the epidemiology ofnosocomialinfection due to bacteria carrying ndm-1 gene in vietduc hospital-hanoi, 2010-2011

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



11
Table 3.5. Univariate analysis of risk factors associated with NDM-1
bacterial infection

No Variables Case Control OR 95% CI p
1 Chronic diseases
15/35
(42.85%)
18/70
(25.71%)
2.16 0.19-5.1 0.06
2 History of treatment
in other hospital
20/35
(57.14%)
22/70
(31.42%)
2.9 1.25-6.72 0.019
3 Admission to
hospital to treat
urinary tract
diseases
20/35
(57.14%)
15/70
(21.42%)
14.22 4.87-41.53 0.0001
4 Treatment in
urology department
19/35
(54.28%)
4/70
(5.71%)

19.59 5.85-65.62 0.0001
5 Urinary tract
infection
20/35
(57.14%)
4 /70
(5.71%)
22.0 6.55-73.85 0.0001
6 Infection with
Enterobacteriaceae
31/35
(88.57%)
8/70
(11.42%)
7.75 3.99-15.03 0.0001

The univariate analysis showed six factors significantly associated with
NDM-1 bacterial infection (table 3.5),


Table 3.6. Multivariate analysis of risk factors associated with NDM-1
bacteria infection

N
o
Variables Case Control
Adjuste
d (OR)
95% CI P
1 Urinary tract

infection
19/35
(54.28%)
4/70
(5.71%)
18.03 6.32-51.43 0,03
2 Infection with
Enterobacteriaceae
31/35
(88.57%)
8/70
(11.42%)
13.26 1.99-88.20 0,008
In the multivariate analysis, using conditional logistic regression, two
factors, treatment in urology (adjusted OR: 18.03; 95% CI: 6.32-51.43)
and infected with Enterobacteriaceae resistant to carbapenem (adjusted
OR: 13.26; 95%CI: 1.99-88.20) remained indipendently associated with
with NDM-1 bacteria infection (table 3.6).
3.2. Contamination of carbapenem resistant bacteria carrying NDM-
1 gene in environment of Vietduc hospital
Among of 200 environment samples, 5 (2.5%) were found positive with
gram-negative bacteria carrying NDM-1 gene (table 3.8; Figure 3.8).


12

Table 3.8. Distribution of positive NDM-1 bacteria by samples (n=200)
Samples No Positive samples %
Patient cabinet 9 0 0
Patient’s sheet bed 46 3 1.5

Floor of patients' room 29 0 0
Toilet areas (toilet cover,
lavabo…)
16 1 0.5
Medical trolley 12 0 0
Vacuum sputum 20 0 0
Medical waste (cover of
medical waste bill, medical
waste)
12 1 0.5
Staff cabinet 9 0 0
Nurse hand 28 0 0
Other site (telephone, office
table )
19 0 0
Total 200 5 2.5


Figure 3.8. PCR to detect NDM-1 gene of Gram-negative bacteria
isolates from environment of Vietduc hospital

Three types of sample were positive with bacteria carrying NDM-
1 gene includes: Patient’s bed sheet 3 (1.5%), cover of medical waste bill 1
(0.5%) and one (0.5%) was toilet cover (table 3.8).


13


Table 3.9. Distribution of positive NDM-1 bacteria of environment

samples by department
Department Bacteria No
%
Positive
Have patient
infected with
NDM-1 bacteria
Urology (n=66)
Acinetobacter
spp.
2
4.54
Yes
E.aerogenes 1 Yes
Hepatobiliary
(n=68)
A. baumannii 1
2.94
Yes
E.aerogenes 1 Yes
Gastrointestinal
(n=66)
No detection
0
0
No

3/66 (4.54%) samples in Urology 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 hospital were resistant to imipenem (4-256mg/L); 4 were
resistant to meropenem (8-128mg/L), 1 E.aerogenes strain was resistant to
meropenem at intermediate level (2mg/L). One E.aerogenes strain was
resistant to ciprofloxacin at 256mg/L. All of strains were resistant to
ceftazidim (512mg/L) and fully susceptible to colistin.
3.3. Molecular characterization of carbapenem-resistant bacteria
carried NDM-1 gene
3.3.1. General characterization of carbapenem-resistant bacteria carried
NDM-1 gene
By using disc diffusion assay, 246 Gram-negative strains resistant
to carbapenem isolated from 240 patients (234 patients were infected with
one and 6 patients were found infections with 2 types of bacteria species).
Seven resistant carbapenem species were found, highest was A. baumannii
(63.8%) and lowest was Providencia rettgeri (P. rettgeri, 0.4%) (Figure
3.9).



