Molecular epidemiology of methicillin-resistant Staphylococcus

Title aureus in patients and their surrounding environment

Author(s) Chan, Chi-fun.; 陳志芬.

Citation

Issued Date 2012

URL http://hdl.handle.net/10722/173920

The author retains all proprietary rights, (such as patent rights)

Rights and the right to use in future works.
 MOLECULAR EPIDEMIOLOGY OF

METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS IN

PATIENTS AND THEIR SURROUNDING ENVIRONMENT

By

Chan Chi Fun

This thesis is submitted to the Department of Microbiology of the University of

Hong Kong in partial fulfillment of the requirements for the Degree of Master of

Medical Sciences

August 2012

Supervisor: Dr. P.L. Ho

 Abstract

Abstract of thesis entitled

MOLECULAR EPIDEMIOLOGY OF METHICILLIN-RESISTANT

STAPHYLOCOCCUS AUREUS IN PATIENTS AND THEIR

SURROUNDING ENVIRONMENT

Submitted by

Chan Chi Fun

for the degree of Master of Medical Sciences

at The University of Hong Kong

in August 2012

Background

Methicillin resistant Staphylococcus aureus (MRSA) is endemic in

healthcare settings in many countries of the world. Patients who have acquired

MRSA serve as a source of transmission by contamination of their surrounding

environments. Numerous studies illustrate that many different inanimate surfaces

in hospitals can become a reservoir for MRSA.

Objectives

The objective of this study is to examine the presence of MRSA on

environmental surfaces and its relationship between patients’ acquisition of

MRSA by studying their molecular characteristics.

Methodology

The near-patient surfaces of 30 MRSA positive patients, 30 control patients

and the ward environments were sampled from June 2011 to September 2011. The

I
swabs were enriched and cultured for the presence of MRSA. The MRSA isolates

obtained from environmental samples and from the clinical samples of the

patients were then characterized by Spa typing.

Results

The MRSA found in case patients and control patients’ environmental

surfaces was 97% (29/30) and 40% (12/30) respectively. Environmental surfaces

that were highly contaminated by MRSA positive patients were bed sheets (70%),

followed by pillows (55%), patient bed frames (52%) and patient lockers (52%).

On the environmental surfaces other than the near-patient areas, ambulatory chair

armrests had the highest amount of MRSA (21%), followed by fax machines

which accounted for 14%. Among the 216 MRSA isolates (30 clinical isolates and

151 environmental isolates), eight spa types were found and the most predominant

spa type was t1081 (63.3%) followed by t032 (17.6%) and t037 (7.4%). 27

patients were found to have the MRSA isolates with same spa type in the clinical

samples and their surrounding environments. The agreement between the MRSA

isolated from the clinical sample of patients and their surrounding environment

was 93.1%.

Conclusion

Identical isolates were recovered from the patient and their environment

(93.1%) which suggests possible environmental contamination of the ward

cubicles, possibly contributing to endemic MRSA. More effective and rigorous

use of current approaches to cleaning and decontamination is required and

consideration of newer technologies to eradicate MRSA.

II
 Declaration

I, Chan Chi Fun, declare that this dissertation represents my own work and

that it has not been submitted to this or any other institution in application for a

degree, diploma or any qualification.

I, Chan Chi Fun, also declare that I have read and understand the guideline

on “What is plagiarism?” published by the University of Hong Kong (available at

http://www.hku.hk/plagiarismf) and that all parts of this work complies with the

guideline.

Candidate: Chan Chi Fun

Signature _______________

Date: 30 August 2012

III
 Acknowledgements

First and foremost I offer my sincerest gratitude to my supervisor, Dr. Ho

Pak Leung, who has guided me throughout my project with his professionalism,

knowledge, patience, kindness, encouragement and support. I attribute the level of

my Master’s degree to his professionalism and support. Without him, this thesis

would not have been written and completed.

I would like to thank Mr. Frankie Chow, for his skilful technical assistance

and guidance. With his great support, I could perform the laboratory work in my

hospital smoothly.

I would like to thank Dr. T.K. Ng, Mr. W.T. Hui and the infection control

team for assisting me in environmental sampling.

I would like to thank all my colleagues from Princess Margret Hospital for

their tremendous support and encouragement.

I show my deepest gratitude to my parents and husband, for their support,

patience and encouragement which have driven me to pass through the toughest

period during these two years. They spent most of the time caring for my beloved

daughter and son so that I could concentrate on my work. Without them, this

would not have been possible.

Finally, I would like to thank my Heavenly Father, for loving me and hearing

my prayer always. He guides me through the hard times and teaches me to be

humble when I am rich. Without Him, my life would not be complete.

IV
 Abbreviations
μg Microgram

μl Microlitre

μm Micromolar

ACCs Aerobic colony counts

ANSORP Asian Network for Surveillance of Resistant Pathogens

AST Active Surveillance Testing

ATP Adeneine Triphosphate

BHI Brain heart infusion

bp Base pairs

CA-MRSA Community-acquired methicillin-resistant Staphylococcus aureus

ccr Cassette chromosome recombinase

CCIDER Central Committee on Infectious Disease and Emergency Response

CDC Centers for Disease Control and Prevention

CFU Colony-Forming Unit

CLSI Clinical Laboratory Standards Institute

cMLS constitutive macrolides, licosamides, streptogramines

CNS Coagulase Negative Staphylococcus

DNA Deoxyribonucleic acid

dNTP deoxyribonucleoside triphosphate

EDTA Ethylenediaminetetraacetic acid

HA Hospital Authority

HAIs Healthcare associated infections

HA-MRSA Hospital-acquired methicillin-resistant Staphylococcus aureus

HCWs Healthcare workers

HICPAC Hospital Infection Control Practices Advisory Committe

V
ICT Infection control team

iMLS inducible macrolides, licosamides, streptogramines

IWG-SCC International Working Group on the Staphylococcal Cassette

Chromosome elements

Log logarithm

mg Milligram

ml Millilitre

mm Millimeter

MLST Multi-locus sequence typing

MRSA Methicillin-resistant Staphylococcus aureus

MSSA Methicillin-susceptible Staphylococcus aureus

ng Nanogram

PBP Penicillin-binding protein

PCR Polymerase chain reaction

PFGE Pulsed-field gel electrophoresis

ppm part per million

PVL Panton valentine leukocidin

RLU Relative light units

ROS Reactive oxygen species

SCCmec Staphylococcal cassette chromosome mec

SHEA Society for Healthcare Epidemiology of America

Spa Staphylococcal protein A

SSR Short sequence repeat

TSB Trypticase soy broth

UV Ultra violet

WHO World Health Organization

VI
 List of Tables

Table 1. Studies investigating the environmental contamination by MRSA

patients in healthcare settings, 2005-2012……………………………...8

Table 2. Studies investigating the environmental contamination of MRSA in

community, 2007-2012………………………………………………..10

Table 3. Frequency of near-patient sites, clinical equipment and far-patient

sites touched by the hands of HCWs………………………………….14

Table 4. The advantages and disadvantages of different hand hygiene

compliance methods…………………………………………………..16

Table 5. Summary of the 5 systems used in monitoring environmental

hygiene………………………………………………………………...21

Table 6. Classification of mec gene complexes...................................................28

Table 7. Classification of ccr gene complexes………………………………….29

Table 8. SCCmec types of MRSA………………………………………………30

Table 9. Sites for environmental samplings…………………………………….36

Table 10. The drugs used in performing susceptibility test according to CLSI….39

Table 11. The demographics of MRSA positive patients………………………...44

Table 12. Spa types found in clinical samples and environmental surfaces……..47

Table 13. The Spa repeats succession of isolated Spa types……………..……....48

Table 14. The antimicrobial profiles of the 5 spa types isolated from patients….51

VII
 List of Figures

Figure 1. Dynamic transmission cycle of MRSA…………………………………4

Figure 2. Five sequential steps for cross-transmission of HAI pathogens e.g.

