Research Article

Molecular Detection of Listeria Species Isolated From Raw Milk with Special Reference to Virulence Determinants and Antimicrobial Resistance in Listeria monocytogenes

Hams M.A. Mohamed1*, Katreen K.G.2, M.W. Abd Al-Azeem1, Faysal A. Wasel2, Ahmed M. Abd-Eldayem3

1Department of Microbiology, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt; 2Department of Microbiology, Animal Health Research Institute, Sohag, Egypt; 3Departement of Pharmacology, Faculty of Medicine, Assuit University, Assuit, Egypt.

Received | August 20, 2022; Accepted | September 20, 2022; Published | November 15, 2022

*Correspondence | Hams M.A. Mohamed, Department of Microbiology, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt; Email: [email protected]; [email protected]

Citation | Mohamed HMA, Katreen KG, Abd Al-Azeem MW, Wasel FA, Abd-Eldayem AM (2022). Molecular detection of listeria species isolated from raw milk with special reference to virulence determinants and antimicrobial resistance in listeria monocytogenes. J. Anim. Health Prod. 10(4): 492-505.

DOI | http://dx.doi.org/10.17582/journal.jahp/2022/10.4.492.505

ISSN | 2308-2801

 

 

Copyright: 2022 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Food contamination may be a vital issue for public health round the world. The foremost perishable and frequently consumed animal food by individuals is milk (Borena et al., 2022). Because of this, microbial contamination is considered to represent a heavy health risk to the public (Akrami-Mohajeri et al., 2018). The Listeria genus is cosmopolitan in nature, and it’s frequent in farms (Tabit, 2018). Listeria spp. is spore-free, facultative anaerobic, rod-shaped, and gram-positive (Odetokun and Adetunji, 2016). There are twenty-one species belongs to this genus, from which, Listeria monocytogenes (L. monocytogenes) and Listeria ivanovii are the 2 species that are recognized as being infective (Quereda et al., 2020).

L. monocytogenes is an important foodborne zoonotic pathogen, according to Reda et al. (2016). The contamination of milk and its products with Listeria spp., especially L. monocytogenes, could lead to severe infections in people. Human listeriosis is a serious or even deadly sickness for particular populations, including newborns, pregnant women, the elderly, and individuals with weakened immune systems, with fatality rates for listeriosis ranging from 20 to 30%. It causes sporadic and epidemic outbreaks of the disease (Phraephaisarn et al., 2017; Sarfraz et al., 2017; Şanlıbaba, 2018).

Typically, Listeria spp. are recognized using a range of diagnostic methods, such as growing on particular media. The required selective medium for the detection of Listeria monocytogenes is Agar Listeria according to Ottaviani and Agosti (ALOA), which has an advantage over other selective media for the isolation of L. monocytogenes and permits the distinction between pathogenic and non-pathogenic Listeria spp. because additional Listeria species frequently co-isolate from L. monocytogenes-positive food samples (Jamali et al., 2013). In addition, the likelihood of finding L. monocytogenes may be decreased if L. innocua is present in foods contaminated with the pathogen (Zitz et al., 2011).

Listeria is usually diagnosed through the time-consuming processes of isolation and organic chemistry identification. However, molecular techniques like polymerase chain reaction (PCR) ought to be employed in laboratories responsible for inspecting foods of animal origin as they permit a faster and correct identification of L. monocytogenes by targeting specific genes (El-Malek et al., 2010). The nucleotide sequence analysis of L. monocytogenes is a good trendy tool for genotyping and investigation of the relation of Listeria species to different native or international lineages.

Numerous molecular markers that are concerned within various stages of the infection, like the virulence genes responsible for the host cells invasion (inlA, inlB and iap), phagosomal escape (hlyA, plcA and plcB) wherever the hlyA gene mediates the discharge of the microorganism cells into the host’s cytoplasm, and positive regulative factor A (PrfA), are usually accustomed assess the virulence potential of L. monocytogenes (Liu et al., 2007). All of those aid the bacterium’s intracellular development and dissemination inside a mammalian host (Tirumalai et al., 2012).

One of the intense risk factors to public health is antibiotic resistance because it makes infections tougher to treat since medication lose their effectiveness, prolongation of hospital stays, raising mortality rates and increasing medical expenses (WHO, 2018). Antibiotic-resistant microorganism has considerably accrued round the world, leading to harder to treat human and animal diseases (SCENIHR, 2009). The flexibility of Listeria to quickly develop antimicrobial agent resistance could be a recently discovered feature that represents a growing danger to each human and animal health.

