Listeria Monocytogenes In Raw Milk Research Articles

Application of MALDI-TOF Mass Spectrometry for the Assessment of Prevalence of Listeria monocytogenes in Raw Milk, Dairy Products and Freshwater Fishes

Background: Foodborne infections like Listeriosis cover several disorders and are a worldwide public health emergency. Listeria monocytogenes has been isolated from various foodstuffs, including milk and fishes. MALDI-TOF mass spectrometry technique is known for its rapid and accurate identification of bacterial organisms. Methods: In the current research, a total of 360 samples comprising raw milk (130), milk products (125) and freshwater fishes (105) were screened for the detection of pathogenic Listeria species by using the USDA method. The recovered Listeria isolates were characterized using conventional set of biochemical analysis along with sugar fermentation tests and further confirmed by MALDI-TOF MS. The virulent nature of pathogenic Listeria isolates was also assessed by in vitro tests like hemolysis on blood agar, CAMP and PI-PLC assay. Result: In current study, on screening 360 animal origin food samples, three isolates were recovered from raw milk samples and identified as Listeria monocytogenes indicating an overall prevalence of Listeria monocytogenes to the tune of 0.83%. Excellent correlation was observed with identification of Listeria species using conventional phenotypic tests and advanced molecular tool Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) technique. The results depict dependability of advanced technique for rapid and reliable identification of Listeria species.

Combination of growth conditions and InlB-specific dot-immunoassay for rapid detection of Listeria monocytogenes in raw milk

The gram-positive bacterium Listeria monocytogenes is an important foodborne pathogen contaminating dairy products. Closely related to L. monocytogenes saprophytic Listeria spp. are also frequent contaminators of food and, particularly, dairy products. To distinguish L. monocytogenes from nonpathogenic Listeria spp. and other bacteria, a dot-immunoassay was developed. The immunoassay is based on the polyclonal antibody to the secreted form of the surface virulence-associated L. monocytogenes-specific InlB protein. To increase InlB production, bacteria were grown on the brain-heart infusion agar supplemented with 0.2% activated charcoal (BHIC agar). Direct plating of artificially contaminated raw milk samples on the BHIC agar followed by the dot-immunoassay allowed a rapid identification of L. monocytogenes in concentrations as little as 10 cfu/mL. Using the developed approach, preliminary results were obtained within 14 h, and the final results were obtained after 26 h. The dot-immunoassay was tested on L. monocytogenes strains belonging to different clonal complexes and phylogenetic lineages, Listeria spp., and other bacterial species. Results showed the exceptional specificity of the developed dot-immunoassay for the rapid identification of L. monocytogenes.

Molecular detection of Listeria monocytogenes in raw milk in Kerbala Province

Listeria monocytogenes was discovered as a source of contamination during sampling of raw milk and its products from local cows at dairy farms, markets, and shipping points. The goal of the current study was to define the accuracy of listeria monocytogenes in raw milk contamination. This study was conducted by randomly collecting samples from various areas in the Karbala province and its surroundings. From January to March 2022, 250 samples of milk and dairy products (yogurt, cheese, and butter) were randomly selected, processed in accordance with Listeria monocytogenes standard procedures, and sent to a lab from individual cows for analysis. Some biochemical experiments were conducted to ascertain the physical and biochemical characteristics of milk and handled in accordance with Listeria monocytogenes standard procedures before being delivered from the individual cows for analysis to the lab, the study includes the isolation and molecular detection of Listeria monocytogenes from animal sources by employing selective media for this bacteria (oxford agar) for growing, as well as the molecular analysis of L. monocytogenes performed by conventional PCR technique using particular 4 primers (Hyl-1-f, Hyl-2-R, List-F, List-R).