14

Figure 3.9. Distribution of Gram
negative bacteria resistant to
carbapenem (n=246)
Figure 3.12. Distribution of Gram-negative
bacteria resistant to carbapenem carrying
NDM-1 gene (n=37)

There were seven species carrying NDM-1 gene, highest was
Enterobacter spp 13 (35.2%); followed by E. coli 9 (24.3%); K.
pneumoniae 6 (16.2%); C. freundii 5 (13.5%); Acinetobacter spp. 2
(5.4%); and one (2.7%) for A. baumannii and P. rettgeri (Figure 3.12).
The highest of resistant carbapenem strains carrying NDM-1 gene
were found in urine 20 samples (57.1%), followed by Bronchial fluid 5
(14.2%) and other samples were from 1 to 3.

These NDM-1 strains were resistant (4-128mg/L) to imipenem
(48.7%) and meropenem (59.5%). However, 33/37 (89.2%) of strains
were resistant to at least one antibiotic of carbapenem. These strains were
resistant to 100% cefotaxime (100%); ceftazidime (86.5%); ciprofloxacin
(89.1%); gentamicin (91.9%); and amikacin were 51.4%. These strains
were still sensitive to colistin 36/37 (97.3%) and 11/37 (29.7%) to
amikacin.



Figure 3.14. New Delhi metallo-beta-lactamase 1(MBL-1) producing of
bacteria (n=37)
Figure 3.14-I shown New Delhi metallo-beta-lactamase 1 producing C.


15
freundii strain carrying NDM-1 gene. By using Eiken Co., Tokyo-Japan
kit, SMA could inhibite this enzyme and make growth-inhibition zone of IMP
and MPM disk close to SMA disk >5mm compared with IMP and MPM disk
alone (Figure 3.14I-A and 3.14 I-B). Similarly, EDTA of MBL E-test (AB
bioMerieux- Nc) was inhibited by this enzyme and made growth-inhibition

zone of IMP plus EDTA >3 time when compared with inhibition zone of IMP
region. 33/37 (89.2%) of NDM-1 strains isolated were positive for New Delhi
metallo-beta-lactamase 1 producing when tested by IMP-, MPM-SMA
and MBL E-test (Figure 3.14-II).

3.3.2. Molecular characterization of carbapenem-resistant bacteria
carried NDM-1 gene
3.3.2.1. PFGE profiles of carbapenem-resistant bacteria carried NDM-1
gene



Figure 3.15. PFGE profiles of E.coli strains carrying NDM-1 gene

Figure 3.16. PFGE profiles of Enterobacter spp. strains carrying NDM-
1 gene

The majority of the PFGE patterns of NDM-1 positive bacteria
showed that these were different and thus limited clonal expansion. Just


16
three Enterobacter cloacae isolates and two Escherichia coli (Figure 3.15
and 3.16) isolates from the urinary tract (Urology department in 2010 and
2011) belonged to a single PFGE profile suggesting clonal spread.

3.3.2.2. Plasmid carrying NDM-1 gene

Figure 3.21. Representative results of plasmid carried NDM-1 gene of
bacteria isolates. A. Pulsed-field gel of S1-treated plasmid DNA of

Vietduc isolates. B. Autoradiogram of Gel A probed with NDM-1
gene

Result showed that individuals in each strain carried NDM-1
plasmid. The plasmid size ranged from 50-150kb. However, E. coli
50VD/2010 strains isolated in 2010 carried 2 NDM-1 plasmids (well 2).
Two strains: E. coli 48VD/2010 and Enterobacter spp. 133VD/2010, NDM-1
gene was located on chromosome. (Lane 1 and 8, Figure 3.12 B).


