Application of MALDI-TOF Mass Spectrometry for the Assessment of Prevalence of Listeria monocytogenes in Raw Milk, Dairy Products and Freshwater Fishes.pdf
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.
International Journal of Engineering and Science Invention (IJESI) is an international journal intended for professionals and researchers in all fields of computer science and electronics. IJESI publishes research articles and reviews within the whole field Engineering Science and Technology, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
Bovine Mastitis due to Coliform Bacteria, and Susceptibility to Antibiotics, ...Premier Publishers
This study was aimed at determining the prevalence of coliform bacteria in bovine milk in Plateau State of Nigeria and their antibiotic susceptibility patterns. A total of 640 milk samples were collected aseptically and 160 questionnaires from where data such as breed, age, parity, lactation stage, floor type, and husbandry system were analyzed. Cows without clinical mastitis were subjected to California Mastitis Test to determine the presence of subclinical mastitis. Bacteriological assays and antibiotic susceptibility tests were conducted according to standard guidelines. Subclinical mastitis with a prevalence of 63.8% was more prevalent in cows than clinical mastitis. Overall, the Friesian breed had the highest mastitis prevalence of 85.7% compared to White Fulani (which is indigenous in Nigeria). Cows aged within 2-4 years old had the least mastitis prevalence of 55.2%. Coliforms isolated from milk samples included E coli, K. pneumoniae, K. oxytoca, C. freundii, E. aerogenes, E. cloacae, and S. marcescens, with E coli having the highest prevalence of 44.8%. The most resistant antimicrobial agent was Streptomycin with 79% prevalence. The principle of One Health approach which targets the environment, animals and humans should be considered important. Good hygienic measures should be intensified among pastoralists.
Characterization and Validation of Point Mutation in Breast Cancer 1 (BRCA1) ...paperpublications3
Abstract: Bovine mastitis is a very common and multietiological disease of dairy cattle which leads to huge economic losses to the dairy industry globally. In this study, the bovine breast cancer 1, early onset gene (BRCA1) was taken as a candidate gene for mastitis resistance. Breast cancer 1 (BRCA1) is one of the genes which predispose organism to early-onset breast cancer, and is involved in DNA damage repair, cell cycle regulation, transcriptional regulation, and other important pathways to suppress tumor and maintain genome stability. A total of 120 Sahiwal cattle were selected to characterize the targeted region of intron 6 of BRCA1 gene for polymorphism screening and their association with mastitis. A 321bp PCR fragment of BRCA1 gene encompassing the targeted region of intron 6 was amplified and digested with Hha I to screen for the reported SNPs having significant association with SCS. Genotype analysis using PCR-RFLP revealed a monomorphic banding pattern. Sequencing was also carried out to explore the in silico screened SNPs which are deposited in dbSNP. The result indicates highly conserved sequence in Sahiwal cattle. Therefore, reported as well as in silico SNPs cannot be considered as a universal marker for mastitis in all the breeds. Since, present study has formulated the results based on a relatively small sample; further studies are required to screen these SNPs in large samples.
The present investigation was done to study the effects of Lactococcus lactis (L. lactis) subsp. lactis on the shelf life of the vacuum-packaged Oncorhynchus mykiss. Fish fillets were prepared and divided into 5 different treatment groups including control (distilled water), 2% and 4% supernatant, and 106 CFU/g L. lactis subspecies lactis. The pH, Thiobarbituric Acid Reactive Substances (TBARS), Total volatile Nitrogen (TVN), and Peroxide Value (PV) of the fillets were determined on days 0, 5, 10, and 15 while maintained at 4˚C. Protein expression and destruction were analyzed using the SDS-PAGE. The organoleptic assessment was done using five expert sensory panelists. Contents of TBARS, TVN, pH, and PV were increased throughout the storage period (P <0.05). An increase in the concentration of supernatant caused a significant decrease in the content of TBARS, TVN, pH, and PV (P <0.05). The highest and lowest contents of TBARS, TVN, pH and PV on 15th day were belonged to the control (3.367±0.04 mg MDA/kg) and pure bacteria (0.70±0.02 mg MDA/kg), control (87.20±6.40 mg/100g) and 4% supernatant (40.79±0.61 mg/100g), pure bacteria (6.23±0.04) and 4% supernatant (5.44±0.07) and control (12.22±0.01 meq/kg) and 4% supernatant (3.08±0.06 meq/kg) groups, respectively. Protein destruction was lower in the fillet samples treated with pure bacteria and 4% supernatant. The highest scores of the odor, flavor, texture, and color were obtained for fillets treated with 4% supernatant, pure bacteria, pure bacteria, and 4% supernatant and pure bacteria, respectively. The results revealed that treating O. mykiss fillets with 4% supernatant and 106 CFU/g of pure L. lactis subsp. lactis can extend the shelf life of O. mykiss fillets.