MRSA via the hands of HCWs………………………………………..13

Figure 3. The “ Five moments for hand hygiene” concept from WHO…………15

Figure 4. The structure of SCCmec gene………………………………………..26

Figure 5. S. aureus Protein A gene map……………….…………………………33

Figure 6. Flow diagram describing the methodology of determination of the

lowest limit of detection of MRSA……………………………………38

Figure 7. MRSA found on the environmental surfaces of case and control

patients………………………………………………………………...45

Figure 8. Numbers of MRSA found on the non-near patients’ environmental

surfaces………………………………………………………………...46

Figure 9. Correlation of the spa types between the patients and their
surrounding environment……………………………………………...50

VIII
 Table of Contents
Abstract……………………………………………………………………………I
Declaration……………………….…………………………………...………….III
Acknowledgements…………………………………………………………........IV
Abbreviations……………………………………………………………………..V
List of Tables………………………………………………………………..….VII
List of Figures……………………………………………………………….…VIII
1. Introduction…………………………………………………………….1
1.1 Epidemiology of MRSA in healthcare setting………………………….2
1.2 The transmission cycle of MRSA………………………………………4
1.2.1 People carry MRSA……………………………………………….5
1.2.2. People shed MRSA into the general environment………………...5
1.2.3. People transmit MRSA to other people……………………………6
1.2.4 MRSA spread between people and the environment……………...7
1.3 The role of HCWs in transmission of MRSA…………………………12
1.4 Hygienic cleaning in healthcare setting……………………………….17
1.4.1 Chemical disinfectants…………………………………………...17
1.4.2 Airborne hydrogen peroxide……………………………………..18
1.4.3 Portable Pulsed Ultra Violet (UV) Radiation Device…………….18
1.4.4 Photocatalytic coatings…………………………………………...19
1.5 Analysis of hygiene practice monitoring tools……………………….19
1.6 Infection control measures of MRS...………………………………..21
1.6.1 Cohorting and contact precaution………………………………..22
1.6.2. Methods of Active Surveillance Testing for MRSA carriers……..22
1.6.3 Decolonization therapy…………………………….…………….25
1.7 Molecular typing methods of MRSA………………………………….25
1.7.1 SCCmec Typing…………………………..………………………25
1.7.2 Plused-field gel electrophoresis (PFGE)……………..…………..31
1.7.3 Multilocus sequence typing (MLST)…………...……..………...31
1.7.4 Spa typing………………………………………………..……….32
2. Objectives of the study……………………………………………..34
3. Methods and Materials…………………………………………….35
3.1 Study design and participants………………………………………….35
3.2. Microbiological methods……………………………………………...37
3.2.1. Determining the lowest limit of detection (LLD) of the laboratory
culture method…………………………………………………....37
3.2.2. Laboratory culture methods and antimicrobial susceptibility
testing of MRSA…………………………………………………39
3.3 Molecular analysis…………………………………………………….40
3.3.1. DNA extraction…………………………………………………..40
3.3.2. Amplification and Gel electrophoresis…………………….…….40
3.3.3. Purification of the PCR product.………………………………....41
3.3.4. Spa typing………………………………………………………...41
4. Results………………………………………………………………….42
4.1 Patients………………………………………………………………...42
4.2 Environments………………………………………………………….42
4.3 Spa types of the MRSA isolates……………………………………….47
4.4 Relationship of the spa types between patients and environment……..48
4.5 Antimicrobial resistance profiles of different spa types………………48
IX
5. Discussion……………………………………………………………..52
5.1 Environmental contamination by patients……………………………..52
5.2 Contamination of environmental surfaces…………………………….55
5.3 Molecular characteristics of the MRSA isolates……………………....57
5.3.1 Molecular characteristics of the MRSA isolates between
patients and their environment…………………………………...58
6. Conclusion…………………………………………………………….59
7. Limitations of the study…………………………………………....61
8. References…………………………………………………………….62
9. Appendix I The result of the method to determine the limit of
detection……………………………………………….80
10. Appendix II The photo of gel electrophoresis of MRSA…………...82
11. Appendix III Photo guidelines for environmental samplings………..83

X
1. Introduction

Staphylococcus aureus is non-motile, facultative gram-positive cocci with

spherical cells of 0.5 to 1.5 um in diameter and form grape-like (Greek staphyle)

clusters. S. aureus is a clinically important species, capable of causing a wide

range of human diseases from minor skin infections to severe diseases such as

toxic shock, bacteraemia, pneumonia and endocarditis. Within health care

facilities, S. aureus strains are transmitted from patient to patient primarily via

hand carriage by medical personnel.

In 1961, two years after the introduction of methicillin into clinical practice,

methicillin-resistant S. aureus (MRSA) strains were first reported. S. aureus

developed resistance to methicillin due to the acquisition of the mecA gene in the

staphylococcal cassette chromosome mec element (SCCmec). The mecA gene

encodes an additional low binding affinity penicillin-binding protein, PBP2 which

confers resistance to all currently available β-lactam agents (Fuda et al., 2004).

Since then, MRSA has spread and caused significant mortality and morbidity in

many hospitals throughout the world.

In the past decades, MRSA has been one of the most common pathogens to

cause healthcare associated infections (HAIs) in hospitals worldwide, i.e. hospital

acquired MRSA (HA-MRSA). MRSA is typically spread in healthcare settings

from patient to patient on the unclean hands of healthcare personnel or through

the improper use or reuse of equipment. Hands may become contaminated with

MRSA by contact with colonized or infected patients, colonized or infected body

sites of the personnel themselves, or devices, items, or environmental surfaces

contaminated by body fluids containing MRSA.

1
 At the end of the 1990s, virulent community-associated MRSA (CA-MRSA)

clones producing Panton-Valentine leukocidin (PVL) were first isolated in the

outpatient clinics and caused skin and soft-tissue infections. It affected healthcare

facilities later and became another impact on the public health burden because it

was disseminated widely in healthcare facilities as well (Boyce, 2008a). At the

moment, the distinction between CA-MRSA and HA-MRSA is beginning to fade

(Lowy, 2003). The prevention and control of infection due to MRSA has become

a national public health priority. In addition, the Centers for Disease Control and

Prevention of the USA (CDC) has guidelines for healthcare workers and the

public to prevent MRSA.

1.1 Epidemiology of MRSA in healthcare settings

MRSA infections result in higher mortality, greater lengths of hospital stay,

and increased cost compared with methicillin sensitive S. aureus (MSSA)

infections (Engemann et al.; 2003; Cosgrove et al., 2005; Robicsek et al., 2008).

Patients with MRSA bacteraemia have a mortality of 1.78%, three times higher

than with MSSA bacteraemia (Wang et al., 2008). Likewise, another study found

that MRSA was independently associated with a three-fold increase in mortality

and increased hospital charges of $14,000 per infection in patients with S. aureus

surgical site infection (in 2000) (Engemann et al., 2003). It has become the most

challenging infection control issue.

A large surveillance program of nosocomial S. aureus bloodstream

infections in the United States showed that the percentage of MRSA isolates

increased from 22% in 1995 to 57% in 2001 (Wisplinghoff et al., 2004). Among

the National Nosocomial Infections Surveillance (NNIS) hospitals in 2003, 64.4%

of hospital associated S. aureus infections occurring in ICUs were caused by

2
MRSA (Rosenthal et al., 2010). In 2010, a CDC published report showed that

invasive (life-threatening) MRSA infections in healthcare settings are declining.

Invasive MRSA infections that began in hospitals declined 28% from 2005

through 2008 (Kallen et al., 2010). However, the increase in the incidence of

CA-MRSA has become another challenge for the center. In Hong Kong, a

prospective surveillance study conducted by the Asian Network for Surveillance

of Resistant Pathogens (ANSORP) from 2004 to 2006 showed that the prevalence

of HA-MRSA among the healthcare associated S. aureus from 2004-2006 was

56.8% (Song et al., 2011).

