Abstract
The aim of the study was the identification and characterisation of coagulase-positive Staphylococcus bacteria obtained from food matrices by mass spectrometry and molecular methods. A total of 46 coagulase-positive Staphylococcus isolates were collected from different foodstuffs. The Staphylococcus isolates were identified by MALDI-TOF MS and confirmed by the presence and sequence analysis of the Staphylococcus protein A gene. Staphylococcal enterotoxin genes were also investigated by multiplex PCR. Based on the identification of strains by the MALDI-TOF MS technique and spa-typing, all strains were identified as Staphylococcus aureus. Based on their MS peak profiles, the isolates matched the spectra of three S. aureus reference strains in the Bruker MALDI Biotyper database, with identification scores higher than 1.999 in the case of all 46 (100%) isolates. The isolates showed great genetic variability. Twenty spa types were identified, from which most lineages are capable of colonizing humans. Fifty percent of the strains harboured at least one of four enterotoxin genes (seg, seh, sei, and ser), but none of the classical enterotoxin genes could be detected.
In the European Union, Staphylococcus aureus (S. aureus) is an important pathogenic agent of food intoxications (Denayer et al., 2017). S. aureus strains have several virulence factors such as lipases, thermonuclease, hyaluronidase, and haemolysins; however, the major virulence factors are the heat-stable enterotoxins that cause staphylococcal food poisoning (SFP) (Ortega et al., 2010). The onset of SFP symptoms is very rapid in contrast to other gastrointestinal infections, generally a short time after ingestion of the contaminated food (Mossong et al., 2015). In outbreaks, the symptoms typically include diarrhoea (89%), vomiting (87%), and abdominal cramps (72%); however, fever (9%) is rarely reported. Usually, pork (41% of pork dishes were ham) or poultry dishes (41% freshly prepared poultry dishes) are implicated (55%) in S. aureus outbreaks (Bennett et al., 2013).
Coagulase-positive Staphylococcus species have several defence mechanisms like immunoglobulin binding proteins - including staphylococcal protein A (SpA) - that assist the evasion of the host immune system (Foster, 2005). Protein A is produced by numerous species of Staphylococcus, and is a highly potent virulence as well as immune evasion factor (Balachandran et al., 2018). The 40-60 kDa cell-wall protein is secreted during the exponential growth phase and is encoded by the spa gene (Björk & Sjöquist, 1972). The synthesised protein is able to bind to the Fc and F (ab’) 2 regions of immunoglobulins (Forsgren, 1970) and thus may interfere with the immunising ability of vaccines that depend on the production of opsonophagocytic antibodies, because these inhibit the functions of B-cells (Daum & Spellberg, 2012).
The spa-typing of S. aureus isolates has two major aims: to analyse genetic microvariation in outbreak investigations and to analyse genetic macrovariation in population- and phylogenetic-based assaying (Hallin et al., 2009). A variable repeat region (spa) in the gene allows a reliable and rapid method to discriminate S. aureus isolates in outbreaks from those presumed to be epidemiologically unrelated (Koreen et al., 2004). Furthermore, the epidemiological definition makes it possible to compare the outbreaks occurring in clinical cases and animal husbandry. Given that spa-typing involves the sequencing of only one gene, it provides significant advantages in terms of time-to-result, standardisation, ease of use, and reproducibility as compared to other molecular biology techniques (Agius et al., 2007).
Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI- TOF MS) allows the identification of microorganisms isolated from food or clinical cases by a low-cost, rapid, easy, high-throughput, and efficient identification technique (Croxatto et al., 2012). Using standardised and developed procedures, MALDI-TOF MS devices have revolutionised the identification at the species level of most Gram-positive and Gramnegative bacteria (Barnini et al., 2015). The present study aims to discriminate and perform molecular characterisation of foodborne coagulase-positive staphylococci typed molecular techniques after identification by MALDI-TOF MS.
1 Materials and methods
1.1 Isolates and culture conditions
The food samples were collected (January 2018-January 2019) in the WESSLING Hungary Ltd. Microbiological Laboratory. During the examination period, 46 coagulase-positive staphylococci were isolated by culturing on non-selective and selective growth media, based on the standard MSZ EN ISO 6888-1:2008. On Baird-Parker selective medium (Biokar, France), Staphylococcus isolates formed typical colonies. All strains were grown at 37 °C for 24±1 h on Columbia Blood agar (Neogen, UK), and all colonies showed a positive coagulase reaction. Samples were collected from different food matrices: poultry (n=8), beef (n=3), pork (n=20), venison (n=1), dried pasta (n=6), dairy products (n=3), ready meals (n=3), and vegetables (n=2).
