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Research Article
 

Identification of Staphylococcus aureus Causing Bovine Mastitis using MALDI-TOF Fingerprinting



Waleed S. Shell, M. L. Sayed, Aatia A. El-Gedawy, Ghada M. El Sadek, A. A. Samy and Abdelhakam M.M. Ali
 
ABSTRACT

Background: Reliable and rapid methods for identification of clinical bacterial isolates are mainly dependent on phenotypic and genotypic characteristics of the bacteria. As an alternative identification methods, mass spectral (proteomics) analysis for identification of clinical bacterial isolates including Staphylococcus aureus has been recognized. This study was aimed to evaluate and compare the performance, reliability and sensitivity of conventional bacteriology, phenotypic methods and MALDI-TOF MS in identification of clinical Staphylococcus aureus isolated from bovine mastitis cases. Materials and Methods: Ninety five milk samples were collected from three dairy farms (Giza governorate-Egypt) with high incidence of contagious bovine mastitis and examined for isolation and detection of the causative agents using classical identification, API-Staph kit and matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF MS). The MALDI-TOF MS were carried out using standard ethanol-formic acid extraction method. Results: Twenty five cases from these 95 cases were detected by conventional methods and API-Staph kit to be due to Staphylococcus aureus. All Staphylococcus aureus isolates with 4 Staphylococcus aureus reference strains (control positive) and one E. coli isolate revealed from bovine mastitic case (control negative) were examined using MALDI-TOF MS. The MADI-TOF MS identified all clinical bacterial samples as Staphylococcus aureus with the exception of the control negative sample which was E. coli with 100% agreement with bacteriological and phenotypical examination. Also, MALDI-TOF MS gave a valid score of 100% when used in identification of tested Staphylococcus aureus, control positive and control negative samples with 100% sensitivity in comparison to results obtained by ABI system and conventional methods. Conclusion: This study concluded that according to its fast, accurate and reliable nature, MADLI-TOF MS could be used as alternative diagnostic tool for routine differentiation and identification of Staphylococcus aureus isolates in the clinical bacteriological laboratory. The MALDI-TOF MS need more verification and validation using more samples to detect reliability, sensitivity and performance of this type of bacterial identification.

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Waleed S. Shell, M. L. Sayed, Aatia A. El-Gedawy, Ghada M. El Sadek, A. A. Samy and Abdelhakam M.M. Ali, 2017. Identification of Staphylococcus aureus Causing Bovine Mastitis using MALDI-TOF Fingerprinting. International Journal of Dairy Science, 12: 105-113.

DOI: 10.3923/ijds.2017.105.113

URL: https://scialert.net/abstract/?doi=ijds.2017.105.113
 
Received: September 06, 2016; Accepted: October 10, 2016; Published: February 15, 2017

INTRODUCTION

A reliable and rapid method of identification of pathogenic bacterial isolates is mainly dependent on phenotypical characters (morphology, culture and serology) and genotypical characters (DNA and RNA) of the bacteria. Traditional characterization and identification of these isolates is often a time waste procedures needing also long incubation periods, biochemical reactions and requires considerable expertise1,2.

Mastitis is a worldwide disease in dairy cows throughout the world and is responsible for significant economical losses to the dairy farms due to loss in milk production, discarded abnormal milk, degrading milk price and quality due to high somatic cell or bacterial count, high treatment cost, increased birth costs, increased percentage of herd replacement, subsequent mastitis and hazardous of antibiotics residues in milk3. Mastitis usually classified according to the causative pathogens, into contagious mastitis caused by Staphylococcus aureus and Streptococcus agalactiae which are widespread from the infected quarters during milking and environmental mastitis caused by Streptococcus dysgalactiae, Escherichia coli and Streptococcus uberis which are present in the environment (droppings, flooring, bedding) and generally transmitted at any period of cow’s life either during milking or during dry period4.

Identification of Staphylococcus aureus isolates revealed from milk of mastitic cows depends on classical phenotypic identification as morphological and cultural methods, Gram stain, antibiotics sensitivity testing, pbp2a latex agglutination test kit, oxacillin agar screen test and cefoxitin disk diffusion test5-7.

As an alternative identification method, mass spectral (proteomics) analysis for identification of clinical bacterial isolates including Staphylococcus aureus has been accepted. Matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF MS) can be used as a sensitive, reliable and rapid procedure for identification of deferent clinical bacterial isolates8, such as Gram-positive bacteria9, mycobacteria10, Brucella, Enterobacteriaceae11, yeast12, mold13 and non-fermenting bacteria14.

