APPLICATION OF MALDI-TOF MASS SPECTROMETRY FOR IDENTIFICATION OF BACTERIA ISOLATED FROM TRADITIONAL SLOVAK CHEESE “PARENICA”

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June – July, 2019, vol. 8, no. 6
pages: 1294-1297
Article type: Microbiology of Microbiology
DOI: 10.15414/jmbfs.2019.8.6.1294-1297
Abstract: In this study, the cultivable population of bacteria from a traditional Slovak cheese “Parenica” made from cow milk were identified using MALDI-TOF mass spectrometry (MS). A total of 100 “Parenica” cheese packages from four producers were examined, including n=50 smoked and n=50 non-smoked cheese samples. The lactic acid bacteria (LAB) were cultured on MRS, APT and MSE agars at 30 °C, coliform bacteria on VRBL agar at 37 °C, total count of bacteria (TCB) on Plate count agar at 30 °C and enterococci on Enterococcus selective agar at 37 °C. Gram-positive and gram-negative strains were subjected to identification by MALDI-TOF MS profiling. MALDI-TOF MS identification revealed four genera belonging to LAB including Lactococcus, Lactobacillus, Enterococcus and Leuconostoc. Lactobacillus was the most represented genus with seven species: Lactobacillus curvatus (L. curvatus), L. delbrueckii, L. fermentum, L. casei, L. paraplantarum, L. plantarum and L. sakei.. Escherichia coli, Enterobacter asburiae, Klebsiella oxytoca and K. pneumoniae were the most identified bacteria species from Enterobacteriales order.
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APPLICATION OF MALDI-TOF MASS SPECTROMETRY FOR IDENTIFICATION OF BACTERIA ISOLATED FROM TRADITIONAL SLOVAK CHEESE “PARENICA”


AUTHORS

Miroslava Kačániová, Simona Kunová, Ľudmila Nagyová, Elena Horská, Peter Haščík, Margarita Terentjeva

ABSTRACT

In this study, the cultivable population of bacteria from a traditional Slovak cheese “Parenica” made from cow milk were identified using MALDI-TOF mass spectrometry (MS). A total of 100 “Parenica” cheese packages from four producers were examined, including n=50 smoked and n=50 non-smoked cheese samples. The lactic acid bacteria (LAB) were cultured on MRS, APT and MSE agars at 30 °C, coliform bacteria on VRBL agar at 37 °C, total count of bacteria (TCB) on Plate count agar at 30 °C and enterococci on Enterococcus selective agar at 37 °C. Gram-positive and gram-negative strains were subjected to identification by MALDI-TOF MS profiling. MALDI-TOF MS identification revealed four genera belonging to LAB including Lactococcus, Lactobacillus, Enterococcus and Leuconostoc. Lactobacillus was the most represented genus with seven species: Lactobacillus curvatus (L. curvatus), L. delbrueckii, L. fermentum, L. casei, L. paraplantarum, L. plantarum and L. sakei.. Escherichia coli, Enterobacter asburiae, Klebsiella oxytoca and K. pneumoniae were the most identified bacteria species from Enterobacteriales order.


KEYWORDS

bacteria, mass spectrometry, identification, Slovak “Parenica” cheese

INTRODUCTION

Manufacture of most of cheese varieties involves a combining of four ingredients: milk, rennet, microorganisms and salt, which are processed. The common steps of cheese-making include gel formation, curd whey expulsion, acid production and salt addition, followed by a period of ripening. A variation in ingredient blends and processing has led to the evolution of cheese varieties. While variations in processing parameters such as processing temperature and curd handling techniques play a major role in production of each cheese type, but the cheese microflora play a critical and pivotal role in the development of the unique characteristics of each cheese variety (Beresford et al., 2001). Traditional raw-milk cheeses are highly valued for their flavors, while large-scale products are often perceived by the consumer as ‘‘boring’’ (Law, 2001). This difference is a consequence of the elimination of raw milk microflora by pasteurization that has a key role in flavor development. To compensate the sensory characteristics of product the food industry looks for alternative LAB (Lactic Acid Bacteria) cultures capable of improving products flavor (Leroy and De Vuyest, 2004). However, the LAB are only a part of the complete microflora of raw milk (Kongo et al., 2007). Complex approach then the addition of LAB is associated to other technological methods such as pressing allows the production of diverse of traditional cheeses (Parguel, 2011). The raw-milk microbiota also represents the contamination from the environment (air, utensils, the animal skin), and the load and its diversity will vary with location, season and livestock species and milking procedures.

