1 INTRODUCTION Hospital-acquired infections (HAI) or nosocomial infection are a leading cause of morbidity and mortality in the world. These infections often caused by multidrug-resistant bacteria not only effect a large number of patients every year, but also have a significant impact in terms of excess costs, prolonged hospital stays, attributable mortality, and other complications. Carbapenem are powerful drugs but their “last-resort antibiotics” status to treat severe HAIs is losing its effectiveness. The most recent concern is carbapenem resistant bacteria carrying New Delhi metallo-beta-lactamase-1 gene (NDM-1). Since their discovery in 2008, NDM-1 has the ability to resistant to almost all of the available antibiotics, including carbapenem, the most important antibiotic in clinical practice. The NDM-1 bacteria are now spread in several countries in the world. In Vietnam, 2 common nosocomial pathogens P. aeruginosa and A. baumannii were assessed for carbapenem resistance in 2008. Twenty percent of P. aeruginosa and almost 50% of A. baumannii strains were carbapenem resistant. Vietduc hospital, a leading surgical hospital in Vietnam and performs approximately 28,000 operations every year. The hospital is overcrowded and inadequately controls infections. Carbapenem common use in the hospital is one of important factor for bacterial resistance to this antibiotic group to emerge. Up to now, studies on antibiotic resistant bacteria, especially on NDM-1, could not provide a whole picture of these problems in Vietnam. There is thus an important need for studies that assess the epidemiology, clinical and risk factors, as well as the molecular characteristics of carbapenem resistant bacteria carrying NDM-1 gene. This will help the medical leadership in Vietnam to have regular prescriptions, antibiotic use and develop intervention strategies in order to control the spread of carbapenem resistant bacteria carrying NDM-1 gene in hospital and community. We therefore carried out a study: “The Epidemiology of nosocomial infections with bacteria carrying NDM-1 gene in Vietduc hospital-Hanoi, 2010-2011” with three objectives: 1. To describe the epidemiology of nosocomial infections with bacteria carrying NDM-1 gene in Vietduc hospital-Hanoi. 2. To describe the contamination of carbapenem resistant bacteria carrying NDM-1 gene in the environment of Vietduc hospital. 3. To determine molecular characterization of carbapenem resistant strains carrying NDM-1 gene. 2 NEW FINDING OF THE THESIS 1. This is one of the first studies on carbapenem-resistant bacteria carrying NDM-1 gene in Vietnam. 2. Epidemiological characteristic of nosocomial patients infected with carbapenem resistant bacteria carrying NDM-1 gene in Vietduc hospital. 3. Contamination of carbapenem-resistant bacteria carrying NDM-1 gene in environment of Vietduc hospital. 4. Molecular characterization of carbapenem-resistant bacteria carrying NDM-1 gene in Vietduc hospital. THESIS STRUCTURE The thesis consists of 134 pages. Introduction: 3 pages; Conclusions: 2 pages; New contributions 1 page; Recommendation: 1 page; Thesis contains 4 chapters: Chapter 1: literature review 42 pages; Chapter 2: Method 18 pages; Chapter 3: Result 37 pages; Chapter 4: Discussion 20 pages. 23 tables, 31 figures, 144 references including 2 in Vietnamese and 142 in English. CHAPTER 1: LITERATURE REVIEW 1.1. Hospital-acquired infection Hospital-acquired infections (HAI) or nosocomial infections are becoming a major global public health problem. According to WHO, HAI from 1995 to 2010, pooled HAI prevalence in mixed patient populations was 76% in high-income countries and from 5.7% to 19.1% in low- and middle-income counties. However, over the past 10 years, the increasing of HAI caused by gram-negative bacteria resistant to some last-resort antibiotics such as cephalosporin and carbapenem are threats to treatment outcome in the hospitals. 1.2. Antibiotics and bacteria resistant to antibiotic The discovery of anti-microbial was one of the greatest medical triumphs of the twentieth century, which played an important role to prevent and reduce the mortality rate of infectious diseases. However, the increase of antibiotic resistant bacteria in hospitals and community now become a major global public health problem. 1.2.1. Bacteria resistant to Antibiotic 1.2.1.1. Development resistant antibiotic of bacteria In nature, strong selective pressures could help bacteria to develop resistance to antibiotics. Therefore, resistance to antibiotic often occurs very quickly after their introduction for treatment. Recently, some of 3 Gram-negative bacteria strains isolated from HAI patients were resistant to all antibiotic classes, including powerful antibiotics such as cephalosporin and carbapenem. 