ISTC # G-597p

BTEP # 10

Molecular Epidemiology and Antibiotic-resistance of Bacterial Infections in Georgia

Annual Report

I. Short Report

1. Name: Molecular Epidemiology and Antibiotic-resistance of Bacterial Infections in Georgia

Project Number: ISTC G-597p

BTEP # 10

2. Leading Institution: National Center for Disease Control (NCDC) of Georgia

3. Other participating Institutions: N/A

4. Project Manager: Lela Bakanidze, Ph.D. Tel.: (995 32) 39 89 46. Fax: (995 32) 94 04 85. E-mail: threatred@ncdc.ge.

5. Project Start: January 1, 2002.

Project Duration: 3 years.

6. Project objectives:

The main objective of the Project is establishing a well-equipped microbiology laboratory/antimicrobial-resistance testing reference center at the NCDC, and providing accurate data on the prevalence and antibiotic-resistance patterns of major bacterial pathogens in Georgia. The project is designed to address several critical aspects of the problem of antimicrobial resistance in Georgia, with the ultimate goal of limiting the development of bacterial resistance in, and its spread from, this developing country. The project is based on a strong research component and involves close collaboration between Georgian researchers and investigators in the United States.

7. Scope of Activities and Scientific – Technical Approach

Surveillance/sample collection activities are limited to three major sites in Georgia, instead of trying to cover numerous Georgian hospitals and private clinics. In eastern Georgia, strains are collected from five hospitals in Tbilisi: Children’s Infectious Diseases Hospital, Children’s Republican Hospital, the Sepsis Center, Republican Clinical Hospital, and Center for Infectious Pathology were chosen. In Central Georgia, samples were taken from the Kutaisi Regional Department of Health laboratory. In south and southwest Georgia, bacterial strains were collected at the NCDC Black Sea Laboratory, Batumi.

The isolates were speciated using standard biochemical tests at the NCDC, and their resistance to various antibiotics was determined by the disc-diffusion method, according to NCCLS guidelines.

One of the subaims of this study is to develop an MLST approach for characterizing enterococci, for which an MLST approach has not yet been developed. During the development of the MLST approach for characterizing enterococci, specific primers will be designed and nucleotide sequences will be determined for up to 12 loci of ca. 20 enterococcal strains (with the forward and reverse primers separated by ca. 500 bp, to facilitate sequencing of amplified fragments). The results will be compared with the PFGE data, and a group of 4-6 loci will be selected (based on the discriminatory ability) and used for MLST of all of the enterococcal strains in our collection. Briefly, the desired genes will be amplified and the amplified fragments will be subjected to cycle sequencing using fluorescent dye-labeled dideoxynucleotides (each of the nucleotides will have a different fluorescent tag, so only a single reaction will be needed for each DNA sequence).

Our educational activities include publishing detailed brochures and handouts on antibiotic resistance issues.

8. Summary of Technical Progress during the Reported Year.

During the reporting period tasks were performed as follows:

Task # 1. According to the workplan, we were gathering materials from hospitals in Tbilisi and regions from Western part of Georgia. The list of hospitals in Tbilisi was - Children’s Infectious Diseases Hospital, Children’s Republican Hospital, the Sepsis Center, Republican Clinical Hospital, and Center for Infectious Pathology were chosen. In Central Georgia, samples were taken from the Kutaisi Regional Department of Health laboratory. In south and southwest Georgia, bacterial strains were collected at the NCDC Black Sea Laboratory, Batumi.

Probes were taken from patients’ pus, wound smear, blood, pleural fluid, eye excretion, throat and nose mucus, cerebrospinal fluid, mouth smear, urine, ear excretion, feces, etc.

More than 2000 samples were brought from all hospitals. Totally 1082 “strains of interest” were isolated.

It must be mentioned, that though the investigating in the scope of the Project is concentrated on Staphylococcus, Pseudomonas and Enterococci, we have isolated and investigated other bacteria as well.

All isolates are kept at the microbial library of NCDC.

Retrospective and prospective studies of histories of patients with infectious complications after surgery were carried out. Together with studies of histories we were studying the literature on antibiotic susceptibility.

We have visited poultry farms in different parts of Georgia. 26 strains of S. aureus, 20 strains of S. epidermidis, 20 strains of E. coli and 15 strains of E. faecalis were isolated from the poultry, farmers and environmental samples.

Task # 2. Epidemiologists visited hospitals in Tbilisi, where data on using antibiotics was gathered.