Food spoilage is an enormous economic worldwide problem. Approximately one-fourth of the world’s food supply is lost through microbial activity alone (Huis in’t Veld, 1998). Milk is highly nutritious food that serves as an excellent growth medium for a wide range of microorganisms (Ruegg, 2003; Rajagopal et al., 2005). The microbiological quality of milk and dairy products is influenced by the initial microbiota of raw milk, the processing conditions, and post-heat treatment contamination (Richter et al., 1992). Undesirable microbiota that can cause a spoilage of dairy products includes Gram-negative psychrotrophs, coliforms, lactic acid bacteria, yeasts, and molds. In addition, the various pathogens of public health concern such as Salmonella spp., Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica, pathogenic strains of Escherichia coli and enterotoxigenic strains of Staphylococcus aureus may also be found in milk and dairy products (Tatini and Kauppi, 2003; Al-Sahlany, 2016; Verma and Niamah, 2017).  This is one of the reasons why the increased emphasis should be focused on the microbiological examination of milk and dairy foods. Microbiological analyses of milk and milk products are critical for assessment of quality and safety, conformation with standards and specifications, and regulatory compliance (Vasavada, 1993).

The aim of this study was to evaluate microbiological quality of the traditional Slovak non-smoked and smoked cheese “Parenica” made from cow milk and to identify bacterial strains with MALDI-TOF MS Biotyper.

MATERIAL AND METHODS

Samples

There were 50 samples of the Slovak national cheese “Parenica“ examined in this study. The cheese samples included non-smoked cheese (n=25) and smoked cheese (n=25). Additionally, a total of 50 cow milk cheese samples from the Slovak producers located in the western and the middle part of Slovakia were collected (Bánovce nad Bebravou, Liptovský Mikuláš, Červený Kameň, Važec). All samples were placed in sterile sample containers and transported to laboratory on ice for microbiological investigations. Samples were kept in a refrigerator (4±1°C) until the testing began.

The primary dilution of the milk products was made for preparing the samples for testing. For that a 5 ml of sample material was added to 45 ml of 0.87 % sterile saline, then the serial dilutions (10−1 to 10−4) were done and a 100 µl of each dilution was plated out.

Isolation of total count of bacteria

Plate count agar (PCA, Sigma-Aldrich®, St. Louis, USA) for total count bacteria enumeration was used. Inoculated plates were incubated at 30 °C for 24-48 h and then examined for the presence of bacterial colonies.

Isolation of coliform bacteria

Violet red bile lactose agar (VRBGA, Sigma-Aldrich®, St. Louis, USA) for enumeration of coliforms bacteria was used. Inoculated plates were incubated at 37 °C for 24-48 h and then examined for the presence of typical colonies.

Isolation of enterococci

Enterococcus selective agar (ESA, Sigma-Aldrich®, St. Louis, USA) for enumeration of enterococci was used. Inoculated plates were incubated at 37 °C for 24-48 h and then examined for the presence of typical colonies.

Isolation of Lactic Acid Bacteria (LAB)

MRS (Main Rogose agar, Oxoid, UK), MSE (Mayeux, Sandine and Elliker in 1962, Oxoid, UK), and APT (All Purpose TWEEN® agar, Oxoid, UK ) agars were used for enumeration of LAB including lactobacilli, leuconostocs and lactic acid streptococci as well as other microorganisms with high requirements for thiamine (Sigma-Aldrich®, St. Louis, USA). Inoculated agars were incubated at 30 °C for 72 h anaerobically and then the bacterial growth was evaluated.