Prevalence of Listeria Monocytogenes in Raw Milk of the Healthy Cattle in Lorestan Province (Iran) by PCR

Background & Objective: The prevalence of Listeria monocytogenes (L. monocytogene) in milk and dairy products, the high complications and damage associated with zoonotic Listeriosis have led to this bacterium being considered as a risky infection for public health.
 Materials & Methods: In the present descriptive and cross-sectional study, to investigate the prevalence of L. monocytogene, 100 samples of raw milk obtained from industrial farms in Lorestan province in winter and spring of 2019-2020 were collected and 16SrRNA and hlyA genes were detected using PCR reaction to identify Listeria genus and L. monocytogene species, respectively.
 Results: Based on the observed results, three samples (3%) were positive for Listeria genus, while no report of L. monocytogens species was observed.
 Conclusion: The results of this study did not show evidence of the presence of L. monocytogenes in raw cow milk, which indicates the sanitation and desirable nutritional conditions in industrial farms. Therefore, due to the presence of Listeria (other species of Listeria) in raw milk, if you consume raw and unpasteurized milk, the risk of human infection with listeriosis should not be ignored.

Short Communication: Prevalence of Listeria monocytogenes in Raw Milk of Healthy Sheep and Goats.

Listeria monocytogenes, one of the most important bacterial pathogens transmitted through milk, causes listeriosis in humans and animals. This study aimed to investigate the prevalence of L. monocytogenes in raw milk of healthy sheep and goats in the west of Iran (Lorestan Province) by touchdown PCR (TD-PCR). Listeria spp. were found in milk samples taken from 21 sheep (29.16%) and 3 goats (10.71%) whereas L. monocytogenes was isolated from milk samples taken from 4 sheep (5.55%) and 1 goat (3.75%). The results showed that there was a significant difference between sheep and goats in the prevalence of Listeria spp. in their raw milk (p < 0.05), but no significant difference was observed between them in the prevalence of L. monocytogenes. The study findings suggested that the raw milk of healthy sheep and goats was infected with L. monocytogenes and warned of the risk of human infection with listeriosis following consumption of raw and unpasteurized milk.

Prevalence and Antimicrobial Resistance of Listeria monocytogenes in Raw Milk in Tehran, Iran

Background and Objective: One of the major sources of Listeria monocytogenes, as the causative agent of invasive listeriosis, is raw milk. The aim of the present study was to detect Listeria monocytogenes in raw milk samples collected from dairy stores in Tehran, Iran, 2019. Material and Methods: A total of 100 raw milk samples were assessed using cultural techniques. Furthermore, antimicrobial resistance profiles of the Listeria isolates were assessed against eight antimicrobials using disc diffusion method. Results and Conclusion: Listeria spp., including Listeria grayi (5%), Listeria ivanovii (3%) and Listeria monocytogenes (2%), were detected in 10% of the samples. Listeria monocytogenes isolates were susceptible to major antimicrobials used for the treatment of listeriosis with no multidrug resistances. The highest frequencies of resistance were seen against streptomycin (60%), gentamicin (50%) and tetracycline (50%). In conclusion, potential risks of listeriosis still threaten consumers of the raw milk in Tehran. Conflict of interest: The authors declare no conflict of interest.

Detection and Pulsed-Field Gel Electrophoresis Typing of Listeria monocytogenes Isolates from Milk Vending Machines in Croatia

The aim of the present study was to evaluate the persistence of Listeria monocytogenes in raw milk from vending machines, based on culture and molecular detection of pathogen and Pulsed-Field Gel Electrophoresis typing. From December 2015 to January 2017, a total of 319 milk samples from 36 vending machines were examined for the presence and count of L. monocytogenes by reference methods ISO 11290:1 and ISO 11290:2. Molecular detection of pathogens was performed by loop-mediated isothermal DNA amplification (LAMP) coupled with bioluminescence (Molecular Detection Assay). L. monocytogenes was detected by MDA in 14 milk samples (4.38%) from four farms, compared to eight positive samples (2.5%) retrieved by a reference ISO method. Cultivable L. monocytogenes isolates were subjected to Pulsed-Field Gel Electrophoresis typing and pulsotypes were compared with those obtained during the previous survey in Croatia (2014‒2015). It was found that identical PFGE patterns of L. monocytogenes occur in milk samples of the same producer over a three-year period, indicating the persistence of pathogens in raw milk vending machines. The results obtained support the need for more effective control of milk in the entire food chain.

