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Year : 2019  |  Volume : 22  |  Issue : 8  |  Page : 1083-1090

Salmonella Spp. and Shigella Spp. detection via multiplex real-time PCR and discrimination via MALDI-TOF MS in different animal raw milk samples

1 Department of Medical Microbiology, Medical Faculty, Beykent University, Istanbul, Turkey
2 Department of Genetic, Faculty of Veterinary Medicine, Harran University, Şanlıurfa, Turkey
3 Department of Food Hygiene and Technology, Faculty of Veterinary Medicine, Harran University, Şanlıurfa, Turkey
4 Department of Medical Microbiology, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey
5 Department of Medical Microbiology, Cerrahpaşa Medical Faculty, Istanbul University-Cerrahpaşa, Istanbul, Turkey

Date of Acceptance07-Jun-2019
Date of Web Publication14-Aug-2019

Correspondence Address:
Dr. M Demirci
Beykent University, Medical Faculty, Department of Medical Microbiology, Istanbul - 34580
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/njcp.njcp_596_18

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Aims: The aim of this study was to provide epidemiological data about the presence of Salmonella spp. and Shigella spp. in raw milk samples collected from different animals. Methods: A total of 231 raw milk samples from 48 cows, 65 goats, 65 sheep, and 53 donkeys were studied. The ISO 6579:2002 and ISO 21567:2004 methods, antimicrobial susceptibility tests, and serotyping were performed. Species and subspecies discriminations were made via matrix-assisted laser desorption/ionization-time of flight mass spectrometry. After DNA isolation from all samples, Salmonella spp. and Shigella spp. were detected using real-time polymerase chain reaction (PCR) kits. Results: Five samples (2.16%) showed positivity out of 231 raw milk samples for Salmonella spp., and 2 (0.87%) samples were detected to be positive by multiplex real-time PCR design. Conclusion: We found that raw milk samples were not free of Salmonella spp. and Shigella spp. and need to be tested routinely to avoid public health problems. Rapid and reliable real-time PCR method can be developed and used for this purposes instead of slow bacterial culture processes.

Keywords: MALDI-TOF MS, qPCR, Salmonella spp., Shigella spp., subspecies discrimination

How to cite this article:
Demirci M, Yigin A, Altun S K, Uysal H K, Saribas S, Kocazeybek B S. Salmonella Spp. and Shigella Spp. detection via multiplex real-time PCR and discrimination via MALDI-TOF MS in different animal raw milk samples. Niger J Clin Pract 2019;22:1083-90

How to cite this URL:
Demirci M, Yigin A, Altun S K, Uysal H K, Saribas S, Kocazeybek B S. Salmonella Spp. and Shigella Spp. detection via multiplex real-time PCR and discrimination via MALDI-TOF MS in different animal raw milk samples. Niger J Clin Pract [serial online] 2019 [cited 2022 Nov 30];22:1083-90. Available from:

   Introduction Top

Foodborne diseases that may arise from the consumption of contaminated foods, especially those of animal origin, may cause serious human health problems.[1] Many persons become ill from foodborne diseases, and many of these cases are not reported.[2]  Salmonella More Details spp.,  Escherichia More Details coli, Shigella, Campylobacter spp., E. coli O157:H7,  Yersinia More Details enterocolitica, Listeria monocytogenes as well as intoxications by Staphylococcus aureus are most frequently encountered causes of human outbreaks due to the consumption of raw milk or products.[3],[4] The conventional culture methods for the detection of Shigella spp. and Salmonella spp. from environmental and human samples were regarded as the gold standard, but these methods require at least 7 days with time-consuming subcultures and biochemical and serological confirmation.[5] In recent years, molecular technologies such as real-time polymerase chain reaction (PCR) method have been used to detect Shigella spp. and Salmonella spp., but they also require specialized laboratories, trained personnel, complicated apparatus, and expensive reagents which are difficult to obtain in lower income countries.[6],[7]

In this study, we aimed to provide epidemiological data for the presence of Salmonella spp. and Shigella spp. contamination using both conventional and molecular techniques, and also to provide the discrimination analysis via matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) in different raw milk samples collected from different animals, which is critically important to human health. In addition, we designed a new multiplex real-time PCR assay, for detecting Salmonella spp. and Shigella spp. simultaneously and comparing the results of conventional culture and commercial real-time PCR kits available.