Listeria monocytogenes has been concerned in many deadly ill health outbreaks. Future outbreaks could also be harder to manage due to the increase of antibiotic resistance among food L. monocytogenes isolates that has been exaggerated particularly for those antibiotics usually accustomed to treat listeriosis like tetracycline, penicillin, ampicillin, and gentamicin (Olaimat et al., 2018) with variable percentages. In 1990, acquired resistance to antibiotics was initial delineated in L. monocytogenes by Poyart-Salmeron et al. (1990). Since then, unpredictable animal and human listeriosis-causing multidrug-resistant (MDR) microorganisms are found in food samples (Haubert et al., 2016). However, early administration of the proper combination of antibiotics will cut back the severity of listeriosis, with a cure rate of 70% (Abdallahzadeh et al., 2016).

In dairy farms, biofilm development could be a common phenomenon and is assumed to be a possible infective agent transmission technique since the setting is contributing to microorganism survival. Biofilms from dairy farm niches are coupled to L. monocytogenes (Latorre et al., 2010). Biofilm formation starts within twenty minutes (Weiler et al., 2013) on completely different food contact surfaces, together with stainless steel and plastic, as L. monocytogenes multiplies quickly on improperly clean dairy farm appliances. This protects the organism from environmental stresses and will increase its resistance to cleaners and sanitizers utilized in the food industry. To lower the chance of tainted milk and human infections, it’s crucial to inhibit biofilm creation on milking instrumentation (Latorre et al., 2010).

Hence, the objective of this study was to determine the incidence of listeria species in raw milk in addition to detection of the virulence, phenotypic and genotypic antibiotic resistance profiles as well as biofilm formation of L. monocytogenes strains.

MATERIALS AND METHODS

Collection of Samples

In Sohag governorate, Egypt, a total of 150 raw milk samples were collected from supermarkets, local vendors, and small-scale producers. In sterile plastic bags, the samples were taken and transferred to the laboratory in refrigerator for bacteriologic investigation.

Isolation and Identification of different species of Listeria

Listeria spp. were isolated using Listeria selective enrichment broth (CM0862, Oxoid), that was supplemented with the Listeria-selective enrichment agent (SR0141, Oxoid) for twenty-four hours at 28 °C and plated onto ALOA (Merck, Germany), that was supplemented with the Listeria-selective supplement (SR0140, Oxoid) and incubated for twenty-four hours at 37 °C as described by Ottaviani and Agosti in ISO 11290–1 (1997). The potential colonies were transferred to Tryptic Soy Agar with 0.6% Yeast Extract (TSA-YE) (Sigma, Germany), wherever they were incubated for 24-48 hours at 37 °C. The biochemical tests comprised; colony morphology, gram staining reaction, catalase test, oxidase test and sugar fermentation test (xylose, rhamnose, mannitol) were carried out (MacFaddin, 2000). Furthermore, Staphylococcus aureus strain (((identified Staph. aureus strain was obtained from South valley university, Department of Microbiology, Qena, Egypt) was treated using Christie Atkins Munch Petersen’s (CAMP) reaction, streaked on blood agar in a straight line across the plate center, then L. monocytogenes strain was streaked in a vertical direction to S. aureus then, incubated at 37 ̊C for 24 h and checked for β-hemolysis appearance as an arrowhead or circle shape in the positive reaction (CFSAN, 2001).

PCR-based confirmation of presumptive Listeria spp.

The QLA amp DNA mini kit (catalogue number 51304) was used for DNA extraction according to the manufacturer’s instructions.

PCR was performed to confirm the Listeria colonies using the Listeria iap gene. 12.5 µl of Emerald Amp GT PCR Master combined (2x premix), 1 µl each of the forward and reverse primers, 5.5 µl of PCR grade water, and 5 µl of template DNA created up all the 25 µl total. Initial denaturation occurred at 5°C for five minutes then there have been thirty-five cycles of 94 °C for 30 seconds, 60 °C for one minute, and 72 °C for one minute, followed by a final extension lasting 12 minutes at 72 °C. PCR products were seen and captured using 1.5% agarose gel electrophoresis stained with ethidium bromide below ultraviolet illumination. The amplicon size and primer sequences are listed in Table (1).

Detection of L. monocytogenes using species specific primers

The PCR amplification was done in a thermal cycler (Applied Biosystem 2720) at 50 µl total volume included; 25 µl the EmeraldAmp GT PCR Master mix (2x premix), 1.5 µl of forward and reverse primers derived from 16SrRNA gene (Table.1), 17 µl of PCR grade water, and 5 µl of template DNA created up the reaction mixture. The template DNA was initial denaturized at 95°C for four min to perform 35 cycles of PCR amplification. Then, the subsequent temperatures were used: DNA extension for one minute at 72°C, deoxyribonucleic acid denaturation for 45 s at 95°C, primer tempering for 45 s at 60°C, and a final extension for 5 minutes at 72°C.