Prevalence and Characteristics of Listeria monocytogenes Isolates in Raw Milk, Heated Milk and Nunu, a Spontaneously Fermented Milk Beverage, in Ghana

Listeria monocytogenes is a gram-positive food-borne pathogen that causes listeriosis in humans. Currently, there is little information on the prevalence of Listeria monocytogenes in raw milk and traditional yoghurt-like milk beverage, nunu, in Ghana. The purpose of this study was to investigate the prevalence of L. monocytogenes isolates in raw cow milk, boiled milk and nunu in Ghana, and to characterize these L. monocytogenes isolates according to their serogroups, virulence potentials and antibiotic susceptibility profiles. A total of 254 samples comprising 114 raw cow milk, 56 boiled milk and 84 nunu were collected from dairy farms and market vendors for detection of L. monocytogenes. The overall prevalence of L. monocytogenes in raw milk, boiled milk and nunu was 5.5% (14/254). Listeria monocytogenes was prevalent in raw cow milk (8.8%; 10/114) and nunu (13.1%; 11/84), while no Listeria spp. was not detected in boiled milk. A total of 62 L. monocytogenes isolates were analysed to belong to molecular serogroups 1/2a-3a (32/62, 51.6%), 1/2b-3b-7 (14/62, 22.6%), 4b-4d-4e (9/62, 14.5%) and 1/2c-3c (7/62, 11.3%). All 62 L. monocytogenes isolates harbored the virulence-associated genes inlA, inlB, inlC, inlJ, plcA, actA, hlyA, iap and prfA. All Listeria monocytogenes in the present study were generally susceptible to the tested antibiotics, except neomycin and tetracycline, for which phenotypic resistance was observed among isolates.

Development and application of a new multiplex real‐time PCR assay for simultaneous identification of Brucella melitensis, Cronobacter sakazakii and Listeria monocytogenes in raw milk and cheese

Brucella melitensis, Cronobacter sakazakii and Listeria monocytogenes are important foodborne pathogens in milk and milk products, which are responsible for a variety of diseases that pose serious hazards to public health and food safety. The objective of this study was to develop a novel multiplex RTi‐PCR for the detection of B. melitensis, C. sakazakii and L. monocytogenes and to characterise the potential risk of these pathogens in raw milk and cheese. The raw milk (n = 25) and cheese samples (n = 20) were analysed by multiplex RTi‐PCR assay and detected for quantification of the three pathogens. In this study, B. melitensis, C. sakazakii and L. monocytogenes were simultaneously identified using BMEII0466, mms operon and hly as target genes, respectively. The multiplex RTi‐PCR assay that was developed showed good sensitivity and selectivity for the pathogenic microorganisms (r2 = 0.986–0.997). Multiplex RTi‐PCR results showed that most of the samples were contaminated with the pathogens screened.

Presence and Antibiotic Resistance of Listeria monocytogenes in Raw Milk and Dairy Products

Presence and Antibiotic Resistance of Listeria monocytogenes in Raw Milk and Dairy Products

