Iranian Journal of Medical Microbiology

Samples Collection and Isolation of Staphylococci Species
Forty-five clinical samples were collected from patients using sterile cotton swabs within Federal Medical Centre, Yola, Adamawa State, Nigeria. The samples were transported on ice packs to the General Laboratory, Department of Biotechnology, Modibbo Adama University, Yola, Adamawa State, Nigeria. Isolation was done by streaking on Mannitol Salt Agar. The plates were incubated at 37˚C for 18 to 24 h aerobically. Individual colonies suspected to be Staphylococci were sub-cultured on freshly prepared Mannitol Salt Agar (MSA) and incubated at 37˚C for 18 – 24 h. The pure cultures of the isolates were recovered and sub-cultured on nutrient agar slant and incubated at 37˚C for 18 – 24 h. These pure culture slants were kept in the refrigerator (Haier Thermocool) at 4˚C until they were needed for other analyses.
Screening for Methicillin and Multi-Drug Resistant Staphylococcal Species
Suspected Staphylococci isolates were screened for methicillin resistance using the disk diffusion method as described by Hudzicki (18). Staphylococcal isolates that were positive for methicillin resistance were further screened for multi-drug resistance using a Gram-positive multi-drug antibiotic disc (Biomark Laboratories, India). Both methicillin and multi-drug resistant screening were carried out on Mueller Hinton Agar. Cefoxitin antibiotic disc (Rapid Lab, UK) was used to screen for methicillin-resistant while antibiotic disc comprising Vancomycin (VAN) 30 µg, Ceftazidime (CPZ) 10 µg, Cephalexin (CP) 1.5 µg, Ampicillin (AMP) 10 µg, Meropenem (MEM) 10 µg, Erythromycin (ERY) 5 µg, Tetracycline (TET) 30 µg, Cotrimoxazole (COT) 25 µg, Cefuroxime (CRX) 10 µg, Gentamicin (GEN) 10 µg, Ciprofloxacin (CIP) 5 µg, and Augmentin (AUG) 30 µg were used for multi-drug antibiotic screening. The results were measured using a metric ruler and interpreted as either resistance (R), intermediate (I), or sensitive (S) using the CLSI standard (19).
Determination of Multiple Antibiotic Resistance Indices of the Four Staphylococci Isolates
The multiple antibiotic resistance index of each of the isolates M. sciuri HFS1, S. haemolyticus HFS2, M. sciuri HFS3, and Staphylococcus spp. HFS4 was determined by dividing the number of antibiotics each of the isolates was resistant to by the total number of antibiotics applied in the study as described by Krumperman (20).
Sugar Fermentation and Biochemical Tests
Biochemical characteristics and sugar fermentation using arabinose, xylose, inositol, mannitol, fructose, lactose, maltose, glucose, sucrose and galactose, catalase, urease, citrate, TSI, oxidase, and indole tests were carried out to have a presumptive identity of the isolates (21).
DNA Extraction
Deoxyribonucleic acid (DNA) of the four selected Staphylococci that were methicillin and multi-drug resistant were extracted using an overnight growth culture in nutrient broth. A DNA extraction kit (model 24700) manufactured by Norgen, Canada was used according to the instructions of the manufacturer.
Amplification and Partial Sequencing of 16S rDNA Gene
The 16S rDNA was amplified using the primers are ST16-F-5’-TTGCTTCTCTGATGTTAGCG-3’, ST16-R-3’- AATCATTTGTCCCACCTT-5’ (22). The mix consisted of NEB one Taq master mix with standard buffer (catalog No MO4825) 10 μL, genomic DNA 1.0 μL, forward and reverse primers each 1.0 μL and nuclease-free water (catalog No E476) 7.0 μL were prepared to run the PCR. The initial denaturation was done at 94˚C for 5 min, followed by 35 cycles of denaturation at 94˚C for 1.0 min. The annealing took place at 50˚C for 30 sec while the extension took place at 68˚C for 1.0 min and the final extension at 68˚C for 1 min.
The integrity of the PCR amplicon was visualized on a 1 % agarose gel (CCL-AG500, Cleaver Scientific Ltd). A molecular ladder of 100 bp (NEB’s fast DNA ladder) was added to serve as a standard. The bands were stained with EZ-Vision Blue light DNA Dye. The amplicon was purified by PCR clean-up and then sequenced in an automated DNA sequencer using the dideoxy chain termination method (23).
Construction of the phylogenetic tree
The sequences of the 16S rDNA gene of three out of four (one failed to amplify) selected Staphylococci were compared with similar sequences of Staphylococci strains deposited at GenBank using the Entrez search engine at the National Center for Biotechnology Information (NCBI). The similarity in sequences of the isolated Staphylococci and deposited Staphylococci strains at GenBank was done using the BLAST program. Additionally, multiple sequence alignment and phylogenetic tree construction were done with molecular evolutionary genetics analysis (MEGA X) (24).
The 16S rDNA gene sequences of three Staphylococci were deposited at GenBank with assigned accession numbers.
Amplification of Coagulase coa, Methicillin mec A, and Enterotoxigenic SEA Genes
Two genes, coa, and SEA were amplified using the respective primers coa F 5’-ATAGAGATGCTGGTACA-GG-3’, coa R 3’GCTTTCCGATTGTTCGATGC-5’ for coagulase gene, and SEA-A2-5’ATTAACCGAAAG-TTCTGTAGA-3’, SEA-U2-3’TTGCGTAAAAGTCTGAATT-5’ for the enterotoxigenic gene in the different reaction mix. The amplicon of each gene was observed on agarose gel of 1.0 % with a molecular ladder of 100 bp (NEB’s fast DNA ladder) (CCL-AG500, Cleaver Scientific Ltd) while the staining of the bands was done with EZ-Vision Blue light DNA Dye.
Statistical Analysis
Data collected in this study were analyzed using simple mean ± standard deviation (Mean±SD) and presented using bar charts.
 