Amplified 16SrRNA sequencing and phylogenetic analysis

The distinct Listeria spp. were identified by the sequencing of amplified 16S rRNA PCR product. Table (1) lists all-purpose primers. The PCR reaction was effectively carried out in 25 µl of the reaction mixture, which contained bacterial DNA (1 µl), each primer (0.5 µl), EmeraldAmp GT PCR Master Mix (12.5 µl), and nuclease-free water (10.5 µl). The PCR settings were first established by Rohwer et al. (2002). On 1.5% agarose gel, electrophoresis was used to analyses the outcome. The PCR products were subsequently cleaned using the QIA Rapid PCR Product Extraction Kit (Qiagen, Valencia, CA). On an automated DNA sequencer, purified PCR products were sequenced using the Big Dye terminator V3.1 kit (Bio system Cat. No.4336917, Applied Bio systems). Isolates were analyzed both forward and reverse for the 16S rRNA gene sequence. MegAlign generated sequence alignment using multiple alignment techniques (DNASTAR, Window version 3.12e).

Phylogenetic analysis

MegAlign from the Lasergene program (version7) was used to create a phylogenetic tree based on the 16S rRNA gene nucleotides sequence for our isolates in order to explore the identity of these isolates with each other and with reference strains registered with GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi? PROGRAM= blastn& BLAST_SPEC=GeoBlast&PAGE_TYPE=BlastSearch).

Evaluation of antimicrobial susceptibility of L. monocytogenes

The Kirby-Bauer disk diffusion assay was used with slight modification in consistent with CLSI (2011), to perform the antimicrobial sensitivity check for L. monocytogenes strains Briefly; 0.1ml of bacterial suspension (1x108 CFU/mL) corresponding to (0.5 McFarland) was plated on Mueller-Hinton agar supplemented with 5% defibrinated sheep blood. The plates were left for five minutes to dry then antimicrobial disks were deposited on the agar surface and incubated at 35ºC±2 for 24-48hrs. Completely different antimicrobials were used like Penicillins: penicillin G (25 μg), amoxicillin/clavulanic acid (20/10 μg), oxacillin (1 μg), ampicillin (25 μg); Aminoglycosides: gentamicin (10 μg), streptomycin (10 μg); Tetracyclines: tetracycline (30 μg), oxytetracycline (30 μg); Macrolides: erythromycin (15 μg), clarithromycin (15 μg) and vancomycin (30 μg); Lincosamides: lincomycin (2 μg); Fluoroquinolones: ciprofloxacin (5 μg), and levofloxacin (5 μg). Zone diameter was interpreted according CLSI (2011).

Biofilm formation

L. monocytogenes isolates were cultured for 24 hours at 37°C in 10 ml of brain heart infusion broth containing 1% glucose, in accordance with Stepanovic et al (2000)’s protocol. A sterile 96-well polystyrene microtiter plate with three wells was filled with 20 µL of each bacterial solution along with 180 µL of BHI with 1% glucose and 200 µL of uninoculated BHI as a negative control. The microtiter plate was incubated at 37°C for 24 hours. After that, the broth was taken out and the wells were cleaned three times with sterile phosphate-buffered saline. Biofilms were turned over and air-dried in a warm place for around 30 minutes after being fixed with methanol for 20 minutes. Biofilms were stained with crystal violet (2%) for 15 minutes. After two rinses with distilled water, the wells were dehydrated. The pigmented adherent cells were resolubilized in 150 µL of 33% acetic acid for 30 minutes at room temperature. Finally, a microtiter plate reader was used to measure the OD of each well at 595 nm (OD595). Based on the absorbance, the bacteria were categorized into four groups: no biofilm producer (OD≤ODc), weak biofilm producer (ODc<OD2X ≤ODc), moderate biofilm producer (2X <ODc ≤OD 4X ODc), and strong biofilm producer (OD>4X ODc). The optical density for the negative control was calculated as ODc (uninoculated broth).

Detection of antimicrobial resistance and virulence genes in L. monocytogenes by PCR

β-lactams antibiotics resistance gene (ampC) along with other drug-resistant genes to the following antibiotics: tetracycline (tetM), aminoglycoside (aad6) and macrolides (mefA) were determined by PCR using the primers displayed in Table 1. Furthermore, L. monocytogenes isolates were screened for the presence of three virulence markers hlyA, inlA and inlB (Table 1).