International Journal of Engineering and Science Invention (IJESI) is an international journal intended for professionals and researchers in all fields of computer science and electronics. IJESI publishes research articles and reviews within the whole field Engineering Science and Technology, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
Bovine Mastitis due to Coliform Bacteria, and Susceptibility to Antibiotics, ...Premier Publishers
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Characterization and Validation of Point Mutation in Breast Cancer 1 (BRCA1) ...paperpublications3
Abstract: Bovine mastitis is a very common and multietiological disease of dairy cattle which leads to huge economic losses to the dairy industry globally. In this study, the bovine breast cancer 1, early onset gene (BRCA1) was taken as a candidate gene for mastitis resistance. Breast cancer 1 (BRCA1) is one of the genes which predispose organism to early-onset breast cancer, and is involved in DNA damage repair, cell cycle regulation, transcriptional regulation, and other important pathways to suppress tumor and maintain genome stability. A total of 120 Sahiwal cattle were selected to characterize the targeted region of intron 6 of BRCA1 gene for polymorphism screening and their association with mastitis. A 321bp PCR fragment of BRCA1 gene encompassing the targeted region of intron 6 was amplified and digested with Hha I to screen for the reported SNPs having significant association with SCS. Genotype analysis using PCR-RFLP revealed a monomorphic banding pattern. Sequencing was also carried out to explore the in silico screened SNPs which are deposited in dbSNP. The result indicates highly conserved sequence in Sahiwal cattle. Therefore, reported as well as in silico SNPs cannot be considered as a universal marker for mastitis in all the breeds. Since, present study has formulated the results based on a relatively small sample; further studies are required to screen these SNPs in large samples.
The present investigation was done to study the effects of Lactococcus lactis (L. lactis) subsp. lactis on the shelf life of the vacuum-packaged Oncorhynchus mykiss. Fish fillets were prepared and divided into 5 different treatment groups including control (distilled water), 2% and 4% supernatant, and 106 CFU/g L. lactis subspecies lactis. The pH, Thiobarbituric Acid Reactive Substances (TBARS), Total volatile Nitrogen (TVN), and Peroxide Value (PV) of the fillets were determined on days 0, 5, 10, and 15 while maintained at 4˚C. Protein expression and destruction were analyzed using the SDS-PAGE. The organoleptic assessment was done using five expert sensory panelists. Contents of TBARS, TVN, pH, and PV were increased throughout the storage period (P <0.05). An increase in the concentration of supernatant caused a significant decrease in the content of TBARS, TVN, pH, and PV (P <0.05). The highest and lowest contents of TBARS, TVN, pH and PV on 15th day were belonged to the control (3.367±0.04 mg MDA/kg) and pure bacteria (0.70±0.02 mg MDA/kg), control (87.20±6.40 mg/100g) and 4% supernatant (40.79±0.61 mg/100g), pure bacteria (6.23±0.04) and 4% supernatant (5.44±0.07) and control (12.22±0.01 meq/kg) and 4% supernatant (3.08±0.06 meq/kg) groups, respectively. Protein destruction was lower in the fillet samples treated with pure bacteria and 4% supernatant. The highest scores of the odor, flavor, texture, and color were obtained for fillets treated with 4% supernatant, pure bacteria, pure bacteria, and 4% supernatant and pure bacteria, respectively. The results revealed that treating O. mykiss fillets with 4% supernatant and 106 CFU/g of pure L. lactis subsp. lactis can extend the shelf life of O. mykiss fillets.
