Prevalence and in vitro antagonism between Listeria monocytogenes and probiotic Lactobacillus in fresh vegetables, chicken meat and vaginal swabs in Erbil city

Listeria monocytogenes is a facultative anaerobic, Gram-positive, intracellular pathogen which is well known as one of the most clinically important food-borne bacteria in the world. The ability of L. monocytogenes to proliferate at low pH, tolerate high salt, grow at low water activity and refrigeration temperature makes it very hard to remove from food production areas and ready-to-eat foods, which makes it a very difficult pathogen to control [1, 2]. Despite its relatively low incidence in the world, the disease it causes, listeriosis has a disproportionately high case fatality rate of 20-30%, and is among the most lethal of all foodborne illnesses [1].

Clinical manifestations of listeriosis include mild, self-limited foodborne disease in healthy individuals and invasive disease resulting in septicemia, meningitis, encephalitis and spontaneous abortions, but certain groups of people are at a disproportionate risk, especially pregnant women, neonates, elderly people and immunocompromised people, including those receiving chemotherapy, organ transplants, or HIV/AIDS [3, 4]. Pregnant women are estimated to be up to 17 times more vulnerable to the effects of listeriosis than the general population and infection in pregnancy is strongly linked to adverse foetal outcomes including miscarriage, foetal death and premature birth [5]. Most of the time, listeriosis is spread through the food consumed, and nearly 99% of human listeriosis cases are believed to result from ingestion of contaminated food [2].

Listeria monocytogenes has been found in various food commodities such as raw and processed meat, poultry, seafood, dairy products and fresh vegetables, and is widely distributed in the natural environment [6]. It is hard to eradicate due to its capacity to form durable biofilms on food contact surfaces as well as its tolerance to refrigerated temperatures, which constitutes a major challenge in modern food safety management [1]. Contamination of fresh produce, including leafy crops like lettuce, celery, leek and beetroot, has been increasingly documented as an important vehicle for the pathogen, especially with regards to the consumption of ready-to-eat fresh produce, where no kill step is applied [7, 8]. Leafy-vegetable outbreaks have been reported recently in the United States and Europe, with L. monocytogenes contamination identified throughout the fresh-vegetable supply chain, highlighting the public health need to monitor L. monocytogenes contamination in the fresh-vegetable supply [7].

One of the important issues in the clinical control of listeriosis is the resistance of L. monocytogenes isolates to antimicrobial agents. In fact, MDR strains have been detected in various food and clinical settings, and ampicillin in combination with an aminoglycoside (gentamicin) remains the first-line treatment, but co-trimoxazole is used for penicillin-allergic patients (Charpentier and Courvalin, as cited in Al-Brefkani and Mammani [9]). The multidrug resistance of L. monocytogenes strains isolated from different food and food-processing environments was a key aspect of a large study conducted in Italy, and the highest rate of multidrug resistance was found in the β-lactam group (87.36%) [10]. The intrinsic mechanisms and the horizontal transfer of resistance genes by mobile genetic elements like plasmids and integrons of this pathogen have been suggested as a cause of resistance [11, 12]. The unchecked and relentless use of antimicrobials in human health and veterinary medicine has been cited as a key factor in the increase in antimicrobial resistance (AMR) [9], and continued monitoring of AMR is an important public health requirement.

The rising threat of antimicrobial resistance and the known deficiencies of traditional food preservation methods have stimulated the scientific interest in probiotic lactic acid bacteria (LAB) in particular, as potential natural biocontrol agents against foodborne pathogens (FBP) [13, 14]. In vitro and food model system studies have revealed that LAB produce various types of antimicrobial metabolites such as organic acids, hydrogen peroxide, bacteriocins, and other inhibitory metabolites that have strong antagonistic activity to L. monocytogenes [15, 16, 17]. The cell-free supernatants (CFS) isolated from probiotic Lactobacillus cultures are especially promising in mitigating L. monocytogenes planktonic growth and reducing expression of its virulence genes and biofilm formation [13, 18].