 

All four isolates HFS1, HFS2, HFS3, and HFS4 were unable to ferment sucrose, inositol, and xylose while only HFS2 and HFS4 utilized arabinose and lactose as their carbon and energy sources respectively. However, they were able to metabolize galactose, glucose, maltose, fructose, and mannitol as their carbon and energy sources. The reported observations in this study in sugar fermentation, enzymes catalase and coagulase production as well as oxidase and indole reaction are similar to the earlier report by Gotz et al. (5). The utilization of any carbon source and production of specific enzymes are under the influence of genes controlling the reaction and environmental factors. Similarly, all four strains were negative for indole and oxidase tests Gotz et al. (5). These two biochemical tests are used to presumptively biochemically identify Staphylococci isolates (25). 
The amplicon of the 16S rDNA was found to be around 1.0 kb. The use of PCR amplification of 16S rDNA specific primers has been mentioned to identify Staphylococcal isolates up to the species and subspecies level (22), who reported an amplicon size of 1.4 kb for 16S rDNA gene of the Staphylococci isolated by them.
Methicillin and multi-drug resistant strains of Staphylococci are becoming a public health burden due to the tediousness and cost of treatment. They are responsible for nosocomial infections which have led to an increase in morbidity and mortality rates (2). Sands et al. (26) reported the isolation of methicillin-resistant strains of S. epidermidis, S. haemolyticus, and M. sciuri that are resistant to routinely prescribed antibiotics. The methicillin resistance could be due to the possession of different mec genes present in the staphylococcal cassette chromosome mec (SCCmec), a genetic mobile element (International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (9).
Another factor that has been mentioned to be responsible for the spread of methicillin and antimicrobial resistance genes is the ease with which the genes are acquired among the family members of Staphylococcaceae as well as wide distribution of species of Staphylococcus and Mammaliicoccus among human and animals (9). M. sciuri is a reservoir for the mecA gene though it may not be expressed in the strain when acquired and mutated within the promoter region, it could be expressed in S. aureus (5) Species of Mammaliicoccus have been implicated to carry genes that make them to be resistant to many antibiotics (6, 27). Bardasheva et al. (15) had previously mentioned the resistance of coagulase-positive S. haemolyticus and S. epidermidis to be methicillin and multidrug-resistant. High resistance of Staphylococcal species to ampicillin, tetracycline, ciprofloxacin, and erythromycin has been mentioned by Osada et al. (27), Marincola et al. (7), Sadiq et al. (28).
The multiple antimicrobial resistance indices for the four isolates studied ranged from 0.5 to 0.8. A similar report of high indices has been recently reported by Mir et al. (29), though with Salmonella sp., while Asante et al. (30) reported a high index of 0.8 with Staphylococci species associated with nosocomial infections. The high values of indices are indications of high-risk contamination which could be responsible for multi-drug resistant strains observed in this study. The values we report in this study are higher than 0.2, which is regarded as the standard (19). The observations we are reporting in this study are similar to a previous report by Gotz et al. (5).