Effects of dietary thyme essential oil on hemato-immunological indices, intes...Inacio Mateus Assane
Thyme (Thymus vulgaris) essential oil (TVEO) is a herbal medicine with one of the highest levels of antimicrobial activity. Although TVEO has been broadly used in poultry and swine production due to its immunostimulatory and growth-promoting characteristics, the effects of TVEO on fish are poorly characterized. In this study, Nile tilapia (Oreochromis niloticus L.) were fed 0, 0.1, 0.5, and 1% TVEO for 15 days. Subsequently, blood parameters, intestinal morphology, and the population of Bacillus bacteria in the intestine were evaluated. The numbers of lymphocytes (p < 0.05) and leukocytes (p < 0.05) significantly increased in the blood of the fish fed the highest dose of TVEO. Based on the normal behavior of the fish and the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, which were not altered (p > 0.05), this study concluded that the diets were safe and showed no negative or toxic effects. Even at doses as high as 1%, TVEO did not alter the population of beneficial Bacillus bacteria in the gut. In conclusion, supplementation with TVEO stimulated the cellular components of the non-specific immune response of Nile tilapia without causing deleterious effects or altering the population of important intestinal bacteria.
Abstract: Johne’s disease (Paratuberculosis) is a chronic enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). MAP is a subspecies of M. avium, effects wide range of animals including domestic cattle, sheep, goats, buffaloes, camelids and wild ruminants resulting in progressive and chronic enteritis known as Johne’s disease. Clinically indected animals show watery diarrhea, emaciation and eventually death due to lack of effective treatment. Clinically as well as subclinically infected animals shed bacteria in feces and milk. Fecal-oral rout is the main rout of transmission. It is economically very important disease in livestock due to decrease in milk yield, working efficiency and culling of infected animals. Enlargement of mesenteric lymph node and transverse corrugation in intestine is characterstic finding in post-mortem. Strategies to control this disease include improved management practices, testing and culling and vaccination.Keywords: Johne’s disease, diarrhoea, corrugation.
Title: Johne’s Disease: A Review Article
Author: Vikash Sharma, Lalit, Sakshi Tiwari
International Journal of Recent Research in Life Sciences (IJRRLS)
ISSN 2349-7823
Paper Publications
Abstract— Coccidiosis swine causes high economic loss, and its prophylaxis is usually performed by the use of chemical drugs. However, these chemical drugs are not allowed in agroecological, organic or biological dynamic systems of production. Additionally, there are concerns about pharmacological resistance and contamination by the presence of chemical residues in the environment and at the food of animal origin. The objective of this study was to evaluate the weight gain and the prevalence of coccidia in piglets submitted to the following treatments: alcoholic extract of propolis 30% (AEP), chemical treatment toltrazuril (CTT), negative control with grain alcohol (NCA) and negative control without treatment (NCT). By means of the individual weights and the number of coccidia at the faeces, it was monitored 216 piglets from commercial farms. Under the conditions of this study none therapeutic intervention provided benefits for weight gain and prevalence of coccidia. Thus, by making it possible the minimization of chemical drug use, these results allow us to suggest the laboratory periodic monitoring as a prophylactic control method for swine coccidiosis.
Prevalence of Salmonella and Staphylococcus aureus from meat in Cambodian mar...ILRI
Poster by Rortana Chea, Delia Grace, Hung Nguyen-Viet, Sothyra Tum, Sinh Dang-Xuan, Ren Theary, Siek Sophary, Sok Koam, So Pheany, Hout Sotheany, Theng Heng, Seng Sarim and Johanna Lindahl presented at a regional symposium on research into smallholder pig production, health and pork safety, Hanoi, Vietnam, 27–29 March 2019.
