A total of the 91 CoNS isolates were collected from various clinical specimens submitted to three teaching hospitals (including Beheshti, Besat, and Farshchian Hospitals) located in Hamedan, Iran, from September 2017 to November 2018. The origins of the isolates were as follows: blood, urine, catheters, and wounds. The included criteria were all patients with: (i) at least one positive blood culture for CoNS (ii) a central line in place for at least 48 h prior to a first positive blood culture (iii) who were treated with either linezolid, vancomycin or other antibiotics, methicillin- resistant and susceptible CoNS. The criteria for Exclusion were patients who transferred to other hospitals during treatment, as we could not assess outcomes such as recurrence or mortality. Staphylococcus aureus, Duplicate samples of same patients.
This study was approved by the ethics committee of the Hamadan University of Medical Sciences (Code No: IR.UMSHA.REC.1396.827).
DNA extraction from isolates
The plasmid DNA Extraction Mini Kit (Favorgen, Taiwan) and Plasmid Extraction Kit (Sinaclon, Iran) were used for plasmid DNA extraction according to the manufacturer’s recommendations(18, 19). CoNS Chromosomal DNA was extracted by boiling method(20). Quality of extracted DNA was assessed by the Nanodrop ND‑1000 (Nanodrop Technologies, Inc., Wilmington, DE, USA).
Antibiotic susceptibility testing
The phenotypic antimicrobial susceptibility testing of various CoNS species was evaluated using a panel of 11 commercial antibiotic discs which belonged to various classes of antimicrobial agents. Disk agar diffusion (DAD) method was conducted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines(21). The antimicrobial agents used in current study were as follows: chloramphenicol (30 µg), cefoxitin (30 µg), clindamycin (2 µg), doxycycline (30 µg), erythromycin (15 µg), gentamicin (10 µg), levofloxacin (5 µg), novobiocin (5 µg), rifampicin (5 µg), trimethoprim-sulfamethoxazole (25 µg) and vancomycin (30 µg) (MAST, Merseyside, UK). S. aureus ATCC33591 was used as a quality control.
Detection of SEs and TSST1 encoding genes
Multiplex PCR was used to analyze the following genes: sea, seb, sec, sed, see, seg, seh, sei, selj, selm, seln, selo, selk, sell, selp, selq, selr, selu and tsst. Multiplex PCR method was performed using five different sets of oligonucleotide primer mixtures (Set 1: sea, seb, sec; Set 2: sed, see, seg, seh; Set 3: sei, selj, selm, seln; Set4:, selk, sell, selq, tsst; Set 5: selo, selp, selr, selu). PCR amplifications were carried out in 25 µL volums, containing 2 µL template DNA, 1 µL of each forward primer, 1 µL of each reverse primer, 7 µL (set 2, 3, 4,and 5) and 9 µL (Set 1) of sterile double distilled water and 8 µL of 2x Taq Premix-Master mix (Parstous Biotech Co, Iran). The primers used to amplify SEs and TSST-1 encoding genes are listed in Table 1(22-24). Each PCR amplification reaction was performed using a Bio-Rad thermocycler (Bio-Rad, USA) with the following cycles: initial denaturation for 5 min at 95ºC and then 35 cycles at 95 ºC for 1 min (denaturation), 52 ºC for 1min (annealing) and 72 ºC for 2 min (extension) and final extension was performed at 72 ºC for 7 min. All PCR products were analyzed by electrophoresis for 50 min at 100V through 1% agarose gel (Invitrogen). S. aureus reference strains were included in all reactions as positive control; ATCC 13565 (sea), ATCC 14458 (seb), ATCC 19095 (sec), ATCC 27664 (see), ATCC 51811 (seh), FRI 472 (sed, seg, sei, selj, selm, seln, selo, selr, selu).