1.2 Identification of Staphylococcus spp. with MALDI-TOF MS
For the identification, the colonies were grown on Columbia Blood agar. A formic acid suspension preparation protocol was used: a single colony was picked up with a sterile inoculation loop and suspended for 30 s in 40 μl of formic acid in a microtube. Then, 40 μl of acetonitrile was added to the suspension and mixed extensively. Finally, from the suspension, 1 μl was transferred onto the target plate; when dried, it was overlaid with 1 μl α-HCCA (10 mg ml-1) matrix solution and left to dry.
Mass spectra were collected by application of a Bruker Microflex LT MALDI-TOF mass spectrometer operating in the molecular mass range of 2.0-25 kDa, in positive linear mode. Staphylococcus isolates were identified by MALDI BioTyper 3.1 software. More than 200 shots were required to give adequate spectra with an appropriate signal-to-noise ratio. Before each measurement, calibration was carried out with the Bruker Bacterial Test Standard using lyophilised Escherichia coli. During the measurement, 640 shots were performed with the aim of analysing the mass spectra of Staphylococcus spp.
The 46 isolates were analysed in parallel; the mass spectra obtained were compared with the Bruker MALDI Biotyper database, where mass spectra of six different Staphylococcus aureus isolates were available. The results are reported as numeric scores based on similarity with the reference spectra based on a proprietary algorithm of the Bruker MALDI Biotyper software comparing the presence and symmetry of peaks in the mass spectra of the unknown strain and the database strain entries. The software sets the following threshold score values for identification: scores between 2.300-3.000 are designated as “highly probable species identification”, scores between 2.000-2.299 as “secure genus identification”, scores of 1.700-1.999 as “probable genus identification”, and scores below 1.699 are reported as “nonreliable genus identification”. The closest matches are listed in order of score value, the highest indicating the highest similarity in the mass spectra.
1.3 Molecular analysis - spa-typing
The cheaper and less laborious spa-typing is a single-locus technique, which offers a subtyping resolution comparable to MLST and PFGE. During spa-typing, the sequence of a polymorphic VNTR (variable-number tandem repeat) sequence was investigated, which is located in the 3’ coding region of the S. aureus-specific SpA. The spa type of a strain is determined by the repeat succession. There are exceptions; however, the special repeat length for the spa VNTR is usually 24 bp. The method used is based on the second version of the Protocol for PCR Amplification of spa Recommended by the EURL-AR (2012).
1.4 Staphylococcal enterotoxin (SE) gene PCR
Genomic DNA was obtained from S. aureus strains using PrepMan Ultra lysis buffer (Thermo Scientific, Biocenter Ltd, Szeged, Hungary) according to the manufacturer’s protocol. The supernatant was diluted five-fold with TE buffer and used for PCR amplification. To detect the major SE genes (sea, seb, sec, sed, and see), multiplex PCR protocols were performed using the primer sets of the European Union Reference Laboratory for Coagulase-Positive Staphylococci (EURL-CPS) as published elsewhere (Bianchi et al., 2014) with a slight modification. Primers detecting the seh and ser genes were run as a third, separate reaction to avoid overlapping of the PCR products of similar size during the gel electrophoresis. Reference strains of S. aureus DSMZ 18586 (SEB), DSMZ 18587 (SEC, SEG, SEH, SEI), DSMZ 18588 (SED, SEG, SEI, SEJ, SER), DSMZ 18589 (SEE), and ATCC 29213 (SEA) were used as positive controls. Amplicons were separated by agarose gel electrophoresis and identified according to their predicted size.
2 Results and discussion
Table 1 includes the results of spa-typing and identification of strains by MALDI-TOF MS. Furthermore, the type of food and the best value and best match of identification are also listed, as are the enterotoxin genes harboured by the isolates. Most of the samples containing coagulase-positive staphylococci were collected from meat (70%) including pork (n=20), poultry (n=8), beef (n=3), and venison (n=1) and food made from them. As the total number and type distribution of sampled materials was not available, further conclusion on the isolation frequency of coagulase-positive staphylococci from different food matrices is not within the aims of this study.
