Identification Of Listeria Species Research Articles

Identification of Listeria species and Multilocus Variable-Number Tandem Repeat Analysis (MLVA) Typing of Listeria innocua and Listeria monocytogenes Isolates from Cattle Farms and Beef and Beef-Based Products from Retail Outlets in Mpumalanga and North West Provinces, South Africa.

In this study, Listeria isolates (214) were characterized as follows: L. innocua (77.10%), L. monocytogenes (11.21%), L. welshimeri (5.61%), L. grayi (1.40%), L. seeligeri (0.93%), and L. species (3.73%) that were not identified at the species level, from beef and beef based products from retail and farms in Mpumalanga and North West provinces of South Africa. MLVA was further used to type Listeria innocua isolates (165) and Listeria monocytogenes isolates (24). The L. monocytogenes isolates were also serogrouped using PCR. The MLVA protocol for L. monocytogenes typing included six tandem repeat primer sets, and the MLVA protocol for L. innocua included the use of three tandem repeats primer sets. The L. monocytogenes serogroups were determined as follows: 4b-4d-4e (IVb) (37.50%), 1/2a-3a (IIa) (29.16%), 1/2b-3b (IIb) (12.50%), 1/2c-3c (IIc) (8.33%), and IVb-1 (4.16%). MLVA could cluster isolates belonging to each specie, L. monocytogenes, and L. innocua isolates, into MLVA-related strains. There were 34 and 10 MLVA types obtained from the MLVA typing of L. innocua and L. monocytogenes, respectively. MLVA clustered the L. monocytogenes isolates irrespective of sample category, serogroups, and geographical origin. Similarly, the L. innocua isolates clustered irrespective of meat category and geographical origin. MLVA was able to cluster isolates based on MLVA relatedness. The clustering of isolates from farms and retailers indicates transmission of Listeria spp. MLVA is an affordable, simple, and discriminatory method that can be used routinely to type L. monocytogenes and L. innocua isolates.

Listeria Species Occurrence and Associated Risk Factors and Antibiogram of Listeria Monocytogenes in Milk and Milk Products in Ambo, Holeta, and Bako Towns, Oromia Regional State, Ethiopia.

A cross-sectional study was conducted to estimate the prevalence and associated risk factors of Listeria species and assess the antibiogram of Listeria monocytogenes (L. monocytogenes) isolated from milk and milk products from Holeta, Ambo, and Bako towns, Ethiopia. A total of 482 samples (384 milk, 35 cottage cheeses, 30 bulk tank milk, and 33 curdle milk) were collected using a systematic random sampling method and isolation and identification of Listeria species were done using standard microbiological techniques. An antimicrobial susceptibility test for L. monocytogenes was performed using the Kirby–Bauer disk diffusion technique. Descriptive statistics were used to summarize the prevalence of Listeria, while the Chi-square test and logistic regression were used to determine the association between the prevalence of Listeria and the risk factors and the magnitude of association, respectively. The overall isolation rate of Listeria species from milk and milk products was 7.67% (37/482; 95% confidence interval (CI): 5.46, 10.42). The highest prevalence of Listeria species (15.15%; 95% CI: 5.11–31.90) was detected in bulk tank milk and the lowest prevalence of Listeria species (6.67%; 95% CI: 0.82–22.07) and L. monocytogenes (0.00; 95% CI: 0.00–1.15) was found in curdled milk. The other species isolated were Listeria welshimeri 0.62% (3/482; 95% CI: 0.13–1.81), Listeria seeligeri 1.04% (5/482; 95% CI: 0.33–2.40), Listeria ivanovi 1.24%, (6/482; 95% CI: 0.45–2.68), and Listeria grayi 2.49% (12/482; 95% CI: 5.46–10.42). Univariable logistic regression showed that study town, herd size, farm size, number of lactating cows, and management system were the factors significantly associated with the isolation of Listeria species at farm level, while the intensive management system was the independent predictor at cow level in the multivariable model (adjusted odds ratio = 3.38, P=0.046). L. monocytogenes isolates showed the highest resistance against oxacillin (100%), amoxicillin (90.91%), and vancomycine (81.82%). L. monocytogenes showed a very high multidrug resistance (MDR) [81.82%]. In conclusion, the current study showed the widespread type of Listeria species MDR L. monocytogenes isolates in cow raw milk and milk products from Ambo, Holeta, and Bako towns, Oromia Regional State, Ethiopia.