Resistance of 654 isolates of Staphylococcus aureus was investigated to antibiotics: penicillin, ampicillin, erythromycin, oxacillin, gentamycin, ciprofloxacin, chloramphenicol, nitrofurantoin, tetracycline, tobramycin, trimethoprim, clindamycin, vancomycin, amikacin, amoxicillin – clavulanic acid and imipenem.

193 strains of S. aureus were resistant to oxacillin. Among these isolates multidrug resistance was seen too.

Resistance of 303 isolates of Pseudomonas aeruginosa to 13 antibiotics was investigated. These antibiotics were: gentamycin, ciprofloxacin, chloramphenicol, tetracycline, tobramycin, trimethoprim, clindamycin, carbenicillin, azithromycin, nitrofurantoin, amikacin, amoxicillin – clavulanic acid, piperacillin, cephepin and imipenem.

43 isolates of P. aeruginosa were resistant to all tested antibiotics. 19 isolates were resistant only to imipenem, 1- to piperacillin, 7- to piperacillin and imipenem, 1 – to piperacillin and ciprofloxacin, and 2 – only to ciprofloxacin.

Resistance of 222 isolates of Enterococcus faecalis to 8 antibiotics was investigated. These antibiotics were: penicillin, erythromycin, ciprofloxacin, chloramphenicol, nitrofurantoin, tetracycline, vancomycin and rifampicin.

Task # 3. During the first year we had studied DNA of 111 strains of Enterococcus faecalis and about 30 strains of Staphylococcus aureus on existence of genes of resistance to some antibiotics.

It was established, that in all resistant to tetracycline strains of E. faecalis only 741 fragment was amplified, corresponding to the resistance gene tet (M). None of investigated cases showed amplification of resistance gene tet (O).

In experiments aiming to find genes of resistant to gentamycin we used pair of primers determining amplification of 880 pair fragment. In our experiments amplification of the 880 bp size fragment was not observed. Fragments of size 450-500 bp were amplified.

In such case assuming that we have found the gene of resistance to gentamycin we do not consider correct, as the size of amplified fragments do not correlate with their theoretical one. The necessity of using other pairs of primers amplifying other specific locuses of the gentamycin - resistance gene is evident.

In investigated strains of S. aureus, resistant to oxacillin, we checked existence of gene of resistance to methicillin. In all strains specific fragment of gene of methicillin – resistance was amplified.

We want on getting acquainted with the literature on the subject, new methods of investigation.

Task # 4. During the second quarter two participants of the Project – Nikoloz Tsertsvadze and Giorgi Kurtsikashvili visited Kutaisi and Batumi, and though this visit was not in the scope of the Project, they took part in seminars on “Infections Control at Hospitals and Use of Antibiotics; Susceptibility of Microorganisms to Antibiotics: Modern Methods of Investigation”. Physicians from hospitals in Kutaisi and Batumi that provide us with materials, took part in these seminars. They got acquainted with our investigation, visited local laboratories where material is gathered befor transporting to NCDC, and showed great interest to our work.

Two participants of the Project – Manager Lela Bakanidze and WhoNet Giorgi Kurtsikashvili visited Kutaisi and Batumi. We had carried out quality control of all documentation, and provided physicians from hospitals in Kutaisi and Batumi materials, Informed Consent forms. We had visited local laboratories where material is gathered befor transporting to NCDC, and showed great interest to our work.

Web page on the project activities is being prepared. It will be linked to NCDC page in the internet.

The leaflet on prejudice use of antibiotics in Georgia is being prepared.

9. Plan for next years – has not been changed.

10. Publications.

The abstract of the poster presentation “ANTIBIOTIC RESISTANCE AMONG CLINICAL S. aureus, P. aeruginosa and Enterococcus ISOLATES IN GEORGIA” - 13^th annual Scientific Meeting of the society for Healthcare Epidemiology of America (SHEA) (See Annex 1).

II. Annual Report

1. Name: Molecular Epidemiology and Antibiotic-resistance of Bacterial Infections in Georgia

Project Number: ISTC G-597p

BTEP # 10

2. Leading Institution: National Center for Disease Control (NCDC) of Georgia

3. Other participating Institutions: N/A

4. Project Manager: Lela Bakanidze, Ph.D. Tel.: (995 32) 39 89 46. Fax: (995 32) 94 04 85. E-mail: threatred@ncdc.ge.

5. Project Start: January 1, 2002.

Project Duration: 3 years.