1.2.1.2. Types of antibiotic resistance: Resistance type, including intrinsic resistance and acquired resistance. This relates to the genetic changes that help the bacteria to survive, or the resistance genes produced in the process are replicated and transferred via plasmids. 1.2.1.3. Mechanism of antibiotic resistant: There are several mechanism of antibiotic resistance, including changes in target sites, production of enzyme hydrolyze antibiotic, prevention of the antibiotic from binding to its site of action, ribosomal mutations or modifications, and production of isoenzymes… 1.3. Carbapenem: The carbapenems are very similar to the penicillin , but the sulfur atom in position 1 of the structure has been replaced with a carbon atom and Carbapenems are active against Extended-spectrum beta- lactamase. 1.3.1. Global Bacterial resistance to carbapenem Class A Carbapenemase: NmcA/IMI, SME, GES and KPC are major types of class A. These enzymes have the ability to hydrolyze a variety of beta-lactams, including penicillins, cephalosporins, carbapenems and aztreonam. However, only the KPC enzyme is a clinical significant enzyme among class A beta-lactamase. The first KPC-producing strain is K. pneumoniae in 1996 in the United States of America. Within few years KPC-producing disseminated widely and have been identified over the entire United States of America, in 2004, about 1/3 of K. pneumoniae strains isolated in Brooklyn-New York carried KPC gene. Today, Hospital-acquired infection caused by KPC producer strains also has been reported in many European and South American countries. Class B metallo-beta-lactamases (MBL): The first MBLs were detected in B. cereus and can hydrolyze beta-lactams including penicillins, cephalosporins and carbapenems. Since then, MBLs producing Gram- negative bacteria isolated from hospital-acquired infection were reported from several studies. The common MBLs enzyme includes IMP (in Japan, China and Greece), VIM (responsible of outbreaks in South-Europe and Taiwan) and the new enzyme NDM-1 that will be described at the end of this chapter. Class D enzyme of the OXA-48 type: The first identified OXA-48 was from K. pneumoniae strains isolated in Turkey 2003. Since then, OXA-48 has been reported in many European countries such as France, Germany 4 and the Netherlands. OXA-181 (a point mutation of OXA-48) has similar carbapenemase activity and has been identified in strain in India. Currently, resistances to carbapenem of Gram-negative bacteria are reported worldwide. However, the actual prevalence of carbapenemase producer is still unknown because many countries lack reports on carbapenem resistant bacteria, as well as antimicrobial surveillance system. The rapid spread of carbapenem resistant bacteria constitutes a threat to public health and treatment policy in the hospital. 1.3.2. Carbapenem resistant in Vietnam Several studies indicated that Gram-negative bacteria, the main causes of nosocomial infections were resistant to antibiotics at high level. Two common nosocomial pathogens, P. aeruginosa and A. baumannii, were assessed for carbapenem resistance in 2008. Twenty percent of P. aeruginosa strains and almost 50% of A. baumannii strains were resistant to carbapenem. However, these data cannot represent the current status of carbapenem resistant in Vietnam, because most of hospitals don’t have report on antibiotic resistant bacteria. 1.4. Methods to detect bacteria resistant to carbapenem: Disk diffusion, MIC and E-test. 1.5. Identification of carbapenemase producers: Modified Hodge test, E-test MBL and molecular techniques (PCR, cloning and sequencing). 1.6. Molecular techniques for resistant-bacteria research: PCR, RAPD-PCR, PFGE, ribotyping, RFLP and plasmid analysis (plasmid- typing, southern-blotting, sequencing and plasmid conjugation). 1.7. Resistance carbapenem bacteria carrying NDM-1 gene 1.7.1. Bacteria carrying NDM-1 gene in the world Discovered in 2008 by Yong et al, the new enzyme NDM-1 have captured the attention of scientists, as well as politicians and the general public because NDM-1 was not only resistant to carbapenem “last resort of antibiotic” but also because these bacteria can disseminate rapidly viva plasmid transmission among the normal human Gram negative intestinal flora. From 2008 to 2010, 77 NDM-1 cases were reported in 13 European countries. Many countries now have reported the presence of NDM-1 bacteria such as Australia, China and United states. In the UK, the patients’ age ranged from 2 to 87 years and the male: female ratio was of 0.62. Several species producing an NDM-1 enzyme were reported such as