Characterisation of Staphylococcus species from subclinical mastitis dairy cattle in Free State Province, South Africa

doi:10.21203/rs.3.rs-2228017/v1

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Characterisation of Staphylococcus species from subclinical mastitis dairy cattle in Free State Province, South Africa

https://doi.org/10.21203/rs.3.rs-2228017/v1

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Background: Mastitis is an important disease in dairy industry causing major economic burden. Subclinical mastitis (SCM) is regarded as one of the most economically important diseases affecting the dairy industry due to the financial constraints incurred by it. Subclinical mastitis as compared to clinical mastitis does not cause visible changes in the udder or physical changes of the milk hence the need for routine testing and surveillance.

Materials and methods: The aim of this study was to assess the prevalence of subclinical mastitis in dairy cows of small-holder farms in the Free State province using California mastitis test (CMT) and somatic cell counts (SCC) and further characterise staphylococcal species isolated by using Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and Polymerase chain reaction (PCR).

Results: The CMT results revealed that out of 166 cows, only 55 (33%) were positive for intramammary infection. The quarter level prevalence based on SCC was 87 (54%) for intramammary infections. Out of 50 bacterial culture isolates, 48 (96%) were identified as S. aureus (76%), S. epidermidis (4%), S. chromogenes (12%) and S. haemolyticus (4%) by MALDI-TOF MS. The 16S rDNA PCR products and obtained sequences indicated that S. aureus was the dominant species infecting tested cows at 76%, followed by S. chromogenes, S. agnetis, S. argenteus and S. devriesei at 10%, 4%, 4%) and 1% respectively.

Conclusions: This study has shown the presence of diverse Staphylococcusspecies infecting subclinical mastitis cows in sampled areas of the Free State province. It further indicated the importance of using various diagnostic tools for bacterial infection surveillance involved in subclinical mastitis to ensure accurate species identification.

Mastitis

raw milk

PCR

MALDI-TOF MS

16S rRNA

Staphylococcus

Mastitis is thought to be one of the causes of antimicrobial resistance in dairy cattle. Pathogen-specific mastitis treatment has been shown to reduce antimicrobial use without affecting clinical or bacteriological cure outcomes [1, 2]. Because intramammary infections are typically diagnosed through bacteriological culturing of milk, quarter milk sampling promotes monitoring of bovine udder health [3]. Due to the time lag between sampling and microbial culture results, treatment of this infection is typically based on bacteriological examination of a single colony. The number of colonies, isolation in pure or mixed cultures, and inflammation signs are all factors in diagnosing intramammary infections [3, 4].

Amongst all pathogens causing mastitis staphylococci are mostly isolated bacteria in raw milk from dairy cows [5, 6]. Staphylococci are typically classified as either coagulase-negative staphylococci (CNS)/non-aureus staphylococci (NAS) or coagulase-positive staphylococci/Staphylococcus (S.) aureus [5]. The CNS are a diverse group, with over 15 species isolated in association with bovine mastitis [7, 8]. Classical bacteriology, which relies solely on phenotypic and biochemical criteria, cannot reliably identify CNS at the species level [9]. While S. aureus is regarded as a major pathogen with a primary mode of transmission via cow-to-cow contact, the epidemiology of CNS mastitis remains unknown [10, 11]. The CNS are opportunistic pathogens that cause mastitis and they have been isolated from extramammary sites such as bovine skin and teats [12]. Some authors speculate that some CNS strains, as a native part of a microflora, may play a beneficial role in udder health maintenance [13].

Research, primarily from European countries, has provided evidence that some CNS, such as S. chromogenes, are more specifically adapted to the bovine udder and can cause more serious infections than other species [8]. Coagulase Negative Staphylococci has generally been thought to cause only a slight increase in SCC and a mild self-limiting clinical inflammatory response, especially in heifers. However, some CNS can mimic S. aureus, causing clinical and subclinical mastitis that can last for months [5]

Recent research suggests that CNS infections may cause more serious harm than previously thought [14]. To date, the classification of staphylococci into S. aureus and CNS has been deemed adequate for field management of bovine mastitis. There are currently 49 validly described species and 22 subspecies in the genus Staphylococcus. At least 24 different CNS have been reported in cases of bovine mastitis [15]. To phenotypically differentiate CNS, a large number of biochemical tests must be performed, which is both time-consuming and expensive [16]. Although commercially available manual and automated bacterial identification systems are available to aid in the identification of Staphylococcus species, many of these systems have been found to produce inaccurate results when tested on veterinary isolates [17]. The reason for this is the small number of veterinary strains evaluated and included in reference databases [18]. Variability in phenotypic expression and the occurrence of phenotypic variants may also lead to the misidentification of Staphylococcus species or subspecies [19].