6. Summary of the Project:

Project goals: establishment of a well-equipped microbiology laboratory/antimicrobial-resistance testing reference center at the NCDC, and providing accurate data on the prevalence and antibiotic-resistance patterns of major bacterial pathogens in Georgia. The project is designed to address several critical aspects of the problem of antimicrobial resistance in Georgia, with the ultimate goal of limiting the development of bacterial resistance in, and its spread from, this developing country. The project is based on a strong research component and involves close collaboration between Georgian researchers and investigators in the United States. More specifically, four major tasks are jointly addressed: (i) a central laboratory-based surveillance will be undertaken in Georgia for three major nosocomial pathogens of particular public health concern (“strains of interest”), including enterococci (with an emphasis on E. faecalis and E. faecium), S. aureus, and P. aeruginosa, (ii) antibiotic-resistance patterns of the strains of interest will be determined using modern antimicrobial susceptibility-testing techniques (conventional and genetic-based), with appropriate quality control performed at the University of Maryland, (iii) molecular subtyping of the strains of interest will be performed by pulsed field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) techniques, and internet-based data exchange capabilities will be established at the NCDC; in addition, a novel typing methodology based on the multilocus sequence typing (MLST) technique will be developed for enterococci, and the enterococcal strains isolated in Georgia and the United States will be comprehensively analyzed for the first time by this novel approach, and (iv) educational materials (e.g., brochures and handouts) about the judicious use of antibiotics will be developed and distributed to all major hospitals and medical centers in Georgia. As a result of these activities, several Georgian scientists previously engaged in research associated with biological warfare will be able to redirect their activities to address one of the most urgent Georgian and global public health concerns, and they will have an opportunity to integrate their talents into the international scientific community. In addition, the successful completion of the project will significantly improve antibiotic-resistance testing capabilities in Georgia and will provide rigorous data about multidrug-resistant bacteria in the country - which will be critical for designing proper control and implementation strategies to prevent the spread of antibiotic-resistant bacteria in Georgia and to other countries from Georgia. Furthermore, a new typing methodology for characterizing enterococci will be developed based on the MLST approach, which will provide a means for (i) better understanding the epidemiology of these bacteria and the mechanisms of spread of antibiotic-resistance (specifically against vancomycin) in enterococci, and (ii) delineating phylogenetic relationships between enterococcal strains isolated in Georgia and the United States. From a basic science standpoint, data generated during the project will (i) broaden our understanding of the mechanisms for the development of antimicrobial resistance, (ii) will provide insight into the possible links between the use of certain antibiotics and development of resistance, and (iii) may lead to the identification of new resistance mechanisms/genes in a bacterial population that has not been previously available to US researchers for detailed analysis.

Expected results: The successful completion of the project will lead to the establishment of a well-equipped microbiology laboratory/antimicrobial-resistance testing reference center at the NCDC, and will provide accurate data on the prevalence and antibiotic-resistance patterns of major bacterial pathogens in Georgia. These data will be critical for the development and implementation of science-based practical control measures in Georgia, by the Georgian Ministry of Health and by various US and/or other agencies and institutions in collaboration with the Georgian Ministry of Health. In addition, data sharing capabilities (via the Internet) will be established at the NCDC, which will enable US and Georgian researchers to track the emergence and spread of antibiotic-resistant bacteria in and from Georgia, as well as to conduct sophisticated molecular epidemiological analysis of disease outbreaks. Also, a new typing methodology for subtyping enterococci will be developed based on the MLST approach, which should provide means for better understanding the epidemiology of these bacteria, and for determining the degree of genetic clonality and phylogenetical relationship between enterococci strains isolated in Georgia and the United States. In addition to having direct public health improvement implications, we believe that the project will generate novel and valuable data worthy of presentation at various national and international meetings, and suitable for publication in several peer-reviewed scientific journals.

Scientific – Technical Approach is described according to Project tasks.