MRSA has the ability to survive for days to weeks on environmental surfaces

in healthcare facilities. It is capable of withstanding a wide range of temperatures,

humidity; and exposure to sunlight, and it is resistant to desiccation. These

properties make it able to contaminate a large variety of hospital items, e.g. chairs,

mattresses, bed frames and computer keyboards (Bures et al., 2000; Bhalla et al.;

2004, Sexton et al., 2006; Lu et al., 2009; Rohr et al., 2009). Environmental

contamination makes an important contribution to the transmission of MRSA

(Dancer, 2008a). It also explains why MRSA outbreaks occur easily (Dietze et al.,

2001; Hardy et al., 2006a ; Huang et al., 2006). When mixed with hospital dust,

MRSA can still be alive more than one year after inoculation (Wagenvoort et al.,

2000). This increases the chance that someone else will acquire viable MRSA

from the environment as a new host. Even deep cleaning of a ward with detergent

and a steam cleaner, followed by use of 1,000 ppm chlorine disinfectant for all

hard surfaces, does not completely eradicate MRSA from the clinical environment

(French et al., 2004; Jeanes et al., 2005). Such persistence is likely to create a

reservoir in hospitals and represent a significant risk of infection to patients. In

3
addition, the hospital setting nowadays provides more hand-touch sites that

require a greater degree of sophisticated cleaning attention. Certain liquid

cleaning agents damage many items of medical and nursing equipment. All of

these differences could have contributed towards an increase in MRSA acquisition

in modern hospitals.

1.2 The transmission cycle of MRSA

The transmission cycle of MRSA is perpetuated by a dynamic transmission

cycle between human beings and their environment (Figure 1). MRSA transfers

from an affected patient to a susceptible host most commonly via the hands of

healthcare workers (HCWs), but contaminated objects, surfaces and air can be

either directly or indirectly involved in the transmission pathway.

(Otter et al., 2011)

Fig 1. Dynamic transmission cycle of MRSA

4
1.2.1 People carry MRSA

MRSA is found to colonize on many sites of the human body and anterior

nares are the most common carriage site. One retrospective, observational study in

the USA showed that the MRSA positive carrier rate was 10.8%. Among them,

85.3% were asymptomatic MRSA nasal carriers (Parvez et al., 2010). A review of

the MRSA prevalence in HCWs in 104 studies with denominator data showed that

the nasal carriage rate of MRSA in HCWs was 4.1% (Albrich and Harbarth,

2008). MRSA colonization has been shown to result in 10 times the number of

nosocomial infections when compared with MSSA colonization (Butterly et al.,

2010). It is not surprising that MRSA carriers will transfer their own strain of

MRSA to any site of the body via their hands and contaminate the environment.

1.2.2 People shed MRSA into the general environment

MRSA carriers shed their strain into the environment in various ways. People

with an upper respiratory tract infection or wound infection produce great

quantities of MRSA and the individual is referred to as a “cloud adult”. Some

individuals shed after antibiotic treatment, some shed depending on which sites

are colonized, and some individuals seem never to shed at all (Sherertz et al.,

2001). Therefore, MRSA can contaminate areas frequently and the frequency of

MRSA contamination correlates with the number of culture-positive body sites

(Rohr et al., 2009). Infected patients usually shed more MRSA than those who are

only colonized. The infectious dose for most environmentally associated

nosocomial pathogens appears to be low. For S. aureus, less than 15 cells were

sufficient to cause infection in experimental lesions (Dancer, 2008a).

Contamination of air has also been reported, one study conducting sequential

5
air sampling before and after bed making showed that MRSA counts remained

elevated for up to 15 minutes after the bed was made (Shiomori et al., 2002). It

has enough time to let MRSA particles to be transported to nearby areas and

adhere to them. However, the debate over the importance of airborne MRSA

continues because most microbiologists argue that patients are more likely to

acquire MRSA from the hands of healthcare workers rather than directly from the

air (Eames et al., 2009).

Contamination of rooms of unaffected patients has been reported; MRSA

was cultured from 43% of beds used by patients not known to be MRSA positive.

It was most likely to be due to the continued viability of MRSA shed by previous

occupants, or resulted from importation by HCWs or asymptomatic carriers

(French et al., 2004).

1.2.3 People transmit MRSA to other people

There are studies showing the MRSA transmission between people in

hospitals and people at home. These cases may relate to outbreak situations

involving HCWs or patients newly transferred to elsewhere (Blok et al., 2003;

Tansel et al., 2003). Other reports documented transmission spread between

patients in the community and transmission between ambulant patients (Borer et

al., 2002; Herwaldt et al., 2003; Teare et al., 2010). These person to person

transmissions point out the importance of MRSA in the environment. In fact,

some may think that indirect transmission through an environment frequented by

both source and recipient should be regarded as direct person-to-person

transmission (Dancer, 2008a).

6
1.2.4 MRSA spread between people and the environment

Patients and carriers are the prime source of contamination; surfaces in the

vicinity of patients that are touched by patients and HCWs frequently are termed

“high-touch surfaces”, which means that they have a higher frequency of

contamination than other sites. A recent study defined high-touch surfaces as the

bed rails, the bed surface, and the supply cart by observation of their contact

frequency (Huslage et al., 2010). People can acquire MRSA from these

hand-touch sites or from the air and transmit it to other environmental sites

(Wertheim et al., 2005). Many studies have shown evidence of dynamic

transmission of MRSA between people and the environment by finding MRSA

contamination in the environment of MRSA infected or colonized patients (Table

1). Also, evidence of MRSA contamination has been found in environments other

than hospitals (Table 2). Contaminated environments act as a reservoir to spread

MRSA when healthcare workers contaminate their hands or gloves by touching

contaminated surfaces. A study showed that MRSA can be acquired on the hands

of HCWs through contact with contaminated environmental surfaces without

direct patient contact (Bhalla et al., 2004). Another study reported that about 12

(17%) of 70 contacts between HCWs and an MRSA-colonized patient resulted in

the transmission of MRSA from the patient to the gloves of the health-care worker

(McBryde et al., 2004).

7
Table 1. Studies investigating the environmental contamination by MRSA patients in healthcare settings from 2005-2012
References Settings, location Study design Culture method Main findings
used
Wang et al., -1,600 bed -Screening for MRSA carriage in the -No broth -A relatively lower environmental
2011 teaching hospital emergency ward and intensive care enrichment step contamination rate of 34.1% (860/2520).
unit -The sites near to the perineum and
-Beijing -Sampling 30 environmental swabs for -Blood agar nasopharynx had the highest contamination
each MRSA carriage rate
Eibicht et al., -A university -Examined ambulances’contamination -Brain-heart -MRSA contamination rate was 9% (9/89
2011 hospital and 3 aid rate after transporting MRSA patients infusion (BHI) transportations)
organizations or carriers broth enrichment -5 cases only of the headrest area and 2
-Two sites at the stretcher, three sites cases only of the handles of the stretcher
-Germany at the bin were swabbed -Baird Parker were contaminated
-Transport time <20 minutes agar - 1 case was found of having MRSA in both
areas
Rohr et al., -248-bed surgery -25 MRSA carriages were placed in -Trypticase soy -Only 10.5% (105/1000) of the surface and
2009 department decontaminated private rooms broth (TSB) 4.0% (4/100) of the airborne samples were
-10 environmental samples and 1 enrichment MRSA positive
-Germany airborne sample were collected on -Bed linen had the highest contaminated
each visit (5 times) -Sheep blood rate of 25%
agar
Oie et al., 2007 -756-bed hospital -Environmental surfaces of 106 -No broth -50.9% (54/106) of the inpatients and 19.0%
patients with MRSA positive body enrichment step (4/21) of the control patients were detected
- Japan sites and 21 control patients that were to have MRSA in their surrounding
nasal negative were examined -Salt egg yolk environment
agar -In inpatients, the highest contaminated rate
was bed linen (40.2%) while it was over bed
tables (15%) in control

