) and South Korea (5.2%), which support our results 22,34 . In different studies conducted in the Kurdistan region/ Iraq, a higher prevalence was reported from red meat at 14% 17 . In contrast to all previously mentioned studies in red meat including the current study, lower prevalence rates were recorded in Spain, Turkey, and India 2.6% and 2.2%, respectively 35,36 . Furthermore, studies took place in India and reported the prevalence of L. monocytogenes 2.4 % and 2.7% in two separate studies 37,38 . High mortality and hospitalization rates are recorded from L. monocytogenes infections due to eating of contaminated and undercooked food. Different factors found to be associated with the incidence of L. monocytogenes in meat such as the ability of this microorganism to form biofilms on the exterior and interior part of the tissue and the optimum temperature and pH of meat 39,40 .
Raw milk and locally produced white soft cheese are other types of samples were investigated for L. monocytogenes identification.
The results showed that out of 118 milk samples examined just 3 (2.54%) samples were positive for L. monocytogenes and only 1(0.82%) out of 121 white cheese samples were found to be positive for L. monocytogenes. This study found low prevalence of L. monocytogenes in dairy products. This is probably due to pasteurization of milk is an obligatory step during soft cheese preparation. However, most of L. monocytogenes contamination in cheese occurs after pasteurization 41 . The differences in prevalence rate in different studies and sources could be due to many reasons such as, sample size, study region, time of study and methods of L. monocytogenes isolation 17 . In addition to the slaughtering process of animals, personal hygiene and storage condition of food samples. The nutrient composition, water content, and pH of the environment are other significant factors that enable proliferation of numerous microorganisms in raw milk and dairy products and other food samples 42 . On the other hand, Gilbert and colleagues highlighted different factors that could affect the incidence rate of L. monocytogenes in milk in different countries; including differences in seasonal variation, location, differences between the milking devices used, and the bacterial capability to survive within different environmental conditions 43 . Fortunately, low frequency of L. monocytogenes was found in ready to eat food represented by cheese and milk compared to chicken and red meat. Although cooking is required for both meat types, dealing with contaminated meat can lead to listeriosis through handling and may cross contaminate other food sources as well 44 .
However 647 human clinical samples (Blood, Urine, and Vaginal swabs) were tested for L. monocytogene. This bacterium was detected from 7/647 (1.1%) samples. Out of 200 vaginal swabs collected from pregnant women only three samples (1.5%) were positive for L. monocytogenes namely among women with history of at least one miscarriage. Whereas, only one (0.5%) L. monocytogenes was isolated from 200 urine samples. The positive sample from patients also had history of stillbirth delivery. The age's distributions of these patients were between 18-38 years old. In addition to that, three samples (1.2%) were positive out of 247 tested immunocompromised patients for Listeriosis, which were diagnosed in their blood samples. Two of the patients were of age >70 years, who suffered from renal failure and experienced hemodialysis for a long time, the third positive case was a 3 years old and diagnosed as meningitis case. No significant relationship between clinical case and L. monocytogenes infection have been recognized (P value=0.21) (Fig. 1). These results are in agreement with previous investigation in human clinical samples that reported L. monocytogenes by 1.3% from vaginal swab 30,45 . The lower and higher infection rate were reported in India from the samples taken from vaginal swabs at 0.8% and 10.28%, respectively 46,47 . Generally, pregnant women were found more sensitive to Listeriosis than other people by 17 fold 48 . This may lead to spontaneous abortions, premature births and stillbirths. In agreement with our results 48 reported 1.0%. Fewer than our outcomes, only 0.3% of clinical samples were found to be infected with L. monocytogenes in a study conducted by 49 . Recently, Al-dorri, 2018 reported 37.93% of positive cases in a study conducted in Tikrit province-Iraq which dis-consistent with the present study results 50 . The differences between the data of the present study and that reported by 47,50 may be due to the time when the samples were collected from the pregnant women (pregnancy trimesters), variations in the samples size involved and the methods used for the bacterial isolation, the type of the samples used, personal hygiene and diet.

Antibiotics sensitivity
L. monocytogenes isolated from the meat products, dairy products and human clinical samples were examined for their antibiotic sensitivity. Majority of the isolates, from food samples were resistant to ampicillin (65.85%), Cephalexin (65.85%), Penicillin (63.41%) and Doxycycline (60.97%), while, variable resistances have been noticed to Gentamicin (56.09%), Clindamycin (53.65%), Rifampin (51.21%), Chloramphenicol (41.46%) and Co-trimoxazol (39.02%). However, these isolates were mostly sensitive to Piperacillin (75.60%), Meropenem (73.17%) and Ciprofloxacin (70.73%). On the other side, the majority of human clinical isolates were resistant to Clindamycin (71.42%) and Doxycycline (71.42%), while some human L.monocytogenes isolates exhibited multi-drug resistance (MDR) to the Ampicillin, Cephalexin, Chloramphenicol, Gentamicin, Penicillin and Rifampin. These isolates were completely sensitive to Co-trimoxazol (85.71%), Meropenem (85.71%), Ciprofloxacin (71.42%) and Piperacillin (71.42%) ( Table 2). Penicillin, ampicillin, gentamycin, rifampin, chloramphenicol, tetracycline, and co-trimoxazol were used widely in human and veterinary Listeriosis treatments 25,47 . Our results are supported by many studies conducted elsewhere 25,47 . In one study in Iraq, 100% of isolates were resistance to clindamycin and 100% of isolates were sensitive to gentamycin and Imipenem which were similar to our results to some extent 23 . Contrary, Osaili, Kalekar, N‫צ‬lkes, Jemal and their collegues reported lower rate of resistance of L. monocytogenes strain against the previous antibiotics [51][52][53] . According to the currently accepted standards if the bacterium resistant to three or more antibiotics of different classes this will be considered as a multi-drug resistant bacterium 54 . Multi-drug L. monocytogenes strains has been reported in different countries 55,56 . In the present study, 45/48(93.75%) strains were resistance to at least 4 antibiotics. In agreement with our findings, multidrug-resistant L. monocytogenes strains were reported in many clinical cases which indicated an extensive health considerations 25,53,57 . In general the antibiotics resistant could be due to the extensive and uncontrolled use of antibiotics for human and veterinary treatment 58 , as ampicillin or penicillin G combined with an aminoglycoside (gentamicin) is considered the standard and the first choice for listeriosis treatment. While, the second line of treatment of this microorganism is the co-trimoxazol 59 . The other possible reasons for antibiotics resistant are the acquisition of antibiotic resistant genes by insertion elements and integrons 60 . Thus, the outcome of current study revealed prevalence of multi-drug resistant isolates of L. monocytogenes in meat, human clinical and milk samples. The results also emphasize the necessity for active and continuous investigation of their antibiotic resistance.