The MALDI-TOF MS profiling for binomial identification of microorganisms has been demonstrated by Holland et al.15 and Claydon et al.16 in the 1990s but has evolved to the first-line identification method just in the past 5 years. The technique, combined with reference peptide databases and advanced software has revolutionized microbial characterization17. It is consistent with 16S rRNA gene sequencing and is expected to substitute for classic biochemical tests18. Its quickness and reliability makes it fit for counter-bioterrorism, epidemiological tracing of field strains and detection of food contamination19. The MALDI-TOF MS is approximately two-thirds less expensive than conventional bacteriological methods20.

Aim of this study is to evaluate and compare the performance, reliability and sensitivity of classical bacteriological and phenotypic methods in comparison to MALDI-TOF MS in identification of clinical Staphylococcus aureus isolated from bovine mastitis cases.

MATERIALS AND METHODS

Physical examination of mastitis
Clinical examination of udder: Udder attachments, any physical abnormalities such as anatomical malformations, presence of lesions, swelling of the udder and tick infestation were recorded21.

Physical examination of milk: The milk was examined for its color, odor, consistency and other abnormalities prior to milking21.

Sampling (milk samples collection): A total of 95 milk samples were collected by Animal Health Research Institute (AHRI) from clinically and physically examined mastitic cows from three dairy farms (Giza governorate-Egypt) with high incidence of contagious bovine mastitis using standard protocols and measures mentioned by the national mastitis council and Suleiman et al.22. Briefly, these measures are based on disinfection of the teats with cotton moistened with 70% alcohol. After discarding the first few milk squirts, about 20 mL of milk sample were collected in sterile universal bottles and kept in an ice box for transportation to the laboratory for bacteriological examination according to Suleiman et al.22.

Bacterial isolation: Bacteriological examination of isolates was performed following standard protocols of the national mastitis council, Quinn et al.5 and Sears et al.6. Briefly, A loopful of each milk sample was cultured on sheep Blood Agar (BA). In parallel, MacConkey agar (MaC) plates were used to isolate Enterococcus species and also any Gram-negative bacteria. Inoculated plates were incubated aerobically at 37°C for 24-48 h.

Classical bacterial identification: A variety of phenotypic tests used for standard and presumptive identification of bacterial isolates. These tests included colony morphologic characteristics on culture plates, Gram stain, haemolytic features on blood agar, oxidation-fermentation test and catalase test5,6,23. Identification of Micrococcus and Staphylococcus species were based on catalase and oxidase tests, coagulase production by using staphylect plus reagent (Oxoid) and their growth characteristics on Mannitol Salt Agar (MSA), where Streptococcus and Gram negative isolates were identified5,6,23.

Oxacillin screening agar test was carried out according to Jain et al.7 and the Mueller-Hinton agar plates, supplemented with 4% (w/v) Nacl containing oxacillin at concentration of 6 ug mL–1, were spot inoculated with a cotton swab according to outlines documented by Clinical and Laboratory Standards Institute (CLSI)24. After 24 h incubation at 35°C, oxacillin resistance was detected by bacterial growth.

API-Staph kit identification (BioMerieux): API-Staph kit was used to confirm the results of classical identification methods of Staphylococcus aureus. The API system strip (BioMe’rieux, Paris, France) consists of 32 cupules, 26 of which contain dehydrated biochemical agents for colorimetric examinations. The tests based mainly on acid production from different reagents as urea, L-arginine, D-glucose, D-fructose and D-mannose.

Bacterial reference strains: Four referances Staphylococcus aureus, as shown in Table 1, obtained from Boehringer Ingelheim Vetmedica, Inc.

Staphylococcus aureus culture conditions: Staphylococcus aureus isolates and reference strains (control positive) and E. coli isolate from bovine mastatic case (control negative) were cultivated onto brain heart infusion agar medium or 7% sheep blood agar. Plates were incubated at 37°C for 24 h till the appearance of streaks5. These fresh pure cultures were used for MALDI-TOF MS identification.