   Methods Top

Sample collection

The study animals were initially screened for mastitis, and animals with mastitis were not included in the study. From 48 cows, 65 goats, 65 sheep, and 53 donkeys meeting the healthy selection criteria and available for sampling, 231 raw milk samples were obtained by random sampling. Milk samples obtained from 48 cows, 65 goats, 65 sheep, and 53 donkeys by milking individually were collected from four different dairy farms between the period of January and April 2016. Udders were cleaned with water and were then wiped with cotton buds soaked in 70% alcohol, after which a 5-mL milk sample for bacteriological culturing was aseptically collected in sterile tubes. Afterward, they were transferred to the laboratory rapidly in a cold chain.

Identification of Salmonella spp. and Shigella spp. with the culture method

The standardized ISO 6579:2002 and ISO 21567:2004 culture method was used for the evaluation of the Salmonella spp. and Shigella spp., respectively.[8],[9] In addition, suspected colonies were identified by biochemical tests using API 20 E (bioMérieux, Marcy l'étoile, France) according to the manufacturer's instructions.

Serotyping of Salmonella spp. and Shigella flexneri isolates

All Salmonella isolates were investigated using direct active slide agglutination method for serological confirmation. Somatic (O), flagella (H), and Vi (capsular) antigens were characterized with hyperimmune sera (Bio-Rad, France). The results were interpreted based on Kauffmann–White scheme.[10] Monovalent antisera kit (MAST, UK) and monoclonal antibody reagents (Reagensia AB, Sweden) were used for S. flexneri isolate to determine the serotypes.[11]

Species and subspecies discriminations via MALDI-TOF MS

Suspicious colonies for Salmonella spp. and Shigella spp. were also removed from agar plates with sterile pipette for MALDI-TOF sample plate (Bruker GmbH, Bremen, Germany). Samples were mixed with matrix solution immediately and MALDI-TOF MS analysis was performed according to the manufacturer's instructions. To determine the species and subspecies discriminations, SARAMIS software (AnagnosTec GmbH, Potsdam, Germany) was used according to peak list after spectra analysis.[12]

The determination of antimicrobial resistance

Antimicrobial susceptibility tests were carried out by standard disk diffusion technique on Mueller–Hinton agar in accordance with CLSI 2016 guidelines.[13] Ten different antibiotics were studied: ampicillin (Amp; 10 μg/mL), amoxicillin–clavulanic acid (AMC; 20/10 μg/mL), amikacin (AK; 30 μg/mL), trimethoprim/sulphamethoxazole (SXT; 1.25/23.75 μg/mL), chloramphenicol (CAM; 30 μg/mL), ciprofloxacin (CIP; 5 μg/mL), ceftriaxone (CRO; 30 μg/mL), nalidixic acid (N; 30 μg/mL), gentamicin (G; 10 μg/mL), and tetracycline (TET; 30 μg/mL).

DNA isolation

DNA isolation was performed for the detection of Salmonella spp. and Shigella spp. by real-time PCR from enriched culture using ShortPrep I kit (S40001; BIOTECON Diagnostics GmbH, Germany) according to the manufacturer's instructions. Shigella sp.ATCC 12038 and Salmonella sp. ATCC 700623 were used as positive controls in both our design and commercial kits.

Detection of Salmonella spp. and Shigella spp. by real-time PCR using commercial kits

For the detection of Salmonella spp. and Shigella spp. by real-time PCR, foodproof Salmonella detection kit (R31027; BIOTECON Diagnostics GmbH) and foodproof Escherichia coli and Shigella detection kit (R30009; BIOTECON Diagnostics GmbH ) were used according to the manufacturer's instruction, respectively. Real-time PCR procedures were performed on LightCycler 480 II (Roche Diagnostics GmbH, Mannheim, Germany) system according to the manufacturer's instructions.

Detection of Salmonella spp. and Shigella spp. by multiplex real-time PCR

The ipaH gene (Genbank accession no. M32063) of Shigella spp. and invA gene (Genbank accession no. NC.003197) of Salmonella spp. were used for multiplex real-time PCR. NCBI primer blast tool was used to check specificity for the ipaH and invA genes ( Primers and probes were brought from Integrated DNA Technologies (IDT, Coralville, IA, USA). The oligonucleotide primers and probes are shown in [Table 1]. All reactions were carried out in a volume of 20 μL using 5 μL of template DNA. The reaction setup of the multiplex real-time PCR was as follows: 10 μL LightCycler 480 Probes Master (Roche Diagnostics GmBH), totally 1 μL (0.5 μM) of each primer, totally 1 μL (0.1 μM) of each probe, and 3 μL of PCR grade water. Amplification was performed on a LightCycler 480 (Roche Diagnostics GmBH) real-time PCR system as a two-step PCR with initial denaturation of 10 min at 95°C, followed by 45 cycles for 10 s at 95°C and for 45 s at 60°C with data acquisition at the end of the 60°C step.
Table 1: Oligonucleotide primers and probes for multiplex real-time PCR