Sample preparation and MALDI-TOF MS measurement

Prior to identification, the bacterial colonies were subcultured on TSA agar (Tryptone Soya Agar, Oxoid, UK) at 37°C for 18-24 h. One colony of each bacterial isolate was selected. Subsequently, the identification was performed using the Maldi TOF MS Biotyper described by Kluga et al. (2017). Totally, a number of 512 isolates were identified with score higher than 2.

RESULTS AND DISCUSSION

Number of isolated bacterial group

Cheeses are fermented dairy products whose manufacturing involves different types of bacteria (Montel et al., 2014; Irlinger et al., 2015). Cheese producing is a process when a nutrient-rich substrate as milk is colonized by adventitious and deliberately inoculated microorganisms. Two different habitats of bacteria in cheese may be considered: the interior of the cheese and the cheese rind. The rind microbiota can be considered as an interesting model system for the field of ecosystems biology (Wolfe et al., 2014).

Total count of bacteria in non-smoked cheese ranged from 5.25 to 5.58 log cfu.g-1. Enterococci were not identified in the studied samples. Coliform bacteria counts ranged from 1.25 to 1.80 log cfu.g-1, but lactic acid bacteria counts ranged from 4.12 to 4.51 log cfu.g-1. Total count of bacteria in smoked cheese ranged from 5.45 to 5.85 log cfu.g-1. Enterococci and coliform bacteria number of bacteria were not identified in the samples studied. Lactic acid bacteria counts ranged from 4.12 to 4.48 log cfu.g-1.

Kačániová et al., (2018) found similar results in cheese samples, and the total count of bacteria in non-smoked cheese ranged from 3.15 to 3.58 log cfu.g-1. Enterococci were not identified in the studied samples. Coliform bacteria counts ranged from 1.12 to 1.52 log cfu.g-1, but lactic acid bacteria counts ranged from 2.12 to 2.51 log cfu.g-1. Total count of bacteria in smoked cheese ranged from 2.14 to 2.58 log cfu.g-1. Enterococci and coliforms bacteria were not identified in the studied samples. Lactic acid bacteria counts ranged from 1.12 to 2.18 log cfu.g-1.

Total counts of bacteria are the most useful indicator for the overall microbiological quality of the cheese. High viable count often indicates a contamination of the raw material, unsatisfactory sanitation, or unsuitable time and temperature during storage and/or production. The attention has been focused on coliform bacteria because of their public health importance. Coliforms are widely distributed in nature. They gain entry to milk and milk products through the water supply, equipment, unhygienic conditions of production and handling (El-Leboudy et al., 2014).

Isolated bacteria with MALDI-TOF MS Biotyper

Table 1 Isolated species of bacteria from smoked and non-smoked cheese “Parenica”

