Halemah Mohamed Abulkasim*, G. S. Shukla,  H K Bajaj and Harrison Masih.

*Department of Clinical laboratory Sciences-Faculty of Health Science, SHUATS, Allahabad-211007. U.P. (India)

(Received: 01 October 2017; accepted: 30 November 2017)

Abstract:

Throat swabs were collected to isolate S. aureus by using blood agar media. Biochemical tests were performed. Genomic DNA was isolated and PCR was done for ermA, ermB and erm C genes followed by bioinformatics analysis. Isolates were screened on erythromycin disc (erythromycin range 1- 125mg/l). All 47 S. aureus isolates found to be resistance to erythromycin by phenotypic methods contained at least 1 were erythromycin resistance gene. That’s mean all S. aureus isolates which were resistance to erythromycin by disc diffusion method were found as positive by PCR for erm A, B, and C genes.

Keywords: Throat swabs, bacteria,ermA, ermB, ermC.

INTRODUCTION

Staphylococci are Gram-positive bacteria, with diameters of 0.5 – 1.5 μm and characterized by single cocci, which divide in more than one level to form grape-like clusters. To date, there are 45 species and eight sub-species in the genus Staphylococcus, many of which preferentially found the human body (Kloos and Bannerman, 1994), however Staphylococcus aureus and Staphylococcus epidermidis are the two most characterized and studied strains.

The staphylococci are non-motile, non-spore form facultative anaerobes that grow by aerobic respiration or by fermentation. They grow easily on many types of media and are active metabolically, fermenting carbohydrates and producing pigments that differ from white to deep yellow. Most species have a relative complex nutritional requirement, However, in general they require an organic source of nitrogen, supplied by 5 to 12 essential amino acids, e.g. arginine, valine, and B vitamins, including thiamine and nicotinamide(Kloos and Schleifer, 1986; Wilkinson, 1997).

Members of this genusare catalase-positive and oxidase-negative, differentiatesthem from the genus Streptococci, which are catalase-negative, and have a different cell wall compositionto Staphylococci (Wilkinson, 1997).

  1. aureus is both a commensal organism and a pathogen. The anterior nares are the main ecological niche for S. aureus. Approximately 20% of individuals are persistently nasally colonized with S. aureus, and 30% are intermittently colonized. However, numerous other sites may be colonized, including the axillae, groin, and gastrointestinal tract. Colonization provides a reservoir from which bacteria can be introduced when host defenses are breached, whether by shaving, aspiration, insertion of an indwelling catheter, or surgery. Colonization clearly increases the risk for subsequent infection (Wertheim et al., 2005; Kluytmanset al., 1997).

Erythromycin is a metabolic product of Streptomyces erythreus and consists of a 14-member lactone ring to which are attached two deoxy-sugars, desosamineandcladinose. The macrocyclic lactone ring is the source of the class name, macrolide.Erythromycin, like most macrolides, appears to act by binding in the ribosomal tunnelthrough which the nascent peptide moves and thus can be considered apeptidyltransferase inhibitor (Schmidt, 1999).

Material and Methods:

Sample collection:

185 samples were collected from healthy students,aged between17-28 years, present at CytoGene Research and development laboratory from January 2016 to May 2016.All specimens collected constitute of both male and female.

Isolation and Identification Bacteria:

The throat swab samples were immediately cultured on 5% sheep blood agar media plates. The culture obtained from primary culture on 5% sheep blood agar plates. It was sub-cultured on brain heart infusion to obtain a pure growth. The isolates were identified based upon their culture characteristic,morphology and by using Biochemical methods (Bergey’s Manual of Systemic Bacteriology)(Holt et al., 1984).

Antibiotic Sensitivity Test

Antibiotic sensitivity test was done by Disc diffusion (Kirby bauer) method using erythromycin, penicillin, Amoxicillin, clindamycin, tetracycline, ciprofloxacin and trimethoprimeagainst all the isolates

DNA Isolation from bacterium

Isolation of genomic DNA was performed using the Phenol chloroform method. Extraction of bacterial DNA was done through broth bacterial culture. Taken 2 ml culture broth in an Eppendorf tube and centrifuged at 10000 rpm for 10 mins only. There after supernatant was discarded and the pellet was dissolved in T.E. buffer. 10% SDS was added and tubes were kept in water-bath for cell lysis. After 1-2 hours tubes were again centrifuged and the supernatant was collected. Phenol: Chloroform: Isoamyl ratio was added and the samples were again centrifuged. An upper transparent layer containing DNA was collected and 3 M sodium acetate was added for precipitation of DNA and kept in ice cold conditions. Again double volume of ethanol added in eppendorf tubes and centrifuged. Discard the supernatant and air dry the tubes and add T.E. buffer to the tubes for loading in Agarose gel electrophoresis.