Task # 1. Conducting a central laboratory-based surveillance in Georgia for major nosocomial pathogens of particular public health concern (“strains of interest”). Surveillance for nosocomial infections is an integral component of infection control programs, and is critical for evaluating both the magnitude of the problem and the efficacy of the implemented infection control measures. However, surveillance systems, both active and passive, tend to become ends in themselves and generate unused and unusable data, and they can become an intolerable burden for the public health infrastructure, especially in developing countries. Therefore, in order to make the proposed antimicrobial-resistance/molecular epidemiology program maximally efficient with the limited resources available (and in a country whose public health infrastructure is severely underdeveloped), we wee focused on a limited number of pathogens that are of critical public health importance, such as S. aureus, enterococci (especially vancomycin-resistant mutants), and P. aeruginosa. The major reason for focusing on these pathogens is that the development of drug-resistance in these pathogens is a major problem worldwide, and it is of the utmost public health importance to determine the prevalence of such strains in Georgia. Surveillance/sample collection activities are limited to three major sites in Georgia, instead of trying to cover numerous Georgian hospitals and private clinics. In eastern Georgia, strains are collected from five hospitals in Tbilisi: (i) Children’s Infectious Diseases Hospital, (ii) Children’s Republican Hospital, (iii) the Sepsis Center, (iv) Republican Clinical Hospital, and (v) Center for Infectious Pathology (which, in total, have ca. 900 beds). In Central Georgia, samples are collected from the Kutaisi Regional Department of Health laboratory (servicing hospitals with approximately 600 beds), and in south and south-west Georgia, bacterial strains are collected at the NCDC Black Sea Laboratory, which services hospitals having approximately 200 beds and is located in Batumi, West Georgia. While this design is centralized and the majority of strains are recovered in the Tbilisi area, it still enables collection of many strains of interest from south/south-west and central regions of Georgia, which, in turn, permits a generalization of the data on the prevalence of antimicrobial resistant strains (and various molecular types) in these regions.

Task # 2. Determining antibiotic-resistance patterns of the strains of interest using modern antimicrobial susceptibility testing techniques (conventional and genetic-based). NCDC microbiologists are proposed to (i) be trained in modern antimicrobial susceptibility testing techniques; this involves training one senior Georgian microbiologist at the University of Maryland (6 months), and in-country training of a large group of Georgian microbiologists, and (ii) provide the NCDC with kits, primers, genetic probes, reagents, and supplies required for antimicrobial susceptibility testing by conventional and genetics-based techniques. If additional, custom-designed primers are required, they will be developed jointly by the US and Georgian investigators, will be synthesized at the University of Maryland, and will be provided to the NCDC investigators on an as-needed basis. In developing new primers, we target the sequences within the open reading frame of the resistance gene (to avoid the possibility of selecting a primer that may contain insertion elements or promoter sequences that may also be present in susceptible strains, and subsequently reduce the risk of obtaining false-positive results). In addition, efforts will be made to select primers that will amplify fragments having distinct lengths (at least 250 bp difference), so that several sets of primers can be combined during a single PCR amplification, which, in turn, will enable detection (and differentiation) of several resistance genes in a single strain during a single PCR experiment. In the same context, the values of combining the primers for vanA and vanC-1 genes also will be examined; the fragments amplified by these primers in a single PCR reaction are sufficiently distinct in size (885 and 467 bp, respectively) to enable their differentiation and, therefore, identification of the amplified product in an agarose gel. A similar approach has been shown to be effective during PCR detection of genes encoding aminoglycoside-modifying enzymes, and it can be of much value in areas having limited resources. Special attention will be paid to quality control, in order to (i) assure the accuracy of the testing, and (ii) evaluate the correlation between the conventional and genetics-based testing approaches at the NCDC. Efforts must made to determine whether there is a correlation between increased resistance to one or more tested antibiotics and the year, source, and place of isolation of the corresponding strain. This should enable us to gain insight into the kinetics of development of resistance against specific antibiotics used in Georgia for more than two decades.

Task # 3. Molecular subtyping of the strains of interest, developing of a novel typing methodology for enterococci, and establishing data-exchange capabilities between the NCDC and other laboratories via the Internet. Nowadays it is becoming increasingly urgent to have a standardized procedure for subtyping bacterial isolates, in order to delineate the routes of global transmission of emerging pathogens and to design science-based, prevention strategies. PFGE is currently the CDC-designated “method of choice” for typing bacteria, and it has been used extensively in molecular-epidemiological investigations of disease outbreaks. PFGE is also the only molecular typing technique that is approved, for outbreak investigations, by the foodborne diseases active surveillance network (FoodNet) administered by the CDC. One of the subaims of this study will be to develop an MLST approach for characterizing enterococci, for which an MLST approach has not yet been developed. During the development of the MLST approach for characterizing enterococci, specific primers will be designed and nucleotide sequences will be determined for up to 12 loci of ca. 20 enterococcal strains (with the forward and reverse primers separated by ca. 500 bp, to facilitate sequencing of amplified fragments). The results will be compared with the PFGE data, and a group of 4-6 loci will be selected (based on the discriminatory ability) and used for MLST of all of the enterococcal strains in our collection. Briefly, the desired genes will be amplified and the amplified fragments will be subjected to cycle sequencing using fluorescent dye-labeled dideoxynucleotides (each of the nucleotides will have a different fluorescent tag, so only a single reaction will be needed for each DNA sequence).