17
3.4.2.4. Conjugation transfer of NDM-1 plasmid assay

Table 3.15. Conjugation transfer of NDM-1 plasmid to E. coli J53
Strain n
(35)
Conjugation-transfer of NDM-1 plasmid
to E. coli J53
yes no
E. coli 8 2 6
K. pneumoniae 6 1 5
C. freundii 5 1 4
Enterobacter spp. 12 1 11
Acinetobacter* 3 0 3
P. rettgeri 1 0 1
Total 35 5 (14.3%) 30 (85.7%)
*Acinetobacter includes 1 A. baumannii and Acinetobacter spp strains.

Results of table 3.15 shown 14.3% (5/35) of strains could conju-
transferred NDM-1 plasmid to E. coli J53 in-vitro.

3.3.2.5. Analysis of plasmid carrying NDM-1 gene


Figure 3.23. NDM-1 Plasmid isolates from Vietduc hospital with NDM-
1 plasmid in the world.
- Figure 2.23 shown 14 NDM-1 plasmids isolated from Vietduc hospital
and 19 NDM-1 plasmids isolated from some countries in the world are
belonging to six groups.
- Group 1: This group have 8 plasmids which are the most characterized
IncA/C type for NDM-1 transmission and have only one NDM-1
plasmid isolates from Vietduc belonging to groups 1
- Group 3: These plasmids are IncN and IncFII types. And only 10


18
plasmids isolated in Vietduc belonging to this group.
- Group 4 and 6: These groups’ contained NDM-1 plasmids from China
and 3 NDM-1 plasmids isolated from Vietduc hospital.
- Groups 2 and 5 include 7 NDM-1 plasmids isolated from Japan, Taiwan,
Morocco, Italy, Australia 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. This is
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 and Orthopedics department, few days
later, others 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 cases were found
in 10 departments and the highest number of NDM-1 cases was in the
urology department (n=19). However, it’s difficult to track the spread of
NDM-1 from the first case. This can be explained by the fact that (1) its
might already have NDM-1 case in the hospital before the study; (2) 35
NDM-1 infected cases with 7 Gram-negative species. Moreover, there
were 2 urinary tract infection cases with two types of NDM-1 bacteria
strains might suggesting vivo transfer of conjugative NDM-1 plasmids
(similar to the first NDM-1 case in the world); (3) The first case was in
Injury and Orthopedics department but we only detect 2 NDM-1 in this
department in nearly 2 years of the study, and most of cases were in
Urology department. Therefore, the spread of NDM-1 need to be
investigated by molecular typing methods.


19
Most of NDM-1 cases were male and over 50 year of age while in 13
European countries ranged from 2-87 year. This result might explain:
20/35 NDM-1 cases were admitted hospital to treatment of urinary tract

diseases, most of them were prostatic hyperplasia and its complications,
therefore will affect to the sex ratio, age and high number of positive in
urine as well as in Urology department.

4.1.2. Risk factor associated with nonsocomial infection of NDM-1
bacteria
Treatment in Urology department and infection with
Enterobacteriaceae were independent risk factors associated with NDM-1
infection in this study. To date, there have been no case-control or cohort
studies looking at risk factors linked to colonization or infection of
humans with NDM-producing Enterobacteriaceae and most of
epidemiological investigations focused on the history of travel to the
Indian subcontinent strongly suggest that contact with healthcare systems
on the Indian subcontinent is a risk factor for the acquisition of NDM-
producing Enterobacteriaceae and some cases in United Kingdom related
to endoscope procedure, therefore it’s difficult to compare. A studied in 2
army hospital has shown the presence of NDM-1 Enterobacteriacae in
stool of the patients. Other study estimated that at least 100 million Indian
residents carry NDM-1-positive bacteria as normal gut flora. 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 gastro-
intestinal 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 carrier status of NDM-1 bacteria in
normal gut flora of patient before and after discharge from hospital. The
results showed that 3/100 stool samples before admission were positive
with NDM-1 bacteria [unpublished data]. When patients carry NDM-1
and given that the urinary tract is close to the intestinal tract, it will
increase the risk for NDM-1 for urinary tract to get infection with NDM-1

bacteria from their gastrointestinal. This result might also support that
infection with Enterobacteriaceae is risk factor for NDM-1 infection.