Advances in the molecular field have resulted in the development and application of DNA-based methods for staphylococcal species differentiation and identification [16, 17, 20]. These methods are not subject to the same pitfalls as phenotyping methods because they do not rely on gene expression [21]. In general, DNA-based methods have been shown to have greater discriminatory power, reproducibility, and typeability than traditional phenotyping methods [23–24]. However, limitations such as low-quality DNA sequence data in databases may pose difficulties. One of the most used methods for differentiating bacterial species is amplification and sequencing of variable regions in the 16S rDNA gene [21]. Therefore, the current study aims to use culture independent methods such as MALDI-TOF MS and PCR amplification of the 16S rRNA gene for identifying staphylococcal species involved in bovine subclinical mastitis cases.

2.1. California Mastitis Test

In this study, California mastitis test was used as a baseline on-farm diagnostic tool of intramammary infection or sub-clinical mastitis. Based on this test, the results showed that out of 166 cow random samples only 55 (33%) were found to have intramammary infection/subclinical mastitis. While on the other hand quarter level prevalence was 90 (56%) in 160 individual quarter samples collected.

Table 1

The cow and quarter prevalence of sub-clinical mastitis based on CMT and SCC.
	No Examined	Sub-clinically infected	Prevalence (%)
Herd size	7
Total number of cows	166	55	33
Total number of quarters (SCC)	160	87	54
Total number of quarters (CMT)	160	90	56

2.2. Somatic Cell Counts

According to the threshold set by National Mastitis Council’s guidelines and Petzer et al. (2018) on normal and abnormal raw milk based on SCCs, cows with SCC of 1x10⁵ cells.ml^− 1 or less indicated an uninfected cow; SCC of 1x10⁵ cells.ml^− 1 − 2x10⁵ cells.ml^− 1 would indicate that a cow had intramammary infection in at least one or more teats; and SCC of 2x10⁵ cells.ml^− 1 − 5x10⁵ cells.ml^− 1 or greater indicated that the cow was infected significantly and probably had high bacterial counts. It is on these thresholds that our results showed that out of 166 cows that were screened for subclinical mastitis, only 55 (33.13%) were considered to have intramammary infection/subclinical mastitis at a cow level. Furthermore, for quarter level prevalence based on SCC this study found that out of 160 individual quarters that were examined, only 87 (54%) were found to have intramammary infection as depicted in Table 1.

2.3. Morphological and bacteriological analysis of isolates

After screening all milk samples for subclinical mastitis, an even number of 10 positive samples from each farm was subjected for microbiological analysis and the results showed that a total number of 70 presumptive isolates i.e. based on preliminary test such as the Catalase test, Mannitol salt agar tests were suspected to be Staphylococcus species and only a representative of 20 out of 70 isolates were confirmed by RapID STAPH PLUS System and the remaining 50 isolates were subjected to MALDI-TOF identification system and PCR. The results of RapID STAPH PLUS System (Supplementary file 1) showed that from 20 samples, 14 (70%) samples with a probability of > 99% were indeed Staphylococcus species with S. aureus and S. xylosus dominating with 3 (21.4%) isolates respectively, followed by S. chromogenes and S. capitis sp. Urealyticus with 2 (14.2%) isolates and S. warneri, S. capitis sp. Capitis, S. intermedius and S. haemolyticus with 1 (7.1%) respectively.

