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Sensitivity of different pathogens to biological antimicrobial agents
7. Протоколи надання медично!' допомоги. Стоматологiя. МОЗ Украши. - Кшв, МВЦ «Медшформ», 2005. C. 95-101.
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Israyelyan Arevik, “Artsakh Scientific Center " SNPO, Stepanakert, Nagorno Karabakh Republic Karapetyan Kristina, Scientific and Production Center "Armbiotechnology" NAS, Yerevan, Republic of Armenia Tkhruni Flora,
Scientific and Production Center "Armbiotechnology" NAS, Yerevan, Republic of Armenia Arstamyan Lilya, ‘Artsakh Scientific Center" SNPO, Stepanakert, Nagorno Karabakh Republic Balabekyan Tsovinar, Scientific and Production Center “Armbiotechnology" NAS,
Yerevan, Republic of Armenia E-mail: [email protected]
Sensitivity of different pathogens to biological antimicrobial agents
Abstract: In recent years methicillin-resistant Staphylococcus aureus (MRSA) and Proteus mirabilis are significant public health problem worldwide. The diversity of multidrug-resistance (MDR) of pathogenic strains to antibiotics, most widely used for treatment of human diseases in the Republic of Armenia and Nagorno Karabakh Republic were examined and it was shown, that difference of resistance of pathogens to antibiotics depends on their isolation sources. It was shown, that bacte-riocin containing partially purified metabiotics, obtained from Lactobacillus rhamnosus BTK 2012, Lactobacillus acidophilus 1991, Streptococcus sp.K13 and Lactococcus sp. M44 strains inhibited the growth of multidrug-resistant bacteria with different efficiency and it depending from properties of bacteriocins.
Keywords: multidrug resistance, pathogens, bacteriocins, endemic lactic acid bacteria
Introduction
Selective pressure exerted by the use of antimicrobials in both human and animal populations over the past several decades has led to the emergence of multidrug-resistant (MDR) bacterial populations that are resistant to many
commercially available drugs. This is an increasing threat to both human and animal health [1, 397411]. In the WHO European Region, the resistance of some pathogens now reaches over 50% in some countries, and new resistant mechanism are emerging and spreading rapidly caused by highly
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resistant bacteria. Some of the more problematic drug-resistant pathogens encountered today include methicillin-resistant Staphylococcus aureus, multidrug-resistant Streptococcus pneumoniae, and vancomycin-resistant Enterococcus spp. among the gram-positive bacteria and multidrug-resistant Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa among the gram-negative bacteria [16, http://www.who. int/healthinfo].
Methicillin-resistant Staphylococcus aureus (MRSA) is a significant public health problem worldwide [6, 5572-5579]. Proteus mirabilis causes 90% of Proteus infections and can be considered a community-acquired infection. Proteus vulgaris and Proteuspenneri are easily isolated from individuals in long-term care facilities and hospitals and from patients with underlying diseases or compromised immune systems [5, 2735-2742]. P.aeruginosa presents a serious therapeutic challenge for treatment of both community-acquired and nosocomial infections, and selection of the appropriate antibiotic to initiate therapy is essential to optimizing the clinical outcome [8, 582-610]. Epidemiological outcome studies have shown that infections caused by drug-resistant P.aeruginosa are associated with significant increases in morbidity, mortality, need for surgical intervention, length of hospital stay and chronic care, and overall cost of treating the infection. P.aeruginosa exhibits the highest rates of resistance for the fluoroquinolones, with resistance to ciprofloxacin and levofloxacin ranging from 20 to 35% [8, 582-610].