8
Table 1. Continued
Boyce et al., -500-bed teaching -10 environmental swabs of 8 patients -Non selective -58.8% (47/80) of the surfaces in the case
2007 hospital submitted for Clostridium difficile enrichment patients and 23.3% (14/60) in the control
toxin with MRSA colonization in the patients were found to have MRSA
- USA gastrointestinal tract and 5 control -Blood agar plate -Bedside rails were found to have the
patients with MRSA colonization were hightest MRSA contamination in both case
examined and control patients
Hardy et al., -Intensive care -Environmental swabs from 9 beds for -BHI broth -MRSA was present in 21.8% (188/864) of
2006 units 14 months were collected enrichment the environmental sites.
-During admission and then 3 times -The highest MRSA contamination was
-UK weekly -Oxacillin found underneath the beds with 37.5%
-In total, the environment of 53 MRSA resistant agar (81/216)
colonized patients were sampled
Sexton et al., -720-bed hospital -6 horizontal surfaces and 1 airborne -No broth -53.6% (269/502) of the surface samples,
2005 sample were collected in the isolation enrichment 28% (70/250) of the air samples were found
-Ireland rooms of 25 MRSA positive patients to be MRSA positive.
up to 4 weeks -Mannitol salt -Both bed and mattress had >50%
agar contamination rate

Lemmen et al., -9 intensive care -20 environmental samples of 136 -TSB enrichment -25.5% (165/648) of the environmental
2005 units of a patients with multi-drug resistant swabs were found to have MRSA
1500-bed tertiary organisms isolated including MRSA -Rodac plate -Bed corners had an MRSA contamination
teaching hospital (50 patients) (Becton rate greater than 35%
Dickinson) -Environmental contamination rate by
-Germany multidrug resistant organisms in the ICU
was showed no difference with regard to
general wards

9
Table 2. Studies investigating the environmental contamination of MRSA in the community in 2007-2012
Study (year) Settings, location Study design Culture methods Main findings

Murphy -10 nursing homes -The centres were categorized -7% sodium chloride -16% (78/500) of the items were MRSA
et al., 2012 into high and low groups based broth enrichment positive
-USA upon the MRSA point -A high proportion of MRSA-positive
prevalence and delta prevalence -MRSA chromagar objects were found in the high than the
-10 high-touch items were low delta prevalence nursing home (19%
sampled on 6 visits vs. 10%)
Simoes et al., 2011 -85 public urban -A single gauze was used to -BHI broth with -26% (22/85) of the buses were found to
buses sample a large surface area of enrichment be MRSA positive
different handrails
-Portugal -MRSA chromagar
Iwao et al., 2011 -349trains -The surfaces of the straps and -Did not mention -2.3% (8/349) of the trains were found to
handrails of 349 trains of 16 be MRSA positive
-Japan train lines were swabbed
Montgomery et al., -10 high school -9 surface categories were -No broth -46.7% (42/90) of the surfaces were
2010 athletic training sampled at each centre enrichment found to be MRSA positive
centres -Water coolers had the highest MRSA
-MRSA chromagar contamination rate of 19% (8/42)
-USA
Stanforth et al., -9 high school -10 surface categories were -No broth -All the 10 surfaces had at least positive
2010 wrestling sampled with multi-swabs enrichment samples of MRSA
environments -The inner and outer circles of the
-MRSA chromagar wrestling mats showed the greatest
-USA prevalence of MRSA (89%)

10
Table 2. Continued
Scott et al., 2009 -35 homes in the -Environmental sampling was -TSB enrichment -MRSA was found in 9 of the 35 homes
metro-Boston area had performed on 32 commonly -2 of the 9 MRSA positive homes had
children in diapers and a touched household surfaces in -Mannitol salt agar HCWs living which showed an
dog or cat in the each home insignificant difference in MRSA
household contamination rate among homes with or
without HCWs
-USA
Scott et al., 2008 -35 homes had children -Environmental samples of 32 -TSB enrichment -26% (9/35) of the homes were
in diapers and a dog or household surfaces were contaminated with a total of 15 isolates
cat in the household collected in each home -Mannitol salt agar of MRSA
-44% (4/9) of the MRSA positive homes
-USA had child care attendance but was not
statistically significant
-78% (7/9) of the MRSA positive homes
had a cat with a positive correlation

11
1.3 The role of HCWs in the transmission of MRSA

Studies had shown contamination of HCWs’ hands with MRSA after contact

with MRSA patients and after contact with environmental surfaces (Bhalla et al.,

2004; McBryde et al., 2004; Stiefel et al., 2011). Transmission of healthcare

associated pathogens from one patient to another via HCWs’ hands is the major

route, requiring five sequential steps (Figure 2). An observational study

categorized the hand-touched events contacted by HCWs’ hands into three

categories and accounted for the frequency of the HCWs touching the surfaces

through monitoring strategy (Table 3). The near-patient surfaces (in room) had a

high frequency of being touched by the hands of HCWs, followed by the

far-patient surfaces (outside room) and the clinical equipment (in room). This

sequence of hand-touch practices by HCWs during patient management provides

a theoretical demonstration of how hospital organisms spread from one

environmental site to another (Smith et al., 2012). HCWs can carry MRSA from

the contaminated surfaces or the infected patients to others. MRSA carriage of

HCWs is another risk factor for the transmission of MRSA. A review of the

prevalence of MRSA in HCWs was performed by Albrich and Harbarth (2008),

the average MRSA prevalence of HCWs being 4.6%. The HCWs can shed the

MRSA from their body or carry the MRSA through their hands after nasal contact

to the environment or to susceptible patients. So, the hand hygiene of HCWs is

considered to be the most beneficial intervention in the control of MRSA and

many other pathogens (Pittet et al., 2000). However, the problem with hand

hygiene compliance is that it is difficult to get everyone to do it at the most

appropriate time. One study has already contrasted the success and relative ease of

instituting and maintaining an environmental cleaning program with the failure of

12
a hand hygiene initiative (Hayden et al., 2006).

Hand hygiene guidelines published by the CDC and World Health

Organization (WHO) emphasize the importance of monitoring hand hygiene

compliance and providing HCWs with feedback regarding their performance

(Boyce and Pittet, 2002; WHO guidelines, 2009). WHO guidelines for hand

hygiene in healthcare settings suggest easily applicable concepts such as the “five

moments for hand hygiene” (Figure 3). Methods for measuring hand hygiene

compliance include direct observation of HCWs, self-reporting by HCWs,

observation by patients, measurement of product usage, and electronic systems for

measuring hand hygiene compliance (Haas et al., 2007; Boyce, 2008b; Gould et

al., 2011). Each of these approaches has advantages and disadvantages and are

summarized in Table 4 (Boyce, 2008b; Boyce, 2011).

1. MRSA is present on the patient's skin or have been shed onto inanimate
objects immediately surrounding the patient

2. MRSA must be transferred to the hands of HCWs

3. MRSA must be capable of surviving for at least several minutes on

HCWs' hands

4. Handwashing or hand antisepsis by the HCW must be inadequate or

omittted entirely, or the agent used for hand hygiene inappropriate.