Uniplex PCR reaction was done in 25 μL PCR reaction mixture containing 1 μL of bacterial DNA, 0.5 μL of each primer, 12.5 μL of EmeraldAmp GT PCR Master Mix, and 10.5 μL nuclease-free water. The cycling conditions used for the uniplex PCR amplification of the ampC β-lactamase gene were: 5 minutes, 94˚C; followed by 35 cycles (30 seconds, 94˚C; 40seconds, 50˚C; 45 seconds, 72˚C); 10 minutes, 72˚C. For Aad6 gene: 5 minutes, 94˚C; 35 (30 seconds, 94˚C; 40 seconds, 55˚C; 50 seconds, 72˚C); 10 minutes, 72˚C. For tetM tetracycline gene: 5 minutes, 94˚C; followed by 35 cycles (30 seconds, 94˚C; 40 seconds, 55˚C; 40 seconds, 72˚C); 10 minutes, 72˚C and for mefA gene: 5 minutes, 94˚C; 35 (30 seconds, 94˚C; 40 seconds, 55˚C; 40 seconds, 72˚C); 10 minutes, 72˚C. For hlyA gene, the initial duration at 94 ̊c for 5 min, followed by 35 cycles of (94 ̊c for 30 s., 50 ̊c for 30s.and 72 ̊c for 30 s.) with a final extension at 72 ̊c for 7min. For the inlA gene, the initial duration at 94 ̊c for 5 min, followed by 35 cycles of (94 ̊c for 30 s., 55 ̊c for 45s.and 72 ̊c for 45 s.) with a final extension at 72 ̊c for 10min and for inlB gene, the initial duration at 94 ̊c for 5 min, followed by 35 cycles of (94 ̊c for 30 s., 55 ̊c for 40s. and 72 ̊c for 40 s.) with a final extension at 72 ̊c for 10min. The PCR products were visualized using ethidium bromide-stained 1.5% agarose gel electrophoresis under UV light and photographed.

Statistical Analysis

The values were compared using SPSS (Version 28) data processing. The Scheffe and Duncan tests were conducted with a p value ≤ 0.05 indicating a significant difference after the one-way ANOVA analysis.

RESULTS

In our investigation, 150 samples of raw milk were analyzed, and 22 isolates (14.6%) were recognized as Listeria spp. by cultural characteristics on ALOA agar plates (bluish green color) and traditional biochemical identification of the suspected colonies. It was noted that the biochemical testing showed catalase positive, oxidase negative, CAMP test was positive for eight isolates and negative for other suspected Listeria isolates and variable results were showed in the fermentation tests particularly rhamnose and xylose sugars, these tests yielded an inconsistent and unreliable findings. Therefore, we employed PCR to validate the findings. PCR confirmed the presence of Listeria DNA using iap gene in 13 out of 22 Listeria isolates that were classified as positive biochemically (Fig.1a). Additionally, the use of species-specific L. monocytogenes primer derived from 16S rRNA gene showed that 8 out of 13 Listeria spp. were L. monocytogenes (Fig.1b). The results of sequencing of the amplified universal 16S rRNA gene showed that the remaining five isolates (5/13) were classified as; L. innocua (3 isolates) and L. welshimeri (2 isolates)

Our isolates were found to have the following relationships with reference strains that were registered in the gene bank (Table 2 and Fig. 2): L. monocytogenes grouped with L. monocytogenes (NR044823) with an identity percentage of 98.8%, L. innocua isolates grouped with L. innocua (FJ774201) with an identity percentage ranging from (99.1 to 99.5%), and L. welshimeri grouped with L. welshimeri (DQ065846) with identity percentage ranged from (99.9-100%) (Table 3).

 

Table 1: Primers sequence

Gene	Primer Sequence 5'-3'	Amplified product (bp)	Reference
iap	ATGAATATGAAAAAAGCAAC	1450-1600 bp	Chen and Knabel, (2007)
	TTATACGCGACCGAAGCCAAC
L. monocytogenes species specific 16S rRNA	GGA CCGGGGCTA ATA CCG AAT GAT AA	1200 bp	Kumar et al., (2015)
	TTC ATG TAG GCG AGT TGC AGC CTA
Universal 16S rRNA	AGAGTTTGATC MTGGCTCAG	1,500-bp	Chèneby et al., (2000|)
	TACGGYTACC TTGTTACGACTT
inlA	ACG AGT AAC GGG ACA AAT GC	800 bp	Liu et al., (2007)
	CCC GAC AGT GGT GCT AGA TT
inlB	CTGGAAAGTTTGTATTTGGGAAA	343 bp
	TTTCATAATCGCCATCATCACT
hlyA	GCA-TCT-GCA-TTC-AAT-AAA-GA	174 bp	Deneer and Boychuk, (1991)
	TGT-CAC-TGC-ATC-TCC-GTG-GT
aad6	AGAAGATGTAATAATATAG	978 bp	Morvan et al., (2010)
	CTGTAATCACTGTTCCCGCCT
mefA	AGTATCATTAATCACTAGTGC	345 bp
	TTCTTCTGGTACTAAAAGTGG
tetM	GTGGACAAAGGTACAACGAG	405 bp
	CGGTAAAGTTCGTCACACAC
ampC	TTCTATCAAMACTGGCARCC	550 bp	SRINIVASAN et al., (2005)
	CCYTTTTATGTACCCAYGA

 

Table 2: Accession numbers of Listeria spp.