Task # 4. Developing and conducting educational activities concerning the judicious use of antibiotics in Georgia. Three examples of possible topics include (i) novel approaches for determining antimicrobial susceptibility, (ii) novel methodologies for bacterial typing and their application to molecular epidemiology, and (iii) the effect of antibiotic misuse on the development of resistance against commonly used antibacterial agents. The emphasis is on conveying the importance of the following four principles of rational antibiotic use (i) accurate diagnosis, (ii) knowledge of the antibiotic susceptibility pattern of the disease-causing bacterium, (iii) selection of the appropriate antibiotic having a narrow spectrum of activity, and (iv) the use of appropriate dose and duration of treatment. Our educational activities include publishing detailed brochures and handouts on antibiotic resistance issues.

7. Summary of Technical Progress for the First year of project implementation – N/A.

8. Summary of Technical Progress during the Reported Year.

During the reporting period tasks were performed as follows:

Task # 1 implies conducting a central laboratory-based surveillance in Georgia for major nosocomial pathogens of particular public health concern (“strains of interest”).

According to the workplan, we were gathering materials from hospitals in Tbilisi and regions from Western part of Georgia. The list of hospitals in Tbilisi was - Children’s Infectious Diseases Hospital, Children’s Republican Hospital, the Sepsis Center, Republican Clinical Hospital, and Center for Infectious Pathology were chosen. In Central Georgia, samples were taken from the Kutaisi Regional Department of Health laboratory. In south and southwest Georgia, bacterial strains were collected at the NCDC Black Sea Laboratory, Batumi.

Probes were taken from patients’ pus, wound smear, blood, pleural fluid, eye excretion, throat and nose mucus, cerebrospinal fluid, mouth smear, urine, ear excretion, feces, etc.

More than 2000 samples were brought from all hospitals. Totally 1082 “strains of interest” were isolated.

Table 1. Isolation of “Strains of Interest” by Quarters of the First Year.

Microorganism	Quarters
Microorganism	I	II	III	IV	Total
Staphylococcus aureus	222	165	117	150	654
Pseudomonas aeruginosa	96	73	79	55	303
Enterococcus faecalis	39	100	20	63	222
Total:	357	338	216	268	1179

Fig 1. Isolation of “Strains of Interest” by Quarters of the First Year of the Project Implementation.

All isolates are kept at the microbial library of NCDC.

Table 2. Isolated microorganisms (other from “Strains of Interest”) by Quarters of the First Year.

Microorganism	Quarters
Microorganism	I	II	III	IV	Total
Proteus mirabilis	4	2	3	1	10
Proteus vulgaris	14	28	14	26	82
Escherichia coli	18	50	53	53	174
Staphylococcus epidermidis	24	84	49	88	245
Gram (-) non-fermenting bacilli	2	5	-	1	8
Saprophyte flora	-	-	-	56	56
Acinetobacter calcoaceticus	2	1	1	-	4
Klebsiella	-	10	-	1	11
Candida albicans	24	65	-	-	89
Salmonella typhimurium	16	4	52		72
Citrobacter	-	-	-	2	2
Aeromonas	-	-	-	2	2
Serratia marcescens	-	-	-	1	1
Shigella species	9	-	-	-	9
Conventionally pathogenic enterococci	67	-	-	-	67
Total:	180	249	172	231	832

Epidemiologists visited hospitals in Tbilisi, where data on using antibiotics was gathered.

Task # 2. Determining antibiotic-resistance patterns of the strains of interest using modern antimicrobial susceptibility testing techniques (conventional and genetic-based)

193 strains of S. aureus were resistant to oxacillin. Among these isolates multidrug resistance was seen too.

