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Research Article | Open Access
Phenotypic Investigation of Vancomycin, Teicoplanin and Linezolid Resistance Among Enterococcus spp. Isolated from Children Diarrhea

Hussein O. M.  Al-Dahmoshi1 , Hussein W. S. Rabeea2, Ali Saleem Abdulridha Aridhee3, Noor S. K. Al-Khafaji1, Mohammed H. Al-Allak1, Anwar M. Lazm4 and Mohammed Sh. Jebur5

1Biology Department, College of Science, University of Babylon, Babylon, Iraq.
2College of Medicine, Jabir Ibn Hayyan Medical University, Kufa, Najaf, Iraq.
3Pharmacy Department, Imam Hassan College for Medical Science, Iraq.
4Department of Biology, Al-Farabi University College, Baghdad, Iraq.
5College of Health and Medical Technology, Middle Technical University, Baghdad, Iraq.
J Pure Appl Microbiol, 2019, 13 (1): 531-536 | Article Number: 5350
https://dx.doi.org/10.22207/JPAM.13.1.59 | © The Author(s). 2019
Received:29/10/2018| Accepted: 15/12/2018 |Published: 07/03/2019
Abstract

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.

Keywords

Enterococcus spp., VRE, MDR, XDR, LRVRE, TRLRVRE.

Introduction

Enterococci werea Gram-positive, non-spore-forming,catalase-negativefacultative-anaerobe, 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 patients1,2. Enterococci, particularly relevant Enterococcus faecium  and Enterococcus faecalis, have arise as objects of importance because of the distinctness of resistant strains of many drugs3-5. 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 one-third of Enterococcus  isolates6-8. There are three main patterns of resistance: Multi-drug resistance (MDR) was indicated as acquired non-susceptibility 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-microbial9-12. 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 precursors13-15. 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 tetracyclines17. 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 Rifampicin-resistance get up from a range of mutations in the rpoB gene that encodes for the polymerase RNA b-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 methylase17-24. 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 agar25.

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 201626 using standard disk diffusion method upon Muller-Hinton agar after normalization of broth to 0.5 McFarland (1×108 CFU/ml at OD=0.08).

Biosafty Aspects
The biosafty aspects include decontamination of swabs, broth, contaminated disposable and culture medium27.

Results and Discussion

The result of Enterococcus sp. Isolation revealed that, enterococcal diarrhea compile 28(48.3%) Fig. 1. Enterococcus spp. is intestinal opportunistic bacterium with virulence possibilities like protease gelatinase (GelE). It is not naturally virulent but their resistance arrays of antibiotics classes leading to infections in susceptible individual like immuno-compromised,  children and elderly and cancer patients. Infected patients with diarrhea can be the source of MDR-enterococci especially VRE28,29.


Fig. 1. Distribution of Enterococcus spp. among children diarrhea.

Enterococcus spp. regards reservoir of intrinsic and inherently resistance to various antibiotics classes. The results of the current study publicized different resistance percentage as rifampin (92.86%), erythromycin (85.71%), nitrofurantion (64.29%), ciprofloxacin (60.71%), tetracyclin (57.14%), penicillin (53.57%), vancomycin (46.43%), linezolid (35.71%), teicoplanin (25%) and doxycyclin (17.86%) table (1).

Table (1):
Antibiotics resistance percentage among Enterococcus spp.

Antibiotic
Symbol 
Potency (μg)
Resistance %
Rifampin
RA
5
92.86
Erythromycin
E
15
85.71
Nitrofurantion
F
300
64.29
Ciprofloxacin
CIP
5
60.71
Tetracycline
TE
30
57.14
Penicillin
P
10
53.57
Vancomycin
VA
30
46.43
Linezolid
LNE
30
35.71
Teicoplanin
TEC
30
25
Chloramphenicol
CHL
30
21.34
Doxycycline
DO
30
17.86

Fig. 2. Antibiotics resistance patterns among Enterococcus spp.

Table (1) show the results for antibiotic resistance among Enterococcus spp. Our results in accordance with many Iraqi studies like Khalid (2016)30, Chabuck et al., (2011)31, Al-Marjani (2013)32 and Al-Halaby AH, Al-Hashimy (2016)33 who found (72-100%) of enterococci resistance to rifampin  respectively.  Many  studies around the world also stated similar results, resistance to enterococci were (76-100%)34-36. The most common mechanism of resistance to rifampin is mutation in b subunit of RNA polymerase (encodes by rpoB)37. Resistance to erythromycin were (85.71%) and it is quite same those stated in another studies30-33,38-40. Co-existence of  triple resistance to vancomycin/teicoplanin/linezolid were present in 7/13 (53.84%) of vancomycin resistant enterococci (VRE) table (2). Our results is the first who stated co-existence of resistance to vancomycin/teicoplanin/linezolid in Iraq while many studies in Iraq and neighboring countries not state such resistance30-40. The most common phenotypes  of  vancomycin resistance among VRE are VanA and VanB which related to vanA and vanB genotypes. Van A characterized by their co-resistance to both vancomycin and teicoplanin while VanB confer only resistance to vancomycin41. Our results stated both phenotypes, VanA in 7/13 (53.84%) while VanB in 6/13 (46.16%).

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 linezolid42-46.

Table (2):
Co-existence resistance among Enterococcus spp.

Antibiotics co-existence
No.( %)
VancomycinR total (VRE)
13/28 (46.42)
TeicoplaninR total  (TRE)
7/28 (25.00)
LinezolidR total  (LRE)
10/28 (35.71)
VancomycinR/TeicoplaninS/ LinezolidS
6/28 (24.00)
VancomycinR/TeicoplaninR/ LinezolidS  (TRVRE)
0/28 (0.00)
VancomycinR/TeicoplaninR/LinezolidR  (TRLRVRE)
7/28 (25.00)
VancomycinS/TeicoplaninR/ LinezolidR  (TRLRE)
0/28 (0.00)
VancomycinS/TeicoplaninS/ LinezolidR
3/28 (10.71)

Concern the resistance patterns, MDR, XDR and PDR, the results revealed that 1/28 (3.6%), 24/28(85.7%), 3/28(10.7%) and 0/28 (0.00%) of enterococci isolates were non-MDR, MDR, XDR and PDR respectively.

Different percentage of MDR-enterococci were stated in many studies (28-63%)47-50. XDR-enterococci 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 infections51,52.

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.

Conflict of Interest

The authors declare that there are no conflicts of interest.

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