Accession Number	No. of isolates
OM897223	L.monocytogens(1)
OM897398	L. innocua(2)
OM897478	L. welshimeri(3)
OM897399	L. innocua(4)
OM897429	L. innocua(5)
OM897482	L. welshimeri(6)

 

Table 3: Identity percentages of our isolates with reference strains registered on gene bank

 

 

 

The Results in Table (4) demonstrated the antimicrobial resistance of eight isolates of L. monocytogenes. All of the isolates tested positive for the greatest levels of resistance to tetracycline (100%), lincomycin (100%) and gentamycin (100%), as well as penicillin, ampicillin, oxacillin, and amoxicillin/clavulanic acid (100%). Vancomycin (50%) and erythromycin (37.5%) both exhibited intermediate resistance, but all isolates shown great sensitivity to ciprofloxacin and levofloxacin (100%). All of the L. monocytogens milk isolates in this investigation (100%) showed multidrug resistance.

 

Table 4: Antimicrobial sensitivity pattern of L. monocytogenes isolated from raw milk.

Type of Antibiotics	No. of L. monocytogenes isolates (n =8)
	Sensitive (S)		Intermediate (I)		Resistant (R)
	N.	%	N.	%	N.	%
Penicillin G (25 μg)	0	0%	0	0%	8	100%
Ampicillin (25 μg)	0	0%	0	0%	8	100%
Amoxicillin/clavulanic acid (20/10 μg)	0	0%	0	0%	8	100%
Oxacillin (1 μg)	0	0%	0	0%	8	100%
Gentamicin (10 μg)	0	0%	0	0%	8	100%
Streptomycin (10 μg)	0	0%	0	0%	8	100%
Tetracycline (30 μg)	0	0%	0	0%	8	100%
Oxytetracycline (30 μg)	0	0%	0	0%	8	100%
Erythromycin (15 μg)	2	25%	3	37.5%	3	37.5%
Clarithromycin (15 μg)	2	25%	2	25%	4	50%
Lincomycin (2 μg)	0	0%	0	0%	8	100%
Vancomycin (30 μg)	3	37.5%	1	12.5%	4	50%
Ciprofloxacin (5 μg)	8	100%	0	0%	0	0%
Levofloxacin (5 μg)	8	100%	0	0%	0	0%

 

Data illustrated in Figure (3) revealed that all the isolates (100%) could form biofilms. After 24 hours incubation, five (62.5%) L. monocytogenes strains formed moderate biofilm while 3 (37.5%) L. monocytogenes isolates formed weak biofilm.

Statistical analysis showed a significant relationship between multidrug resistance and biofilm formation (P < 0.05) among L. monocytogens isolates (Figure 4).

 

The inspection for three key virulence genes (hlyA, inlA and inlB) in L. monocytogens isolates by conventional PCR revealed that hlyA and inlB genes were detected in 100% of the isolates and inlA gene is detected in 50% of L. monocytogenes isolates of raw milk samples as shown in (Figure 5).

The molecular detection of antimicrobial resistance genes in L. monocytogenes isolates revealed that the resistance to β-lactam antibiotics (penicillin, ampicillin, oxacillin and amoxicillin/clavulanic acid), tetracycline, gentamycin, and erythromycin was associated with the presence of ampC β-lactamases (100%), tetM (100%), aad6 (100%) and mefA (37.5%) genes in the identified strains respectively (Figure 6).

 

DISCUSSION

In addition to being the third most frequent cause of food poisoning-related fatalities in humans, Listeria monocytogenes is one of the most significant foodborne bacteria that is linked to food-borne disease outbreaks and high hospitalization rates globally (Osman et al., 2020; Luque-Sastre et al., 2018). Because of its potential to cause severe and occasionally fatal listeriosis disease, its widespread distribution in the environment and food (Gasanov et al., 2005), its ability to survive in environmental niches by producing biofilms (Lee et al., 2017), and its capacity to grow and endure challenging conditions like low temperature and high salt concentrations (NicAogáin and O’Byrne, 2016), it poses serious health risks.