Serotypes identification
Due to the importance of serotyping in determining the sporadic and epidemic strains of L. monocytogenes, we dedicated a major part of our study for that purpose. The method used by Doumith et al. (2004) was successfull in separating our 48 strains of L. monocytogenes into three distinguished groups based on specific multiplex PCR 11 . All food and human isolates gave positive results for species specific gene for L. monocytogenes (16sRNA). All human isolates (7) belonged to serogroup 1/2a (or 3a) which is known to be more prevalent in food and food related environments 44 . However, three different serogroups were found in food samples, sixteen frozen chicken meat, eight fresh red meat, three raw milk samples and one white soft cheese isolates belonged to serogroup 1/2a or 3a. The second serogroup was (4b) in which 6 frozen chicken and 2 fresh meat isolates were found to be in this serogroup. Five fresh red meat isolates belonged to third group, 1/2b (Table  3). All our strains belonged to serotypes (1/2a, 1/2b, and 4b) which are mostly associated with human listeriosis such as 61 . Serotyping profile of human isolates, revealed that about 98% of the strains diagnosed among patients and food samples belonged to serotype 1/2a, 1/2b, 1/2c and 4b 12 . These data are in contrast to the other study reported elsewhere that found most food born listerial strains belonging to serogroup 4d (or 4b, 4e) and particularly 4b 62 . These results show the necessity of proper handling to prevent outbreaks of listeriosis in Kurdistan. Also, routine sampling from supermarkets and butcher stores is recommended as this bacterium can survive for long periods if favorable temperature and nutrients are available 61 .

Detection of Virulence genes
Strains of L. monocytogenes vary in their pathogenicity according to the number of virulence genes. Potential correlation between hlyA, plcA, actA and iap virulence associated genes from L. monocytogenes and their pathogenicity was detected 63 . Virulent strains have been found to produce more phagosomal membrane disruptors particularly hlyA and plcA as compared   64 . Unexpectedly, all L. monocytogenes strains isolates from both food and human samples were positive towards four virulence genes tested (iap, hlyA, ActA and plcA), (Table 3). Three out of 4 milk samples isolated in a study from India were found to be positive towards all virulence genes tested which might also refer to human source contamination 46 . Such results were found only in pathogenic strains isolated from human samples and not from food samples 55 .

Sequencing of 16rRNA and phylogeny analysis
Out of 48 isolated strains from food and human samples, 15 isolates were selected for sequencing, analyzed and examined for detection of any genetic diversity within the isolated samples and compared with the data base isolates. All isolated samples exhibited amplification of 16sRNA up to expected size 1600bp. All food and human sequences were blasted (http://blast.ncbi.nlm. nih.gov/Blast.cgi) and showed a sequence identity of 98-100% for the 16S rRNA gene, based on the sequence similarity, more than 90 isolates from the data base were found to be strictly related to our L. monocytogenes isolates. These isolates were mainly from soil, vegetables, milk, water and human. However, genetic diversities were found within the isolated samples of food and human as show in (Fig. 2). Phylogenetic analysis of our results divided our isolates in to three different groups. On the basis of phylogeny analysis of 16SrRNA, it is found that there is a genetic relationship between human clinical isolates; the same pattern was determined between the foods isolates as a second group, while the third group which was of milk isolates was genetically further away and grouped out from both human and food samples. Comparing with the databases isolates, it is clear that the Listeria monocytogenes isolated from fresh red meat of this study is closely related to the strains isolated from prepared meat products in South Africa and to that isolated from patient in USA. L. monocytogenes isolates from different sources, food and clinical samples showed to be identical with the isolates from the milk product recorded in India and sludge, waste water reported in France 65 .

CONCLUSION
Taken together, the data in the present article confirmed that the virulent strains of L. monocytogenes are widely distributed within animals' food products with a high incidence of the bacterial infection among the human population in Duhok province. Authorities should be notified to take their role in maintaining and controlling the further spreading of the diseases, strict hygienic measurements should be applied to control entrance of the contaminated food to the area. The current study confirmed the existence of potential virulent strains of L. monocytogenes in food and human clinical samples, and the study stated that the prevalence rate of L. monocytogenes in this study was higher in food samples compared with human samples. The prevalence rate of multi-drug resistant isolates of L. monocytogenes in meat, human clinical and milk samples, and highlighted the necessity for active and continuous investigation of their antibiotic resistance.
The most serotypes found in our isolates were 1/2a (or 3a); also, the molecular serotyping is irreplaceable for better understanding the routes of L. monocytogenes dissemination and the origin of the contamination. In term of antibiotic susceptibility profile, the study determined that the majority of our isolates from both food and human were resistant to the most antibiotics tested in this study. Furthermore, milk isolates were found to be genetically diverse from food and human isolates.