5. The contaminated hand(s) of the caregiver must come into direct contact
with another patient or with an inanimate object that will come into direct
contact with the patient

(Pittet et al., 2006)

Fig 2. Five sequential steps for cross-transmission of HAI pathogens e.g.
MRSA via the hands of HCWs
13
Table 3. Frequency of near-patient sites, clinical equipment and far-patient
sites touched by the hands of HCWs
Category of surface
touched or handled Total no. of times % touched of all
Site
during 40 X 30 min touched sites handled
observed sessions
Near-patient
Patient console 8 5%
surfaces (in room)
Notes 37 22%

Bed frame 19 11%

Locker 4 2%

Curtains 6 4%

Category total (%) 74 44%

Clinical equipment
Hoist 0 0%
(in room)
Commode 4 2%

BP stand 10 6%

Stethoscope 3 2%

IV drip 23 14%

Category total (%) 40 24%

Far-patient surfaces
Computer 20 12%
(outside room)
Filing cabinet 0 0

Notes trolley 18 11%

Telephone 16 169%

Category total (%) 54 32%

Total items
168 100%
touched
(Smith et al., 2012)

14
(Navarro et al., 2008)

Fig 3. The “ Five moments for hand hygiene” concept from WHO

15
Table 4. The advantages and disadvantages of different hand hygiene
compliance methods
Method Description Advantages Disadvantages

Observations -Direct observation -Only method that -Time consuming

by trained of hand hygiene by can detect whether -The performance of
observers trained observers. HCWs have the observers may
-WHO developed performed hand vary
the concept of the hygiene practice -Cannot compare the
“My 5 Moments of appropriately, compliance rates
Hand Hygiene” to -To ensure the between facilities due
serve as a frame HCWs follow the to differences in the
work for training five major survey methods
observers indications for hand
hygiene
recommended by
WHO
Self- The hand hygiene -Requires little time
-HCWs often tend to
reporting by practice is reported -Easy to perform over-estimate their
HCWs by HCWs level of compliance
-Reporting format
varies between
healthcare settings, so
it is hard to compare
the compliance rate
Patient Patients are invited -HCWs have higher -Patients always
observers to be an observers awareness of the forget to observe the
of HCW hand need to clean their hand hygiene practice
hygiene practices hands always of HCWs

Measuring Recording the -Requires fewer -There is no way to

product amount of hand resources determine if hand
consumption hygiene products, -The measurements hygiene has been
volume or weight are easy to conduct performed under the
to determine the and are feasible in appropriate conditions
frequency of hand various healthcare -No information on
hygiene settings compliance by HCWs
is generated
Electronic Electronic motion -The electronic -No information is
monitoring sensors that detect monitoring includes provided if hand
with voice entry/exit of anyone who enters hygiene was
prompts persons into the room (including conducted at the
patient rooms have visitors) appropriate time,
also been used to - The hand hygiene before patient contact
estimate hand compliance will be or during patient care
hygiene increased
compliance rates in
healthcare settings

16
1.4 Hygienic cleaning in healthcare settings

To improve environmental hygiene in healthcare settings, the CDC published

guidelines for Environmental Infection Control in Healthcare

Facilities-Environmental Surfaces in 2003. It recommended that hospitals clean

and disinfect “high-touch surfaces” (Sehulster and Chinn, 2003). The guideline

for isolation precautions preventing the transmission of infectious agents in

healthcare settings 2007, from the CDC, recommends routine environmental

decontamination of non-critical surfaces in the patient-care area by cleaning and

disinfecting as a part of the standard precautions (Siegel et al., 2007).

1.4.1 Chemical disinfectants

There is evidence to support the role of domestic cleaning in hospitals as an

important intervention in the control of MRSA (Datta et al., 2009; Dancer, 2009a).

The traditional liquid chemical disinfectants, such as sodium hypochlorite,

phenolic disinfectants and hydrogen peroxide are usually used to decontaminate

environmental surfaces, and the use of these compounds is periodically reviewed

by the Hospital Infection control Practices Advisory Committee (HICPAC).

However, these compounds are usually toxic to humans, labour intensive,

inefficient in the eradication of organisms and require long contact times for

disinfection (French et al., 2004; Jeanes et al., 2005). Rutala and Weber (2004)

criticized the effectiveness of using disinfectant for environmental hygiene in

hospitals. They concluded that the routine use of disinfectants to disinfect hospital

floors and other non-critical items is questionable. A study used non-disposable

wipes to clean the environmental surfaces with disinfectants, the number of

bacteria was raised if rinsing was not conducted in between. Accumulations of

17
organisms were resulted in increasing the chance of cross transmission of MRSA

(Cheng et al., 2011a). So, various newly developed technologies have been under

evaluation to achieve more rapid and effective decontamination of the hospital

environment.

1.4.2 Airborne hydrogen peroxide vapour

This newly developed technique is increasingly used by healthcare

institutions. Hydrogen peroxide is a broad-spectrum disinfectant, considered

active against the majority of pathogens implicated in nosocomial infections

(Falagas et al., 2011). The commercial systems for airborne hydrogen peroxide

disinfection run automatically by using preprogrammed robots (Otter et al., 2006).

By applying the correct concentration of the disinfectant and adequate contact

time, the disinfection process can be achieved without reliance on cleaning

personnel (Rutala and Weber, 2004). The time required for a cycle of airborne

hydrogen peroxide disinfection is proportional to the size of the area to be

disinfected. Most importantly, the gas can reach sites that are inaccessible for

cleaners such as clinical equipment (Anderson et al., 2006). It appears to have low

toxicity and has good compatibility with most of the inanimate materials as it

degrades into oxygen and water. However, patients should be moved to other

areas before using this machine.

1.4.3 Portable Pulsed Ultra Violet (UV) Radiation Device

Continuous ultraviolet-C irradiation is known to be effective in the

disinfection of a broad range of bacteria. However, it is not portable and the

irradiation needs to last as long as 10 to 50 minutes. Pulsed UV requires 5 seconds

18
of radiation to attain bactericidal activity with more than 2Log growth inhibition

of all bacterial species. It was proven to have a bactericidal activity against critical

nosocomial bacteria after short irradiation, and found to be practical as a method

for disinfecting housekeeping surfaces and decreasing the labour burden

(Umezawa et al., 2012).

1.4.4 Photocatalytic coatings

Titanium dioxide, a photo-excited semiconductor, is coated on the

contaminated surface and achieves photocatalytic disinfection by production of

reactive oxygen species (ROS) which result in disruption of lipopolysaccharides

and phospholipids within the cell wall and cell membrane. It causes leakage of

cellular components and direct ROS attack of organelles and genetic material

(Kiwi and Nadtochenko, 2005). In one study, MRSA inactivation required the

shortest photocatalytic exposure time with 99.8% inactivation observed following

40 min (Dunlop et al., 2010).

1.5 Analysis of hygiene practice monitoring tools

There are five systems that are currently used in enhanced programmatic

monitoring. Covert monitoring environment contamination provides an objective

assessment of individual staff performance and compliance with cleaning

protocols. Trained research observers have to covertly monitor daily disinfection

cleaning. The thoroughness of environmental cleaning was improved by

application of this method in a study from 48% to 87% (Hayden et al., 2006).

Swab cultures of surfaces have been commonly used in epidemiological

studies of hospital acquired pathogens as well as in the evaluation of outbreaks

19
related to specific organisms (Carling and Bartley, 2010). Although swab cultures

are easy to use, the cost of processing, including isolate identification, the delay in

analyzing results, and the need to develop baseline values for comparisons limit

its use.

Agar slide cultures have been used in environmental surface monitoring to

assess the limitations of the visual audit of environmental contamination. Several

studies have used agar-coated slide to evaluate cleaning practices as well as to

compare cleaning regimens by quantifying aerobic colony counts (ACCs) per

square centimetre (Griffith et al., 2007; Dancer et al., 2009b). It can provide an

easy method for quantifying viable microbial surface contamination. The same as

the swab cultures, both systems need to develop baseline values for accurate

interpretation of the study findings.

The fluorescent gel system is a method using invisible transparent gel that

dries on surfaces following application and resists abrasion and was developed

specifically to evaluate the thoroughness of environmental cleaning in health care

settings. It was found to improve in the thoroughness of disinfection from 48% to

77% by applying this system in a study (Carling et al., 2008b). However, the

fluorescent gel system cannot be used to measure the actual cleanliness of

surfaces but only the thoroughness of cleaning practices.