MRSA strains were resistant also to various mixtures of all tested antibiotics, except vancomycin. These variations are:

1. penicillin – ampicillin (5),

2. penicillin - ampicillin – gentamycin (2),

3. penicillin - ampicillin – tobramycin (4),

4. penicillin - ampicillin – erythromycin – (2);

5. penicillin - ampicillin – erythromycin - tetracycline – clindamycin (2);

6. penicillin - ampicillin – chloramphenicol – nitrofurantoin – trimethoprim (2),

7. penicillin - ampicillin –gentamycin - chloramphenicol - tobramycin – (2);

8. penicillin - ampicillin – erythromycin - chloramphenicol - clindamycin – (1);

9. gentamycin – tetracycline - tobramycin - clindamycin - amikacin (2),

10. penicillin - ampicillin – gentamycin – chloramphenicol - tobramycin - amikacin (4);

11. penicillin - ampicillin – tetracycline - tobramycin - clindamycin – amikacin – (3);

12. penicillin - ampicillin – gentamycin – chloramphenicole - tobramycin - amikacin – clindamycin (14),

13. penicillin - ampicillin – erythromycin - gentamycin –tetracycline - tobramycin – trimethoprim (24),

14. penicillin - ampicillin – gentamycin – tetracycline - tobramycin - amikacin – clindamycin (18),

15. penicillin - ampicillin – erythromycin - gentamycin – tetracycline - tobramycin - amikacin – clindamycin (12),

16. penicillin - ampicillin – gentamycin – tetracycline - tobramycin – clindamycin - amikacin – imipenem (2),

17. penicillin - ampicillin – erythromycin - gentamycin – ciprofloxacin - chloramphenicole - tobramycin – trimethoprim - clindamycin - (2),

18. penicillin - ampicillin – erythromycin - gentamycin – chloramphenicol - tetracycline - tobramycin – amikacin (3),

19. penicillin - ampicillin – erythromycin - gentamycin – ciprofloxacin - tetracycline - tobramycin – clindamycin – imipenem(2),

20. penicillin - ampicillin – erythromycin - gentamycin – ciprofloxacin – chloramphenicol -tetracycline - tobramycin – clindamycin – amikacin (4),

21. penicillin - ampicillin – erythromycin - chloramphenicol - trimethoprim - clindamycin - 1;

22. penicillin - ampicillin - erythromycin - tetracycline - nitrofurantoin - clindamycin – amikacin - 6;

23. penicillin - ampicillin – erythromycin - gentamycin – tetracycline - tobramycin – imipenem - 1;

24. penicillin - ampicillin – gentamycin – chloramphenicol - tetracycline - tobramycin – trimethoprim - 1;

25. penicillin - ampicillin – erythromycin - gentamycin – chloramphenicol - tobramycin - amikacin -9;

26. penicillin - ampicillin - gentamycin - tetracycline - tobramycin - trimethoprim -amikacin – 1;

27. penicillin - ampicillin – erythromycin - gentamycin – chloramphenicol - tetracycline - clindamycin - amikacin -6;

28. penicillin - ampicillin – erythromycin - chloramphenicol - nitrofurantoin -tetracycline - tobramycin – trimethoprim – 3;

29. penicillin - ampicillin - erythromycin - tetracycline - nitrofurantoin - clindamycin – amikacin - 6;

30. penicillin – ampicillin - erythromycin - gentamycin - chloramphenicol – amikacin - amoxicillin - clavulanic acid – 3;

31. penicillin - ampicillin – gentamycin – ciprofloxacin - tetracycline - amoxicillin - clavulanic acid - imipenem – 4.

32. penicillin - ampicillin - erythromycin - gentamycin – tetracycline - tobramycin - amikacin - trimethoprim – clindamycin -1;

33. penicillin – ampicillin - erythromycin - gentamycin – ciprofloxacin – chloramphenicol - tetracycline - tobramycin – trimethoprim - clindamycin - amikacin - 4;

34. penicillin - ampicillin – erythromycin - gentamycin – chloramphenicol - tetracycline - tobramycin – amikacin (3),

35. penicillin - ampicillin – erythromycin - gentamycin – ciprofloxacin - tetracycline - tobramycin – clindamycin – imipenem(2),

36. penicillin - ampicillin – erythromycin - gentamycin – ciprofloxacin – chloramphenicol -tetracycline - tobramycin – clindamycin – amikacin (4).

Fig.2. Ratio of MRSA among S. aureus isolates by Quarters of the First Year of the Project Implementation.