Foodborne disease hazards and burden in Ethiopia: A systematic literature rev...ILRI
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Microbial Quality of Raw and Pasteurized Milk Samples Collected From Differen...iosrjce
Milk is the fluid normally secreted by female mammals for the nourishment of their young ones. It is
a compulsory part of daily diet for the expectant mothers as well as growing children and also serves as good
medium for microbial growth and contamination 240 raw milk samples and 72 pasteurized milk samples from
different places of Madurai District for a period of six months were analysed for microbial quality. Among the
raw milk samples only 19.1% of samples were good quality and 28.3% are very poor quality. In the pasteurized
milk samples 81.9% of samples were good for human consumption. The bacteria isolated from milk samples
includes Lactobacilli, Staphylococcus aureus, Escheritia coli, Bacillus subtilis , Salmonella typhi, and feacal
coliforms
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Deep Behavioral Phenotyping in Systems Neuroscience for Functional Atlasing a...Ana Luísa Pinho
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Application of MALDI-TOF Mass Spectrometry for the Assessment of Prevalence of Listeria monocytogenes in Raw Milk, Dairy Products and Freshwater Fishes.pdf
1. Volume Issue 1
DR-2095
[1-5]
RESEARCH ARTICLE Asian Journal of Dairy and Food Research
Application of MALDI-TOF Mass Spectrometry for the
Assessment of Prevalence of Listeria monocytogenes in Raw
Milk, Dairy Products and Freshwater Fishes
Rahul Suryawanshi1
, Ashok Bhosale2
, Gopal Bharkad3
, Onkar Shinde1
, 10.18805/ajdfr.DR-2095
Aishwarya Jogdand1
, Niraj Hatwar1
, Hrishikesh Kamat1
ABSTRACT
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.
Key words: CAMP, Listeria monocytogenes, MALDI-TOF MS, PI-PLC assay.
INTRODUCTION
Listeriosis is an important foodborne disease in humans
because it is associated with the ingestion of contaminated
food and water with pathogenic Listeria spp. (Low and
Donachie, 1997). It leads to severe invasive illness in
humans; the main signs are septicemia, abortion, stillbirth,
perinatal infections, meningitis, gastroenteritis and
meningoencephalitis, particularly in aged and immuno
compromised individuals (Posfay-Barbe and Wald 2004).
Pregnant women are more vulnerable to infection than non-
pregnant women, which can result in abortion, stillbirth, or
perinatal illnesses (Jackson et al., 2010). The incidence of
listeriosis caused by this bacterium has skyrocketed in
recent years. Most human listeriosis occurs when
contamination levels of 102-106 CFUs/ml or/g of Listeria
are present in food (Dawson et al., 2006). Listeria
monocytogenes has been isolated from various foodstuffs,
including milk (Barbuddhe et al., 2002). Several incidences
of foodborne listeriosis have been reported caused by
consuming contaminated meat (Lunden et al., 2003;
Bhandare et al., 2007). Matrix-assisted laser desorption/
ionization-time of flight (MALDI-TOF) mass spectrometry
(MS) is a single identification and source-tracking tool
for L. monocytogenes (Jadhav et al., 2015). 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 and the
fingerprinting has the potential for bacteria identification and
subtyping (Barbuddhe et al., 2008). It has become the
method of choice for bacterial species identification in clinical
diagnostics due to its little hands-on and turnaround time,
low costs and high accuracy (Angeletti and Ciccozzi, 2019;
Rodrıìguez-Saìnchez et al., 2019). The present study was
1
Department of Veterinary Public Health, College of Veterinary and
Animal Sciences, Udgir-413 517, Latur, Maharashtra, India.
2
Department of Veterinary Microbiology, College of Veterinary and
Animal Sciences, Udgir-413 517, Latur, Maharashtra, India.