Adeneinetriphosphate (ATP) bioluminescence is the measurement of organic

ATP on surfaces using a luciferase assay. A specialized swab is used to sample a

standardized surface area, which is then analyzed using a portable handheld

luminometer. The amount of ATP is quantified and expressed as relative light

units (RLU). Very low readings are typically associated with low ACCs while

very high RLU readings may represent either viable organisms or organic debris

20
including dead bacteria. It is impossible to use the system when a bleach-based

disinfectant is being used for cleaning (Boyce et al., 2010). Table 5 shows a

summary of the five methods used in evaluating environmental hygiene.

Table 5. Summary of the 5 systems used in monitoring environmental

hygiene
Method Ease of Use Identifies Useful for Directly
pathogens individual Evaluates
teaching cleaning
Covert Low No Yes Yes
Practice
Observation
Swab cultures High Yes Not studied Potentially

Agar slide Good Limited Not studied Potentially

cultures

Fluorescent High No Yes Yes

gel

ATP system High No Yes Potentially

(Adopted from Carling and Bartley, 2010).

1.6 Infection control measures of MRSA

Besides the hand hygiene of HCWs and housekeeping of hospital

environments, cohorting with contact precaution for MRSA positive patients is

also a guideline of the CDC to prevent the spread of MRSA from healthcare

settings. Active surveillance Testing (AST) is a method to identify that appropriate

contact precautions can be instituted in a timely manner to reduce the frequency

of cross-transmission events to other patients. Some hospital organizations now

screen all hospital admissions for MRSA (Weber et al., 2007).

21
1.6.1 Cohorting and contact precaution

The physical isolation of a MRSA patient in either a single room or a cohort

unit is a guideline to prevent the spread of MRSA. The physical barrier between

an MRSA-positive patient and other patients helps to interrupt transmission and

the psychological message that this barrier gives to HCWs by highlighting the

necessary precautions (Humphreys et al., 2009). Cohorting care of infected and

colonized patients should intuitively have a substantial impact on nosocomial

transmission of MRSA. In an outbreak situation, keeping infected and colonized

patients separate from uninfected and non-colonized patients provides a powerful

tool to prevent transmission (Henderson, 2006). Contact precautions for the

cohorted MRSA patients are usually the major infection control guidelines. The

Society for Healthcare Epidemiology of America (SHEA) and CDC guidelines

recommend contact precautions for all patients known to be colonized or infected

with MRSA, including hand hygiene and use of gloves and gowns before

interacting with these patients or entering their environments. Studies designed to

limit the spread of MRSA by implementation of contact isolation as a part of

infection control generally showed to be effective in controlling further spread

within hospitals (Marshall et al., 2004).

1.6.2 Methods of Active Surveillance Testing for MRSA carriers

The rationale for active surveillance testing is to identify all colonized

patients so that additional precautions can be applied (e.g. Contact Precautions).

Today, studies evaluating AST have had mixed results, the incidence of MRSA

infections not decreasing in the intervention group of the study by Harbarth et al.

(2009). Some studies have demonstrated efficacy in reducing MRSA infection

rates when AST was initiated before patients were admitted to hospitals (Harbarth

22
et al., 2006; Robicsek et al., 2008).

AST of patients for methicillin-resistant Staphylococcus aureus (MRSA)

carriage at hospital admission is widely accepted as an essential part of MRSA

control programs and had been well reviewed (Dancer, 2008b). Screening for

MRSA carriage is generally performed by swabbing the anterior nares or multiple

superficial sites, including nares, throat, and perineum (Brown et al., 2005).

Conventional MRSA detection methods rely on selective culture on solid media.

Recent chromogenic, cefoxitin-based selective agar media have demonstrated

superior selectivity, specificity and faster time to detection for MRSA screening

than non-chromogenic media (Brown et al., 2005), and several chromogenic

commercial media have performed well in clinical evaluations (Malhotra-Kumar

et al., 2008). MRSA is detected in 20–48 h with most of these media, and

negative results are usually reliable at 24 h. The use of a pre-enrichment step in

selective broth medium increases the sensitivity of these media by 15–30%

(Struelens MJ, 2009). By adding an indicator of bacterial growth, these broths can

produce a definitive negative result overnight, with possible positives being

confirmed by further tests and cultures (Gurran et al., 2002). The addition of an

enrichment broth culture has been reported to increase sensitivity, ranging from

2% to 23% (Paule et al., 2007). However, it increases the cost and delays the

results by an additional 18–24 h. Therefore, the practical value and

cost-effectiveness of using an enrichment step needs further study. Both culture

method and pre-enrichment take longer time (up to 48-72h) to identify MRSA

carriers. If pre-emptive isolation is not employed, it may allow transmission of

MRSA prior to recognizing MRSA carriers. However, these two methods can

culture the MRSA isolates for antimicrobial epidemiological study and further the

23
investigation for the presence of vancomycin intermediate or vancomycin

resistant S. aureus.

Rapid methods for molecular detection of MRSA-colonised patients have

recently been developed (Jeyaratnam et al., 2008). PCR primers can be used to

amplify the extremity of the SCCmec genetic element, which contains mecA, and

orfX, an open reading frame that is specific to S aureus. High sensitivity and

specificity have been reported in several studies that have analysed nasal samples,

with results available in 2 h (Jeyaratnam et al., 2008). However, PCR systems that

use unlinked primers targeting an S. aureus species-specific gene and mecA, such

as the LightCycler Staphylococcus and MRSA detection kit (LC Assay; Roche

Diagnostics, Mannheim, Germany) and the Hyplex StaphyloResist PCR (BAG,

Lich, Germany), may give false-positive results with such mixtures

(Malhotra-Kumar et al., 2008). To resolve this problem, the BD GenOhm system

(previously known as IDI-MRSA) (BD Diagnostics, San Diego, CA, USA)

amplifies MRSA-specific sequences by targeting a single locus that includes the

right extremity of SCCmec downstream of mecA, and part of the adjacent S.

aureus-specific orfX gene. Five primers target SCCmec sequences corresponding

to types I, II, III, IVa, IVb, and IVc, and one primer and three molecular beacons

are specific for orfX (Huletsky et al., 2004). The test is performed in real time

with fluorescence detection. Similar assays are the GeneXpert MRSA (Cepheid,

Sunnyvale, CA, USA) and the GenoType MRSA Direct (Hain Lifescience,

Nehren, Germany), the latter using hybridization to oligonucleotides on a

cellulose strip for detection and identification. (Rossney et al., 2008). In contrast,

qMRSA 2003 is an in-house triplex quantitative PCR assay that amplifies mecA

and femA from S. aureus and Staphylococcus epidermidis to identify the origin of

24
the mecA signal (Francois et al., 2003). Although the molecular methods can

identify MRSA carriers within a day and is benefit to infection control, the cost is

needed to take into consideration and some PCR methods have high false positive

rate.

1.6.3 Decolonization therapy

Colonization by MRSA is often asymptomatic, but MRSA carriage is also a

risk factor for the transmission of MRSA. Therefore, decolonizing of patients or

HCWs identified as carriers is regarded as useful in preventing the transmission of

MRSA. Topical mupirocin applied on the anterior nares in combination with a

chlorhexidine bath is commonly used to decolonize the MRSA carriage. Some

studies supported the beneficial effect of decolonization therapy with reduction in

MRSA infection (Robicsek et al., 2008; Bode et al., 2010). Although the

effectiveness of decolonization with mupirocin has been documented, mupirocin

resistance may develop relatively quickly and is increasingly reported as a clinical

concern (Henderson, 2006).

1.7 Molecular typing methods of MRSA

In order to prevent the spread of MRSA, effective strategies have been

required and various molecular typing methods have been developed. The most

commonly used typing methods are Staphylococcal cassette chromosome mec

(SCCmec) typing, pulsed-field gel electrophoresis (PFGE), multilocus sequence

typing (MLST), Stahpylococcal Protein A (Spa) typing.