Isolates of Enterococcus faecalis were resistant to:

1. tetracycline – 1;

2. erythromycin - chloramphenicol (1);

3. penicillin – erythromycin – ciprofloxacin (4);

4. penicillin – ciprofloxacin – tetracycline (2);

5. penicillin – chloramphenicol - rifampicin (1);

6. penicillin – chloramphenicol - tetracycline (3);

7. erythromycin – ciprofloxacin – tetracycline (3);

8. penicillin – erythromycin – ciprofloxacin – tetracycline (7);

9. penicillin – erythromycin – ciprofloxacin – rifampicin (3);

10. penicillin – erythromycin – chloramphenicol - tetracycline (1);

11. penicillin – erythromycin – ciprofloxacin – chloramphenicol (1);

12. erythromycin – ciprofloxacin – chloramphenicol - tetracycline (1);

13. erythromycin –chloramphenicol – tetracycline - rifampicin (1);

14. erythromycin – ciprofloxacin – chloramphenicol - tetracycline – rifampicin (2); etc.

Task # 3. Molecular subtyping of the strains of interest, developing of a novel typing methodology for enterococci, and establishing data-exchange capabilities between the NCDC and other laboratories via the Internet.

During the first year we had studied DNA of 111 strains of Enterococcus faecalis and about 30 strains of Staphylococcus aureus on existence of genes of resistance to some antibiotics.

In experiments of finding genes of resistance to tetracycline two pairs of primers were used. One of them is specific for the resistance gene tet (M), it is amplifying 741 pair fragment of this gene. Another is specific for the resistance gene tet (O), amplifying 519 pair fragment. The experiment was held on the “Techne” thermocycler under conditions given below: 95°C – 5 min, 35 cycles (95°C- 1 min, 55°C- 1 min, 72°C- 1 min) and at last 72°C- 5 min. Electrophoresis was carried out in 1,5 % agarose in TBE buffer. DNA fragments were stained with ethidium bromide and results were fixed with the camera “Polaroid GelCam”.

tet (O).

Fig. 1. Lines 1, 3, 5, 7 of the upper part and 2, 4 of the lower part – amplification of tet (O) gene. Lines 2, 4, 6 of the upper part and 1, 3, 5 of the lower part – amplification of tet (M) gene. Lines 6, 7 of the lower part – tetracycline – sensitive strain of E. faecalis.

Fig. 2. Upper part – amplification of tet (O) resistance genes; lower part – amplification of tet (M) resistance genes of the same strains of E. faecalis.

According to all above mentioned we can conclude, that in majority of resistant to tetracycline strains of E. faecalis isolated from hospitals in different parts of Georgia resistance to tetracycline was determined by existence of tet (M) gene.

Fig. 3. Amplification of gentamycin – resistance genes of E. faecalis.

Fig. 4. Amplification of methicillin – resistance genes of S. aureus.

We want on getting acquainted with the literature on the subject, new methods of investigation.

Task # 4. Developing and conducting educational activities concerning the judicious use of antibiotics in Georgia

During the second quarter two participants of the Project – Nikoloz Tsertsvadze and Giorgi Kurtsikashvili visited Kutaisi and Batumi, and though this visit was not in the scope of the Project, they took part in seminars on “Infections Control at Hospitals and Use of Antibiotics; Susceptibility of Microorganisms to Antibiotics: Modern Methods of Investigation”. Physicians from hospitals in Kutaisi and Batumi that provide us with materials, took part in these seminars. They got acquainted with our investigation, visited local laboratories where material is gathered befor transporting to NCDC, and showed great interest to our work.

Epidemiologists and the Project Manager together with WhoNet link Giorgi Kurtsikashvili had entered the data, and analyzed antibiotic resistance by regions, by hospitals, hospital departments, etc. The data ia already analyzed, and is prepared as a publication. The abstract of the poster presentation “ANTIBIOTIC RESISTANCE AMONG CLINICAL S. aureus, P. aeruginosa and Enterococcus ISOLATES IN GEORGIA” is already sent to the 13^th annual Scientific Meeting of the society for Healthcare Epidemiology of America (SHEA) (See Annex 1).

Web page on the project activities is being prepared. It will be linked to NCDC page in the internet.

The leaflet on prejudice use of antibiotics in Georgia is being prepared.

9. Current State-of –the-art

Project is implemented according to Work Plan. No correction of the schedule is needed.

10. Collaboration with US Partners

We had permanent contact with our US counterpart. All project issues were discussed with Dr. Alexander Sulakvelidze – via e-mail by phone, etc. The collaborator is synthesizing primers for molecular-biological studies. In June project manager Lela Bakanidze visited our University of Maryland (non-project funds were used for this visit) in Baltimore. She and counterpart (Dr. Alexander Sulakvelidze) discussed the project progress and problems, particularly problems of procurement.