Identification of strains by MALDI-TOF MS and by spa-typing, and occurrence of staphylococcal enterotoxin (SE) genes
| Category of food | ID number | Type of food sample | Best score | Organism (best match)* | spa type | SE genes typed detected | |
| Dairy products | SA-1 | Cheese | 2.458 | 1 | t091 | selp | |
| SA-10 | Cheese | 2.440 | 1 | t091 | selp | ||
| SA-18 | Milky dessert | 2.513 | 1 | t084 | - | ||
| SA-2 | Dried pasta | 2.476 | 1 | t127 | seh | ||
| SA-49 | Dried pasta | 2.308 | 1 | t127 | seh | ||
| Dried pasta | SA-7 | Dried pasta | 2.204 | 1 | t084 | - | |
| SA-21 | Dried pasta | 2.486 | 1 | t084 | - | ||
| SA-22 | Dried pasta | 2.448 | 1 | t084 | - | ||
| SA-11 | Dried pasta | 2.429 | 1 | t084 | - | ||
| SA-12 | Drumstick | 2.465 | 1 | t1491 | sei | ||
| SA-13 | Drumstick | 2.345 | 2 | t002 | sei | ||
| SA-16 | Marinated chicken | 2.332 | 2 | t3478 | sei | ||
| Poultry | SA-44 | Duck meat | 2.401 | 1 | t1491 | - | |
| SA-17 | Goose liver | 2.447 | 1 | t706 | sei, selp | ||
| SA-20 | Duck meat | 2.432 | 1 | t1491 | seh | ||
| SA-28 | Duck leg | 2.353 | 3 | t1491 | seh | ||
| SA-29 | Duck greaves | 2.571 | 1 | t11807 | - | ||
| SA-4 | Bacon | 2.119 | 3 | t963 | - | ||
| SA-31 | Bacon | 2.546 | 1 | t7673 | seh | ||
| SA-5 | Pork sausage | 2.477 | 1 | t1491 | seh | ||
| SA-6 | Pork chops | 2.166 | 1 | t091 | selp | ||
| SA-32 | Pork chops | 2.466 | 1 | t011 | - | ||
| SA-25 | Pork sausage | 2.429 | 1 | Unknown | seh | ||
| Meat | SA-33 | Pork chops | 2.329 | 1 | t213 | selp | |
| SA-24 | Pork shoulder | 2.243 | 1 | t213 | - | ||
| SA-34 | Pork chops | 2.350 | 1 | t1778 | seh | ||
| Pork | SA-9 | Pork shoulder | 2.425 | 1 | t1778 | seh | |
| SA-35 | Pork shoulder | 2.500 | 1 | t1451 | - | ||
| SA-36 | Pork shoulder | 2.287 | 3 | t213 | selp | ||
| SA-37 | Pork shoulder | 2.429 | 1 | Unknown | - | ||
| SA-15 | Pork greaves | 2.342 | 3 | t091 | selp | ||
| SA-38 | Pork sausage | 2.474 | 1 | t084 | - | ||
| SA-02 | Pork shoulder | 2.283 | 3 | t15546 | _ | ||
| SA-01 | Pork sausage | 2.295 | 2 | t189 | - | ||
| SA-39 | Pork knuckle | 2.562 | 1 | t084 | - | ||
| SA-40 | Pork knuckle | 2.516 | 1 | t084 | - | ||
| SA-41 | Pork cheek | 2.403 | 2 | t10349 | seh | ||
| SA-3 | Beef | 2.333 | 1 | t091 | - | ||
| Beef | SA-8 | Beef | 2.484 | 3 | t330 | - | |
| SA-14 | Beef offal | 2.362 | 3 | t084 | - | ||
| Venison | SA-42 | Rabbit | 2.371 | 1 | t656 | - | |
| SA-43 | Burgers | 2.282 | 3 | t363 | sei | ||
| Ready meals | SA-47 | Grilled chicken | 2.305 | 2 | t1491 | - | |
| SA-45 | Chocolate | 2.039 | 1 | t330 | seg, sei | ||
| Vegetables | SA-46 | Fresh salad mix | 2.525 | 1 | t330 | - | |
| SA-27 | Carrot paste | 2.421 | 1 | t091 | selp | ||
* 1: S. aureus subsp. aureus DSM 799; 2: S. aureus subsp. aureus DSM 20231T; 3: S. aureus ATCC 33862 THL.
Based on the presence of the spa gene and on the best match and best score, all strains were identified as S. aureus. Based on the MALDI-TOF MS data, the isolates matched the spectra of three S. aureus reference strains in the Bruker MALDI Biotyper database. The number of the best-matching isolate is indicated in Table 1.
Using the formic acid suspension preparation method, it was possible to obtain identification scores higher than 1.999 in the case of all 46 (100%) isolates by comparison with the Bruker MALDI Biotyper database.The vast majority, 83% of the isolates, gave MS scores ≥2.300, so the species were reliably identified. Identification at the genus level was secure in 17% of isolates, and no isolates had a probable genus level identification with a score in the 1.700-1.999 range. Manukumar and Umesha (2017) obtained similar results when investigating foodstuffs: 34 out of 36 coagulase-positive Staphylococcus isolates were identified with a score >2.000, when using another method of direct sample preparation; however, no results of identification with a score over 2.300 were demonstrated.