1.7.1 SCCmec Typing

SCCmec elements commonly carry several characteristics (Figure 4). A mec

gene complex, which is 2.1 kb in length, is encoded for the 78-kDa

25
penicillin-binding protein (PBP)2a and causes resistance to methicillin and all

other β-lactam antibiotics. This gene is regulated by two proteins, mecI (a

repressor protein) and mecR1 (a signal transducer protein) (Deurenberg and

Stobberingh, 2008). In the presence of a β-lactam drug, mecR1 is autocatalytically

cleaved and protease activity is released to the metalloprotease domain of mecR1

which results in the increased transcription of mecA and production of PBP2a

(Berger et al., 2002). These two genes are sometimes truncated and deleted by the

integration of insertion sequences of IS431 or IS1272. These gene alleles are

regarded as mecA gene complexes and there are six classes of mec gene

complexes defined by the International Working Group on the Classification of

Staphylococcal Cassette Chromosome (SCC) Elements (IGW-SCC) (Table 6).

IGW-SCC was set up in 2009 to define consensus rules for a nomenclature system

for SCCmec elements classification.

(Iwao et al., 2011)

Fig 4. The structure of SCCmec gene

26
 A cassette chromosome recombinases (ccr) gene complex is composed of

one or two Site–specific recombinase genes and is responsible for the

mobilization of cassette (Katayama et al., 2000). It can catalyze precise excision

and integration of SCCmec elements at specific sites. Today, two distinct ccr gene

complexes have been reported; one carries ccrA and ccrB and the second carries

ccrC. The ccrA and ccrB genes have been classified into four and five allotypes

respectively. Currently, there are eight types of ccr gene complexes which have

been found in staphylococci (Table 7) (http://www.sccmec.org ).

The regions that are not part of the mec complexes and ccr genes are called J

(junkyard) regions. The junkyard regions comprise three parts: J1, J2 and J3.

These regions are considered less important in terms of SCCmec function;

However, they are epidemiologically significant since they may serve as targets

for plasmids or transposons, carrying additional antibiotic and heavy metal

resistance determinants (Turlej et al., 2011). Variations in the J regions within the

same mec-ccr gene complex are used for defining SCCmec subtypes. (Shore et al.,

2005; Chongtrakool et al., 2006).

SCCmec typing is one of the methods used to study the epidemiology of

MRSA by classification of the SCCmec element such as mec complex, ccr

complex and J region. The most commonly used method is multiplex PCR assays

(Oliveira and de Lencastre 2002; Milheirico et al., 2007; Stephens et al., 2007).

Different SCCmec types are identified according to the combination of (1) the

type of ccr gene complex, which is represented by the ccr gene allotype, and (2)

the class of the mec gene complex. “Subtypes” are based on the variations in the J

regions within the same mec-ccr gene complex (http://www.sccmec.org). Today,

there are more than 10 SCCmec types identified along with various subtypes

27
which have been distinguished among MRSA strains (Table 8). Variations in these

SCCmec types have provided the basis for differentiation among MRSA strains. A

combination of two typing methods like SCCmec typing along with MLST is

suggested for reliable typing for inter-hospital, multicentre surveillance, and the

international transmission and evolution of MRSA strains (Struelens et al., 2009).

Table 6. Classification of mec gene complexes

Class mec gene complex

Class A IS431-mecA-mecR1-mecI

Class B IS431-mecA-ΔmecR1-IS1272

Class C1 IS431-mecA-ΔmecR1-IS431
(both insertion sequence IS431 are in the same direction)

Class C2 IS431-mecA-ΔmecR1-IS431
(both insertion sequence IS431 are in reverse direction)

Class D IS431-mecA-ΔmecR1 *
(* no IS element downstream)

Class E blaZ-mecALGA251-mecR1LGA251-mecILGA251

(http://www.sccmec.org last accessed on 1/8/2012)

28
Table 7. Classification of ccr gene complexes

ccr gene complexes ccr genes

Type 1 A1B1

Type 2 A2B2

Type 3 A3B3

Type 4 A4B4

Type 5 C1

Type 6 A5B3

Type 7 A1B6

Type 8 A1B3

(http://www.sccmec.org last accessed on 1/8/2012)

29
Table 8. SCCmec types of MRSA

SCCmec Genebank ccr gene mec gene Origin References

type/ accession complexes complexes
subtype no.
I AB033763 Type 1 Class B UK Ito et al., 2001
(A1B1)

II D86934 Type 2 Class A Japan Ito et al., 1999

(A2B2)

III AB037671 Type 3 Class A New Ito et al., 2001

(A3B3) Zealand

IVa AB063172 Type 2 Class B USA Ma et al., 2002

(A2B2)

V AB121219 Type 5 Class C2 Australia Ito et al., 2004

(C1)

VI AF411935 Type 4 Class B Portugal Oliveira et al.,

(A4B4) 2006

VII AB373032 Type 5 Class C1 Sweden Berglund et al.,

(C1) 2009

VIII FJ670542 Type 4 Class A Canada Zhang et al.,

(A4B4) 2009

IX not Type 1 Class C2 Thailand Li et al., 2011

available (A1B1)

X not Type 7 Class C1 Canada Li et al., 2011

available (A1B6)

XI not Type 8 Class E not http://www.scc

available (A1B3) available mec.org

(Turlej et al., 2011, http://www.sccmec.org last accessed on 1/8/2012 )

30
1.7.2 Pulsed- Field Gel Electrophoresis (PFGE)

PFGE is considered the most common method for investigating outbreaks of

MRSA in hospitals as well as hospital to hospital transmission. PFGE is based on

the digestion of the chromosomal DNA by the restriction enzyme Smal, followed

by agarose gel electrophoresis in an alternating voltage gradient. The PFGE

banding patterns can be analyzed by using different software programs

(Rementeria et al., 2001). Some attempts have been made to standardize PFGE

protocols and to set up a common nomenclature, but they had only limited success

when assessed for cost of analysis, reproducibility and speed. Since strict

adherence to standardized protocols is needed for a common nomenclature, they

have only been realized at national level in some countries, e.g. the USA. On an

international level, it is not successful to attempt to create a common

nomenclature. (Murchan et al., 2004; Goering, 2010).

1.7.3 Multilocus sequence typing (MLST)

MLST is a useful tool for studying the clonal evolution of MRSA. This is a

method based on sequence analysis of several housekeeping genes and each is

approximately 500-bp in length. The different sequences of each housekeeping

gene are assigned as distinct alleles, and each MRSA strain lineage is defined by

the alleles of the seven genes resulting in an allelic profile called Sequence Type

(ST). MLST is usually used in conjunction with PCR analysis of the SCCmec

typing method to define the clonal type of MRSA strain. The Microbiological

Societies' Subcommittee on S. aureus typing has accepted an international

nomenclature for these clonal types, e.g. the EMRSA-16 clone is referred to as

ST36-MRSA SCCmec type II (Cookson et al., 2007). A web-based database for

MLST typing is available (www.mlst.net) for comparison of results. However, this

31
method is expensive, laborious and time-consuming due to the detection of

various genes. A good degree of concordance has been reported between results

obtained by MLST, PFGE and Spa typing (Hallin et al., 2007; Melles et al., 2007;

Tavares et al., 2010).

1.7.4 Spa typing

Spa typing is a single-locus sequence typing technique commonly used to

compare sequence variation of a single target gene. The sequence of the

polymorphic region X of the S. aureus Protein A (spa) is a short sequence repeat

(SSR) region that is sufficiently polymorphic to provide a useful resolution

(Narukawa et al., 2009). The gene for protein A having tandem repeats, 24bp in

length, have been studied extensively and it is reported that MRSA strains can be

discriminated by determining the repeat sequence numbers within the X region of

the spa gene through its point mutations, deletions and duplications (Figure 5)

(Kahl et al., 2005; Deurenbery and Stobberingh, 2008). Its discriminating ability

lies between PFGE and MLST and is useful for studying both the molecular

evolution and hospital outbreaks of MRSA (Koreen et al., 2004, Malachowa et al.,

2005). It has been increasingly used in epidemiology studies (Ho et al., 2008b, Ho

et al., 2009, Soliman et al., 2009, Cheng et al., 2011). The advantage of spa

typing over MLST is its simplicity because it only involves a single locus.