Occurrence and growth of Listeria monocytogenes in packaged raw milk

The increased availability of packaged raw drinking milk necessitates the investigation of the occurrence and growth of Listeria monocytogenes in raw milk during distribution and storage. The occurrence of L. monocytogenes in 105 retailed raw milk bottles, 115 bulk tank milk samples, 23 in-line milk filter socks and in 50 environmental samples collected from an on-farm dairy establishment were investigated. Growth of inoculated low-level L. monocytogenes contamination was also investigated in two types of raw milk packaging, namely in 1-litre plastic bottles and 3-litre bag-in-boxes, both stored at three different storage temperatures of 6, 8 and 10°C. The occurrence of L. monocytogenes was higher (4.8%) in bottled raw milk stored until the use-by-date of the package compared to fresh bulk tank milk (1.7%). L. monocytogenes counts were ≤13CFU/ml in bottled raw milk and ≤1CFU/ml in bulk tank milk. L. monocytogenes was not detected in the packaging facility, but occurred very frequently (39%) in the milk filter socks. Subtyping of L. monocytogenes isolates using pulsed-field gel-electrophoresis revealed seven pulsotypes, of which two occurred in multiple samples. Targeted inoculum levels of 1–2CFU/ml yielded L. monocytogenes counts≥100CFU/ml within seven days of storage in 22% of the raw milk packages stored at 6°C, and in all of the raw milk packages stored at 8°C. The frequent occurrence of L. monocytogenes in raw milk and the ability of a low-level L. monocytogenes contamination to grow at refrigeration temperatures highlight the importance of consumer education regarding the appropriate raw milk storage and handling.

Detection of Brucella spp., Campylobacter spp. and Listeria monocytogenes in raw milk and cheese of uninspected production in the metropolitan area of São Paulo

Foodborne diseases are a major public health issue but their overall incidence is underestimated due to insufficient report. The present study aimed to investigate the presence of Brucella spp., Campylobacter spp. and Listeria monocytogenes in raw milk and cheese of uninspected production obtained from cattle bred on the polluted banks of the Tietê River. Generally, milk from these animals is used to prepare fresh cheese, which is then commercialized by the producers themselves or in local markets. We analyzed 81 samples consisting of 38 samples of cheeses, 15 samples of raw milk and 28 samples of water collected from the Tietê River. These samples were evaluated for the presence of the three pathogens using bacteriological methods and the conventional polymerase chain reaction (PCR), with primers specific for each bacterial genus. In the bacteriological examination, all samples were negative for Brucella spp., Campylobacter spp. and Listeria monocytogenes. In the PCR test, Brucella spp. was detected in 5/38 (13.16%) cheese samples. Campylobacter spp. was present in 18/38 (47.37%) cheese samples, 1/15 (6.66%) raw milk samples and in 12/28 (42.86%) water samples. Listeria monocytogenes was not detected by PCR. The detection of Brucella spp. DNA in cheese and Campylobacter spp. DNA in cheese, milk and water may reflect inadequate animal sanitary management and deficiencies in good manufacturing practices. The presence of these pathogens in the food and water may pose a threat to the health of the consumer and increase the incidence of zoonosis.

Listeria monocytogenes in raw milk, milking equipment and dairy workers: Molecular characterization and antimicrobial resistance patterns.

The aim of this study was to evaluate the genetic relatedness and patterns of antimicrobial resistance amongst L. monocytogenes isolated from raw milk, milking equipment, and hand swabs from workers in dairy farms. A total of 300 samples of raw milk, milking equipment, and hand swabs were collected from four dairy farms to examine the presence of Listeria species. Suspected isolates were further identified by VITEK-2 system and Polymerase Chain Reaction (PCR). Antimicrobial susceptibility of the L. monocytogenes isolates was determined, and genotyping analysis was performed by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). Listeria spp. was isolated from 79 (26.3%) of the 300 samples, including 29 (36.7%), 32 (40.5%), and 18 (22.8%) isolates found in raw milk, milking equipment, and hand swabs, respectively. L. monocytogenes was the most common isolated (87.3%) species, while the remaining Listeria isolates were L. innocua (12.7%). Among the 69 L. monocytogenes isolates, 42 (60.8%) showed the mutual presence of hlyA, prfA, inlA, and inlB virulence-associated genes. L. monocytogenes isolates from raw milk, milking equipment, and hand swabs showed high genetic relatedness. The potentially virulent L. monocytogenes isolates were most frequently resistance to tetracycline and clindamycin (81%, each) followed by rifampicin (71.4%), whereas, antimicrobial susceptibility was most frequently observed for ampicillin, levofloxacin, moxifloxacin, linezolid, and tigecycline (100%, each). Furthermore, 88% of L. monocytogenes isolates showed multidrug-resistance. The findings of this study show that the contamination of dairy farms with L. monocytogenes is relatively high, and highlight the emergence of multi-drug resistant L. monocytogenes in dairy farms. However, ampicillin is a good choice for treatment of listeriosis in the study area.

