Phenotypic Investigation of Vancomycin, Teicoplanin and Linezolid Resistance Among Enterococcus spp. Isolated from Children Diarrhea

Vancomycin-resistant Enterococci(VRE) were common among Enterococcus. faecalis and Enterococcus faecium. Teicoplanin resistance or sensitivity can determine the VRE phenotypes whether VanA (VanR/tecR) or VanB (VanR/ tecs). Linezolid resistance among VRE regards an newly emerged health problem. Infection with LRVRE or TRLRVRE pushan hazardous alert for hard to heal illness. Twenty eight Enterococcus spp. isolates were recovered from children diarrhea after their inoculation on m-EI chromogenic agar. Antibiotics susceptibility and phenotypic detection of antibiotics resistance were performed according to CLSI 2016. The results revealed 92.86% resistance to rifampin, 85.71% to erythromycin. VRE were 46.42%, TRE were 25% and LRE were 35.71% while co-existed resistance for Vancomycin/Teicoplanin/Linezolid(TRLRVRE) were detected 25% in. concern antibiotics resistant patterns, the MDR compile (85.7%) while XDR compile (10.7%) and there is no PDR among Enterococcus. spp. isolates were PDR. The presentstudy conclude that VanA and VanB phenotypes were common among MDR and XDR and although there is no using of linezolid but the emergence of TRLRVRE isolates were stated.


INTRODUCTION
Enterococci werea Gram-positive, non-spore-forming,catalase-negativefacultativeanaerobe, Which normally dwellthe alimentary tract of humans. even if Enterococcus spp. is a coexistence organism of the intestinal tract. However although, It may be the causative agent of diarrhea in the elderly and children and immune compromised patients 1,2 . Enterococci, particularly relevant Enterococcus faecium and Enterococcus faecalis, have arise as objects of importance because of the distinctness of resistant strains of many drugs 3-5 . Enterococcus which includes some of nosocomial multidrug-resistant organisms. Vancomycin-resistant enterococci (VRE) is now one of the leading causes of nosocomial infections and represent approximately onethird of Enterococcus isolates 6-8 . There are three main patterns of resistance: Multi-drug resistance (MDR) was indicated as acquired nonsusceptibility to asminimumas one agent in three or more antimicrobial classes, extensive-drug resistance (XDR) was clear as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories and pan-drug resistance (PDR) was defined as resistance to all classesof anti-microbial 9-12 . Vancomycin and teicoplanin resistance via one or more of nine genes (vanA-vanE, vanG, vanL, vanM and vanN) which express for enzymes needed for the synthesis of new peptidoglycan precursors and enzymes that disrupt the normal d-Ala-d-Ala-ending precursors [13][14][15] . Enterococci resistant to erythromycin by main two mechanism: enzyme production like ribosomal methylases (coded for by erm genes) methylate the bacterial ribosome, impairing the binding of macrolide and macrolide efflux, coded for by mef genes 16] . Eflux pumps encoded by tetKand tetLwere responsible for tetracyclines 17 . Three mechanisms were described well includes mutation in gyrA gene, production of NorA efflux pump and encoding for Qnr proteins which guard DNA-gyrase by diminishing DNA binding of the quinolone and succeeding formation of the quinolone-gyrase complex. Tell yet Rifampicinresistance get up from a range of mutations in the rpoB gene that encodes for the polymerase RNA β-subunit. Two mechanism of resistance were well described among Enterococcus sp. to linezolid : genes in which mutations occurencoding the 23S rRNA, (which is an important part of the drug-binding site on the ribosome) and Enzymatic modification of the 23S rRNA by methylase [17][18][19][20][21][22][23][24] . In this study aims to check the antibiotic resistance patterns along with resistance phenotypes of diarrheal Enterococcus faecalis and Enterococcus faecium.

MATERIALS AND METHODS Sample Collection and Processing
Fifty eight stool samples (diarrhea) were collected from children with diarrhea with age ranged from 1-7 years. Swab were used to take the sample and put it in brain heart infusion broth for transportation and incubated at 37°C for 24 hrs. and then inoculated to mEI chromogenic agar 25 .

Cultivation on m-EI chromogenic agar
Agar chromogenic mEI is a chromogenetic agar for recovery and distinctionof faecalis and faecium enterococci. It containnutrients and cycloheximidefor fungi inhibition. Incubation for 18-24 hours and then Enterococcus faecium itGrowth will appears greenish-blue, while give blue colonies for Enterococcus faecalis.

Antibiogram
Antibiotic susceptibility test were done according to CLSI 2016 26 using standard disk diffusion method upon Muller-Hinton agar after normalization of broth to 0.5 McFarland (1x10 8 CFU/ml at OD=0.08).

Biosafty Aspects
T h e b i o s a f t y a s p e c t s i n c l u d e decontamination of swabs, broth, contaminated disposable and culture medium 27 .
Resistance of enterococci to linezolid (LRE) were very rare and single cases documented around the world. Also the Co-existed resistance to vancomycin and linezolid ( LRVRE) and vancomycin, teicoplanin and linezolid (TRLRVRE) were note documented yet in Iraq and this study seem the first to report TRLRVRE phenotypically. The results revealed that 7/28 (25%) of enterococci were TRLRVRE or LRVRE table (2). Linezolid resistance may be appear after treatment with linezolid while many cases reported the resistance in patients without prior use of linezolid [42][43][44][45][46] .
Concern the resistance patterns, MDR, XDR and PDR, the results revealed that 1/28  Different percentage of MDR-enterococci were stated in many studies (28-63%) [47][48][49][50] . XDRenterococci were also stated in many studies and compile ( 8-35%) of isolated enterococci. The resulted multidrug or extensive drug resistance, due to many factors such as antibiotic pressures or antibiotics abuse, can leads to costly, hard to cure, prolonged illness and high mortality infections 51,52 .

CONCLUSION
The current study conclude that VanA and VanB phenotypes were common among MDR and XDR and although there is no using of linezolidbut the emergence of TRLRVRE isolates were stated.