2.4. MALDI-TOF MS identification

For the identification of species using MALDI-TOF MS log score between 0 and 3 was calculated by the Biotyper algorithm (Bruker Daltonics, Bremen, Germany). The analysis for each isolate was run in duplicate. After 2 rounds of MALDI-TOF, the unidentified isolates were considered as no possible identification. A cutoff score ≥ 1.7 was regarded as a reliable threshold for the bacterial identification of Staphylococci at species-level according to Cameron et al., (2017a, 2018). Out of 50 bacterial culture isolates, 48 (96%) were identified to species level by MALDI-TOF as Staphylococcus species i.e., S. aureus (36/72%), S. epidermidis (2/4%), S. chromogenes (6/12%), S. haemolyticus (2/4%) while two samples were mixed cultures between S. aureus and S. hyicus 2 (4%) whereas, the remaining 2 (4%) were identified as enterococcus faecalis (1/2%) and Cytobacillus oceanisediminis (1/2%) after 2 submissions to MALDI TOF (Table 2).

Table 2

Bacterial isolates from subclinical bovine mastitis identified by MALDI-TOF MS
Bacteria	Log score¹	Bacteria	Log score¹	Bacteria	Log score¹	Bacteria	Log store¹	Bacteria
S. aureus	≥ 2	S. chromogenes	≤ 2	S. chromogenes	≥ 2	S. aureus	≥ 2 ≤ 2	S. aureus	≤ 2
S. aureus	≥ 2	S. chromogenes	≤ 2	S. aureus	≤ 2	S. aureus	≤ 2 ≥ 2	S. aureus	≤ 2
S. aureus	≥ 2	S. aureus	≥ 2	E. faecalis	≥ 2 ≤ 2	S. aureus	≤ 2 ≥ 2	S. aureus /S. hyicus	≤ 2
S. aureus	≥ 2	S. aureus	≤ 2	S. aureus	≥ 2	S. aureus	≥ 2	S aureus	≥ 2
S. aureus	≤ 2	S. aureus	≤ 2	S. aureus	≥ 2	S. haemolyticus	≥ 2	S. aureus /S. hyicus	≤ 2
S. aureus	≥ 2	S. aureus	≤ 2	S. aureus	≥ 2	S. haemolyticus	≤ 2	S. aureus	≥ 2
S. aureus	≥ 2	S. chromogenes	≤ 2	S. aureus	≥ 2	S. chromogenes	≤ 2	S. aureus	≤ 2
S. aureus	≥ 2	S. aureus	≤ 2	S. aureus	≥ 2	S. aureus	≥ 2	S. aureus	≥ 2
S. aureus	≤ 2	S. epidermidis	≤ 2	S. aureus	≤ 2	S. chromogenes	≤ 2	C. oceanisediminis	≤ 2
S. aureus	≥ 2	S. epidermidis	≤ 2	S. aureus	≥ 2	S. aureus	≥ 2	S. aureus	≥ 2

A log score between 0 and 3 is calculated by the Biotyper algorithm (Bruker Daltonics, Bremen, Germany). Log scores < 1.7 provide no identification. Log score 1.7 ≥ x < 2.0 indicates genus identification, and log score ≥ 2 indicates species identification. All identifications ≥ 2.0 expressed specific species identification. The next closest score always specified the same species if the score was ≥ 2.0.

2.5. The detection of Staphylococcus species using 16S rDNA.

Staphylococcus isolates were further confirmed using the conventional PCR targeting the 16S rDNA. A total number of 50 isolates were confirmed to be Staphylococcus species by PCR, and they were identified as S. aureus at 38/50 (76%), S. chromogenes, S. agnetis, S. argenteus and S. devriesei at 5/50 (10%), 4/50 (4%), 2/50 (4%) and 1/50 (1%) respectively (Table 3 and Fig. 2).