Accurate classification of Listeria species by MALDI-TOF mass spectrometry incorporating denoising autoencoder and machine learning

Listeria monocytogenes belongs to the category of facultative anaerobic bacteria, and is the pathogen of listeriosis, potentially lethal disease for humans. There are many similarities between L. monocytogenes and other non-pathogenic Listeria species, which causes great difficulties for their correct identification. The level of L. monocytogenes contamination in food remains high according to statistics from the Food and Drug Administration. This situation leads to food recall and destruction, which has caused huge economic losses to the food industry. Therefore, the identification of Listeria species is very important for clinical treatment and food safety. This work aims to explore an efficient classification algorithm which could easily and reliably distinguish Listeria species. We attempted to classify Listeria species by incorporating denoising autoencoder (DAE) and machine learning algorithms in matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). In addition, convolutional neural networks were used to map the high dimensional original mass spectrometry data to low dimensional core features. By analyzing MALDI-TOF MS data via incorporating DAE and support vector machine (SVM), the identification accuracy of Listeria species was 100%. The proposed classification algorithm is fast (range of seconds), easy to handle, and, more importantly, this method also allows for extending the identification scope of bacteria. The DAE model used in our research is an effective tool for the extraction of MALDI-TOF mass spectrometry features. Despite the fact that the MALDI-TOF MS dataset examined in our research had high dimensionality, the DAE + SVM algorithm was still able to exploit the hidden information embedded in the original MALDI-TOF mass spectra. The experimental results in our work demonstrated that MALDI-TOF mass spectrum combined with DAE + SVM could easily and reliably distinguish Listeria species.

Listeria Species Occurrence and Associated Factors and Antibiogram of Listeria monocytogenes in Beef at Abattoirs, Butchers, and Restaurants in Ambo and Holeta in Ethiopia.

PurposeListeriosis is one of the globally distributed foodborne diseases with the highest fatality rate. The objectives of this study were to isolate and identify Listeria species, assess factors for contamination of beef, and antibiogram of Listeria monocytogenes in Ambo and Holeta towns, Central Ethiopia.Materials and MethodsA total of 450 meat samples were collected from abattoirs (n=150), butchers (n=150), and restaurants (n=150) for isolation and identification of Listeria species. Logistic regression analysis was used to assess the association between the occurrence of Listeria species in meat and potential risk factors. The antimicrobial susceptibility test was done using the Kirby Bauer test.ResultsThe overall occurrence of Listeria species in Ambo and Holeta towns was 28.4% (128/450; 95% confidence interval [CI]: 24.3–32.9%). The isolation rate of Listeria monocytogenes was 4.4%, Listeria ivanovii 2.2%, Listeria seeligeri 1.8%, Listeria welshimeri 3.8%, Listeria innocua 6.2%, and Listeria grayi 10.2%. The probability of contamination of meat in butchers and restaurants was higher in Holeta than Ambo [OR=3.4; 95%; p=0.001], in dry than wet season [OR=5.2; p=0.009], and where the hygiene of cutting boards was poor (OR=7.7; p=0.008). Of the 20 Listeria monocytogenes isolates, 80%, 70%, 60%, and 55% were resistant to oxacillin, amikacin, and nalidixic acid, chloramphenicol, and tetracycline, respectively. The Listeria monocytogenes isolates were 95%, 90%, and 85% susceptible to amoxicillin, vancomycin, and clindamycin, respectively. About 95% of Listeria monocytogenes isolates were multidrug-resistant. One isolate (5%) had developed resistance to 10 classes of antimicrobial drugs.ConclusionListeria species are widespread and study towns, season, and hygiene of cutting boards are independent predictors of isolation of Listeria species. Multidrug resistance among Listeria monocytogenes was very high. Therefore, adequate cooking of meat, regular training of beef handlers, prudent use of drugs, and further molecular studies on Listeria species are important.

İnci Kefalinde (Chalcalburnus tarichi, Pallas 1811) Listeria Türlerinin Varlığı ve Yaygınlığı

Lake Van is not a very convenient environment for life of both sea and freshwater fish due to its high alkaline (pH 9.8) and salty (0.21%) water and only pearl mullet (Chalcalburnus tarichi, Pallas 1811) are present in the lake. In this study, the presence and prevalence of Listeria species in samples of the pearl mullet (Chalcalburnus tarichi, Pallas 1811) living as an endemic species in Lake Van and taken a total of 160 times for 12 months were examined. The method recommended by the United States Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) was used for the isolation and identification of Listeria species. Among the pearl mullet samples examined, Listeria spp. were isolated in 20 (12.50%) and of them L. innocua in 10 (6.25%) and L. welshimeri in 8 (5.00%) and L. ivanovii in 2 (1.25%) were identified. In this study, the presence of Listeria species in pearl mullet samples examined according to the seasons was revealed for the first time. Failure to identification of L. monocytogenes in any of the samples that is an important pathogen for humans, was assessed as an indicator that these fish did not constitute a public health risk in terms of listeriosis. As a result, Listeria spp. and other pathogenic microorganisms not to pose a risk to public health and in order to prevent the deterioration of fishes and reduce the economic losses, the transportation, storage and marketing of the caught fishes should be done under hygienic and cold conditions.