Family Genera Species
Moraxellaceae Acinetobacter Acinetobacter pittii
Moraxellaceae Acinetobacter Acinetobacter baumannii
Moraxellaceae Acinetobacter Acinetobacter junii
Bacillaceae Bacillus Bacillus cereus
Bacillaceae Bacillus Bacillus pumilus
Bacillaceae Bacillus Bacillus thuringiensis
Brevibacteriaceae Brevibacterium Brevibacterium casei
Flavobacteriaceae Chryseobacterium Chryseobacterium oranimense
Enterobacteriaceae Citrobacter Citrobacter braakii
Enterobacteriaceae Citrobacter Citrobacter youngae
Burkholderiaceae Cupriavidus Cupriavidus metallidurans
Enterobacteriaceae Enterobacter Enterobacter asburiae
Enterobacteriaceae Enterobacter Enterobacter cloacae
Enterobacteriaceae Enterococcus Enterococcus durans
Enterococcaceae Enterococcus Enterococcus faecalis
Enterococcaceae Enterococcus Enterococcus faecium
Enterococcaceae Enterococcus Enterococcus italicus
Enterobacteriaceae Escherichia Escherichia coli
Enterobacteriaceae Ewingella Ewingella americana
Acetobacteraceae Gluconobacter Gluconobacter cerinus
Enterobacteriaceae Hafnia Hafnia alvei
Micrococcaceae Kocuria Kocuria kristinae
Enterobacteriaceae Klebsiella Klebsiella oxytoca
Enterobacteriaceae Klebsiella Klebsiella pneumoniae
Lactobacillaceae Lactobacillus Lactobacillus curvatus
Lactobacillaceae Lactobacillus Lactobacillus delbruckii
Lactobacillaceae Lactobacillus Lactobacillus fermentum
Lactobacillaceae Lactobacillus Lactobacillus paracasei
Lactobacillaceae Lactobacillus Lactobacillus paraplantarum
Lactobacillaceae Lactobacillus Lactobacillus plantarum
Lactobacillaceae Lactobacillus Lactobacillus sakei
Streptococcaceae Lactococcus Lactococcus lactis
Leuconostocaceae Leuconostoc Leuconostoc mesenteroides
Staphylococcaceae Macrococcus Macrococcus caseolyticus
Micrococcaceae Micrococcus Micrococcus luteus
Enterobacteriaceae Pantoea Pantoea agglomerans
Lactobacillaceae Pediococcus Pediococcus pentosaceus
Enterobacteriaceae Pluralibacter Pluralibacter gergoviae
Pseudomonadaceae Pseudomonas Pseudomonas rhodesiae
Enterobacteriaceae Rahnella Rahnella aquatilis
Enterobacteriaceae Raoultella Raoultella ornithinolytica
Enterobacteriaceae Raoultella Raoultella planticola
Rhizobiaceae Rhizobium Rhizobium radiobacter
Enterobacteriaceae Serratia Serratia liquefaciens
Sphingomonadaceae Sphingomonas Sphingomonas melonis
Sphingomonadaceae Sphingomonas Sphingomonas parapaucimobilis
Staphylococcaceae Staphylococcus Staphylococcus epidermidis
Staphylococcaceae Staphylococcus Staphylococcus haemoliticus
Staphylococcaceae Staphylococcus Staphylococcus saprophyticus
Staphylococcaceae Staphylococcus Staphylococcus succinus
Staphylococcaceae Staphylococcus Staphylococcus warneri
Streptococcaceae Streptococcus Streptococcus equinus
Streptococcaceae Streptococcus Streptococcus salivarius_ssp._thermophilus

A total of 53 species of 30 bacterial genera (18 gram-negative G and 12 Gram positive G+) were identified in smoked and non-smoked cheese by MALDI-TOF Mass Spectrometry. The percentage representation of each bacterial group (Gand G+) were 42.58% for G(218 isolates) (and 57.42% for G+ (294 isolates). (). Isolated species of bacteria from smoked and non-smoked cheese “Parenica” are shown in Table 1.

Percentages of the number of isolates of each species for Gand G+ are shown in Table 2. The most abundant G bacterium was Escherichia coli, Klebsiella oxytoca and Klebsiella pneumoniae. Lactobacillus was the most abundant within 12 different species of G+ bacteria with Lactobacillus casei, L. delbrueckii and L. sakei were the most distributed.

Nevertheless, many LAB species were found in both kinds of French cheeses, e.g. L. plantarum, L. paracasei, L. curvatus, L. rhamnosus, L. fructivorans, L. parabuchneri, L. brevis (Nacef et al., 2017). As previously were reported, some Lactobacilli are present in the natural microflora of dairy products and arise from animals, farms and dairies: L. casei ssp. casei/L. paracasei ssp. paracasei, L. rhamnosus, L. plantarum, L. fermentum, L. brevis, L. buchneri, L. curvatus, L. acidophilus and L. pentosus (Corbo et al., 2001, Gobbetti et al., 2002, Medina et al., 2001). Lactobacilli, especially L. curvatus, represents a type of milk microbiota that is resistant to pasteurization. Moreover the presence of LAB could be also attributed to contamination occurring after pasteurization (Martley and Crow, 1993).

Kačániová et al. (2018) found in microbiological analysis of 50 cheese samples three main groups of microorganisms: gram-negative and gram-positive bacteria and fungi. Althogether, 47 species of 18 bacterial genera (17 Gram negative G and 12 Gram positive G+) and 10 species of yeasts of 5 genera were identified with MALDI-TOF Mass Spectrometry. The percentage representation of each microbial group (G, G+ and yeasts) from a total of 669 isolates, reached the following values: 166 isolates of G (24.81%), 297 isolates of G+ (44.39%), and 206 isolates of yeasts (30.79%).