Table 3

*Staphylococcus* species identified and their accession numbers
Isolates ID	Sequence length	16S rRNA results	Accession number
1.1	1450	S. aureus	OP341380
1.2	1453	S. chromogenes	OP341381
1.3	1464	S. aureus	OP341382
1.4	1451	S. chromogenes	OP341383
1.5	1443	S. aureus	OP341384
1.6	1453	S. aureus	OP341385
1.7	1456	S. aureus	OP341386
1.8	1454	S. aureus	OP341387
1.9	1452	S. aureus	OP341388
1.10	1455	S. argenteus	OP341389
2.1	1455	S. chromogenes	OP341390
2.2	1430	S. aureus	OP341391
2.3	1438	S. aureus	OP341392
2.4	1454	S. aureus	OP341392
2.5	1456	S. aureus	OP341393
2.6	1454	S. aureus	OP341394
2.7	1437	S. aureus	OP341395
2.8	1435	S. chromogenes	OP341396
2.9	1455	S. argenteus	OP341397
2.10	1445	S. aureus	OP341398
3.1	1461	S. aureus	OP341399
3.2	1329	S. agnetis	OP341400
3.3	1445	S. chromogenes	OP341401
3.4	1448	S. aureus	OP341402
3.5	1418	S. aureus	OP341403
3.6	1461	S. aureus	OP341404
3.7	1459	S. aureus	OP341405
3.8	1454	S. agnetis	OP341406
3.9	1450	S. agnetis	OP341407
3.10	1451	S. aureus	OP341408
4.1	1441	S. aureus	OP341409
4.2	1450	S. aureus	OP341410
4.3	1434	S. aureus	OP341411
4.4	1458	S. aureus	OP341412
4.5	1423	S. aureus	OP341412
4.6	1458	S. aureus	OP341414
4.7	1451	S. aureus	OP341415
4.8	1455	S. derviesei	OP341416
4.9	1439	S. aureus	OP341417
4.10	1425	S. aureus	OP341418
5.1	1451	S. aureus	OP341419
5.2	1451	S. aureus	OP341420
5.3	1423	S. aureus	OP341421
5.4	1455	S. aureus	OP341422
5.5	1450	S. aureus	OP341423
5.6	1456	S. aureus	OP341424
5.7	1430	S. aureus	OP341425
5.8	871	S. agnetis	OP341426
5.9	1448	S. aureus	OP341427
5.10	1454	S. aureus	OP341389

One of the major ailments plaguing the world's dairy sector is bovine mastitis. Whether it is clinical or subclinical, this infection can result in financial losses owing to decreased milk production, milk discarding, and treatment costs [8, 22–23]. It is crucial to be constantly aware that organisms found in raw milk samples can act as pathogens, commensals, or contaminants. The S. aureus and CNS can both be found in raw milk without increasing SCC, and as a result, they can also be found on the extramammary sites, which is why they are frequently associated with intramammary infection (IMI) [24–25]. This study used a variety of culture, biochemical, and PCR approaches to identify several staphylococcal species in raw milk samples from dairy cows with subclinical mastitis.

According to CMT and SCC at the quarter level, subclinical mastitis prevalence in the current research was 56 and 54%, respectively. According to [26–27] this observed prevalence is comparatively lower than the previously recorded in other regions of Africa, such as Uganda, Kenya, and Ethiopia, at 86.2%, 64%, and 59.2% respectively.

In addition to the aforementioned, further analysis bases on bacterial culture approach of the raw milk samples determined the predominant staphylococcal species isolates from dairy cows with subclinical mastitis. The analysis revealed that t every sample had staphylococcal species. The MALDI-TOF MS and biochemical tests were employed to identify these species. According to the findings, a total of 62 (68.8%) isolates were discovered in 90 quarter milk samples using both the culture technique and the RapID STAPH plus system. The isolates were identified using the MALDI-TOF Ms method and 16S rDNA. The overall findings are consistent with those of Wald et al., [8] and Hosseinzadeh and Dastmalchi Saei [28], who used similar approaches to analyze the staphylococci presence in quarter samples of raw milk.

Our MALDI-TOF MS findings revealed that S. aureus (38/76%) was a dominating species occurring in most of the samples, followed by S. epidermidis (2/4%), S. chromogenes (6/12%), and S. haemolyticus (2/4%). The research conducted by Braga et al., [9] and Nonnemann et al., [29], who both sought to discover subclinical mastitis pathogens in raw milk, supports these findings. The current study's findings are consistent with the assertion made by Braga et al., [29], that using of MALDI-TOF MS in conjunction with the Biotyper software is a superior approach for identifying Staphylococcus species. Furthermore, the current study supported Mahmmod et al., [30] findings, indicating that we may utilize this technique to further detect CNS. However, despite the effectiveness and efficiency of the MALDI-TOF system, Banach et al., [31] reported that some staphylococci strains, including S. sciuri (3 strains), S. xylosus (2 strains), and S. equorum (1 strain), cannot be identified to the species level but can instead be identified using other techniques. This was consistent with numerous studies reporting on this MALDI-TOF system limitation [32]. It is also proposed that sample preparation, number of reference strains, bacterial growing conditions, and the software version of Biotyper may all contribute to the identification of CNS [33–34].