In August Dr. Alexander Sulakvelidze has visited Tbilisi. He had a meeting with project participants and discussed the project progress and problems. Further plans and publication drafts were drawn.

In October project manager Lela Bakanidze and Principal Investigator Paata Imnadze visited our University of Maryland (non-project funds were used for this visit) in Baltimore. Together with Dr. Alexander Sulakvelidze they discussed the project progress and problems.

11. Problems, ways of overcoming.

The main problem was the great amount of “strains of interest” isolated, that need to be studied by molecular – biological methods, and lack of specialists in the group of molecular biology. But the problem will be solved in the nearest future – we have already applied to ISTC Office with request for recruiting new participant in this group.

12. Future Perspectives of Development

Project progress ensures further improvement of isolation, identification, investigation of antibiotic resistance and molecular – biological features of bacterial pathogens.

_________________________ ___________________________

Paata Imnadze Date

Director

National Center for Disease Control of Georgia

_________________________ ___________________________

Lela Bakanidze Date:

G-597 p Project Manager

Annex 1.

ANTIBIOTIC RESISTANCE AMONG CLINICAL S. aureus, P. aeruginosa and Enterococcus ISOLATES IN GEORGIA

Paata Imnadze¹, Lela Bakanidze¹, George Kurtsikashvili¹, Ekaterine Tsertsvadze¹, Mira Ghadua¹, Tsaro Gomelauri¹, Natela Mchedlidze¹, and Alexander Sulakvelidze²

National Center for Disease Control, Tbilisi, Georgia;¹ University of Maryland School of Medicine, Baltimore, Maryland. U.S.A.²

The objective of the present study was to determine the patterns of antibiotic resistance of selected bacterial pathogens in hospital patients. The study was conducted for 8-months (January – August 2002), during which time 367 isolates of S. aureus, 178 isolates of P. aeruginosa, and 89 isolates of E. faecalis were isolated from clinical specimens obtained from 7 hospitals in various regions of Georgia. The isolates were speciated using standard biochemical tests at the NCDC, and their resistance to various antibiotics was determined by the disc-diffusion method, according to NCCLS guidelines.

Among the S. aureus strains, 98% were resistant to penicillin and ampicillin, 67% to amoxicillin and azithromycin, less than 50 % to tetracycline, oxacillin, gentamycin, tobramycin, erythromycin, clindamycin, trimethoprim, less than 10 % to ciprofloxacin, nitrofurantoin, and imipenem. S. aureus strains resistant to chloramphenicol, vancomycin, and amikacin were not identified during the study. Very high rate of resistance of P. aeruginosa strains was seen to clindamycin, tetracycline, chloramphenicol, trimethoprim, nitrofurantoin, azithromycin, gentamycin, carbenicillin, tobramycin, amikacin, piperacillin, ciprofloxacin, and imipenem. E. faecalis strains were resistant to ciprofloxacin, tetracycline, erythromycin, rifampicin, trimethoprim and gentamycin. E. faecalis strains resistant to penicillin, chloramphenicol, nitrofurantoin, and vancomycin were not identified during the study. These data suggest high rates of antibiotic-resistance in Georgia. In general, rates of antibiotic-resistance appeared to be higher among strains isolated in urban areas, which may reflect increased antibiotic usage in those areas. Resistance to vancomycin appeared to be very low or absent.

Appendix 1. Summary of the Personnel Commitments

During the reported period a total of 17 specialists of the 1^st category, 3 persons of the 2^nd category and 2 person of the 3^rd category participated in the project implementation .

Table 3. Personnel Commitments by Quarters

Category	Quarters				Total
Category	I	II	III	IV
1st Category	476	476	701.5	701.5	2355
2nd Category	122.5	122.5	123.5	123.5	492
3rd Category	12.5	12.5	12.5	30.5	68
Total:	611	611	837.5	855.5	2915

Total: 2915 man/days

Appendix 2.

Equipment, Received during the Reported Period

The following items were received:

1. Computer Pentium-III – 4;

2. Computer Pentium-IV – 1;

3. LifeBook Fujitsu Siemens – 1;

4. Thermostat DB-3D – 1;

5. Hybridization Incubator HB-3D – 1;

6. VorTech mixer – 2;

7. TechFreezer – 1;

8. Genofuge microcentrifuge – 1;

9. Thermocycler FLEXIGENE-02T – 1;

10. Eppendorf pipettes – 9.

Besides, chemicals for molecular biology, nutrition media and antibiotic biodisks were received.