Sugar Fermentation and Biochemical Characteristics of the Staphylococci
Biochemically, the four isolates M. sciuri HFS1, S. haemolyticus HFS2, M. sciuri HFS3 and Staphylococcus spp. HFS4 exhibited characteristics of the Staphylococci with little differences among them. They all fermented most 6-C sugars and were unable to utilize the 5-C sugars such as xylose, inositol arabinose, and mannitol  (15). These observations are similar to the previous report by Gotz et al. (5). All four isolates were catalase and coagulase enzyme producers making them potential virulent and pathogenic strains (15, 31). Coagulase for instance is produced by Staphylococci to initiate blood plasma clotting in host plasma (31).
Amplification of Coagulase coa and Enterotoxigenic SEA Genes
Amplification of the coagulase gene, coa was seen in S. haemolyticus HFS2 and M. sciuri HFS3, but not in the remaining two isolates, though all four strains had a positive reaction to the coagulase biochemical test. The positive reaction to the coagulase enzyme test could be attributed to the presence of genes for the enzyme in these strains while the negatives recorded in the PCR amplification could be due to experimental error or nonbinding of the primers to the specific gene. None of the isolates studied had their enterotoxigenic gene SEA amplified after four attempts running the PCR, showing that they all lack this particular gene. The presence of coa gene has been a measure of virulence in S. aureus. In this study, an amplicon size of about 600 bp was generated for the coa gene in S. haemolyticus HFS2 and M. sciuri HFS3. Varied values of amplicon sizes that ranged between 681 bp to 891 bp have been earlier reported by Sadiq et al. (28). A similar study carried out in India and the United Kingdom showed that the amplicon size of the coa gene could range between 510 bp and 1000 bp when same primers were used (31).

 

This study showed that Staphylococci species of Staphylococcus and Mammaliicoccus genera are methicillin, multidrug-resistant, and coagulase as well as catalase positive. They are potential pathogens with multiple genes that have been reported to be resistant to most commonly available drugs in developing countries like Nigeria. All four isolates studied M. sciuri HFS1, S. haemolyticus HFS2, M. sciuri HFS3, and Staphylococcus spp. HFS4 were resistant to not less than five antibiotics that cut across most classes of antibiotics. The multiple antimicrobial indices ranged from 0.5 to 0.8. Isolation of these four strains from clinical samples is a great public health concern, as their presence indicates a high-risk area and may worsen antimicrobial resistance in immune-compromised patients. These may lead to an economic burden on treatment, high morbidity and mortality rates, and death. Therefore, there is a need for regular antimicrobial surveillance within the hospital, timely detection of methicillin and multi-drug resistant Staphylococci, and administration of a cocktail of more potent antibiotics in the treatment of wounds and other patients within the hospital environment.

 

All the authors sincerely appreciate the support received from the management of Federal Medical Centre, Yola, (FMC, Yola) the staff of the Medical Microbiology Department, FMC, Yola, the technical staff of the Biotechnology Department, Modibbo Adama University, Yola.

 

The authors jointly funded this research.

 

Conflicts of Interest

No conflict of interest was declared by the authors.