4.2. Contamination of carbapenem resistant bacteria carried NDM-1
gene in environment of Vietduc hospital
Among of the 200 environment samples, 5 (2.5%) were detected


20
positive with NDM-1 bacteria strains. Three types of bacteria strains
including A. baumannii (n=1), E. aerogenes (n=2) and Acinetobacter spp
(n=2) were detected in 2 departments which have positive NDM-1
patients before and all of positive samples were at high risk of
contamination with bacteria including NDM-1 strains such as patient’s
bed sheet, cover of medical waste bill and toilet cover. However, some
high-risk samples were given negative results. This might be explained by
the facts that nurse often wears gloves, floor of patient room were clean
with 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 wastewater 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 NDM-
1 gene
Among the 246 carbapenem-resistant Gram-negative bacteria isolated
from 240 patients, 37 strains isolated from 35 patients were positive by
PCR. The sequence of NDM-1 genes was 100% matched with standard
NDM-1 gene from genbank, which indicated NDM-1 gene of our study,
were correct.
Seven species were positive for NDM-1, highest was Enterobacter spp.
(n=13), followed by E. coli (9), K. pneumoniae (6) and C. freundii (5).
The results were similar to other studies. However, some countries highly
positive with NDM-1 was found with K. pneumoniae and E. coli. The
highest NDM-1 positive was also found in urine (57.1%), which detection
rate similar to those of EU (57.14%).
4.3.1.2. Antibiotic susceptibility of carbapenem-resistant bacteria
carrying NDM-1 gene
NDM-1 positive strains isolated in Vietduc were resistant to antibiotics
that are commonly used to treat nosocomial infection in Vietnam. 89.2% of


21
the strains were resistant to at least one antibiotic of carbapenem. The
results differ from those of the UK but are similar with NDM-1 isolates
from Chenai and Haryana India. The accurate identification of carbapenem
resistant is an important first step for prevention. Early studies have
demonstrated that some carbapenemase-producing isolates have
carbapenem MICs that remain in the susceptible range and result in a failure
to detect these organisms. To improve the detection of carbapenem-
producing Enterobacteriaceae, the Clinical Laboratory and Standard
Institute recommended lowering the carbapenem breakpoint (Table M100-

S20; CLSI, 2010). These new breakpoints were established to more
accurately predict carpabenem treatment outcome without the need for a
special test to detect carbapenem production. NDM-1 isolates were also
resistant to cephalosporin, fluoroquinolone and aminoglycoside at high
level that is similar to NDM-1 isolates from UK and India. However, 29.7%
of our isolates were still susceptible to amikacin while isolates from the UK
and India were fully resistant; this might be due to the fact that the antibiotic
used in Vietnam has created new type of resistant in Vietnam.
4.3.1.3. New Delhi Metallo-beta-lactamase1 producing
Of the 33 isolates, 37 had the ability to produce New Delhi Metallo-
Beta-Lactamase 1, which is similar with other studies. However, 4
Enterobacter spp carrying NDM-1 gene were negative. Our hypothesis
that these negative strains might have been owing to the coproduction of
OXA-48 carbapenemase is member of the serine-beta-lactamases, whose
activities are not inhibited by SMA and EDTA [unpublished data].
4.3.2. PFGE profiles of carbapenem-resistant bacteria carried NDM-1
gene
PFGE analyzed showed that most of carbapenem-resistant bacteria
carrying NDM-1 gene had different PFGE profiles. However, 2 NDM-1-
positive E. coli and 3 Enterobacter spp. isolates in the Urology
department belonged to a singles PFGE profiles suggesting clonal spread.
The results was similar with other studies in the world and also indicated
that most NDM-1 positive isolates do not belong to a single PFGE profile
and could not prove significant strain relatedness between Indian and UK
isolates, however PFGE was approved that 26 K. pneumonieae isolated
was clonal spread in Haryana-India, which cannot be identified by
traditional epidemiological method.
4.3.3. Analysis plasmid carrying NDM-1 gene
4.3.3.1. Detect plasmid carrying NDM-1 gene
NDM-1 plasmids size in our study range form 50-150kb, most of