Research of last decades in the field of antimicrobial technology is the use of biological antimicrobial agents (AMPs), which have bactericidal effect (cell killers) or bacteriostatics (bacteria growth inhibitors) and, therefore, might not induce the resistance. Nonantibiotics such as phytochemical flavonoids (galangin, quercetin and baicalein), bacteriocins, metabiotics and peptides of lactic acid bacteria (LAB) AMPs represent bactericides have a broad range of activity and are excellent candidates for development of new prophylactic and therapeutic substances to complement or replace conventional antibiotics [7, 751-754]. Metabiotics of LAB can contains bacteriocins and
other low molecular weight antimicrobial molecules, short chain fatty acids, various other fatty and organic acids, polysaccharides, peptidoglycans, antioxidants, different proteins including enzymes, peptides with various activities, amino acids and other [13,16]. Some of their properties (significant potency, high stability, low toxicity, broad spectrum ofactivity) make them suitable compounds for using them as a basis for development ofantimicrobial agents of new generation [6, 5572-5579]. Thus, the increasing interest in these compounds has stimulated the isolation of new strains ofLAB -bacteriocin producers and the characterization of novel peptides [4, 95-105].
The aim ofthis studywas comparativeinvestigation and evaluation of multidrug resistance of pathogens isolated from patients of different regions. Investigation of inhibitory activity of metabiotics from L.rhamnosus 2012, L.acidophilus 1991, Streptococcus sp. K13 and Lactococcus sp. M 44 LAB strains and evaluation of their efficiency.
Materials and Methods
Microbial strains and growth media. L. rhamnosus BTK 2012 (MDC 9631) was isolated from salted cheese from rural household of Republic of Armenia (RA). Streptococcus sp. K13 and Lactococcus sp. M 44 were isolated from dairy products from alpine households of Nagorno Karabakh Republic (NKR). LAB strains were characterized by cell morphology, carbohydrate fermentation, identified by using of 16S rRNA sequencing method. LAB strains deposited with the Department of Center of Microbial Depository (CMD) at the SPC “Armbiotechnology” NAS RA.. Strain is available in the collection of the Laboratory of Microbial Preparations of SPC “Armbiotechnology” NAS RA.. L.acidophilus 1991 (VKPM-6527) strain taken from above mentioned culture collection. Pure cultures of LAB were maintained as frozen stocks at -20 oC in the MRS broth containing 40% Glycerol. Strains were cultivated at 37 oC in MRS broth (Merck, Germany).
Test cultures growth. Pathogenic bacteria were isolated from patients from Infection Hospitals (Armenia and Nagorno Kharabakh). Antibioticresistance of pathogenic strains was determined in the Institute of Epidemiology, Microbiology and
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Sensitivity of different pathogens to biological antimicrobial agents
Parasitology of Ministry of Health of Armenia and in the Stepanakert Center for Hygiene and Epidemiology. Bacteria were grown in Nutrient agar (Himedia, India) at pH 7.2 for 16 hours and at 37С, then harvested and suspended in the Nutrient broth at 2.2x106 CFU/ml.
Determination of resistance to antibiotics.
To determine the resistance of isolated pathogens to antibiotics, the method with standard antibiotic disks (Oxoid, UK) was applied [2, 493-496].
Determination of antimicrobial activity. The antimicrobial activity calculated according to [11, 95-108] and expressed in arbitrary units (AU/ml).
Obtaining of metabiotics. Cell free culture broth of LAB strains was purified by combination of adsorbtion-desorbtion and ion-exchange chromatography methods [17, 3355-3359].
Results
Previously we have shown the morphological and physiological features, carbohydrate fermentation,
probiotic properties, specific growth rate and broad spectrum ofantimicrobial activity ofsome investigated LAB strains (from Armenia and Nagorno Kharabakh Republics) against Gram-positive and Gram-negative organisms [14, 109-115].
More than 60 strains of human pathogens were isolated from infected patients (feces, urine, wounds, blood, throat, etc) in the Infection Hospitals of Yerevan (Group 1) and Stepanakert (Group 2). The multidrug resistance ofhuman pathogenic strains of Salmonella spp, Staphylococcus spp, Pseudomonas aeruginosa strains, Proteus spp., Klebsiella pneumoniae В and E.coli strains to antibiotics, widely used in medical practice in Armenia and NKR was determined. Comparative results of resistance of human pathogens isolated from different sources and from patients from different Republics are shown in the Table 1. As it seen from the given results, the differences of resistance to antibiotics are present between pathogens of two Groups.