As validated by Kosecka-Strojek et al., [35], our 16S rRNA Sanger sequencing indicated that S. aureus (76%) was the dominantly isolated staphylococcal species, followed by 38/50, S. chromogenes (10%), S. agnetis (4%) S. argenteus (4%) and S. devriesei (1%). Using a variety of diagnostic methods (Biochemical, MALDI-TOF and 16S rDNA sequencing), the current investigation found that S. aureus was the predominant bacterium identified from subclinical mastitis cows. It is important to remember that the CNS were also segregated. In humans, S. aureus is widespread [14, 36]. According to evidence from Egypt, S. aureus is the pathogen most likely to cause mastitis, followed by S. agalactiae and E. coli [37–38]. Other authors, however, who isolated S. aureus with incidences of 77.1% and 52.5%, respectively, reported greater detection rates [39–40]. Numerous publications have utilized various PCR-based techniques for the identification of S. aureus isolates from different sources [41–42]. According to Brakstad et al. [43], all the S. aureus isolates examined had an amplicon size of more than 1,250 bp when the gene encoding a S. aureus-specific portion of the 16S rRNA was amplified, which supports the current study.

The epidemiology of CNS mastitis and the effects of certain species are more debatable in comparison to the contagiousness of the majority of S. aureus and effective programs to control S. aureus IMI. Identification of species may be important for mastitis control programs, management practices, and decisions about therapeutic techniques when CNS are found in quarter milk samples [8, 44]. Eleven distinct Staphylococcus species isolated from samples of cow's milk were identified using RapID STAPH PLUS system, MALDI-TOF MS and 16S rDNA Sanger sequencing. These include S. chromogenes, S. epidermidis, S. haemolyticus, S. aureus, S. capitis sp. Urealyticus, S warneri, S. intermedius, S. agnetis, S. argenteus, S. devriesei and S. xylosus [45].The major CNS species identified in the current analysis were S. chromogenes, which is consistent with studies conducted in Switzerland and Austria, with an average herd size 30 cows [46]. Furthermore, Taponen and Pyörälä [5], showed that CNS species (apart from S. chromogenes) isolated from teat surface, apex, and canal predominantly differ from the CNS species isolated from milk.

Although this study contributes to our knowledge of different Staphylococcus strains, the relative relevance of each strain is still up for discussion. The prevalence of Staphylococcus hyicus IMI was extremely low, and the order of frequency did not match that of studies conducted in other areas. This was attributed to a variety of factors influencing pathogen distribution, including variations in management practices and housing, study design, schematic milk sampling, the definition of IMI, and not using molecular methods for species identification [10]. According to a recent example by Calcutt et al. [47], a number of isolates that had previously been classified as S. hyicus would probably be reclassified as S. agnetis. Historically, Staphylococcus hyicus was listed as one of the frequently isolated CNS species, in part due to misclassification and the fact that S. chromogenes was erroneously classified as a subspecies of S. hyicus. [10].

It is highly desirable to accurately identify staphylococci at the species level in order to determine host-pathogen relationships more precisely and to better understand the pathogenic potential of various staphylococcal species in light of the growing clinical significance of CNS [14]. Therefore this indicates that phenotypic identification of CNS is inadequate, unreliable, and non-replicable [48–52]. Therefore, to enhance the identification process, genetic approaches must be used in conjunction to the conventional microbiological diagnostics. For the majority of staphylococcal species, the 16S rRNA gene is an acceptable target for sequencing; nevertheless, for other species, inter-species distinction is challenging or impossible due to a lack of or inadequate heterogeneity within the 16S rRNA. Most studies demonstrate that for closely related Staphylococcus species, 16S rRNA gene sequencing's discriminatory strength is extremely poor [48, 53–55]. However, the poor quality of many of the sequences uploaded to public databases hinders the accuracy of bacterial species identification using 16S rRNA gene sequencing. Compared to 16S rRNA gene sequencing, other high throughput sequencing techniques may have a better identification potential, but they are frequently restricted to a small number of taxa [16]. In the future, whole-genome sequencing might replace current conventional methods for characterizing CNS species [51].