Culture techniques based on colony shape, sugar fermentation, and hemolytic characteristics were used in traditional identification. In our investigation, 14.6% of samples were considered to be contaminated with Listeria spp. based on ALOA and traditional biochemical assays. These techniques constitute the gold standard (Gasanov et al., 2005). Despite the fact that ALOA is the first chromogenic media to be developed to yield presumptive identification of pathogenic Listeria species in a shorter amount of time after initial sample examination (Ottaviani et al., 1997) and enables more efficient isolation of L. monocytogenes from foods contaminated with L. monocytogenes in addition to other Listeria spp., they can occasionally result in false positive results (Nwaiwu, 2015). In order to assess L. monocytogenes contamination in food samples, we must instead employ more precise molecular techniques like PCR (Angelidis et al., 2012).

The Listeria iap gene, a highly specific housekeeping gene used for genus-level identification of Listeria, was used in the PCR findings to establish the presence of Listeria spp. DNA in (13 out of 22 isolates) (Atil et al., 2011). The most often recovered species (8 out of 13) was L. monocytogenes. Similar to this, L. monocytogenes was discovered in Egypt from several raw milk samples (Khedr et al., 2016). By employing the 16S rRNA species-specific gene, some publications are successful in identifying L. monocytogenes (Haggag et al., 2019; El-Banna et al., 2016; Osman et al., 2016).

On the other hand, 16S rRNA gene sequencing revealed the identities of the last 5 Listeria spp. as 3 isolates of L. innocua and 2 isolates of L. welshimeri, where its function is irreplaceable and may be regarded as the definitive method for species identification (Clarridge, 2004; Srinivasan et al., 2015).

Other incidences were recorded by Borena (2022) (7%) where L. monocytogenes was 1.82% and Atil et al. (2011) (1.19%) and 0.19% of L. innocua were detected. Another research, such as those by Aygun and Pehlivanlar S. (2006) and Kobayashi et al. (2017) did not find any L. monocytogenes strains. Contrarily, EL-Naenaeey et al. (2019) discovered a higher incidence of Listeria spp. (19%) in which both L. monocytogenes, L. welshimeri, and L. innocua were all identified from 4% and 2% of raw milk samples, respectively. Additionally, Albastami et al. (2020) found Listeria spp. in 25% of raw milk samples, with 2% and 5% of those samples isolating L. monocytogenes and L. innocua, respectively.

The variation in the prevalence of L. monocytogenes in milk observed in various reports could be caused by a number of factors, including geographic location, seasonal variations in milk sample collection, types of samples, isolation techniques, management system, time allocated for the study, and the hygienic status of the milk production and processing (Teshome et al., 2019). Conversely, listeriosis is largely a ruminant illness, thus it seems logical that bovine hosts may be important in maintaining the pathogen predominance in cattle ranches. Additionally, the incidence of L. monocytogenes may be significantly impacted by the use of untreated animal waste as fertilizer, polluted irrigation water, and inadequate hygiene measures during milking (Shiwakoti, 2015). All studies show that raw cow milk can be a source of Listeria infections in people, despite the widely known variance in L. monocytogenes prevalence in milk (Buchanan et al., 2017).

The identity and provenance of our isolates were validated by phylogenetic analysis, which revealed that they belonged to a group with reference strains of Listeria spp. that were recorded in the gene bank under the accession numbers (NR044823, FJ774201, and DQ065846) and came from food samples (Volokhov et al., 2006).

Raw milk contamination with antibiotic-resistant foodborne bacteria is a significant public health concern that might endanger human food safety. Recent investigations have demonstrated a decrease in the sensitivity of L. monocytogenes to a number of antibiotics (Korsak et al., 2012). All L. monocytogenes strains from raw milk used in this study had 100% resistance to gentamycin, tetracycline, ampicillin, ampicillin/clavulanic acid, penicillin and oxacillin. Our findings are important since penicillin or ampicillin and gentamicin make up the standard treatment for listeriosis and are often used to treat animal and human diseases (Aras & Ardiç, 2015). Our results supported prior research by Marian et al. (2012), AL-Ashmawy et al. (2014), Akrami-Mohajeri et al. (2018), Olaimat et al. (2018), and Borena et al., (2022). These findings demonstrate that penicillin, the first line antibiotic used to treat active listeriosis infections, may not be successful and that second line antibiotics may be necessary. Our strains’ penicillin resistance may be a result of the suggested use of penicillin for treating infectious illnesses like mastitis in ruminants (EVIRA, 2018). However, the study’s findings also demonstrated that all of the isolates were extremely susceptible to ciprofloxacin, which was supported by earlier data (Tahoun et al., 2017; Amajoud et al., 2018).

During the last few decades, multidrug resistance (MDR) among foodborne pathogens, including L. monocytogenes, has emerged (Zhang et al., 2007). MDR now poses a public health risk because it may result in unsuccessful treatment, which could increase hospitalizations costs, lengthen the time antibiotics must be administered, and increase the number of people who die from foodborne illness.