Table 1. - Comparative data of resistance of pathogens to different classes of antibiotics
Classes of antibiotics Antibiotics Resistance of pathogens
RA N1 KR
N % N %
Amino-glycosides Amikacin 30 fig 23 52.2 42 9.52
azithromicin 15 fig 22 59.1 20 30
gentamicin 120 fig 15 20.0 37 10.8
Beta-lactams augmentin 30 EG 5 100 34 55.8
cefalotin 30 fig 16 81.25 51 37.25
oxacillin 5 fig 9 77.8 Nd Nd
cefazolin 30 fig 23 56.5 52 46.15
ceftazidime 30 fig 22 50 25 48
cefuroxime 30 fig 23 47.8 Nd Nd
Tetracyclines doxycyclin 10 fig 23 78.3 28 75
Fluoroquinolones ofloxacin 5fig 23 17.4 35 48.57
ciprofloxacin 5 fig 22 18.2 49 32.65
Legend:
N - absolute amount of pathogenic strains % - average% of resistance Nd - not determined
As it seen from the given results, the studied strains, isolated from patients from Group 1 showed low resistance to fluoroquinolones — 17.4-18.2% (respectively to ofloxacin and ciprofloxacin). Strains from Group 2 were more resistant to fluoroquinolones (32.6 -48.6%). Pathogenic strains from Group 1 were highly resistant to a beta-lactams
(50-100%). At the same time, pathogenic bacteria from Group 2 were less resistant to antibiotics of same class. Pathogenic bacteria of two groups reveal high resistance to doxycyclin. The bacteria from Group 2 were essentially less resistant to aminoglycosides, when pathogens from Group 1.
So, strains of pathogenic bacteria, isolated from patients from Armenia and Nagorno Karabakh Republic hospitals differ by their sensitivity to investigated antibiotics. This can be explained by different ecological conditions of two countries
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and by the fact, that traditionally in NKR antibiotics are used less than in Armenia. So often use of antibiotics in Republic of Armenia brings to development of multidrug resistance of investigated pathogens.
P. aeruginosa infection can lead to urinary tract infections, sepsis (blood stream infection), pneumonia, pharyngitis, and many other medical problems. Proteus found in multiple environmental habitats, including long-term care facilities and hospitals. Proteus mirabilis causes 90% of Proteus infections and can be considered a community-acquired infection [5, 2735-2742]. The food supply, including poultry products, may transmit antimicrobial drug-resistant
Escherichia coli to humans. Acquired resistance to first-line antimicrobial agents increasingly complicates the management of extra-intestinal infections due to Escherichia coli, which are a major source of illness, death, and increased healthcare costs [12, 449-456] http: / / dx.doi.org/10.1016/ S1286-
4579%2803%2900049-2.
The investigation of differences in antibiotic resistance ofhuman pathogens, isolated from different sources was of interest. Results of investigation are summarized in Figure 1 and shown on the example of analysis of multidrug resistant pathogens isolated from patients from Nagorno Karabakh Republic hospitals.
I blood
feces
I unite
saliva
I wound
I throat.
Fig.1 Comparative data of resistance of pathogens isolated from different sources
Obtained results showed, that antibiotic resistance of bacteria belonging to different species depends on their isolation sources (blood, faces, urine, et str). Highest number of bacteria was isolated from blood, urine and faces. The Ps. Aeruginosa and Klebsiella sp. Were more resistant to the action of antibiotics (about 50%), then strains of E.coli species.