K. pneumonia, E. coli and Enterobacter spp Whereas those from the UK were healthcare-associated, acquired following hospital admissions in India, Pakistan or stay in departments that had patient with treatment 5 history in India were risk factors for NDM-1 infections. However, cases of NDM-producing Enterobacteriaceae in India were community-acquired studies from the India and Pakistan showed that NDM is widely disseminated among key species of Enterobacteriaceae in the community, hospitals and the environment (most notably sewage and tap-water) It is likely that indirect faecal-oral inter-human transmission plays a major role via contaminated hands, food or water. Clinical symptoms of infected patients with NDM-producing Enterobacteriaceae appear similar to that described for infections with other types of CPE in this population of patients. Several molecular assays were performed indicating that NDM-1 gene was generally located on plasmid and could be transferable by conjugation into E. coli and other genera of the Enterobacteriaceae family, as well as clonal spread of NDM-producing K. pneumoniae strains in India and to the UK. 1.7.2. Bacteria carrying NDM-1 gene in Vietnam In Vietnam, there was no study conducted on carbapenem- resistant bacteria carrying NDM-1 gene. And researches on NDM-1 were only started in Vietnam after the second article on NDM-1 was published in Lancet in August 2010. Currently most of NDM-1 research groups collaborated with National Institute of Hygiene and Epidemiology in order to investigate the prevalence, clinical characterization and treatment for nosocomial infection with carbapenem-resistant bacteria carrying NDM-1 gene. Risk factors, environmental contamination, carrier status and molecular characterization of these bacteria in order to track the source of infection, transmission routes and provide intervention strategies to control the spread of carbapenem resistant bacteria carrying NDM-1 gene. CHAPTER 2: METHODS 2.1. Study site: Vietduc hospital-Hanoi 2.2. Study design: Descriptive and analytic Epidemiology 2.3. Study time: Objective 1 (8/2010 to 12/2011), objective 2 (7 to 12/2011) and objective 3 (1/1/2012 to 30/6/2013) 2.4. Study subject 2.4.1. Subject for objective 1: Nosocomial infections with bacteria carrying NDM-1 gene were confirmed by PCR and sequencing. Subjects for case-control study: Cases (nosocomial infections with bacteria carrying NDM-1 gene). Control (1 case was selected 2 controls, 6 negative with NDM-1 gene and were staying in the patient list of the study and unmatched sex and age) 2.4.2. Subject for objective 2: Surface of table, floor of patient’s room, patient's bed, toilet, vacuum sputum, and medical waste 2.4.3. Subject for objective 3: Bacteria strains resistant to carbapenem carrying NDM-1. 2.5. Samples size 2.5.1. Sample size for objective 1: - Samples size for descriptive study: Total 240 patients infected with carbapenem resistant bacteria found in the study. - Sample size for case control study: Case: 35 patients infected with carbapenem resistant bacteria carrying NDM-1 gene, for each case were selected 2 controls. 2.5.2. Sample size for objective 2: 200 samples in different site of 3 departments: Urology, Hepatobiliary and Gastrointestinal were selected 2.5.3. Sample size for objective 3: Total bacteria strains resistant to carbapenem carrying NDM-1 gene of the study. 2.6. Sample collection and procedure 2.6.1. Sample collection - Sample for objective 1: Samples were collected from suspected infectious sites of patients with clinical diagnosis of nosocomial infections and then were examined for bacterial infections, following the standard operation procedures of the microbiology department of Vietduc hospital. - Sample for objective 2: Sterile cotton tipped swabs were used to swab on the surface of the collection sites and put into collection tube. - Sample for objective 3: Bacteria strains resistant to carbapenem carrying NDM-1 gene collected from objective 1 and 2. 2.6.2. Procedure 2.6.2.1. Identification of patients infected with carbapenem resistant bacteria carrying NDM-1 gene - Isolation and identification of nosocomial bacteria by standard operation procedure of Vietduc hospital. - PCR and sequencing (following Yong et al), using primers: Kp-ndm1- F: 5’-ttcgacccagccattggcggcga-3’ and Kp-ndm1-R: 5’- atgcacccggtcgcgaagctgag-3’. - Identification of carbapenem resistant bacteria in hospital environment: enrichment environment samples in LB broth containing imipenem. Sub-cultured to MacConkey agar-imipenem, selection of 5-7 colonies, PCR for NDM-1 gene and identification of NDM-1 7 bacteria by API-20E. - Antibiotic susceptibility by MIC, followed by CLSI, 2010 guideline. - Molecular analysis of NDM-1 bacteria strains: MBL produced by Eiken kit (Wachino, J &et al) and MBL E-test (AB bioMerieux- Nc). Genotyping by PFGE; NDM-1 plasmid by Southern-Blotting, transformation NDM-1 plasmid by Karamunsary et al, 2010; and sequencing of NDM-1 plasmids. 