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   Results Top

The distribution of Salmonella spp. and Shigella spp. positivity according to the type of raw milk samples is given in [Table 2]. In bovine and sheep milk, two samples tested positive for Salmonella spp. on conventional culture and two PCR methods. In goat milk, only one sample tested positive by the three different methods. Salmonella spp. was not detected in donkey milk samples. While one bovine and goat milk samples were positive for Shigella spp. by two methods (both multiplex real-time PCR and conventional culture method), two bovine milk samples were positive by the commercial real-time PCR method. We defined one of them as false positive, presumably resulting from high sensitivity of the real-time PCR method. Salmonella spp. serotypes detected in raw cow, sheep, and goat milk samples are shown in [Table 3]. Antimicrobial susceptibility results of isolated Shigella spp. and Salmonella spp. are shown in [Table 4].
Table 2: Distribution of the Salmonella spp. and Shigella spp. positivity according to the type of raw milk samples

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Table 3: Salmonella spp. and Shigella spp. serotypes detected in raw bovine, sheep, and goat milk samples

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Table 4: Antimicrobial susceptibility results of isolated Shigella spp. and Salmonella spp.

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No difference was found between the detection results of commercial real-time PCR and multiplex real-time PCR methods. Five samples (2.16%) showed positivity out of 231 raw milk samples for Salmonella spp. On the other hand, three (1.3%) raw milk samples were positive for Shigella spp. by the commercial kit, but only two (0.87%) samples were positive by multiplex real-time PCR design [Figure 1].
Figure 1: Comparing Comparison of results from diffrent techniques used

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In this study, using antibody and automated MALDI-TOF MS system, Salmonella enterica serovar Agona and S. enterica serovar Dublin were identified in two bovine raw milk samples. Salmonella enterica serovar Infantis was identified in one sheep and one raw goat milk samples. Salmonella enterica serovar Haifa was identified only in one raw sheep milk sample. Shigella flexneri serotype 2a and Shigella sonnei were identified in one bovine and sheep raw milk samples, respectively [Table 3].

All our study strains were susceptible to AMC, CIP, CRO, G, and AK. All strains were also resistant to TET except S. enterica serovar Haifa with intermediate resistance. Most of the strains were resistant to Amp except S. enterica serovar Infantis (goat milk sample G13) and S. sonnei with intermediate resistance. Co-trimoxazole susceptibility was varied due to the various strains. While S. enterica serovar Agona and S. enterica serovar Infantis (goat milk sample G13) were susceptible to co-trimoxazole, other study strains were resistant except S. enterica serovar Dublin with intermediate resistance. All the study strains were susceptible to CAM except S. enterica serovar Infantis (goat milk sample G13) and S. flexneri 2a with resistance to CAM. All strains were susceptible to N, except S. enterica serovar Infantis (sheep milk sample S27) with intermediate resistance to N.

   Discussion Top

The raw milk quality deteriorates more frequently in the developing countries due to technical and economic conditions being unfavorable than developed countries.[14] Thus, it is important to routinely control foodborne pathogens such as Salmonella spp. and Shigella spp. in the developing countries, where raw milk samples are commonly used.[15],[16] Most of the studies that detected Salmonella and Shigella were from developing countries probably due to poor sanitation conditions. Fotou et al.[17] reported the isolation of Salmonella spp. in 5% of the 240 sheep milk samples in Greece. Tajbakhsh et al.[18] reported Salmonella spp. in 4% of cow milk samples in Iran. In a study by Yagoub et al.,[19] 14 (20%) Shigella spp. and 1 (1.43%) Salmonella spp. were isolated from 90 raw milk samples. Reta et al.[20] isolated 4 (3.3%), Salmonella spp. and 21 (17.5%) Shigella spp. from 120 raw milk samples. Junaidu et al.,[21] Tajbakhsh et al.,[18] and Yagoub et al.[19] reported isolation rates similar to our study with relatively low detection rates for Salmonella spp., as 2.17%, 4%, and 1.43% prevalence, respectively. On the other hand, in some studies, such as the study by Addis et al.,[15] a higher prevalence of 10.7% Salmonella spp. detection from raw milk samples was noted. Akoachere et al.,[22] in Cameroon, also reported a high prevalence (27%) of Salmonella spp. in cattle milk samples. Kivanç et al.[23] detected Salmonella and Shigella in two milk samples obtained from Turkey markets. Salmonella spp. and Shigella spp. were also detected in 6 and 11 out of 100 raw milk samples in a study by Rahman et al.,[24] respectively.