The partially purified metabiotics from different strains of LAB, suppressing pathogenic bacteria growth, were obtained by us. It was approved, that Lactobacillus rhamnosus BTK 2012 contains 2 active bacteriocins (BCN). Mass spectrograms demonstrate that molecular weight of BCN 1 and BCN 2 are 1,427 Da and 602.6 Da respectively.
Lactobacillus acidophilus 1991 (VKPM-6527)
produces a small peptide with a molecular weight of 1.1 kDa, designated acidocin LCHV This peptides showed broad spectrum of activity against Grampositive and Gram-negative organisms [14, 109-115; 9, 208-213].
The influence of partially purified metabiotics, obtained from L.acidophilus 1991, L.rhamnosus BTK 2012 (RA) and metabiotics from Streptococcus sp.K13, Lactococcus sp. M44 (NKR) on the growth of pathogenic bacteria were examined. The investigation results in comparison with antibiotics are presented in the Table 2.
As it seen from the given results, the biological metabiotics of different LAB strains inhibited
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Sensitivity of different pathogens to biological antimicrobial agents
the growth of pathogenic bacteria with different efficiency and it depends on sources of pathogens isolation. Average statistical results proved, that metabiotics, obtained from LAB of RA., do not concede to antibiotics by their antibacterial activity. Strains of LAB from NKR shown less
efficiency against pathogens in comparison with antibiotics. Staphylococcus aureus strains 44% sensitive to Lactobacillus rhamnosus 2012 metabiotic and 85% sensitive to L.acidophilus-1991 metabiotic in concentration 100AU/ml. The sensitivity of Proteus mirabilis to investigated LAB metabiotics was 50%.
Table 2. Comparative antimicrobial activity of metabiotics (100AU/ml) and different antibiotics
Antibiotic resistant strains N L.rhamnosus BTK 2012 L.acido philus 1991 Streptococcus sp. K13 Lactococcus sp. M 44 Antibiotics N=19
Sensitivity,%
Staph. aureus sp. 28 44,5 38,8 25,0 0 48,7
Ps. aeruginosa 21 80,0 80,0 57,1 50,0 49,8
Pr. mirabilis 23 50,0 50,0 28,6 50,0 55,0
Klebsiella sp. 8 50,0 50,0 0 0 53,4
Pr. vulgaris 7 57,1 57,2 0 0 61
E.coli sp. 15 42,9 42,9 14,2 50,0 71,5
We have previously shown that the LAB strains showed different efficiency against several species of Salmonella and E.coli genius [9, 208-213]. Obtained data are consistent with the published data of several authors. It was shown that the probiotic culture Enterococcus faecium L3 possess diverse ability to inhibit the growth of Streptococcus sp. belong to different genus [18, 363-366]. However, the literature data on the antagonistic activity of LAB against streptococcus sp. bacteria are single scattered reports [3, 82-100; 15, 43-56].
It should be noted that the differences in the sensitivity of Listeria, Clostridium, Propionibacterium, enterococcus sp., oral Streptococcus sp. to the action of lactic acid bacteria bacteriocins, described by other authors [10, 214-220; 15, 43-56]. Above all, they explain it with the presence of specific receptor proteins required for binding to bacteriocins and their transport into the bacteria [10, 214-220]. The diverse efficacy of growth inhibition may probably relate to
the different mechanisms of action of the substances towards bacteria cell membrane.
This can be explained by different ecological conditions of two countries and by the fact, that traditionally in NKR antibiotics are used less than in Armenia. In NKR for treatment of gastrointestinal disorders used different dairy products instead of antibiotics. So often use of antibiotics in Republic of Armenia brings to development of multidrug resistance of investigated pathogens.
Thus, prospect of the use of certain metabiotics against pathogenic strains is obvious. These metabiotics can be applied for long term use against different etiology antibiotic resistant pathogens for prevention or treatment of infectional diseases as an alternative to antibiotics.
Acknowledgments. This work was realized with financial support of the Biochem-3820 research grant from the Armenian National Science and Education Fund (ANSEF) based in New York, USA”.
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