3
Department of Veterinary Parasitology, College of Veterinary and
Animal Sciences, Udgir-413 517, Latur, Maharashtra, India.
Corresponding Author: Rahul Suryawanshi, Department of
Veterinary Public Health, College of Veterinary and Animal Sciences,
Udgir-413 517, Latur, Maharashtra, India.
Email: rahulvph@gmail.com
How to cite this article: Suryawanshi, R., Bhosale, A., Bharkad,
G., Shinde, O., Jogdand, A., Hatwar, N. and Kamat, H. (2023).
Application of MALDI-TOF Mass Spectrometry for the Assessment
of Prevalence of Listeria monocytogenes in Raw Milk, Dairy
Products and Freshwater Fishes. Asian Journal of Dairy and Food
Research. doi:10.18805/ajdfr.DR-2095.
Submitted: 18-03-2023 Accepted: 17-05-2023 Online: 02-06-2023
2. Asian Journal of Dairy and Food Research
2
Application of MALDI-TOF Mass Spectrometry for the Assessment of Prevalence of Listeria monocytogenes in Raw Milk, Dairy...
planned to explore the prevalence of pathogenic Listeria
species in animal origin foods like raw milk and freshwater
fishes using conventional as well as advanced molecular
tool like MALDI-TOF MS for the confirmation of reliable and
rapid identification of organism.
MATERIALS AND METHODS
Sample collection
In current research work, a total of 360 samples comprising
raw milk (130), milk products (125) and freshwater fishes
(105) were collected from Udgir city of Maharashtra and
screened for microbiological evaluation during the period
of 2017-18 at College of Veterinary and Animal Sciences,
Udgir. Milk products and fish samples were collected in a
sterile zip lock bags while, raw milk samples were collected
in sterile milk sampling bottles (35 ml, International Scientific
Supplies Ltd., UK).
Bacterial strains
The standard strains of L. monocytogenes (ATCC 19115),
Staphylococcus aureus (ATCC 12600) and Rhodococcus
equi (ATCC 6939) were used in the present study which
were obtained from Himedia, Mumbai.
Isolation and phenotypic characterization of Listeria
species
The samples were collected aseptically and processed
immediately after collection, for the isolation of Listeria
species as per the protocol suggested by the USDA method
as described by Curtis and Lee (1995) with suitable
modifications, which includes two-step enrichment with the
University of Vermont (UVM-I and II) and subsequent
streaking onto polymyxin-Acriflavin-Lithium chloride
Ceftazidime Aesculin-Mannitol (PALCAM) medium as a
selective agar. Phenotypically, isolates were characterized
by employing battery of biochemical and sugar fermentation
tests. The results were validated along with standard strain
of L. monocytogenes (ATCC 19115). Biochemical testing
comprised catalase, oxidase Methyl Red-Voges Proskauer
(MR-VP) and nitrate reduction tests, while sugar
fermentation tests were carried out with Alpha-Methyl-D-
Mannoside, Rhamnose and Glucose (Dextrose).
In vitro pathogenicity testing
In order to assess the pathogenic potential of recovered
isolates of L. monocytogenes phenotypically, the isolates
were subjected to in vitro pathogenicity tests like hemolysis
on 7% sheep blood agar (SBA) (Courtieu, 1991), Christie,
Atkins, Munch-Petersen (CAMP) Test (Christie et al., 1944)
and Phosphatidylinositol-specific Phospholipase-C (PI-PLC)
assay (Notermans et al., 1991).
Confirmation of isolates by MALDI-TOF-MS
The ionization method known as matrix-assisted laser
desorption ionisation (MALDI) produces ions from big
molecules with the least amount of fragmentation.