2.8. Study indicators 2.8.1. Indicators for objective 1: Prevalence of nosocomial patients infected with carbapenem resistant bacteria carrying NDM-1 gene. Isolation of strains carrying NDM-1 gene and antibiotic susceptibility results. Demographic data (age, sex, occupation, reason admitted to hospital and history of treatment…). Surgery methods, clinical information, medical intervention, chronic diseases… antibiotic used before and during hospitalized… 2.8.2. Indicators for objective 2: Surface of table, floor, patient’s bed, medical and non-medical trolley, waste toilets…Samples positive with NDM-1 bacteria. 2.8.3. Indicators for objective 3: Genotyping of NDM-1 bacteria, NDM-1 plasmids, transmisson ability of NDM-1 plasmid. 2.9. Method to collect information: Questionnaire form 2.10. Data analysis: Excel and SPSS 21.0 (SPSS: An IBM Company), DNA-Blast, Bionumeric- 6.5 and Inter plasmid Analyzing software. 2.12. Ethics Statement: Ethical approval was obtained from the Ethical Committee of the National Institute of Hygiene and Epidemiology in 2010. 8 CHAPTER 3: RESULTS 3.1. Epidemiology of nosocomial infections due to bacteria carrying NDM- 1 gene 3.1.1. General characterization of study subjects Figure 3.1. Distribution of patients infected with carbapenem strain/total infection patients Figure 3.2. Distribution of patients infected with NDM-1 strain/patient infection with carbapenem strains Among the 6841 nosocomial infections, 240 (3.51%) patients were infected with Gram-negative bacteria resistant to carbapenem (figure 3.1). Figure 3.2 shown that 35/240 (14.58%) of patients were infected with bacteria strains resistant to carbapenem carrying NDM-1 gene. Two patients (2/35; 5.7%) in urology department were infected with 2 strains resistant to carbapenem carrying NDM-1 gene (C. freundii and Enterobacter spp; Enterobacter spp. and P. rettgeri). None of the patients had a travel history to India or Pakistan or contact and treatment in the same department with foreign patients. 3.1.2. Distribution of patients infected with carbapenem resistant bacteria carrying NDM-1 gene by sex and age Figure 3.4. Distribution of patients infected with NDM-1 bacteria by sex (n=35) Figure 3.5. Distribution of patients infected with NDM-1 bacteria age (n=35) The proportion of males infected with NDM-1 strain was higher 9 than the proportion of females, 88.6% (31/35 and 11.4% (4/35) (Figure 3.4). Most of cases were in age groups 60-69; >70 and 50-59 years (Figure 3.5). Table 3.4. Distribution of patient infected with NDM-1 bacteria by department (n=35) Department Patient % Urology 19 54.3 Infectious surgery 4 11.43 ICU 3 8.58 Gastrointestinal emergency 2 5.72 Injury and Orthopedic surgery 2 5.72 Hepatobiliary 1 2.85 Better private care surgery 1 2.85 Cardiothoracic and vascular 1 2.85 Heamodialysis 1 2.85 Oral and Maxillofacial surgery 1 2.85 The highest prevalence of NDM1 was detected in the urology department, 19/35 (54.3%), followed by the infectious surgery department 4 (11.43%), ICU 3 (8.58%) and other departments were found 1-2 NDM-1 cases. Figure 3.6. First detected NDM-1 case per department over time The first patient infected with NDM-1 positive pathogen was 10 isolated from the injury and orthopedics department on the 17/8/2010 (Figure 3.6) and a few days later were found in the Urology department. A month later NDM-1 has spread to adjacent departments (infectious surgery, gastrointestinal emergency and Hepatobiliary department) and on the 26/8/2011 in Oral and Maxillofacial surgery. At the end of 2011, NDM-1 infections were found in10 departments of Vietduc hospital. Figure 3.7. Distribution of patients infected with NDM-1 strains by month (n=35) The highest NDM-1 infected patient were found in November 2010 (6 cases), followed by December 2010 (5 cases), 4 cases were found in 8/2010 and 8/2011. However, no NDM-1 infected patient was found from 2 to s4/2011 (Figure 3.7). Twenty NDM-1 infected patients (57.14%) were admitted to the hospital for reasons related to urinary tract diseases. All of the positive patients were typical of hospital-acquired infections. Some cases had a severe infection due to multiple injuries and blood infection. One death was attributed to septic shock from blood infection, femoral neck fracture, and other infected patients were recovered and discharged from hospital. 3.1.3. Risk factor associated with nosocomial infections of NDM-1 bacteria [...]