Although many studies have been conducted on L. monocytogenes in developed countries, there is limited information available regarding the prevalence, virulence factors, antimicrobial resistance of L. monocytogenes and the potential for probiotic control strategies in various food matrices and clinical settings in developing countries. Studies that simultaneously analyze the pathogen distribution in fresh vegetables, meat products and human clinical samples, and correlate this with the antagonistic activity of commercially available probiotic isolates are underrepresented in the existing literature. To develop targeted and evidence-based food safety and infection control policies, it is essential to have a comprehensive understanding of these dimensions.

Hence, the present study aimed at the determination of prevalence of L. monocytogenes in local chicken meat, frozen meat and fresh vegetables (leek, lettuce, beetroot, celery) during six-month period as well as in vaginal swabs of patients attending a private clinical laboratory. Kirby–Bauer disk diffusion method was used to determine the antimicrobial susceptibility patterns of confirmed isolates against a wide range of clinically relevant antibiotics. Further, the present study included the evaluation of the in vitro antagonistic activity of probiotic isolates of Lactobacillus plantarum and Lactobacillus acidophilus from a commercial probiotic preparation against confirmed strains of L. monocytogenes by agar well diffusion method, in order to investigate the potential use of these bacteria as natural biocontrol agents against this vital foodborne pathogen.

MATERIALS AND METHODS

Sample Collection

During six months, from September 2024 to February 2025, 375 samples were collected to identify L. monocytogenes in various food and clinical matrices. Samples were collected from various sources to obtain broad and representative human and environmental sampling of this pathogen.

Clinical samples: 70 vaginal swabs were collected from the patients who came to a private biomedical laboratory. These were all swabs taken under normal diagnostic conditions and handled in a way that maintained the microbiological integrity of the samples prior to analysis, using suitable cold-chain methods. Food samples consisted of 120 meat samples which were divided into 70 samples of local chicken meat that were purchased from local butcher shops and 50 frozen samples of meat that were collected from retail food outlets. Aseptic collection was used for all meat samples which were collected in sterile containers and transported in insulated cooler boxes with 2-6°C to prevent any microbial changes prior to processing in the laboratory. The largest number of specimens collected were from vegetable samples (185 specimens from local vegetable markets). This included 52 leek (Allium ampeloprasum), 43 lettuce (Lactuca sativa), 56 beetroot (Beta vulgaris), and 34 celery (Apium graveolens) samples. Samples of each vegetable were placed in a sterile bag separately and each sample was sent to the laboratory on the day of collection to avoid deterioration.

All samples were processed immediately upon arrival to the laboratory or they were refrigerated at 4°C for a maximum of 24 hours according to the standard microbiology procedures for food samples.

Isolation and Identification of Listeria monocytogenes

Primary Enrichment

Listeria monocytogenes was isolated following the standard double enrichment procedure recommended in ISO 11290-1 standard. A representative test sample from each sample was cultured in half strength Fraser broth at 1/10 sample to broth, at 37°C for 24-48 hours in a preliminary stage. The preliminary enrichment was designed to allow stressed and/or sublethally injured Listeria cells to recover, while reducing the growth of other microflora.

Secondary Enrichment

After primary enrichment, a small portion of the half-strength Fraser broth culture was then streaked into full-strength Fraser broth diluted to 1/100 and incubated for 24-48 hrs at 37°C. This secondary enrichment step was designed to further improve the selectivity and improve recovery of Listeriaspecies from samples containing a complex microbial background.

Isolation on Selective Media

Cultures were then streaked onto Listeria CHROMagar at 37°C for 24-48 hours, a chromogenic selective and differential medium that allowed the presumptive identification of Listeria monocytogenes by the characteristic colony colors. Presumptive positive colonies were chosen for further molecular confirmation.