(Hillenkamp et al., 1991). The L. monocytogenes isolates
recovered in this study were also subjected to advanced
molecular technique like MALDI-TOF-MS for species level
confirmation. In this investigation, the samples were
prepared by full protein extraction and processed as per
the directions provided by Bruker Daltonics. The mass
spectra were acquired and analyzed using MALDI Biotyper
automation control and the Bruker Biotyper 2.0 software and
library (version 2.0, 3,740 entries; Bruker Daltonics). Using
the manufacturer’s suggested bacterial test standard and
following the manufacturer’s instructions, calibration was
completed. Identification score criteria were performed as
recommended by Bruker Daltonics, which evaluated as per
the protocol described by Shell et al., (2017). A score of
2.000 indicated species-level identification, a score of 1.700-
1.999 indicated identification to the genus level and a score
of 1.700 was interpreted as no identification.
RESULTS AND DISCUSSION
Prevalence of Listeria monocytogenes in animal origin
foods
In this study, on microbiological analysis of 360 food samples
comprising raw milk, milk products and freshwater fishes,
three presumptive Listeria isolates were recovered and
identified as Listeria monocytogenes (all from raw milk)
giving an overall occurrence of Listeria monocytogenes to
the tune of 0.83%. The other samples comprising milk
products and freshwater fishes showed negativity for the
presence of any of the Listeria species. The low prevalence
of Listeria spp. in milk detected in the present research is in
agreement with the results stated by Kalorey et al. (2008)
with a 0.1% (2/2060) prevalence and with Aurora et al. (2006)
who noted 1.69% prevalence of Listeria species in milk
samples. The results also coincide with Sharma et al. (2017),
Khan et al. (2013), Shakuntala et al., (2019) and Shantha
and Gopal (2014), who reported 0.8%, 1.7%, 0.76% and
1.69% prevalence of listerial organisms in milk respectively.
However, certain studies have reported quite a higher
recovery of Listeria spp. in milk which includes Mary and
Shrinithivihahshini (2017) with 52.7% (219/415) and
Gebretsadik et al. (2011) with 22% (22/100) prevalence.
Listeria spp. typically affects raw milk bycontamination caused
by unsanitary conditions in the environment, gastrointestinal
tract and teat skin of animals. Besides, other factors like lack
of hygiene, environmental contamination and poor milking
practices also contribute to the Listeria contamination.
Biochemical characterization and in vitro pathogenicity
testing of Listeria isolates
On analysis, three presumptive isolates showing the typical
greyish green, glistening, iridescent and pointed colonies
of about 0.5 mm diameter surrounded by a diffuse black
zone of aesculin hydrolysis (Curtis and Lee 1995) also
revealed characteristics of Gram-positive coccobacilli
morphology detected under the microscope on Gram
staining, tumbling motility in hanging drop technique as
described by Islam et al., (2016) and positivity towards
3. Volume Issue 3
Application of MALDI-TOF Mass Spectrometry for the Assessment of Prevalence of Listeria monocytogenes in Raw Milk, Dairy...
catalase, MR-VP and nitrate reduction test, while negativity
to oxidase test. On sugar fermentation tests, isolates
fermented only the Alpha-Methyl-D-Mannoside, Rhamnose,
Glucose (Dextrose) sugars, suggestive of species
confirmation of all three isolates as Listeria monocytogenes
(OIE Terrestrial Manual 2021; Nayak et al., 2015). All three
L. monocytogenes isolates were further tested for virulence
character by in-vitro pathogenicity testing such as haemolysis
on 7% sheep blood agar, CAMP and PI-PLC assay. All isolates
showed characteristic beta (β) haemolysis on 7% sheep blood
agar, CAMP positive reaction against S. aureus and typical
blue-green colonies with a clearly defined opaque halo on
ALOA (Agar Listeria Ottavani and Agosti) medium in PI-PLC
assay, revealing their pathogenic nature.