Table 2 Number of isolates identified with MALDI-TOF MS Biotyper in cheese

Microorganisms Non-smoked cheese Smoked cheese Total
Acinetobacter pittii 5 2 7
Acinetobacter baumannii 10 10 20
Acinetobacter junii 2 2 4
Chryseobacterium oranimense 2 2 4
Citrobacter braakii 4 4 8
Citrobacter youngae 4 4 8
Cupriavidus metallidurans 2 1 3
Enterobacter asburiae 8 9 17
Enterobacter cloacae 4 3 7
Escherichia coli 15 12 27
Ewingella americana 2 2 4
Gluconobacter cerinus 1 5 6
Hafnia alvei 8 2 10
Klebsiella oxytoca 10 12 22
Klebsiella pneumoniae 12 12 24
Pantoea agglomerans 2 2 4
Pluralibacter gergoviae 2 2 4
Pseudomonas rhodesiae 2 2 4
Rahnella aquatilis 3 2 5
Raoultella ornithinolytica 3 3 6
Raoultella planticola 1 1 2
Rhizobium radiobacter 2 2 4
Serratia liquefaciens 4 4 8
Sphingomonas melonis 3 3 6
Sphingomonas parapaucimobilis 2 2 4
Gram-negative bacteria 113 105 218
Microorganisms Non- smoked cheese Smoked cheese Total
Bacillus cereus 5 2 7
Bacillus pumilus 4 2 6
Bacillus thuringiensis 1 2 3
Brevibacterium casei 3 3 6
Enterococcus durans 3 5 8
Enterococcus faecalis 4 4 8
Enterococcus faecium 5 5 10
Enterococcus italicus 1 1 2
Kocuria kristinae 2 2 4
Lactobacillus curvatus 15 10 25
Lactobacillus delbruckii 15 15 30
Lactobacillus fermentum 10 10 20
Lactobacillus paracasei 15 15 30
Lactobacillus paraplantarum 10 10 20
Lactobacillus plantarum 10 10 20
Lactobacillus sakei 15 12 27
Lactococcus lactis 10 9 19
Leuconostoc mesenteroides 5 5 10
Macrococcus caseolyticus 3 3 6
Micrococcus luteus 4 4 8
Pediococcus pentosaceus 2 2 4
Staphylococcus epidermidis 2 2 4
Staphylococcus haemoliticus 2 2 4
Staphylococcus saprophyticus 2 3 5
Staphylococcus succinus 1 2 3
Staphylococcus warneri 1 2 3
Streptococcus equinus 2 2 4
Streptococcus salivarius ssp. thermophilus 8 2 10
Gram-positive bacteria 149 145 294
Total 262 250 512

Figure 1 Percentage of isolated bacterial species in cheese samples

Mounier et al. (2006) found out that the microorganisms that developed on the cheese surface were an adventitious microflora from the cheese environment (brine, ripening shelves, and personnel) which rapidly outnumbered the commercial cultures. Several hypotheses have been advanced to explain these findings. These ripening cultures may be unfit for the cheese habitat, or negative interactions may occur between them and the adventitious microflora (Maoz et al., 2003).

CONCLUSION

Microbiological analysis of 100 cheese samples revealed the two main groups of microorganisms comprising 53 species of 30 bacterial genera (18 Gram-negative G and 12 Gram-positive G+) identified with MALDI-TOF Mass Spectrometry. The percentage representation of each bacterial group (Gand G+) were 42.58% (218 isolates of G) (and 57.42% (294 isolates of G+). Fast microbial identification is becoming increasingly necessary in industry to improve microbial control, reduce biocide consumption, to avoid cost-intensive recall of contaminated products and damage to brand reputation. While MALDI-TOF-MS has revolutionized speed and precision of microbial identification for clinical isolates, in contrast few performance studies have been published so far focusing on suitability for particularly industrial applications.

Acknowledgement: Work was supported by the grants APVV-16-0244 “Qualitative factors affecting the production and consumption of milk and cheese”.

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