Table 1. Primers used in this study
Gene targets
|
Primer sequences (5' to 3')
|
amplicon
size (bp)
|
References
|
sea
|
F: TTGGAAACGGTTAAAACGAA
R: GAACCTTCCCATCAAAAACA
|
120
|
(22)
|
seb
|
F: TCGCATCAAACTGACAAACG
R: GCAGGTACTCTATAAGTGCC
|
478
|
(22)
|
sec
|
F: GACATAAAAGCTAGGAATTT
R: AAATCGGATTAACATTATCC
|
257
|
(22)
|
sed
|
F: CTAGTTTGGTAATATCTCCT
R: TAATGCTATATCTTATAGGG
|
317
|
(22)
|
see
|
F: TAGATAAAGTTAAAACAAGC
R: TAACTTACCGTGGACCCTTC
|
170
|
(22)
|
seg
|
F: TGCTATCGACACACTACAACC
R: CCAGATTCAAATGCAGAACC
|
704
|
(22)
|
seh
|
F: CGAAAGCAGAAGATTTACACG
R: GACCTTTACTTATTTCGCTGTC
|
495
|
(22)
|
sei
|
F: GACAACAAAACTGTCGAAACTG
R: CCATATTCTTTGCCTTTACCAG
|
630
|
(22)
|
selj
|
F: CAGCGATAGCAAAAATGAAACA
R: TCTAGCGGAACAACAGTTCTGA
|
426
|
(22)
|
selm
|
F: CCAATTGAAGACCACCAAAG
R: CTTGTCCTGTTCCAGTATCA
|
517
|
(22)
|
seln
|
F: ATTGTTCTACATAGCTGCAA
R: TTGAAAAAACTCTGCTCCCA
|
682
|
(22)
|
selo
|
F: AGTCAAGTGTAGACCCTATT
R: TATGCTCCGAATGAGAATGA
|
534
|
(22)
|
selk
|
F: TAGGTGTCTCTAATAATGCCA
R: TAGATATTCGTTAGTAGCTG
|
293
|
(23)
|
sell
|
F: TAACGGCGATGTAGGTCCAGG
R: CATCTATTTCTTGTGCGGTAAC
|
383
|
(23)
|
selp
|
F: TGATTTATTAGTAGACCTTGG
R: ATAACCAACCGAATCACCAG
|
396
|
(23)
|
selr
|
F: GGATAAAGCGGTAATAGCAG
R: GTATTCCAAACACATCTAAC
|
166
|
(23)
|
selu
|
F: AATGGCTCTAAAATTGATGG
R: ATTTGATTTCCATCATGCTC
|
215
|
(24)
|
selq
|
F: GGAAAATACACTTTATATTCACAGTTTCA
R: ATTTATTCAGTTTTCTCATATGAAATCTC
|
539
|
(24)
|
tsst
|
F: AAGCCCTTTGTTGCTTGCG
R: ATCGAACTTTGGCCCATACTTT
|
447
|
(23)
|
Identification of genes conferring resistance to fluoroquinolone
The Quinolone resistance determining regions (QRDRs) of gyrA, gyrB, grlA, grlB genes were investigated by PCR amplification using specific primer sequences (25, 26). Each 20 μL PCR reaction mixture contained: 10 µL of 2x Taq Premix-Master mix, 6 µL of sterile distilled water, 1 µL of forward primer, 1 µL of reverse primer and 2 µl of template DNA. PCR conditions for amplification of grlA and grlB genes: 95 ºC, 4 min; 95 ºC, 1 min; 48 ºC (52 ºC for grlB), 1 min; 72 ºC, 60 s; 72 ºC, 4 min; for gyrA: 95 ºC, 1 min; 95 ºC,45 s; 54 ºC, 60 s; 72 ºC, 1 min; for gyrB: 94 ºC, 2 min; 94 ºC, 1 min; 48 ºC, 60 s; 72 ºC, 1 min;72 ºC, 5 min.
Detection of insertion sequence elements IS256 and IS257 among CoNS isolates
Multiplex PCR assay was performed to identify insertion sequence elements IS256 and IS257, the appropriate oligonucleotide primers were selected as follows; for IS256 (762 bp), the 5' primer AGTCCTTTTACGGTACAATG and the 3' primer TGTGCGCATCAGAAATAACG; for IS257 (576 bp), the 5' primer CTATCTAAGATATGCATTGAG and the 3' primer TTAACTTGCTAGCATGATGC(17). 25 µl of PCR mixture contained 2 µl of template DNA, 1µl of each primer for IS256 and IS257, 10 µl of Master Mix, and 9 µl of sterile distilled water. The PCR conditions included an initial denaturation at 94 ºC for 3 min, followed by amplification; 35 cycles at 94 ºC for 1 min, 54 ºC for 1 min, 72 ºC for 2 min and 72 ºC for 5 min.
Statistical analysis
Cramer's V, Phi and Chi-Square test were performed to assess of variables correlation. Phi and Cramer's V have ranges from 0 to 1, where 1 indicates a significant association and 0 indicates no relationship. Interpretation of the Phi and Cramer's V results; > 0; No or very weak, > 0.05 weak; > 0.10 moderate; > 0.15 strong; > 0.25 very strong. The Chi-Square test was done by SPSS software version 20. P value < 0.05 was considered as statistically significant.