2. ENVIRONMENTAL ChEMISTRy & TEChNOLOGy 2.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Environmental Chemistry & Technology
P71 ANTIbIOTIC EFFECTS OF ThE
NAPhTOQuINONIC DERIVATIVE ON GRAM-
POSITIVE AND GRAM-NEGATIVE GERMS
RADU TAMAIAn a , nADIA PăUn a , VIOLETA
nICULESCU a , AnDREEA IORDACHE a , RALUCA
VREMERă a and şTEFAnIA BROSCăŢEAn b
a Research and Development Department, National Research
and Development Institute for Cryogenics and Isotopic Technologies
– ICIT Râmnicu Vâlcea, Uzinei Street No. 4 240050,
Râmnicu Vâlcea, Romania,
b S.C. IMOFARM S.R.L., Piscului Street No. 15 040403, Bucu-
reşti, Romania,
tradu@icsi.ro
Introduction
Intestinal motile bacteria Enterobacter aerogenes and
nonmotile coccus Enterococcus faecalis are known as being
contaminating pathogenetic agents of aliments 1–3 and drinking
water 4 . Pathological agents: coccus and bacteria have a
significant multiple-antibiotic resistance at natural drugs and
also at chemically obtained drugs. 5–10
The naphthoquinones are a wide class of plants’ metabolites,
which are currently used for manufacturing cosmetics,
foods or for medicinal purposes 11,12 . Also the natural
naphthoquinones were tested for antitumoral 13 , antiinflamatory
14 and antimicrobial 15,16 activity. For antibiotic activity
is being responsible the compound structure, therefore the
aim of any structure-activity relationship should be to find
the most potent and least toxic compound. In this respect,
we studied the antibiotic effect of dichlor derivative of 1,4naphthoquinone:
2,3-dichloro-1,4 naphthoquinone (dichlone
– Fig. 1.) on Gram-positive nonmotile coccus Enterococcus
faecalis, and Gram-negative motile bacteria Enterobacter
aerogenes.
Fig. 1. Dichlone structure
In order to identify dichlone’s grade of remanence, in the
inoculated culture mediums, it was used HPLC technique.
Experimental
M a t e r i a l s a n d E q u i p m e n t s
Dehydrated culture media: primary culture media TSA
(tryptic soy agar) from Fluka (Buchs, Switzerland) and M-H
broth (Müeller-Hinton broth) from Fluka (Buchs, Switzerland)
for antibiotic susceptibility testing.
s479
Both culture media were rehydrated with ultrapure
water made with water ultra-purifier TKA Smart2Pure UV6
from TKA Wasseraufbereitungssysteme GmbH (niederelbert,
Germany).
Used microbial strains were made by MicroBioLogics
Inc. (Saint Cloud, USA), as following:
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•
•
KWIK-STIK: Enterococcus faecalis (ATCC 29212);
KWIK-STIK: Enterococcus faecalis (ATCC 19433);
KWIK-STIK: Enterobacter aerogenes (ATCC
13048).
Culture media were sterilised with a Raypa AES-75
autoclave from R.ESPInAR, S.L. (Barcelona, Spain).
All cultures were incubated in a Raypa Incuterm ID-50
incubator from R.ESPInAR, S.L. (Barcelona, Spain).
Barium chloride from Utchim S.R.L. (Râmnicu Vâlcea,
Romania) and sulfuric acid from Utchim S.R.L. (Râmnicu
Vâlcea, Romania) were used for preparation of McFarland
turbidity standards.
Odyssey DR/2500 spectrophotometer from Hach Company
(Loveland, USA) was used for inoculums’ and prepared
McFarland turbidity standards’ OD (optical density) determination.
For antimicrobial susceptibility testing it was used dichlone
from Merck (Darmstadt, Germany).
Dichlone’s grade of remanence was detected with HPLC
equipment: Thermo Finnigan Surveyor system from Thermo
Fisher Scientific Inc. (Waltham, USA). PDA detector set to
254 nm. Hypersil GOLD Column 100 × 4.6 mm.
Dichlone standard for HPLC: dichlone standard from
Supelco (Bellefonte, USA).
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•
Other reagents for HPLC technique:
Acetonitrile from Merck (Darmstadt, Germany);
Water Chromasolv from Honeywell International Inc.,
Riedel-de Haën (Seelze, Germany).
M e t h o d s
Primary and secondary cultures: the KWIK-STIK
lyophilized microorganisms’ strains (Enterococcus faecalis
ATCC 29212 and ATCC 19433; Enterobacter aerogenes
ATCC 13048) were transferred on Petri dishes with sterile
TSA and incubated at 35 °C for 24 hours (primary cultures);
then, subcultures were made from primary cultures and incubated
also on sterile TSA for 24 hours at 35 °C.
Antimicrobial susceptibility testing by broth microdilution
technique: dichlone’s MIC (minimal inhibitory concentration
– defined as drug concentration at which no growth is
visible) was determined by broth microdilution technique. A
sterile culture tube with M-H broth was inoculated with an
aliquot from the three types of secondary growth. Inoculated
broth’s OD was adjusted at 0.5 McFarland standard with prepared
turbidity standards – aproximatively 1 × 10 8 CFU ml –1 .
Standardised inoculums’ aliquots from all three types of
secondary culture were transferred on culture tubes with sterile
liquid medium: M-H broth. Dichlone’s MIC was tested
on those standardised inoculums, on M-H broth.