Rapid detection of Listeria monocytogenes in raw milk and soft cheese by a redox potential measurement based method combined with real-time PCR.

The incidence of outbreaks of foodborne listeriosis has indicated the need for a reliable and rapid detection of the microbe in different foodstuffs. A method combining redox potential measurement and real-time polymerase chain reaction (PCR) was developed to detect Listeria monocytogenes in artificially contaminated raw milk and soft cheese. Food samples of 25 g or 25 ml were homogenised in 225 ml of Listeria Enrichment Broth (LEB) with Oxford supplement, and the redox potential measurement technique was applied. For Listeria species the measuring time was maximum 34 h. The absence of L. monocytogenes could reliably be proven by the redox potential measurement method, but Listeria innocua and Bacillus subtilis could not be differentiated from L. monocytogenes on the basis of the redox curves. The presence of L. monocytogenes had to be confirmed by real-time PCR. The combination of these two methods proved to detect < 10 cfu/g of L. monocytogenes in a cost- and time-effective manner. This method can potentially be used as an alternative to the standard nutrient method for the rapid detection of L. monocytogenes in food.

Quantitative detection of Listeria monocytogenes in raw milk and soft cheeses: Culture-independent versus liquid- and solid-based culture-dependent real time PCR approaches

A culture-independent and two culture-dependent real time (rt-) PCR approaches were developed to quantitatively identify Listeria monocytogenes in raw milk and soft cheeses. The optimised rt-PCR revealed 100% inclusivity and exclusivity. DNA- and cell-based standard curves showed a good linearity of response (R2 ≥ 0.987 and R2 ≥ 0.998, respectively) for five orders of magnitude (39×105 – 3 × 100 genome equivalents and 106–101 CFU equivalents, respectively) with about 100% relative accuracy and inter-assay variability ≤0.90%. Up to 1 genome equivalent/and 10 CFU/reaction were quantified in the DNA and cell standard curves, respectively. The rt-PCR was then combined with a liquid- (MPN technique) or a solid- (ALOA and PALCAM) based enumeration. The diagnostic sensitivity of the different approaches was investigated in artificially contaminated raw milk and soft cheeses. The rt-PCR culture-independent approach performed well in raw milk and (with a lower sensitivity) in stracchino cheese-based standard curves. MPN/rt-PCR was the best approach to enumerate low levels of L. monocytogenes in raw milk and stracchino cheese, while the ALOA-based rt-PCR quantification was more effective than the PALCAM-based. These performances were confirmed when 23 real samples of raw milk and soft cheeses by both the rt-PCR approaches were assayed.

Review. Dairy farm management and production practices associated with the presence of Listeria monocytogenes in raw milk and beef