PCR amplification-

Template DNA was used in each primer (forward and reverse primer) by adding dNTP and four deoxyriboneucliotide tri phosphate. Taq polymerase was used in each sample. Amplification was carried out in a thermal cycler. Temperature for Initial denaturation at 96⁰C for 3 minutes followed by 30 cycle, each cycle consisted of denaturation of DNA for 1 second at 95⁰C, annealing of the primers for 30 seconds at 35⁰C and elongation 72⁰C for7 minutes.

Primer sequences

Table 1: primers of ermA,B, C and 16SrRNA

Primers Sequences (5– 3’) Conc (Unit/µl) Tm Value Length (bp)
erm A-F TATCTTATCGTTGAGAAGGGATT 3 57oC 24
erm A-R CTACACTTGGCTTAGGATGAAA 3 53oC 22
erm B-F TATCTGATTGTTGAAGAAGGATT 3 57oC 24
erm B-R GTTTACTCTTGGTTTAGGATGAAA 3 57oC 24
erm C-F CTTGTTGATCACGATAATTTCC 3 53oC 22
erm C-R ATCTTTTAGCAAACCCGTATTC 3 53oC 22
16S-F GGAGGAAGGTGGGGATGACG 3 57Co 20
61S –R ATGGTGTGACGGGCGGTGTG 3 57Co 20
Analysis by bioinformatics:

The comparison of the nucleotide sequences of the unique fragment with the sequences available in the Genbank database was carried out by using the NCBI BLAST program (http//www.ncbi.nlm.nih.gov/blast). Phylogenetic tree were generated on the basis of sequences of Myelo peroxidase gene sequences using Clustal W 1.8 (Thompson et al., 1994). A method poly genetic tree was constructed using the sequences obtained from the samples.

Results and Discussion

Isolation and Identification of S.aureus: Current study was performed in5 months period from January to May in the year 2016. The study was conducted among 185 healthy students of CytoGene Research and Development Laboratory in Lucknow. Spreading of the swab samples were done on blood agar media followed by streaking on Brain heart Infusion agar media. Out of 185 throat swabs samples 87(47%) of samples were positive i.e., β hemolytic colonies and98(52.9%) were non- hemolytic(no zonearound the colonies).In this study, from total throat swab samples 47% of sample showed positive for β hemolysis like Devapiryet al. (2012)work where a similar percentage of β-hemolytic population was obtained on blood agar media. The differences in presence of S.aureus(F.Cal=17.552) in relation to different age group was statistically significant.

Figure 1: Distribution of samples showing positive and negative for β-hemolysis on blood agar media

The isolates were identified based on their morphological and biochemical characteristics. The biochemical tests for identification of isolates and their results are shown in the table (table 2) given below. The characteristic properties of theisolates determined the species as Staphylococcus aureus.

Table 2: Biochemical test results for the isolates from throat swab

Bacteria Catalase Test Coagulase Test Gelatine hydrolysis Mannitol fermentation Test MR Vp Indole Test Citate utilization Test
S.aureus + + + + + + +

* “+” = Positive; “-” = Negative

All the identified S.aureuspopulations were tested for their antimicrobial susceptibility patterns. The isolates showed high resistance to erythromycin, penicillin, Amoxicillin and clindamycin (100%, 100%, 97.87%, 91, 48%) respectively. Antibiotic resistance rates for gentamicin, vancomycins were moderate (53.19%, 78.72%) respectively, while S.aureus isolates showed low resistance against tetracycline, ciprofloxacin and tri methoprime(19.14%, 6.38% and 44.68%) respectively. These results were found similar to earlier studies of Agneet al. (2010), Nizamiet al. (2012) and Kaur and Chate (2015).  However this study showed dissimilarity with previous studies including Ahmad and Srikanth (2013) and Adebayo et al. (2011).