The number of Shigella spp. in milk from tanks and output milking machine was higher (80.7%) than in milk from hand milking (16.5%) and groceries (2.8%) as seen in a study by Oueslati et al.,[25] and they concluded that high percentage of Shigella in the milking machine may arise from the excessive use of water for cleaning milking equipment. Raw milk is really an ideal niche for microorganisms and outbreaks may cause from the contaminated milk. Moreover, Muehlherr et al.[26] did not detect Salmonella spp. in 63 sheep and 344 goat milk samples in Switzerland. Donkor et al.[27] detected no Salmonella spp. or Shigella spp. in raw cow milk samples, and Islam et al.[28] also detected no Shigella spp. and Salmonella spp. in a total of 438 raw milk samples. As seen in all the aforementioned studies, our low Salmonella and Shigella detection rates are in parallel with other studies from the developing countries except the studies by Addis et al.[15] and Akoachere et al.,[22] with high Salmonella spp. detection rates.

In an extensive literature research, we could only find limited studies including serotypes of identified milk-borne pathogens. In a very recent study, eight (0.9%) Salmonella spp. were detected in 902 raw drinking milk samples of cows, goat, sheep, buffalo, and camel. The majority of samples (613/902; 68.0%) belong to the cows' milk samples and eight Salmonella isolates were identified as S. enterica serovar Mbandaka (n = 3) and S. Butantan (n = 5). Salmonella enterica subspecies enterica serovar Dublin (S. Dublin) is the most frequently detected Salmonella in cattle as found in our study in the European Union, and between 2002 and 2010, S. Dublin was the most common isolated Salmonella.[28],[29] In another study by Ahmed and Shimamoto,[30] S. enterica serovar Infantis, S. enterica serovar Typhimurium, and S. enterica serovar Enteritidis were detected in 2 (0.25%), 1 (0.13%), and 1 (0.13%) of raw milk samples, respectively. In another study by Jayarao et al.,[31] Salmonella spp. was detected in 15 (6%) of raw milk samples and Salmonella isolates were identified as S. enteric serotype Typhimurium (n = 10) and S. enterica serotype Newport (n = 5). In England and Wales, S. enterica was commonly detected in milk and milk-product-borne outbreaks and 85% of them were identified as S. Typhimurium [32] and 0.3% of the raw milk samples had S. enteric.[33] S. Dublin is also a public health problem as a zoonotic infection and can cause severe infections such as gastrointestinal disease in humans.[34] It can be transmitted to humans via dairy products.[35] New cases of S. Dublin associated with raw milk cheeses were reported from Europe in 2016. For these new cases, there is still concern for S. Dublin.[36] S. flexneri was identified in one cow raw milk sample by Ahmed and Shimamoto.[30] Robinson et al.[37] analyzed outbreak cases resulting from drinking raw milk between 2001 and 2010 in the United States and identified 36 Salmonella isolates (16 serotypes, most frequently S. enterica serotype Typhimurium, 10 isolates; S. enterica serotype Montevideo, 6 isolates; and S. enterica serotype Newport, 5 isolates). Our Salmonella enterica subtype is also the commonly detected Salmonella subtype in raw milk samples of similar studies.

Different types of milk are produced, including goat milk, for consumption. Goat milk is consumed less than other types of milk and represents ~2% of global milk production.[38] Recently, goat milk has raised concern for its iron bioavailability, higher concentration of fatty acids, and lower allergenicity.[39] Tadesse and Gebremedhin [40] detected lower Salmonella contamination in goat meat than cow meat, and similarly, we isolated Salmonella spp. only in one raw goat milk sample. Contamination may also occur during milking from animals even trying to comply with hygiene conditions. No such study was found on donkey milk. Our data seem to be consistent with international data, yet it suggests that these data may differ among countries.