Human listeriosis is a severe foodborne disease caused by Listeria monocytogenes. It is a zoonosis that represents a significant concern for the food industry due to the high mortality rate it causes and the fact that the organism is capable of growing at refrigeration temperatures. Dairy products and ready-to-eat meats are among the foods most often involved in listeriosis outbreaks. Listeria is a common contaminant in the dairy environment, both on the farm and in the processing plant. The main sources of L. monocytogenes in dairy farms are manure and improperly fermented silage. If silage crops are grown on contaminated land, a new cycle of silage contamination and faecal shedding by ruminants that consume such silage may ensue. High loads of L. monocytogenes produced in farm environments may thus represent a primary source for the introduction of this pathogen into the human food supply chain; dairy cows would represent a reservoir for the bacterium, and raw milk and beef would represent the main vehicles for its transmission from dairy farms to humans. Even if contamination originates in post-processing environments, contaminated raw foods may still represent a vehicle for introducing L. monocytogenes into food processing plants.Molecular typing methods have confirmed that common strains of L. monocytogenes are present in dairy farm-associated isolates and isolates from both human epidemic and sporadic cases. Pre-harvest (on-farm) control of listeriosis should be basedmainlyon the control of manure, silage, herd health and milking practices.

SITE-DIRECTED MUTAGENESIS, ISOLATION AND PURIFICATION OF LISTERIA MONOCYTOGENES LISTERIOLYSIN O AND ITS IMMUNOGENICITY

ABSTRACT Listeria monocytogenes is a food-borne pathogenic bacterial species that can cause diseases in humans and animals. A rapid and reliable method for detection of L. monocytogenes is crucial in disease control. Listeriolysin O (LLO), encoded by the hly gene, is the most important virulence factor of L. monocytogenes and a useful identifying factor. We studied the protein structure of LLO with the aim of developing an antibody to be used in an enzyme-linked immunosorbent assay (ELISA) for detection of L. monocytogenes. By in silico analysis, a Leu52Phe modification of LLO sequence was predicted to increase its immunogenicity. The corresponding modification in the hly gene sequence was achieved by site-directed mutagenesis. The wild-type and mutated hly genes were cloned and expressed in Escherichia coli; the expression levels were 10 and 12%, respectively. Highly purified protein preparations of LLO and its Leu52Phe variant (∼37 kDa) were obtained and their purity values were both greater than 90%. Both proteins were used independently to immunize rabbits. The average optical density (OD) value obtained with the anti-Leu52Phe variant antibody (P2) was higher by 0.463 OD units compared with the corresponding value for the anti-LLO antibody (P1) by ELISA, which indicated that the mutation had indeed resulted in enhanced immunogenicity of LLO. PRACTICAL APPLICATIONS In this paper, the enzyme-linked immunosorbent assay method developed by using listeriolysin O variant provided a powerful tool for detection of Listeria monocytogenes in raw milk with specificity, sensitivity and rapidity.

DEVELOPMENT AND EVALUATION OF A LOOP‐MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) METHOD FOR DETECTING LISTERIA MONOCYTOGENES IN RAW MILK

ABSTRACT The performance of a Loop‐mediated isothermal amplification (LAMP) assay for detecting food‐borne pathogen Listeria monocytogenes was presented in this paper. Three pairs of primers were specially designed for recognizing eight distinct sequences of iap gene (P60 extracellular protein, invasion associated protein IAP). Time and temperature conditions for amplification of L. monocytogenes were optimized to be 40 min at 63C. Detection limit level for artificially contaminated raw milk samples by the LAMP assay was 186 cfu/mL corresponding to 8–10 cells per reaction tube, while the detection level by conventional PCR was 1.86 × 105 cfu/mL. Data on natural raw milk samples indicated that the LAMP method was highly specific and sensitive, giving 91.67% concordance with the ISO 10560 reference method for the samples without enrichment and 100% concordance after enrichment.PRACTICAL APPLICATIONThe LAMP method in this paper provided a powerful tool with the specificity, sensitivity and rapidity for detection of Listeria monocytogenes in raw milk.

Occurrence of Listeria monocytogenes in Raw Milk and Dairy Products in Noorabad, Iran

Occurrence of Listeria monocytogenes in Raw Milk and Dairy Products in Noorabad, Iran

First results on the detection of Listeria monocytogenes in raw milk: comparison of two different analytical methods

First results on the detection of Listeria monocytogenes in raw milk: comparison of two different analytical methods