22
isolates carried 1 plasmid and 1 strain carried 2 NDM-1 plasmids. The
plasmid size was similar with NDM-1 plasmids isolated form India and
UK (50kb and 118kb); however some plasmids from these countries have
a bigger size (350kb-500kb). Two isolates in 2010 were negative with
NDM-1-plasmid by Southern-Blotting, might NDM-1 gene of these
strains are located on the chromosome. Kumarasamy et al 2010 reported
that NDM-1 was located on the chromosome. Currently two of negative
NDM-1-plasmid in this study is under examination in order to confirm the
location of the NDM-1 gene.

4.3.3.2. NDM-1 plasmid conjugation
Conjugation-transfer rate to E. coli J53 in our study was 14.3%, which
is a result similar to other studies in the world (10%). The results showed
that NDM-1 plasmid could conjutransfer among Enterobacteriaceae
populations and also explained the first case of NDM-1, Yong et al in
2009 detected both E. coli and K. pneumoniae carried by NDM-1 plasmid.
The result also approved that the spread of NDM-1 of this study is mainly
due to conjugative plasmids, which cannot be explained by
epidemiological and PFGE data.
4.3.3.3. Analysis of plasmid carrying NDM-1 gene
The most attractive finding was group 3 which have only 10
plasmids isolates in Vietduc. It might explain that group 3 was created and
evolved in Vietnam under excess antibiotic pressure. And other important
finding were: 3 NDM-1 plasmids isolates in this study were in groups 4
and 6, which have NDM-1 plasmids isolates from China (neighboring
country), and one plasmid of study was in group 1 with other NDM-1
plasmid which is mostly characterized IncA/C type for NDM-1

transmission. This might explained that these plasmids have already
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 carbapenem-
resistant 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 groups >50 years. And males were more


23
numerous than females (88.6% and 11.4%).
1.3. Ten departments were found to have patients infected with
carbapenem-resistant bacteria carrying NDM-1 gene and 56.7% of
cases were in Urology department
1.4. Two independent risk factors associated with NDM-1 bacteria
infection were treatment in Urology and infected with
Enterobacteriaceae resistant to carbapenem.
2. Contamination of carbapenem resistant bacteria carried NDM-1
gene in environment of Vietduc hospital
2.1. Two point five percent (5/200) of environment samples were
positive with carbapenem-resistant bacteria carrying NDM-1 gene.
The positive samples were found: in 2 departments (Urology and
Hepatobiliary), patient’s bed sheet, cover of medical waste bill and
toilet cover.
2.2. The carbapenem-resistant bacteria carrying NDM-1 gene in

environment including A. baumannii (1), E. aerogenes (2) and 1
was Acinetobacter 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 spp., A. baumannii and P. rettgeri.
 The highest positive of carbapenem-resistant bacteria carrying NDM-
1 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 strains
produced New Delhi metallo-beta-lactamase 1.
3.2. Molecular characterization of carbapenem resistant bacteria
carrying NDM-1 gene
 Two E. coli and 3 Enterobacter spp. strains isolated in Urology
department were clonal
 Most of strains carried 1 NDM-1 plasmid, one was found to have 2
NDM-1 plasmids. And two strains were negative with NDM-1
plasmid.


24
 Conjugation-transfer NDM-1 plasmid of carbapenem-resistant
bacteria carrying NDM-1 gene to E. coli J53 was 14.3%.
 Ten NDM-1 plasmids in this study were different with NDM-1

plasmid in the world. And 4 plasmids were similar with NDM-1
plasmid isolated in some countries in the world.

RECOMMENDATION

Vietduc hospital need:

1. The guidelines for selection, storage and antibiotic treatment in
different levels are needed. These guidelines should be based on
results of studies, antimicrobial resistant network.
2.
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.
6. Continue investigating resistant bacteria carrying NDM-1 gene in
Vietnam and molecular typing of NDM-1 bacteria representative
for North-Mid-South of Vietnam.
7. Apply epidemiological investigation of NDM-1 patient like other
infectious diseases such as: influenza A virus and polio virus…