... and Singapore There were no NDM-1 plasmids of our study belonging to these groups CHAPTER 4: DISCUSSION 4.1 Epidemiology of nosocomial infection due to bacteria carrying NDM-1 gene 4.1.1 Epidemiological characterization of nosocomial infection due to bacteria carrying NDM-1 gene The first patient infected with NDM-1 positive pathogen was detected from the Injury and Orthopedics department on the 17/8/2010... disseminated in our neighboring countries CONCLUSION 1 Epidemiological characterization of nosocomial infections due to bacteria carrying NDM-1 gene in Vietduc hospital 1.1 There were high numbers of patients infected with carbapenemresistant bacteria carrying NDM-1 gene (14.58%) in Vietduc hospital from 2010-2011 1.2 NDM-1 infected patients were in different age groups, but most of patients were in age... important finding to give the evidence of NDM-1 in Vietnam By the end of 2011, 35/240 (15.58%) NDM-1 cases were found in this study, while only 77 cases were reported in 13 European countries from 2008 to 2010 The results indicated that the potential of NDM-1 in Vietnam is great, and the surveillance should be strengthened to enable monitoring of NDM-1 pathogens in Vietnam After the first case in Injury... Creating intervention studies to treat NDM-1 patients and control the dissemination carbapenem-resistant bacteria carrying NDM-1 gene in hospital and community 3 Establish study on plasmids carrying NDM-1 gene in hospital and community Ministry of Health need: 4 Creating the National antimicrobial resistant network in Vietnam 5 Establish test to screen of carbapenemase-producing bacteria strains in hospital... and drinking water Therefore, improve capacity for infection control and monitoring the emergence and spread of NDM-1 bacteria in hospital and in environment are needed 4 3 Molecular characterization of carbapenem-resistant bacteria carrying NDM-1 gene 4.3.1 General characterization of carbapenem resistant bacteria carrying NDM-1 gene 4.3.1.1 Detection rate of carbapenem-resistant bacteria carrying NDM1... department bacteria and 2/68 (2.94%) samples collected in Hepatobiliary department were found positive with bacteria strains resistant to carbapenem carrying NDM-1 gene, these departments have also been DNM-1 infected patients Three types of bacteria were carrying NDM-1 gene include 2 Acinetobacter spp one A baumannii and 2 were E aerogenes All of 5 bacteria strains resistant to carbapenem isolated in environment... spp These strains were resistant to antibiotic at high level, but still susceptible to colistin 3 Molecular characterization of carbapenem resistant bacteria carrying NDM-1 gene 3.1 General information’s of carbapenem resistant bacteria carrying NDM-1 gene  Seven species of carbapenem resistant bacteria carrying NDM-1 gene were found, including Enterobacter spp., E coli, K pneumoniae, C freundii, Acinetobacter... Acinetobacter spp., A baumannii and P rettgeri  The highest positive of carbapenem-resistant bacteria carrying NDM1 gene were found in urine sample (20/35 samples, 57.1%)  Carbapenem-resistant bacteria carrying NDM-1 gene strains were resistant to antibiotics at high level However, 29.7% strains were sensitive to amikacin and 97.3% to colistin  89.2% of carbapenem-resistant bacteria carrying NDM-1 gene. .. And NDM-1 was produced both by a K pneumoniae isolate from urine and a feacal E coli from the first case NDM-1 in the world Therefore our hypothesis was the patient himself might already carry NDM-1 bacteria in their gastrointestinal tract as a source of the infection To prove this hypothesis, the second study (in collaboration with Oxford university) was conducted in this hospital, in order to investigate... disinfectant twice a day and other sites such as table, cabinet not regular contact with source of infection The presence of positive NDM-1 bacteria in environment hospital will increase the risk for dissemination of these bacteria not only in the hospital but also to the community Isozumi et al 2012 reported NDM-1 bacteria in environment of Hanoi and a study in New Delhi also found NDM-1 bacteria in . NDM-producing K. pneumoniae strains in India and to the UK. 1.7.2. Bacteria carrying NDM-1 gene in Vietnam In Vietnam, there was no study conducted on carbapenem- resistant bacteria carrying NDM-1 gene. . resistant bacteria carrying NDM-1 gene in hospital and community. We therefore carried out a study: The Epidemiology of nosocomial infections with bacteria carrying NDM-1 gene in Vietduc hospital-Hanoi,. resistant bacteria carrying NDM-1 gene in the environment of Vietduc hospital. 3. To determine molecular characterization of carbapenem resistant strains carrying NDM-1 gene. 2 NEW FINDING