The current study is one of the few studies in Southern Africa to characterise Staphylococcus species from subclinical mastitis cases using both MALDI-TOF MS and PCR. Our findings suggest that for effective control and prevention of contagious mastitis, there should be regular monitoring of all staphylococcal species associated with this infection. This study also provides an insight on the usage of MALDI-TOF MS as one of the diagnostic tools as it provides a valuable and efficient platform for identification and typeability of staphylococcal species from aseptically collected quarter milk samples; However, confirmation using nucleic acid-based tools is essential for accurate identification of some Staphylococcal species and strains. Knowledge generated in this study will contribute to improvements in prevention and control strategies to minimize the risk of bovine mastitis caused by staphylococcal species.

5.1. Study design and setting

This is a cross-sectional study which was conducted on seven small-scale farms belonging to three Local Municipalities namely: Mantsopa (Latitude: 29.1283°S, Longitude: 27.2676°E), Setsoto (Latitude: 28.5302°S, Longitude 27.6435°E) and Maluti-a-Phofung (Latitude: 28°16′21.94″S Longitude: 29°07′46.06″E) within Thabo Mufutsanyana District Municipalities in the Free state Province (Fig. 1).

5.2. Study population and sampling strategy

5.2.1. Screening of cows

All experiments including screening, detection and microbiological analysis were conducted according to the National Mastitis Council guidelines. The sample size was determined using Thrusfield’s formula [52], with a 95% confidence interval, a absolute precision of 5%, and a 50% predicted prevalence. Prior to the collection of samples, twenty-five cows were randomly screened for intramammary infections/subclinical mastitis using California Mastitis Kit according to manufactures instructions (DeLaval, South Africa). Briefly, a squirt of milk, about 2 mL from each quarter, was placed in each of four cups in the CMT paddle and an equal amount of the commercial CMT reagent was added to each cup. The mixture was manually mixed by a gentle circular motion in a horizontal plane for 15 seconds. The CMT results were scored as zero (negative), trace, one (weakly positive), two (distinct positive), and three (strongly positive) based on gel formation. All CMT scores of 0 and trace were considered as negative while CMT scores of one, two, and three were considered indicators of subclinical mastitis. Positive cows were defined as having at least one quarter with a CMT score of 1 +. Thereafter, a total of 166 milk samples that were suspected to be infected with subclinical mastitis, were collected in duplicate where one batch of samples was transported to the University of the Free state (Faculty of Natural and Agricultural Sciences. PO Box 339, Bloemfontein 9300, Republic of South Africa) for analysis of somatic cells using EKOMILK® Horizon UNLIMITED (18, Promishlena Str., Industrial Area 19, Stara Zagora, 6000, Bulgaria) and the other batch was transported to the Centre for Applied Food Sustainability and Biotechnology for microbiological analysis.

5.2.2. Microbiological analysis

All isolates were cultured according to [56] with modifications. Briefly, 1 ml of milk samples from respective farms was inoculated in 9 mL of Brain Heart Infusion Broth (BHI) and incubated for 24 hrs at 37°C. Thereafter, aliquots of 10 𝜇l were streaked out on Baired Parker Agar (BPA) plates and incubated 24–48 hours at 37°C. Thereafter, the colonies were identified by colony morphology (BPA: Staphylococci produce dark gray to black colonies due to tellurite reduction; staphylococci that produce lecithinase break down the egg yolk and cause clear zones around respective colonies (S. aureus positive). Presumed staphylococcal colonies were sub-cultured on nutrient agar plates (NAP) and incubated at 37°C for 24–48 hrs to get pure culture. Subsequently, presumptive identification of staphylococci was done using gram staining and catalase testing and further differentiated using Mannitol Salt Agar (MSA) (S. aureus - yellow colonies with yellow zones. CNS - Colourless or Red colonies with red zones) and by means of RapID STAPH PLUS System according to manufacturer’s instructions. Furthermore, all isolates were later stored at -80°C in BHI with 15% glycerol until further analysis.