Egypt has reported the multi-drug resistance L. monocytogenes isolates from raw milk to certain conventional antibiotics (Aksoy et al., 2018). In our investigation, L. monocytogenes strains found in milk samples had a significant incidence of MDR phenotypes (100%) against the antibiotics examined. Our findings were consistent with those noted by Sharma et al. (2017). It was shown to be greater than the results that had previously been published by Jamali et al. (2013) (71.4%), Kevenk and Gulel (2016) (37%), and Kayode et al. (2022) (38.10%).

The predominance of MDR milk strains in our investigation may have come from dairy animals or acquired from repeated contact with antibiotics used in animal husbandry. However, given that biofilm-forming bacteria have been shown to have a higher tolerance to clinical antimicrobials and disinfectants due to regular exposure to sanitizers below the required doses, such resistance may be connected to the isolates’ capacity for biofilm-formation (Doulgeraki et al., 2017).

The propensity of L. monocytogenes to infect and spread through food is associated with its capacity to create biofilms, which provide the microbe sticky and protective qualities (Oliveira et al., 2010). Because these biofilms are persistent for several months or even years, food can be contaminated repeatedly (Markkula et al., 2005). Our research confirms the findings of Skowron et al. (2019), who showed that L. monocytogenes of milk origin frequently exhibit biofilm activity and possess a number of genes linked with virulence and antibiotic resistance. Additionally, L. monocytogenes only moderately or weakly produced biofilm on different surfaces in earlier investigations by Djordjevic et al. (2002) and Harvey et al. (2007). Also, Conficoni et al. (2016) found that even under harsh settings including arid environments, high salt concentrations (10%, wt/vol), at refrigeration temperatures and a wide pH range (4.7-9.2), L. monocytogenes develops and lives in a variety of habitats. It is challenging to regulate the genus Listeria in food processing facilities due to its capacity to survive and grow in the food environment, even by forming biofilms.

Our findings showed a substantial connection between the development of biofilm and multidrug resistance in Listeria monocytogenes isolates (P <0.05). The bacteria that form biofilms are naturally resistant to many antibiotics, increasing antibiotic resistance by up to 1000 times, and requiring high antimicrobial dosages to make these organisms inactive (Uruen et al., 2021; Thien-fah et al., 2001; Stewart et al., 2001). This is done so that biofilms, which are assemblages of surface-attached bacteria encapsulated in an extracellular matrix, may tolerate antimicrobial treatments far better than non-adherent, planktonic cells can. Therefore, biofilm-based illnesses are exceedingly challenging to treat (Clayton et al., 2017).

The discovery of several virulence markers, including internalins (inlA, inlB), listeriolysin O (hlyA), and others that are crucial in L. monocytogenes infection and pathogenesis, provides the basis for the detection of pathogenic L. monocytogenes (Di Ciccio et al., 2012). The identification of pathogenic L. monocytogenes strains carrying these genes has also been done using PCR-based techniques (Swetha et al., 2012). The crucial and well-known L. monocytogenes gene hlyA generates listeriolysin O (LLO), which facilitates bacterial entry into the host’s cytoplasm and promotes pathogen proliferation both within and outside of host cells (Poimenidou et al., 2018). Both host cell adhesion and invasion as well as L. monocytogenes internalization inside host epithelial cells are regulated by the inlA and inlB genes.

The presence of the hlyA gene in all strains examined for our study’s virulotyping analysis was consistent with research by Osman et al. (2016), Şanlıbaba et al. (2018), Owusu-Kwarteng et al. (2018), El-Demerdash et al. (2019) and Skowron et al. (2019). Additionally, it was found that L. monocytogenes’ ability to form biofilms is impacted by the presence of the hlyA virulence gene (Price et al., 2018), as this organism is better able to withstand ineffective disinfection during cleaning procedures, which results in the continued presence of L. monocytogenes in the final product.

According to Kayode et al. (2022), Indra-wattana et al. (2011), and Sant’Ana et al. (2012), practically all tested L. monocytogenes isolates from food samples included the internalin gene inlB. Additionally, the 50% of L. monocytogenes isolates that have the inlA gene support previous research by Coroneo et al (2016). The presence of these crucial virulence genes clearly suggested that L. monocytogenes from the examined raw milk may contribute to the pathogenesis of human listeriosis, according to Cotter et al (2008)’s explanation.