Identification of Listeria species by Fourier-transform infrared spectroscopy

Differentiation of the genus Listeria is significant for food industry, but only few reliable methods are available so far. In the present study, 56 strains isolated from 345 samples of cow raw milk were used. The isolated pure cultures were defined by PCR-based method using specific primers of 16S-23S IGS region of DNA. Bacterial strain samples were submitted to spectroscopic measurements by the trans-mission method at a wavelength of 3000–700 cm–1 using Fourier-transform infrared (FTIR) spectro-photometry. Hierarchical cluster analysis (HCA) was performed based on the identification of the 56 isolated strains. The utilisation of HCA in univariate-FTIR spectral analyses as the most progressive chemometric method was supported by the correct identification of 86.9% bacteria of the genus Listeria at the species level. These results explained the ability of univariate-FTIR spectrum analysis for determination of suspected Listeria species.

Lineage and serotype identification of Listeria monocytogenes by matrix-assisted laser desorption ionization-time of flight mass spectrometry

The identification of Listeria species, lineages and serotypes remains a crucial issue not only in epidemic surveys, but also in monitoring of the diversity of bacteria in the food chain. The aim of this study was identification of L. monocytogenes strains at lineage and serotype level using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The performance of MALDI-TOF MS was tested to identify L. monocytogenes into two lineages (I and II) and four serotypes (1/2a, 1/2b, 1/2c and 4b) the most commonly found in humans and food. Total of 227 L. monocytogenes strains from different sources were subjected to the study. Some of strains (112) were used for main spectrum profile (MSP) library creation. Other strains of interest (115) were then correctly identified on the lineage level comparing with the library by MALDI-TOF MS analysis using Biotyper (90%) and ClinPro Tools (100%) software. The serotype identification with 55.7% (Biotyper) and 67.8% (ClinPro Tools) accuracy is rather a proof that under given conditions the method has not big potential to be used for serotyping. However, MALDI-TOF MS has a potential to identify lineages of L. monocytogenes of food and human origin.

Changes in the Matrix Markedly Enhance the Resolution and Accurate Identification of Human Pathogens by MALDI-TOF MS

The mass spectral profiles of three major/emerging Gram positive pathogens belonging to the genera Clostridium, Listeria and Propionibacterium were investigated using four MALDI-TOF (Matrix-Assisted Laser Desorption/ Ionization- Time of Flight) Mass Spectrometers from Waters, Shimadzu, Bruker and Ciphergen Biosystems. These instruments have been extensively used for developing a diagnostic platform for high throughput, low cost, near sample-free preparation for microbial identification. The results demonstrate the marked effect of spectral quality obtained by altering the matrix and the added value of simple preparative extraction procedures. While microbial spectral data are generally collected in the mass range 2 to 20 kDa for diagnostic signatures, most of the significant mass ions are <10 kDa. The change from the traditional use of CMBT (5-chloro-2-mercaptobenzothiazole) to DHB (2-(2, 5-DiHydroxyBenzoic acid) enabled successful identification of C. difficile whereas the substitution of CHCA (alpha-cyano-4-hydroxycinnamic acid) for DHB (2,5-dihydroxybenzoic acid) improved the identification of Listeria species. By contrast, the use of SELDI-TOF-MS (Surface Enhanced Laser Desorption/Ionization- Time of Flight- Mass Spectrometry), which employs affinity pre-capture of specific classes of molecules and analysed using sinapinic acid, showed a fivefold extension of the mass range of analytes, extending the potential for biomarker discovery and higher resolution strain typing of P. acnes.