Confirmation of isolates by MALDI-TOF-MS
In present investigation, three Listeria monocytogenes
isolates recovered from milk samples were characterized
using conventionally with battery of biochemical tests and
sugar fermentation tests. These isolates were further
subjected for confirmation by MALDI-TOF MS. On the basis
of the score obtained and species identified by MALDI-TOF
MS, all three isolates were designated as Listeria
monocytogenes as shown in Table 1 and analyte ID 34, 35
and 36 in supplementary materials. The results obtained in
molecular charecterization by MALDI-TOF MS were in
complete agreement with the results of phenotypic
biochemical characterization. This concurrence resembles
with the study reported by Thouvenot et al., (2018), wherein
researchers carried out a validation study of similar Bruker
Daltonics system of MALDI-TOF MS Biotyper with
phenotypic based identification of Listeria species. The
authors further also confirmed its accuracy using average
nucleotide identification identity BLAST (ANIb) of whole
genome sequences of organisms. There are other several
studies reported depicting use of MALDI-TOF MS for the
identification of Listeria (Ojima-Kato et al., 2016; Jadhav et al.,
2015; Hsueh et al., 2014; Rychert et al., 2013; Barbuddhe
et al., 2008). However, The MALDI-TOF MS system from
Bruker Daltonics (Bremen, Germany) has been successfully
employed, albeit only on a small number of phenotypically
characterized strains and to create mass spectral fingerprints
of some reference strains (Barbuddhe et al., 2008; Hsueh
et al., 2014). It depends on the MS database, which captures
even single amino acid substitutions, to provide strain- or
serotype-level microbial classification at a better resolution
than that of conventional fingerprinting analysis, moreover
we frequently observe small variations in MS peaks made
up of the same proteins in closely related bacteria (Tamura
et al., 2013; Ojima-Kato et al., 2015). The target bacteria’s
culture and/or growing conditions may still have a significant
impact on fingerprinting analysis, which suggests that
genetic sequence analysis be done to support the
prototyping data for accurate identification (Wieme et al.,
2014). The present results of MALDI-TOF MS for bacterial
identification were accurate, with good repeatability and less
time consumption (Seng et al., 2009; Valentine et al., 2005;
van Baar, 2000). MALDI-TOF MS provided no identification
in mixed cultures but reported identification in single major
bacterial cultures. (Croxatto et al., 2012).
CONCLUSION
To summarize, in current research, the prevalence of
pathogenic Listeria species in animal origin foods like raw
milk, milk products and freshwater fishes was noted to the
tune of 0.83%. Besides, all the isolates examined for
confirmation by MALDI-TOF MS, lead to the resemblance
of results obtained by biochemical characterization
designating all three isolates as Listeria monocytogenes
suggesting the excellent utility of techniques like MALDI-
TOF MS for rapid detection of significant bacterial
pathogens. This study illustrates the entire dependability of
MALDI-TOF mass spectrometry as a quick method for
identifying Listeria species that are pathogenic, with 100%
accuracy. However, the challenges like incomplete
databases, close relatedness of species of interest and
quality of spectra needed to be addressed.
ACKNOWLEDGEMENT
The authors are thankful to the Associate Dean, College of
Veterinary and Animal Sciences, Udgir for his help and
support.
Conflict of interest
Authors declare that there is no conflict of interest.
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Angeletti, S. and Ciccozzi, M. (2019). Matrix-assisted laser desorption
ionization time-of-flight mass spectrometry in clinical
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j.meegid.2019.104063.
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pathogenic Listeria monocytogenes in raw milk and
ready-to-eat milk products in Agra city, India. Indian
Journal of Comparative Microbiology, Immunology and
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Table 1: MALDI-TOF MS analysis of Listeria isolates.
Source MALDI-TOF MS Result Strain Score value
Raw milk L. monocytogenes L. monocytogenes CCUG 59664 2.246
Raw milk L. monocytogenes L. Monocytogenes CCUG 53269 2.308
Raw milk L. monocytogenes L. monocytogenes CCUG 33548 2.216
4. Asian Journal of Dairy and Food Research
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Application of MALDI-TOF Mass Spectrometry for the Assessment of Prevalence of Listeria monocytogenes in Raw Milk, Dairy...
Barbuddhe, S.B., Chaudhari, S.P. and Malik, S.V.S. (2002). The
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