MALDI-TOF MS protocol for Staphylococcus aureus isolates: The two standard methods recommended by Bruker Daltonics for the bacterial identification using MALDI-TOF MS: The direct transfer procedure25 and the standard ethanol-formic acid extraction method26 which was carried out in this study as follow: Approximately 10 colonies of fresh culture on brain heart infusion agar were harvested using sterile needles into tube containing 300 μL of sterile distilled water. These tubes were vortexed then 900 μL of absolute ethanol was added. Vortexing to homnogenize the sample then the tubes were centrifuged at 13,000×g for 2 min. The supernatant was removed by pipetting. Samples were dried for 2 min at room temperature, each pellet was suspended in 150 μL of a solution containing 50% acetonitrile and 1% aqueous trifluoroacetic acid in a 2 mL sterile tube containing 20 mg of acid washed glass beads. The cells were lyses mechanically. The supernatants were transferred to new tubes after centrifugation at 13,000×g for 2 min. Two extractions were performed for each strain and of each extract 1 μL was deposited on MALDI-TOF steel target plate in six replicates (Bruker daltonics). The plates were dried at room temperature and then overlaid with 1 μL of matrix solution containing-cyano-hydroxycinnamic acid saturated with high-performance liquid chromatography (HPLC)-grade water, 2.5% trifluoroacetic acid and 50% acetonitrile. The sample was allowed to co-crystallize with this mixture. Results were analyzed with MALDI-TOF MS spectrometer using flex control software (Bruker Daltonics). According to the guidelines of the manufacturer, a score of ≥2 depicts identify the sample to the species level and an intermediate log score between <2 and ≥1.7 for identify the sample to the genus level. A low score of <1.7 was considered as unreliable for identification.

RESULTS AND DISCUSSION

All collected milk samples (95 samples) were examined for detection of bacterial causes of contagious bovine mastitis. From the milk samples, Gram-positive cocci (Staphylococcus and Streptococcus species) were the most causative pathogens isolated from bovine milk samples in addition to Escherichia coli species. Twenty five S. aureus were isolated from bovine mastitis cases of 35.7% from total isolated bacteria. These isolates were identified phenotypically using classical methods, oxacillin screening agar test and API-Staph kit (BioMerieux). Comité de l'antibiogramme de la société française de microbiologie27 suggested using oxacillin instead of methicillin in identification of Staphylococcus aureus. Ten isolates out of 25 S. aureus isolates were methicillin-resistant. Four Staphylococcus aureus reference strains were used in this study as a control positive (Boehringer Ingelheim Vetmedica, Inc.) (Table 1), these references strains were used in production and evaluation of mastitis vaccine.

Escherichia coli isolate revealed from bovine mastatic case were used in this study as a control negative.

Table 1:Staphylococcus aureus references’ strain

Table 2: Identification of Staphylococcus aureus reference strains (R) and field isolates (F) using MALDI-TOF

Out of 95 mastitic cases, 20 cases were caused by E. coli where Streptococcus agalactiae and Pseudomonas arigenosa were recovered from 11 and 5 cases, respectively. Escherichia coli and Streptococcus agalactiae was the causative agent of mastitis in 9 mixed infection cases. Twenty five cases were due to non bacterial cause.

Using MADI-TOF MS, all phenotypical and bacteriological identified Staphylococcus aureus and control negative and positive samples were checked. The MADI-TOF MS identified all clinical bacterial samples as Staphylococcus aureus with the exception of the control negative sample which was E. coli with 100% agreement with bacteriological and phenotypical examination (Table 2).

The MALDI-TOF MS has successfully been used successfully for identification of many bacterial species. However, only a few researches have evaluated the efficacy of MALDI-TOF MS based identification to be used as routine in diagnostic laboratory28. In this study, MALDI-TOF MS could accurately identify all ATCC strains, references strains (positive control) and E. coli isolate recovered from bovine mastitis case as a negative control as well as tested samples.

Fig. 1:Overview of the MALDI-TOF mass spectra of four Staphylococcus aureus: reference strains 596510 and 596535 and field isolate No. 1 and 2, respectively revealed from bovine mastitic cases

The results obtained from this study concluded that identification of different Staphylococcus aureus strains isolated from bovine mastitis cases was possible using MALDI-TOF MS. The samples being processed and run in duplicates for more results accuracy so the reproducibility of the apparatus was examined and subsequently found to be consistent for all samples. The MALDI-TOF MS mass spectral patterns were also reproducible for bacterial strains and isolates belonging to the same genus and species29,30.

By examination of staphylococcal isolates and strains revealed from cases of bovine mastitis by MALDI-TOF MS, 10-20 prominent ion peaks were detected in the mass spectra. These prominent ion peaks were from the region between 3,000 and 11,000 Da, with the highest-intensity peaks being consistently in the range of 4,000-10,000 Da. On this basis, the score values obtained by MALDI-TOF MS correctly identified all Staphylococcus aureus isolates at the species level (score ≥2.0). Inspection of mass spectra (Fig. 1) reveals strain-specific peaks at 4240, 6900 and 9700 Da for all Staphylococcus aureus.