A clear proliferation of antibiotic determinants was seen by several researchers, particularly in food isolates where the incidence rate of MAR strains reached 30% or higher (Keet et al., 2021). It was discovered that all of the L. monocytogenes strains from milk samples utilized in this investigation contained the ampC B-lactamase gene. Consistent findings by Njagi et al. (2004) and Hassan et al. (2018) revealed ampC genes in 100% of the L. monocytogenes isolates recovered from milk and dairy products in Egypt. In contrast, a previous investigation discovered that none of the L. monocytogenes isolates recovered from milk samples have the ampC gene (Harshani et al., 2022). According to Meletis (2016), this may be related to mutations in «penicillin-binding proteins» (PBPs) that cause B-lactam resistance, contradicting other studies that claimed that the high percentage of ampicillin-resistant strains suggested amoxicillin/clavulanic acid as a better treatment option (Rezai et al., 2018; Fischer et al., 2020).

Beta-lactam antibiotics are the most frequently used in veterinary medicine because of their low toxicity and effectiveness; however, the emergence of beta-lactam resistant pathogenic bacteria poses a serious threat to their widespread use (Ghazaei, 2019).

The most often reported resistance in Listeria species is to tetracyclines (Luque-Sastre et al., 2018). It has mostly been linked to the ribosomal protection proteins-granting tetM and tetS genes (Granier et al., 2011). Although greater than that was also noted by (Elsayed et al., 2022) (41.6%), the prevalence of tetM in our study was in agreement with that found by (Granier et al., 2011; Hassan et al., 2018). Additionally, among clinical isolates of L. monocytogenes, the tetM gene was reported by (Morvan et al., 2010).

In this investigation, 100% of the L. monocytogenes strains had high prevalence of the (aad6) gene. Additionally, 37.5% of the L. monocytogenes strains in our investigation had Macrolides, which are encoded by the (mefA) gene, amplified. In contrast, research by Hassan et al. (2018) found that 100% and 71.4% of L. monocytogenes strains isolated from milk and dairy products in Egypt, respectively, had the (mefA) and (aad6) genes. Efflux pumps like mef(A) identified in Streptococcus pneumonia can mediate the resistance to macrolides, notably erythromycin (Granier et al., 2011). This is a major discovery since, when combined with penicillin, gentamicin is a first-choice medication for treating listeriosis. Additionally, erythromycin is a second-choice antibiotic used to treat listeriosis in persons who are penicillin-sensitive or pregnant (Hof, 2004).

Antibiotic resistance in L. monocytogenes is brought on by a number of mechanisms, including the acquisition of mobile genetic elements, self-transferable, mobilizable plasmids, conjugative transposons, and target gene alterations, such as those in genes producing efflux pumps (Luque-Sastre et al., 2018). Additionally discovered to be a potential scenario was the transfer of erythromycin and tetracycline ARGs from lactic acid bacteria (LAB) to L. monocytogenes in fermenting milk (Toomey et al., 2009).

However, the differences in the susceptibility patterns of L. monocytogenes isolates may be due to the management of the farm and the broad, indiscriminate use of antibiotics to treat diseases and increase animal development (Lungu et al., 2011). Pre-exposure adaptation may be brought on by L. monocytogenes’ ongoing exposure to low doses of antibiotics in the food manufacturing chain, allowing the organism to withstand larger antibiotic concentrations (Olaimat et al., 2018). Additionally, L. monocytogenes food isolates were exposed to various environmental variables, such as salt, heat, and cold, and they adapted to those settings, which increased their resistance to different antibiotics (Al-Nabulsi et al., 2015).

In addition, L. monocytogenes resistance to several antimicrobial medicines has evolved and altered during the past few decades (Olaimat et al., 2018). Tetracycline and ciprofloxacin resistance in L. monocytogenes isolates from humans, high prevalence of clindamycin and oxacillin resistance in food from meat and fish production chains, and significant percentages of resistance to ampicillin, penicillin G, and tetracycline in L. monocytogenes strains isolated from meat, fish, and dairy production chains were all reported in the environment (Caruso et al., 2019).

CONCLUSION

The capacity of these strains to resist various antibiotics as well as the finding that their ability to build biofilm was higher among multidrug resistant strains played a significant role in the identification of Listeria spp. and emphasized the pathogenicity function of L. monocytogenes. Therefore, to combat the issue of resistance and biofilm development, antibiotics must be administered in accordance with a certain regimen and at precise dosages.

ACKNOWLEDGMENTS

The authors are appreciative to all staff members in Department of Microbiology, Faculty of Veterinary Medicine, South Valley University, Egypt , Department of Microbiology, Animal Health Research Institute, Sohag, Egypt and Department of pharmacology, Faculty of Medicine, Assuit University, Assuit, Egypt.

conflict of interest

The authors declared that no conflict of interest.

novelty statement

This study focus on the role of 16SrRNA in identification of Listeria species also directed the light on virulence determinants harbored by L. monocytogenes specially multidrug resistance and its relationship with biofilm formation among these isolates, which makes these isolates pose a threat on public health.

authors contribution

All authors contributed in this work equally.

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