A Study on Detection of Pathogenic Listeria monocytogenes in Ovine’s of Kashmir Region Having Abortion or History of Abortion

The present investigation delineates the role of listeric infection in aborted ewes or those with history of abortion from organized farms of Kashmir region. A total of 141 clinical samples was analyzed for the isolation and identification of Listeria species. On analysis four isolates were identified as Listeria monocytogenes, while 24 were non-pathogenic Listeria species. Of the four L. monocytogenes isolates, two were isolated from brain tissue of aborted fetus (BrS10 and BrG36) while one each was isolated from vaginal swab (Vd13) and rectal swab (RS11) of the ewe. An overall isolation rate of 2.83 % was observed for L. monocytogenes and 17.02 % for non-pathogenic Listeria species. Further to reveal the pathogenic potential, the recovered L. monocytogenes isolates were subjected to the battery of in vitro pathogenicity test such as hemolytic activity on Sheep Blood Agar, Phosphatidyl Inositol-Phospholipase C activity on Agar Listeria according to Ottaviani and Agosti medium, multiplex PCR targeting virulence markers genes viz., prfA, plcA, actA, hly, inlC and in vivo chick embryo inoculation test. All the L. monocytogenes isolates recovered in the present study were potentially pathogenic and on comparison, a good correlation was observed among in vitro and in vivo pathogenicity test including multiplex PCR targeting virulence associated genes.

Rapid Identification and Typing of Listeria Species by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Listeria monocytogenes is a food-borne pathogen that is the causative agent of human listeriosis, an opportunistic infection that primarily infects pregnant women and immunologically compromised individuals. Rapid, accurate discrimination between Listeria strains is essential for appropriate therapeutic management and timely intervention for infection control. A rapid method involving matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) that shows promise for identification of Listeria species and typing and even allows for differentiation at the level of clonal lineages among pathogenic strains of L. monocytogenes is presented. A total of 146 strains of different Listeria species and serotypes as well as clinical isolates were analyzed. The method was compared with the pulsed-field gel electrophoresis analysis of 48 Listeria strains comprising L. monocytogenes strains isolated from food-borne epidemics and sporadic cases, isolates representing different serotypes, and a number of Listeria strains whose genomes have been completely sequenced. Following a short inactivation/extraction procedure, cell material from a bacterial colony was deposited on a sample target, dried, overlaid with a matrix necessary for the MALDI process, and analyzed by MALDI-TOF MS. This technique examines the chemistry of major proteins, yielding profile spectra consisting of a series of peaks, a characteristic "fingerprint" mainly derived from ribosomal proteins. Specimens can be prepared in a few minutes from plate or liquid cultures, and a spectrum can be obtained within 1 minute. Mass spectra derived from Listeria isolates showed characteristic peaks, conserved at both the species and lineage levels. MALDI-TOF MS fingerprinting may have potential for Listeria identification and subtyping and may improve infection control measures.

&lt;i&gt;Listeria monocytogenes&lt;/i&gt; and other Listeria species in retail meat and milk products in Addis Ababa, Ethiopia

Abstract Background: Listeriosis is one of the important emerging bacterial zoonotic infections worldwide. Among the different species of the genus Listeria, Listeria monocytogens is known to cause listeriosis in humans and animals. Information on the occurrence and distribution of Listeria monocytogenes and other Listeria species is very limited both in the veterinary and public health sectors in Ethiopia. Objectives: The present study was undertaken to determine the occurrence and distribution of Listeria monocytogenes and other Listeria species in retail meat and milk products in Addis Ababa, Ethiopia. Methods: A total of 316 food samples were collected using a cross-sectional study design from September 2003 to April 2004. The techniques recommended by the International standards Organization (ISO 11290-1, 1996) and the French Association for Standardization (AFNOR, 1993) were employed for the isolation and identification of Listeria species. Serotyping of Listeria monocytogenes was carried out at the French Authority for Food Safety (AFSSA), Ploufragan, France. Results: Out of the total of 316 samples examined, 103 (32.6%) were found to be positive for Listeria. Listeria species were isolated in 69.8% (37/53), 47.5% (29/61), 43.5% (20/46), 18.6%, (8/43), 15.4% (8.52) and 1.6% (1/61) of the pork, minced beef, ice cream, fish, chicken and cottage cheese samples respectively. Listeria monocytogenes was detected in 5.1% of the samples analysed. It was isolated mainly from ice cream (19.6%) and pork samples (7.5%) followed by minced beef (1.6%), fish (2.3%) and chicken samples (1.9%). The serotypes of Listeria monocytogenes identified belonged to 1/2b, 4b and 4e. In addition to Listeria monocytogenes, other Listeria species identified were Listeria (L). innocua (65%), L. seeligeri (8.7%), L. welshimeri (6.8, L.murrayi (L. ivanovii and L. grayi (each 0.9%). Conclusion: This study demonstrated the widespread occurrence and distribution of L. monocytogenes and other Listeria species in retail meat and milk products in Addis Ababa, Ethiopia. [Ethiop.J.Health Dev. 2004;18(3):208212]