5.2.3. Matrix assisted laser desorption ionisation‑time of flight (MALDI‑TOF) mass spectrometric analysis of the isolates

5.2.3.1. Isolates culture procedure

Species or genus identification was performed by using the Biotyper 3.1 software (cite). Mass spectra were obtained using an Autoflex Speed instrument (Bruker Daltonics, Billerica, MA) calibrated with the Escherichia coli DH5α Bacterial Test Standard (BTS).

The briefly, the isolates were stricked out and grown on nutrient agar (Calle Forja, Madrid) distributed by Separation Scientific SA (Pty) Ltd (1480 Cranberry St, Honeydew, Roodepoort, 2040). Thereafter, a visible quantity of the fresh growing cells (between one single colony and 5–10 mg) from the agar plates were harvested and used to create a homogenous suspension in 300 µl sterile distilled water in an Eppendorf 1.5 ml microtube. Then, 900 µl of 100% HPLC grade ethanol was added to the above-prepared suspension and by vortexing. Subsequently, the tubes were stored in a freezer at -20°C until shipped.

5.2.3.2. Bacterial identification using MALDI‑TOF spectroscopy

Single pure colonies (n = 2–5) were transferred to individual 1.5 mL Eppendorf Safe-Lock Tubes (Sigma- Aldrich, Brondbyvester, Denmark) containiwng 300 µL of water, ultra-HPLC grade (Merck, Hellerup, Denmark), using sterile 1.0 - µL disposable plastic inoculating loops. Tubes were vortexed briefly to create a homogeneous suspension, followed by the addition of 900 µL of 100% ethanol, liquid chromatography solvent grade (Merckmillipore) and an additional 15 s of vortex mixing. Tubes were centrifuged for 3 min at 12,200 × g at room temperature (RT), supernatants were poured and discarded, and tubes were centrifuged again for 3 min at 12,200 × g at RT. The was carefully aspirated using a micropipette. Cell pellets were air-dried for 3 min before the addition of an appropriate volume (15–50 µL) of 70% formic acid. The optimal volume of formic acid was determined by visually sizing the pellet. After up to 3 min, an equal volume of 100% acetonitrile was added to each sample and mixed carefully. Samples were centrifuged for 3 min at 12,200 × g at RT. Eight 1-µL volumes of supernatant were carefully placed on a ground steel target plate and allowed to air dry before each spot was overlaid with 1.0 µL of HCCA matrix (Bruker Daltonics) dissolved in 50% acetonitrile with 2.5% trifluoroacetic acid (Sigma-Aldrich) [9, 57].

5.2.4. Molecular Identification of Staphylococci

The genomic DNA of the isolates were extracted for 16S rDNA analysis using Quick-DNA™ Fungal/Bacterial Miniprep Kit (Zymo Research, Catalogue No. D6005) (17062 Murphy Ave. Irvine, CA 92614, USA) distributed by Inqaba Biotechnical Industries (Pty) Ltd in South Africa according to manufactures instructions. Primers 27F 5’-AGA GTT TGA TCC TGG CTC AG-3’ and 1492R 5’–TAC CTT GTT ACG ACT T-3’ (Li et al., 2012) were used for identification of Staphylococcus species. The PCR reaction contained NEB OneTaq 2X Master Mix with Standard Buffer (New England Biolabs, USA), Genomic DNA (10–30 ng/µl), Forward primer (10 µM), Reverse primer (10 µM) and Nuclease free water (Catalogue No. E476). The thermocycler conditions used were 94°C for 5 min, 94°C for 30 sec then 50°C for 30 sec, 68°C for 1 min for 35 cycles and 68°C for 10 min then lastly, held at 4°C. The PCR amplicons were visualized on a 1% agarose gel (CSL-AG500, Cleaver Scientific Ltd) stained with EZ-vision® Bluelight DNA Dye. Furthermore, fragments were enzymatically purified using the ExoSAP procedure (NEB M0293L; NEB M0371). The amplicons were purified for sequencing (Zymo Research, ZR-96 DNA Sequencing Clean-up Kit™, Catalogue No. D4050), and sequenced in the forward and reverse direction (Nimagen, BrilliantDye™ Terminator Cycle Sequencing Kit V3.1, BRD3-100/1000) using the ABI 3730xl Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific).