Self-medication and administration of subtherapeutic dose of antimicrobials can be used for prophylactic purposes.[41] Antimicrobial use in animal production systems has long been suspected to be a cause of the emergence and dissemination of antimicrobial resistant Salmonella.[18] The highest resistance rate for Salmonella spp. was seen in the study by Reta et al.,[20] to amoxicillin (50%). Tajbakhsh et al.[18] reported that Salmonella spp. isolates were resistant to Amp (42.58%), TET (42.58%), and N (78.57%). Addis et al.[15] reported a high resistance rate for Salmonella isolates to Amp (100%). This resistant Salmonella spp. against Amp and TET in these studies are in parallel with our study results but conversely for N. Strains of our study were detected to be susceptible to N except S. enterica serovarInfantis. The higher antimicrobial resistance of the Salmonella isolates was most likely due to the irregular use of antimicrobials.[42] In the study by Reta et al.,[20] the highest resistance rate for Shigella spp. was observed to Amp (38.1%), but our all Shigella spp. strains were resistant to Amp. Similar to the results of our study, Yagoub et al.[19] reported that Shigella isolated from raw milk were sensitive to G (64.3%) followed by CAM (92.1%), and the highest antimicrobial resistant pattern was observed in Amp and amoxicillin (92.9%, 92.9%). In agreement with our result, Reda et al.[43] reported that Shigella isolates were 100% resistant to Amp and amoxicillin but sensitive to CAM, G, and norfloxacin (41.2%, 88.2%, and 94.1%), respectively. All our Shigella spp. strains were susceptible to G, but only S. flexneri 2a had resistance to CAM and AK. Shiferaw et al.[44] reported that 74% Shigella isolates were resistant to Amp, but all our Shigella spp. strains were resistant to Amp. In another study, all the Shigella isolates were resistant to Amp, 94% to TET, and 82% to CIP similar to our results reported by Bhattacharya et al.,[45] but conversely all Shigella spp. strains in our study were susceptible to CIP.

Shao et al.[46] investigated Salmonella spp. and Shigella spp. in raw milk using multiplex loop-mediated isothermal amplification-restriction fragment length polymorphism. They have seen the DNA bands of Salmonella spp. and Shigella spp. within 60 minutes and with a 5 CFU/10 mL sensitivity. This PCR-based method has been confirmed to be beneficial for detecting these pathogens in a large number of food samples by facilitating sensitivity and time according to conventional detection methods. Wang et al.[47] accomplished detection of Salmonella spp. and Shigella spp. strains' multiplex in food samples by real-time PCR. In the quantitation analysis of this study, similar to our design, TaqMan (hydrolysis) probes were used and Salmonella spp.was identified between the detection limits of 103 and 109 CFU/mL, whereas Shigella spp. between 101 and 108. Wang et al.[6] reported that they have developed a highly sensitive method using the same genes in our study, invA and ipaH for Salmonella spp. and Shigella spp. detection by multiple endonuclease restriction real-time loop-mediated isothermal amplification technique (MERT-LAMP). They even reported that the MERT-LAMP technique was 10 times more sensitive than the qPCR technique. We suggest that the difference they observed may be due to the specific gene region design, and with our qPCR optimization. Gokduman et al.[48] developed a recombinant plasmid-based quantitative real-time PCR analysis method for milk samples with invA and ttrRSBC genes that are common and specific to Salmonella. The quantification threshold was identified as 101 CFU/mL for the invA gene. They reported similar findings as a lower quantitation limit with our study. They also stated that the real-time PCR method is the fastest method for identification and detection, and favorable even in challenging specimens such as milk.

Finally, it is known that even in optimal conditions, milk always contains microorganisms which are derived from the milk ducts in the udder, and other microorganisms from milking utensils and human handlers.[49] The bacteriological quality of water used for watering the animals and cleaning the milking equipment may also play an important role for milk contamination.[50] Litter, feces, soil, and water are important sources of milk contamination.[25] Although contamination of dairy products currently accounts for a small percentage of foodborne illness, it is clear that raw milk consumption and the consumption of products made with raw milk present some risk. Although the levels of Salmonella and Shigella in the milk samples may be in very low quantities and the infectious dose for organism low, the potential for this organism to grow in improperly stored raw milk and in products made from raw milk presents a public health risk, particularly to susceptible members of the population.[20] Raw milk production must be strictly controlled for contaminant bacteria which may cause possible risk for human health and these strains may have acquired multiple resistance to antibiotics.[25]

   Conclusion Top

In a conclusion, Salmonella spp and Shigella spp could be found in raw milk samples from different animals except donkey. To avoid public health problems via foodborne pathogen such as Salmonella spp and Shigella spp need to be tested routinely. Instead of slow bacterial culture processes, rapid and reliable real-time PCR method can be developed and used for this purposes. Subspecies discriminations is important for different antibiotic susceptibility profiles and MALDI-TOF MS could be used for subspecies discriminations in routine labs.

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Conflicts of interest

There are no conflicts of interest.

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  [Figure 1]

  [Table 1], [Table 2], [Table 3], [Table 4]


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