In this study, MALDI-TOF MS gave a valid score of 100% when used in identification of Staphylococcus aureus causing bovine mastitis with 100% sensitivity in comparison to results obtained by ABI system and conventional methods this agree with Da Motta et al.31, Barreiro et al.32 and Dubois et al.33 which reviewed that MALDI-TOF is suitable assay for the differentiation of Staphylococcus isolates at the species level. Also, similar finding agreed with that recorded by Rohokale et al.34 who mentioned that MALDI-TOF MS technique proved equally (100%) effective in identification of Staphylococcus aureus with highest accuracy as compared with bacterial culture and PCR assay which demonstrating the reliability and accuracy of MALDI-TOF MS in Staphylococcus aureus and other bacterial identification. Other study35 revealed that out of 222 Staphylococcaceae isolates from 29 species, 165 (74.3%) were assigned correctly using MALDI-TOF MS. Only one isolate was incorrectly identified to Staphylococcus aureus while it actually belonged to the just recently described coagulase-positive species S. argenteus. The remaining isolates species (56 isolates) decision was not achieved. For all S. aureus isolates, a 100% match rate was obtained with the commercial and the extended database. Also, detection and typing of S. aureus strains recovered from bovine mastitis cases were carried out by proteomic analysis (MALDI-TOF MS) and the frequency of precise identification at the genus and species levels was 97.97%36.

Valid identification scores as explained by the manufacturer is 2.0 or more were enough for a reliable identification to the species level (green color) (i.e., species score cutoff of 2.0) and scores of ≥1.7 but <2.0 were reliable and accepted for detection to the genus level (yellow color). Scores under 1.7 were considered unsatisfactory and unreliable identification (red color)37,38 as shown in Fig. 2 and Table 2.

This technique is depend upon the presence of highly abundant proteins in a mass range between 2 and 20 kDa by calculating their mass (m) to charge (z), m/z values. Thus, for each clinical bacterial isolates a standard fingerprint is generated which is used for comparison with the stored reference spectra and thereby providing identification for the sample.

Fig. 2: Meaning of MALDI-TOF MS score value

The most important advantage of mass spectral approach over phenotypic and genotypic procedures is a simple straight forward sample preparation procedure and the short time required for analysis as it can carried out using one of these two methods: Identification from direct culture or after extraction of samples using ethanol: Formic acid method29,30.

The identification using MALDI-TOF MS method could analyze samples in short time (may be within minutes) after culture positivity especially when direct cultural identification methods used rather than ethanol: formic acid extraction method. However, identification by conventional methods needs more facilities, chemicals, time and experiences. In contrast, the non requirement of high technical expertise, the simple extraction procedure and low running cost provide MALDI-TOF MS provide more advantages over other methods for identification. But the applications have to be carefully carried out, as the results accuracy decrease by using of too much of materials and chemicals. The samples have to be overlaid with the matrix solution with care to avoid incidence of the liquid smear between spots, which increase possibility of cross contamination39.

CONCLUSION

A successful management and plan for diagnosis of contagious bovine mastitis in Egypt can be established with accurate, strong and effective monitoring system for all dairy flocks and farms in a parallel with an accurate and fast identification of microorganisms that cause bovine mastitis. Mass spectral (proteomics) identification tool of bacterial isolates by MALDI-TOF-MS is fast and took about 30 min per isolate from target plate to gain the final results and also requires little effort for sample preparation. In this study, MALDI-TOF-MS is demonstrated to be a most fast and sensitive tool for identification of S. aureus isolates causing contagious bovine mastitis when compared with the results of classical identification and ABI system. Also, from above results, It could be concluded that S. aureus is one of the major pathogen causing bovine mastitis in Egypt so, according to its fast, reliable and accurate nature, MADLI-TOF MS could be introduced as a regular diagnostic tool for routine identification and differentiation of Staphylococcus aureus isolates and bacterial causing bovine mastitis in the clinical bacteriological veterinary laboratory in Egypt in order to provide more precise identification on clinical specimens. Anyhow, this assay needs more verification and validation using more samples to detect reliability, specificity, sensitivity and performance of this type of bacterial identification.

ACKNOWLEDGMENT

Appreciation is expressed to Microbiology Department, Medicine Faculty, Alexandria University and all the technical staff of Microbiology Departments of Central Laboratory for Evaluation of Veterinary Biologics (CLEVB) and Animal Health Research Institute (AHRI) for their contribution in the practical part of this study.

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