5.2.5. Data Analysis

FinchTV (https://finchtv.software.informer.com/1.4/) was used to view the raw chromatogram files (.abi). CLC Bio Main Workbench was used to assemble sequence reads to construct consensus sequence for each sample. BLAST analysis (with default parameters) (Altschul et al., 1997) was performed on the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine if a sequence in the database matches the query sequence above a certain threshold (99% query coverage: 99% identity).

5.2.6. Phylogenetic analysis

The obtained sequences of 16s rDNA isolates were aligned separately by MEGA 11 (Molecular Evolutionary Genetics Analysis) software and compared with all existing sequences of S. aureus and CNS annotated in GenBank database. Phylogenetic trees obtained from DNA sequences using the neighbor-joining method and Kimura’s two-parameter distance correction model with 1,000 bootstrap replications supported by the MEGA 11 software (Tamura et al., 2011).

BHI : Brain heart infusion broth
BPA : Baired parker agar
BTS : Bacterial test standard
CMT : California mastites test
CNS : Coagulase negative staphylococcus
IMI : Intramammary infection
MALDI-TOF MS : Matrix-assisted laser desorption/ionization time off light mass spectrometry
MAS : Mannitol salt agar
NAS : Non-aureus staphylococci
NAP : Nutrient agar plates
PCR : Polymerase chain reaction
SCC : Somatic cell counts
SCM : Subclinical mastitis

7.1. Ethics approval and consent to participate

This study was approved by the scientific committee of the Centre for Applied Food Safety and Biotechnology of the Central University of Technology (Republic of South Africa) and The Department of Agriculture, Forestry and Fisheries (Republic of South Africa) under Section 20 of the Animal Diseases Act 35 of 1984 [permit no. 12/11/1/12A (1650KL) (JD)]. An ethical clearance was approved by Animal Research Ethics Committee of the University of Free State, South Africa (UFS-AED2020/0060/21). An informed consent or permission was given by the Farmers and state veterinarian before sample collection. Furthermore, the experiments conducted in this study were done according to the National Mastitis Council guidelines and followed ARRIVE guidelines.

7.2. Consent for publication

Not Applicable

7.3. Acknowledgements

The authors would like to thank the Department of Agriculture, Fisheries and Forestry for the study section 20 permit, the University of the Free State for ethical clearance approval, the Central University and the National Research Foundation for their funding and the farmers for their approval to participate in this study.

7.4. Availability of data and materials

The datasets used and analyzed in the current study are available from the
corresponding author on reasonable request.

7.5. Competing interests statement

The authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article.

7.6. Author contributions

K.N.G., the main author, was involved in study design, data collection, data analysis and article write-up. O.M.M was involved in study design and reviewing the article. K. M was involved in data curation and reviewing the article. K.Z.T.H and N.S.J were involved in article reviewing.

7.7. Funding

This work is based on the research supported in part by the National Research Foundation (NRF) of South Africa (Grant specific unique reference number (UID) 74426 and 134137.

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No competing interests reported.

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Characterisation of Staphylococcus species from subclinical mastitis dairy cattle in Free State Province, South Africa

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Abstract

Figures

1. Background

2. Results

2.1. California Mastitis Test

2.2. Somatic Cell Counts

2.3. Morphological and bacteriological analysis of isolates

2.4. MALDI-TOF MS identification

2.5. The detection of Staphylococcus species using 16S rDNA.

3. Discussion

4. Conclusion

5. Materials And Methods

5.1. Study design and setting

5.2. Study population and sampling strategy

5.2.1. Screening of cows

5.2.2. Microbiological analysis

5.2.3. Matrix assisted laser desorption ionisation‑time of flight (MALDI‑TOF) mass spectrometric analysis of the isolates

5.2.3.1. Isolates culture procedure

5.2.3.2. Bacterial identification using MALDI‑TOF spectroscopy

5.2.4. Molecular Identification of Staphylococci

5.2.5. Data Analysis

5.2.6. Phylogenetic analysis

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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