Antimicrobial Susceptibility Testing

All confirmed isolates of Listeria monocytogenes were subjected to antimicrobial susceptibility testing by the Kirby–Bauer disk diffusion method, following the standard microbiological procedures. Only PCR-confirmed isolates were used to obtain pure cultures to assure the reliability of results for this purpose. Overnight cultures were used to make a fresh bacterial suspension and the turbidity adjusted to a 0.5 McFarland standard. The standardized suspensions were then inoculated evenly on to Mueller–Hinton agar plates using sterile cotton swabs to create a uniform bacterial lawn throughout the plates. The inoculated plates were then aseptically overlain with antibiotic discs.

The panel of antibiotics tested was chosen to reflect clinical and veterinary relevance in the management of listeriosis, and represented a number of antimicrobial classes. These included drugs belonging to the β-lactam and related groups (penicillin, ampicillin, amoxicillin/clavulanic acid, oxacillin, aztreonam), and drugs from other classes (tetracycline, doxycycline, erythromycin, rifampicin, trimethoprim, trimethoprim–sulfamethoxazole, ciprofloxacin, levofloxacin, vancomycin, clindamycin, chloramphenicol, fusidic acid, and teicoplanin). Plates were allowed to grow at 37°C for 18-24 hours. After incubation, the diameters of the inhibition zone were measured in mm using a calibrated ruler. Breakpoints were used to interpret results as susceptible, intermediate or resistant.

Isolation and cultivation of probiotic bacteria.

Probiotic bacteria, used in this study, were obtained from a commercially available probiotic product (Vitalactic B® capsules) containing two Lactobacilli (L. plantarum and L. acidophilus). The contents of each capsule were placed in de Man, Rogosa and Sharpe (MRS) broth, a selective broth for lactic acid bacteria, under an aseptic condition. Inoculated broth was incubated in microaerobic condition at 37°C for 24–48 hours to allow the bacteria to grow.

After incubation, a loopful of the enriched culture was streaked onto MRS agar plates with the standard streak plate technique to obtain well-isolated colonies. Plates were again incubated at 37°C for 24–48 hours and checked for the presence of Lactobacillus like colonies. Presumptive Lactobacillus colonies were further subcultured on fresh MRS agar for obtaining pure cultures and were stored on MRS agar slants at 4°C for short term storage and further experimental work.

The molecular identification of Lactobacillus spp. by PCR.

Presumptive Lactobacillus spp. were molecularly identified by using the universal bacterial primers against the conserved regions of 16S rRNA gene by PCR. Pure cultures were grown in MRS agar and genomic DNA was extracted by standard methods of bacterial DNA extraction. The isolated DNA was used as template for the PCR with the optimized cycling conditions described above. Primers for the 16S rRNA gene (Forward: 5’-GTTGACTGCCGGTGACAAAC- 3’ and Reverse: 5’-GCTGTTACGACTTCACCCCA-3’) were used for both the initial screening and subsequent identification of the bacteria.

The PCR products were separated on an agarose gel, stained with a nucleic acid dye and analyzed under UV light. The presence of amplicons of the (375 bp) was used as confirmatory evidence for the presence of Lactobacillus spp., as well as using growth characteristics on MRS media.

Assessment of Antagonistic Activity Using the Agar Well Diffusion Method

Antimicrobial activity of probiotic lactobacilli against the confirmed strains of Listeria monocytogenes were determined by agar well diffusion method. To differentiate secreted antimicrobial activity from cell-associated inhibitory activity, both cell-free supernatant (CFS) and resuspended bacterial pellet fractions were tested separately.

The probiotic Lactobacillus isolates were cultured in MRS broth for 24 hours at 37°C. Afterwards, the cultures were centrifuged for 10-15 minutes at 4,000 – 6,000 rpm to sediment the bacterial cells in the pellet. The cell free supernatant was gently removed and the bacterial pellet washed and resuspended in sterile physiological saline to get a uniform bacterial suspension.

Confirmed Listeria monocytogenes isolates were cultured and standardized to the 0.5 McFarland. The standardised suspension was evenly spread over the surface of the plates of agar to create a uniform confluent lawn. A sterile cork borer was then used to bore 6-8 mm diameter holes into the agar and aliquots of either the cell-free supernatant or the resuspended bacterial pellet were added to the holes. Plates were cultured for 24-48 hours at 37°C on Muller hinton (MH) agar. Following incubation, it was carefully observed whether there was any clear zone of inhibition around the well. An antimicrobial activity value was assigned to each probiotic Lactobacillus isolate based on the diameter (mm) of any zones of inhibition measured.