*Ery = Erythromycin; Amo = Amoxicillin; Pen = Penicillin; Clin = Clindamycin; Gen = Gentamicin; Van = Vancomycins; Tet = Tetracycline; Cip = Ciprofloxacin and tri = Trimethoprime

Figure 2: Antibiotic resistance profile of Staphylococcus aureus

Primers for internal regions of erm(A), erm(B), erm(C) have been described previously in table (Table 1).Genomic DNA from the resistant isolates which possessed either erm(A), erm(B) or erm(C) were isolated and subjected to PCR analysis with primers specific for erythromycin resistant genes i.e., erm(A), erm(B) and erm(C). The results of isolation of DNA from the bacterial samples are shown below:

Figure 3: Agarose Gel Electrophoresis (a) 0.8% Agarose gel (b) Buffer used- 1X TAE (c) Visualization dye- EtBr, Lane 1,2,3,4,5,6,7,8,9,10,11 Genomic DNA of S. aureus isolates compared with Lane 12-1kb DNA Ladder.

Genomic DNA was isolated from the five isolates of S. aureus and 16S rRNA region of DNA was amplified using universal 16S rRNA primers in thermal cycler.All the five isolates of S.aureus obtained as genomic DNA were amplified by PCR and characterized by sequencing. The BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) analysis of the sequences was done followed by CLUSTAL OMEGA(http://www.ebi.ac.uk/Tools/msa/clustalo/). Thus the isolate were confirmed as S.aureusas its maximum identity to staphylococcus species was obtained.  In this study 16SrRNA detected in 45 (95.74 %/) out of 47 S. aureus samples.

Figure 4: Agarose gel electrophoresis of PCR product for 16S rRNA amplified gene (size more than 1.4 Kb) compared with (1Kb) DNA ladder

The result obtained showed the aligned sequences with my query sequence. The sequences with maximum identity were selected to construct the phylogenetic tree. The resistance patterns of S.aureus results determined by conventional methods were compared with the results of the (erm) genes which observed by PCR technique. The isolates which showed positive result with conventional methods were subjected to PCR-based detection of erythromycin resistance genes i.e., erm(A), erm(B) and erm(C).

Figure 4: Agarose gel electrophoresis of PCR amplification for ermA gene, erm, B gene erm C gene (amplified size 737bp)compared with (1Kb) DNA ladder lane 1, 2, 3 shows sample 4, 5, 6 for erm A gene which shows positive results of band .DNA ladder lane 4, 5, 6 shows sample 8,9,10 for erm B in which only lane 10 showed positive results of band.DNA ladder lane 7, 8,9,10 shows sample 1,2,3,4 for erm C in which 7,9,10 only showed positive result of band.

Table 3: Distribution of erm genes among S.aureus resistance to erythromycin

Total No of S. aureusisolates
NO &(%) of S.aureus
 

ermA
 
% ermB % ermC %
47  29 61.7 7 14.89 11 23.40
P value=7.815

Figure 5: Distribution of erm genes among S.aureus resistance to erythromycin

Erythromycin resistance in Staphylococci was predominantly mediated by erythromycin resistant methylase encoded by erm genes (Zamantaret al., 2011). Fourty seven (47) of S. aureuswere resistance to erythromycin; these isolates contain any of erythromycin resistance genes tested (erm A, B, C).In our study, the most prevalent resistance gene in S. aureus was erm A (29/47; 61.7%), followed byerm C (11/47; 23.40%). Less common wereerm B which occurring in (7/47; 14.89%) of the erythromycin resistant S. aureus isolates tested. The differences in prevalence of erm A, B and C (p=7.815) in S. aureus was statistically significant (Table 4.10, Figure 4.10).In general our observations are in line with the findings of (Linaet al., 1999; Franz et al., 2000). They found that theermA gene was more common in S. aureus, similar findings to our results were responsible for erythromycin resistance in 60% among erythromycin resistance S. aureusisolated (Saderiet al., 2011). The ermA gene was solely responsible for erythromycin resistance until 1971, while erm C became dominant between 1984 and 1988 (Westhet al., 1995).Although erm B was present in only a minority of strains, it was formerly found only in animal strains (Eadyet al., 1993). In contrast to (Linaet al; 1999;Nicolaet al; 1998) we found an association between different erm genes, namely ermA in combination with ermC, in S. aureusisolates.

Conclusion

All 47 S.aureus isolates found to be resistance to erythromycin by phenotypic methods contained at least 1 were erythromycin resistance gene. That’s mean all S. aureus isolates which were resistance to erythromycin by disc diffusion method were found as positive by PCR for erm A, B, and C genes. From the above study it is concluded that the throat has to be considered as an important carriage site for S. aureus and should be included when screening for S. aureus. The antibiotic resistance grow day by day it is important to use suitable antibiotic after proper laboratory diagnosis such as culture and antibiotic susceptibility tests and treatment should  depend on it. The resistance mainly to erythromycin in S. aureus was due to the presence of ermA, erm B and ermC.

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