The strains showed great genetic variability: 20 spa types were identified among the 46 isolates. The distribution of the spa types in food matrices is shown in Table 2. The most common spa type was t084 with an isolation frequency of 19.6% (9/46), belonging to human- related clonal complex (CC)15, and was found mostly in dried pasta and meat. Other frequent types were t091 (ST7) and t1491, both with an isolation frequency of 13.0% (6/46). These spa types are also frequently isolated from humans, and t1491 is associated with CC1. This lineage is widely distributed and consists of methicillin-susceptible and -resistant strains with low host specificity, thus frequently isolated from livestock and related products (Cuny et al., 2015).
Distribution of spa types in food matrices
| spa type | Category of food | ||||||||
| Dairy | Dried | Meat | Ready meals (n=3) | Vegetables (n=2) | Total number of isolates | ||||
| (n=20) | products (n=3) | Pasta (n=6) | Poultry (n=8) | Venison (n=1) | Pork (n=20) | Beef (n=3) | |||
| t002 | 1 | 1 | |||||||
| t011 | 1 | 1 | |||||||
| t084 | 1 | 4 | 3 | 1 | 9 | ||||
| t091 | 2 | 2 | 1 | 1 | 6 | ||||
| t127 | 2 | 2 | |||||||
| t189 | 1 | 1 | |||||||
| t213 | 3 | 3 | |||||||
| t330 | 1 | 1 | 1 | 3 | |||||
| t363 | 1 | 1 | |||||||
| t656 | 1 | 1 | 1 | ||||||
| t706 | 1 | 1 | |||||||
| t963 | 1 | 1 | |||||||
| t1451 | 1 | 1 | |||||||
| t1491 | 4 | 1 | 1 | 6 | |||||
| t1778 | 2 | 2 | |||||||
| t3478 | 1 | 1 | |||||||
| t7673 | 1 | 1 | |||||||
| t10349 | 1 | 1 | |||||||
| t11807 | 1 | 1 | |||||||
| t15546 | 1 | 1 | |||||||
| Unknown | 2 | 2 | |||||||
The connection between the Bruker MALDI Biotyper database closest match reference strain and the spa type cannot be directly inferred: the 20 spa types were distributed among the three database reference strains, and several spa types were found to be assigned to several database entries (t084, t091, t213, t330, t1491).
Among the strains examined, 50% of the isolates contained at least one enterotoxin gene. In two strains (SA-17 and SA-45), two different types of SE were detected (Table 1). Four different enterotoxin genes were identified among the 46 isolates: sei, seg, seh, and selp, of which seh was the most prevalent at 17% (8/46) and which was found mostly in strains from pork meat. In SFP outbreaks, the predominance of SEA is well documented at 70-80% prevalence, independent of the region where the investigation has taken place (Argudín et al., 2010). Though other SEs and SE-like toxins (SEl), like SEH, may also be involved in clinical cases, their role in SFP is still controversial (Fisher et al., 2018). None of the major enterotoxin genes were present in our isolates, and only other SE genes of minor significance occurred in lower numbers, which may reflect favourable conditions in comparison with other studies on the matter (Li et al., 2015; Qi & Sangma, 2019).
3 Conclusions
Based on the results of spa-typing and the best match and best score with the MALDI-TOF MS technique, all strains were identified as S. aureus. The isolates matched the spectra of three S. aureus reference strains in the Bruker MALDI Biotyper database; 83% of the isolates gave scores ≥2.300, so the species were safely identified by mass spectrometry.
The MALDI-TOF MS technique is suitable for rapid and cost-effective identification of S. aureus among foodborne coagulase-positive Staphylococcus species in routine diagnostics. The correlation between the three MALDI Biotyper database Staphylococcus aureus reference strain entries and the spa types and enterotoxin genes could not be unambiguously established. This can be attributed to the fact that the six Staphylococcus aureus MALDI Biotyper database entries do not represent the genetic diversity within the species, and though it is enough for strain identification, but strain typing is not feasible due to such limitations.
Within the scope of the study, 4 different enterotoxin genes and 20 different spa types were identified among the 46 isolates. Around 50% of the isolates harboured enterotoxin genes (sei, seg, seh, and selp), but these are the SEs of lesser significance.
The study was supported by the EFOP-3.6.1-16-2016-00022 project, co-financed by the European Union and the European Social Fund. We would like to thank Dr. Judit Gasparik, Éva Róka, Ákos Pásztor, and Edit Pocklán for the support in the microbiology laboratory, and Dr. András Helenkar for his valuable insights on the MALDI TOF-MS technique.
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