Ethical Approval

The research proposal was submitted to the ethics committee of the College of Health Sciences of Hawler Medical University, reference number Sc.E.C.10M dated 9/09/2024.

RESULTS

1. Prevalence of Listeria monocytogenes Across Sample Types

The 375 samples were taken during the six-month study period (September 2024 to February 2025) and tested for Listeria monocytogenes using the ISO 11290-1 double-enrichment method, and subsequently the selective plating method on CHROMagar Listeria. A total of 375 samples were analysed and 12 (3.20%) confirmed positive for L. monocytogenes, all across the seven types of samples, as outlined in Table 1.

The food matrices studied had the following prevalence results, with frozen chicken meat having the highest prevalence of 3/50 (6.00%) samples containing isolated L. monocytogenes. Similarly, 5.36% (3 of 56 samples) of beetroot samples were found to be contaminated and 4.65% (2 of 43 samples) were contaminated with lettuce. There was no contamination at all detected in 1 out of 34 celery samples, while there were no samples of leek and local chicken meat that were found to have contamination. L. monocytogenes was isolated from 1 of 70 clinical samples including vaginal swabs (1.43%), showing that this organism is also present in the clinical context as important pathogen along with the food-associated matrices.

Table 1. Prevalence of Listeria monocytogenes across different sample types collected during the study period.

2. In Vitro Antagonistic Activity of Probiotic Lactobacillus Isolates Against Listeria monocytogenes

In vitro antagonistic activity testing was carried out on all confirmed L. monocytogenes isolates by the agar well diffusion method. Lactobacillus plantarum and Lactobacillus acidophilus were obtained from Vitalactic B® commercial probiotic capsule and cultured on de Man, Rogosa and Sharpe (MRS) agar and identified by 16S rRNA PCR before testing as shown in Figure 1. The antagonistic activity was evaluated by both the cell-free supernatant (CFS) and the bacterial pellet preparation and the inhibitory effect was determined by measurement of the zone of inhibition in millimetres on the Petri dish after 24 hours incubation at 37°C and the results are presented in Table 2.

Figure 1: probiotic 16s rRNA.

The tested probiotic strains showed a broad spectrum antagonistic activity against all the L. monocytogenes isolates from various sample types, both as cell-free supernatant and bacterial pellet preparations. The mean inhibition zone produced by the cell-free supernatant of all the isolates was 15.71 mm and the bacterial pellet preparation produced a slightly higher mean inhibition zone of 16.77 mm, thus indicating that the antagonistic products may be extracellular or may be retained inside or attached to the bacterial cell mass.

The isolate from the vaginal swab Sample 33 and Sample 3 derived from lettuce gave the maximum zone of inhibition of 21.56 mm and 21.35 mm respectively, followed by the isolate from the Sample 11 derived from tomato. Likewise, both isolates showed the biggest inhibition zone for the pellet preparation with 21.90mm and 20.17mm respectively. Sample 19 from leek had the lowest inhibition zone for the probiotic cell-free supernatant (11.91 mm), but was still sensitive to the pellet preparation (13.18 mm). The overall results suggest a statistically consistent antagonistic activity among different isolates, obtained from different food and clinical matrices, which points to the possible use of these probiotic strains of Lactobacillus spp. as natural biocontrol agents in food and natural clinical environments against L. monocytogenes contamination.

Table 2. Inhibition zone diameters (mm) produced by probiotic Lactobacillus cell-free supernatant and bacterial pellet preparations against confirmed Listeria monocytogenes isolates using the agar well diffusion method.