Listeria: growth, phenotypic differentiation and molecular microbiology

The identification of Listeria species is based on a limited number of biochemical markers, among which absence or presence of hemolysis and arylamidase are used to differentiate between L. monocytogenes and L. innocua. The CAMP (Christie, Atkins, Munch-Petersen) test must be interpreted with caution. Chromogenic media are based on both the specific chromogenic detection of phosphatidylinositol phospholipase C and the xylose fermentation and give specific and direct identification of L. monocytogenes and L. ivanovii. Isolates of L. monocytogenes with atypical properties require tools of molecular biology for final identification. Serotyping, although not allowing speciation, serves a useful purpose for confirming the genus diagnosis Listeria. Polymerase chain reaction is particularly useful when prior administration of antimicrobial agents compromises culture. For clinical specimens the importance of trying to isolate the pathogen as a prerequisite for an epidemiological work-up and finally for prevention of further cases cannot be overstressed.

The development of an efficient and rapid enzyme linked fluorescent assay method for the detection of Listeria spp. from foods

Conventional isolation methods, including the Health Products and Food Branch (HPFB), Health Canada method used for the isolation and identification of Listeria species and Listeria monocytogenes from foods and environmental samples, can take a week or more to complete and are usually labor-intensive. This has led to the development of various rapid methods which attempt to generate results comparable to standard methods but in a reduced time-frame with less hands-on operation. Our previous work with rapid detection systems indicated that the recommended enrichment protocols failed to grow the Listeria to detectable levels in a reliable and consistent manner. In the present study, a novel enrichment protocol is described and consists of samples being pre-enriched in Palcam broth (incubated at 35 °C for 26 h), enriched in UVM 2 (30 °C for 26 h) and then plated and analysed by a rapid detection kit, with results being generated after 52 h of incubation. In total, 200 naturally contaminated samples were analysed by both the HPFB standard method and the Palcam method. The results showed that the Palcam method is comparable to the HPFB method. Further analysis involved a rapid detection system, which applies ELISA techniques and automation in an enzyme-linked fluorescent assay (ELFA) system. This system, referred to as the Vitek Immuno Diagnostic Assay System or VIDAS, can identify Listeria to the genus or species ( L. monocytogenes) level. In this comparison, an additional 324 naturally contaminated samples were analysed by both the Palcam and ELFA methods. The sensitivity and specificity of the ELFA method were 98.1% and 97.0%, respectively, while the efficiency was 97.5%. False-negative and false-positive rates were 1.9% and 3.0%, respectively. These results show that the ELFA method (when using the Palcam method for pre- and secondary enrichment) was efficient and gave reliable results after 52 h of incubation, and met Health Canada's criteria for approval as a rapid method.

Identification of Listeria species by a semi-nested polymerase chain reaction

Three primers derived from the iap gene were used to distinguish Listeria monocytogenes from L. innocus and other Listeria species. L. monocytogenes and L. innocua yielded a PCR product of 600 and 300 bp, respectively, whereas a typical pattern of three amplimers was observed with L. ivanovii, L. seeligeri and L. welshimeri.

Physiological Characterization and Identification of Listeria Species

A total of 136 strains of the genus Listeria were physiologically characterized using 329 miniaturized tests. Overall similarities of all strains were determined by numerical taxonomic techniques using the UPGMA algorithm and the SSM and SJ coefficients. A total of 10 clusters (2 or more strains) and 1 single-member cluster were defined at the 95.1 to 95.8% similarity levels (SSM). The same clusters were found at the 76.8% to 77.8% similarity levels (SJ). Strains of L. seeligeri, L. welshimeri, L. ivanovii, L. murrayi and L. grayi were found in separate clusters and formed homogeneous taxospecies. In contrast, strains of L. monocytogenes and L. innocua formed five heterogeneous clusters and one single-member cluster. For differentiation of the phena the following tests are useful: utilization of D-xylose, D-xylitol, D-ribose, D-galactose, alpha-methyl-D-mannoside, methyl-alpha-D-glucopyranoside, inulin and L-rhamnose, acid production from D-glucuronate, alpha-D-galacturonate, alpha-methyl-D-mannoside, methyl-alpha-D-glucopyranoside, D-xylose, L-lyxose, D-sucrose, alpha-D-melizitose, D-tagatose, and D-ribose and hydrolysis of pNP-alpha-D-glucopyranoside, pNP-alpha-D-mannopyranoside and 2-deoxythymidine-5'-pNP-phosphate. Independent of the assignment of some strains of L. monocytogenes and L. innocua to the same cluster, members of these species can be separated by hydrolysis of D-alanine-p-nitroanilide.