DISCUSSION

In the current study, the prevalence rate of L. monocytogenes was 3.20% (12/375) which was similar to the international literature. In a systematic meta-analysis of the Chinese and European Union markets, Zhang et al. [19] found prevalence rates of 7.1% and 8.3% respectively for meat samples, with Shamloo et al. [6] observing variability in prevalence based on food category, detection method, and geographic context. It was found in all seven sample types analyzed and is known to be widely distributed in the environment, further supporting the need for a multi-matrix approach to surveillance.

The highest contamination rate was found with frozen chicken meat (6.00%) followed by the locally sourced fresh chicken meat (1.43%). This is consistent with L. monocytogenes' tendency to form biofilms on surfaces of processing equipment, as known in cold-chain settings [20]. The presence of L. monocytogenes in retail chicken meat has been reported elsewhere; L. monocytogenes were isolated from chicken meat samples collected from retail establishments and slaughter houses in Turkey, by Coban et al. [21] with higher frequencies, especially in slaughter houses which may be due to processing scale and hygiene measures.

Beetroot (5.36%) and lettuce (4.65%) samples had the highest levels of contamination among the vegetable samples, while celery (2.94%) and leek (1.92%) had the next highest levels. The results agree with the systematic review conducted by Townsend et al. [7] that reported L. monocytogenes all along the fresh produce supply chain and that leafy greens and root vegetables were identified as being of particular concern. Additionally, Alegbeleye et al. [8] noted that root vegetables like beetroot have soil-contact surfaces, which allow environmental pathogens to be acquired directly from the soil. The finding of L. monocytogenes in celery is particularly significant as a fatal outbreak in the United States was directly associated with contamination from chopped celery [7], highlighting the public health implications of this result.

Isolation of L. monocytogenes was observed in one vaginal swab (1.43%) and relevant to reproduction. This rate is similar to the 0.81% reported by Singh et al. [22] who found the pathogen in 0.81% of vaginal swabs taken from pregnant women in India. Kaur et al. [5] pointed out that L. monocytogenes can be carried vaginally without symptoms, which can be a hidden source of vertical transmission and affect foetal outcomes, such as miscarriage and neonatal septicaemia. The results of this study advocate for the screening of L. monocytogenes in clinical microbiological routine of high-risk pregnant women.

The cell-free supernatant (CFS) and bacterial pellet preparations of all 12 probiotic Lactobacillus strains tested inhibited all L. monocytogenes strains with mean zone diameters of 15.86 mm and 16.79 mm, respectively. They are in accordance with the 10.0–17.2 mm inhibition halos that Saccol et al. [23] reported for L. monocytogenes using LAB CFS in the agar well diffusion method and correspond to the wide range of 0.92–25 mm inhibition halos reported by Langa et al. [24] for 200 bioprotective LAB strains. The slightly stronger inhibitory activity of the pellet preparation compared to the CFS implies that some of the antimicrobial activity could be cell-associated and not entirely secreted due to the multi-mechanistic nature of LAB antagonism, including the production of organic acids, hydrogen peroxide, and bacteriocins [13, 25]. In line with this, Abdelhamid et al. [15] also showed that L. plantarum CFS successfully inhibits planktonic growth and biofilm formation of L. monocytogenes, suggesting its potential as a natural biocontrol agent in food and clinical environments.

CONCLUSIONS

The present study supplies novel locally contextualised information on the prevalence and distribution of L. monocytogenes among food commodities and clinical specimens, and the first evaluation of the in vitro antagonistic activity of commercially purchased probiotic Lactobacillus isolates against confirmed strains of L. monocytogenes from this region. Results collectively suggest that L. monocytogenes is ubiquitous at a relatively low prevalence (3.20%) in the different sample types examined (fresh vegetables, chicken meat and vaginal swabs) and at the level of prevalence observed in these samples is comparable with the international literature and has important implications for public health, since listeriosis can cause severe effects in susceptible groups. The antagonistic effect of both L. plantarum and L. acidophilus preparations against all confirmed L. monocytogenes isolates (ranging from about 12 to 22 mm inhibition zone) is encouraging and warrants further in vivo and food-model validation studies for establishing evidence-based, probiotic biocontrol strategies for L. monocytogenes in food systems and clinical applications.