Another advantage is that laboratories all over the world can use various

sequencing methods and analyze the resulting sequence chromatograms by using

a central SpaServer (http://spaserver.ridom.de) (Friedrich et al., 2006). At present,

the Spaserver database is one of the largest typing databases worldwide and is

continuously collecting spa typing data for infection control purposes.

32
Fig 5. S. aureus Protein A gene map
(a)Primers are numbered from the 5′ end of the primer on the forward strand of S.
aureus (GenBank Accession no. J01786). Region S indicates for gene coding for
signal sequence, A-D are immunoglobulin G binding region, E is a region
homologous to region A-D. The region X includes SSRs (Xr) and the cell wall
attachment sequence (Xc) (Shopsin et al., 1999). (b) The repeat structure of the
Xr region. The spa type illustrated is t008 (Ridom-Harmsen et al. nomenclature)
or YHGFMBQBLO (Kreiswirth nomenclature). (c) The DNA sequence of the spa
repeat 21 (Ridom) or -F 1 (Kreiswrirth) repeat (Hallin et al., 2009).

33
2. Objectives of the study

The studies on the environmental contamination by MRSA above evidenced

that the environment plays an important role in the spread of MRSA in hospital

and non-hospital areas. The reason of higher contamination rate of MRSA may be

due to its ability to survive a long period of time and withstand well in hospital

fomites. Besides, the transmission mode of MRSA is another reason. Patients with

MRSA colonization or infection frequently shed MRSA, resulting in

contamination of their skin, clothing, bedding, and nearby environmental surfaces

(Chang et al., 2009). In hospitals, the hands of the healthcare workers are

considered one of the major vectors to spread MRSA from patients with MRSA

infection or colonization to others after patient management. There is evidence

showing that contamination of the environment with MRSA is sufficient to

contaminate the gloves of HCWs and therefore, lead to transmission to patients

(Boyce et al., 1997). In our hospital setting, patients diagnosed with either MRSA

infection or carriage will follow the guideline for infection control published by

the Central Committee on Infectious Disease and Emergency Response of the

Hospital Authority for patient management and for environmental

decontamination (HA CCIDER, 2012). In addition, nasal carriers are prescribed

decolonization therapy which includes 2% mupirocin ointment applied on anterior

nares three times per day for five days and 4% chlorhexidine gluconate for skin

and hair decolonization for 5 days. All these measures are to avoid MRSA

contaminating people and the environment.

In this study, we examined the presence of MRSA on the environmental

surfaces and its relationship with patents’ acquisition of MRSA by studying their

molecular characteristics.

34
3. Methods and materials

3.1 Study design and participants

This study was conducted from June 2011 to September 2011, at an acute

hospital in Hong Kong. During the study period, 30 MRSA positive patients were

selected randomly, regardless of age, sex, length of hospital stay, site of infection

or colonization. When the Infection Control Team (ICT) received a laboratory

report of a MRSA positive patient, the staff who collect the environmental

samples would be informed and swabs would be taken within the day. A total of

15 and nine environmental swabs surrounding the patient and the ward were taken

respectively (Table 9). At the same time, environmental samples of a patient

without MRSA infection were collected as a control. Then the swabs were sent to

the laboratory for culturing MRSA within 2 hours. The swabs were enriched and

cultured on Chromogenic MRSA agar (bioMérieux, France) for isolation of

MRSA. All the MRSA isolates were undergone susceptibility testing according to

the Clinical and Laboratory Standard Institute (CLSI) guideline 2011 and

polymerase chain reaction (PCR) to amplify the X-region of the spa gene for

sequencing.

35
Table 9. Environmental surfaces sampled from MRSA positive, control
patients and Nurse workstations

Environmental surfaces of case patients and control patients

bedside table monkey pull bed sheet pillow bed sheet that
top covering the patient sleeps
patient on
curtain by the patient bed patient locker cubicle oxygen pump
patient frame partition ledge knobs

bed tilt patient folder locker handle patient table infusion pump
adjustment bars height adjustment
panel adjustment panel
knob
Ward’s environmental surfaces

door handle computer telephone barcode fax machine

inside and keyboard and reader
outside ward, mouse
toilet/ water tap ambulatory
bathroom handle chair armrest
outside handle

36
3.2 Microbiological methods

3.2.1 Determining the lowest limit of detection (LLD) of the laboratory

culture method

To estimate the lowest limit of detection (LLD) of the laboratory isolation of

MRSA, a qualitative evaluation method was performed before collecting the

environmental samples. Swabs were spiked with various dilutions of MRSA

(ATCC 43300) and named Suspension A1, A2, A3, A4, A5 and A6. A bacterial

mixture of E. coli (ATCC 25922) and coagulase negative Staphylococcus (CNS)

(ATCC11490) with various dilutions were prepared and named B1, B2, B3, B4,

B5, B6. They were added to simulate the environment commensals. Figure 6

shows a flow diagram to explain the methodology. The LLD of this culture

method was 5 x 103 CFU mL-1 and the optimum incubation time of the enrichment

broth was 48 hours. In summary, these findings confirm that the swabbing method

was suitable for detecting MRSA contamination qualitatively, but not

quantitatively. The detailed results of the spiking experiment are shown in

Appendix I.

37
Fig. 6 Flow diagram describing the methodology of determination of the
lowest limit of detection of MRSA.
(CFU-colony forming unit, OX-MSA- Mannitol salt agar with 4μl oxacillin,
r.t-room temperature)

38
3.2.2 Laboratory culture method and antimicrobial susceptibility testing of
MRSA

The environmental samples were collected with sterile rayon swabs (Copan

Diagnostic Inc., USA). The swabs were broken into 4ml Mannitol-salt broth

(Oxoid, Oxoid Ltd., UK) and vortexed slightly. They were incubated aerobically

at 35℃ for 48 hours. All the broths were subcultured on Mannitol-salt agar

(Oxoid, Oxoid Ltd., UK) with 4 μg oxacillin and ChromID MRSA agar

(bioMérieux, France). The plates were incubated aerobically at 35 ℃ and

examined after 48 hours of incubation. Bacterial colony morphology, Gram stain,

latex agglutination test (Slidex Staph Plus, bioMérieux, France), tube coagulase

test were used to identify organisms as S. aureus. Antimicrobial susceptibility

testing of the drugs in Table 10 was performed using the disc diffusion method in

accordance with CLSI (2011) recommendations.

Table 10. The drugs used in performing susceptibility test according to CLSI

Tetracycline Ciprofloxacin Minocycline

Fusidic acid Cefoxitin Chloramphenicol

Clindamycin Mupirocin Rifampicin

Erythromycin Co-trimoxazole Gentamicin

Cefoxitin discs were used for phenotypic detection of methicillin resistance.

The D-test was used to detect inducible resistance to clindamycin. A control strain

S. aureus ATCC25923 was included on each day of testing. All the MRSA isolates

were kept in MicroBank (Pro-lab, Diagnostics, Neston, UK) at -70 ℃ for

molecular analysis.

39
3.3 Molecular analysis

3.3.1 DNA extraction

Bacterial isolates were grown on 5% horse blood agar at 35℃ for 24 hours.

One microliter bacterial isolate was suspended in 100 μl RNase and DNase free

water and incubated at 100℃ for 15 minutes. The tubes were then centrifuged at

13,200 rpm for 1 minute. Eighty microliter of the supernatant was kept at -20oC

for further use.

3.3.2 Amplification and Gel electrophoresis

The primers SpaF1 and SpaR1 were used to amplify the polymorphic X

region of protein A as previously described (Oliveira et al., 2001). The sequences

of the primer SpaF1 and SpaR1 were 5’-GACGATCCTTCG GTGACG-3’ and

5’-CAG CAGTAGTGCCGTTTGC-3’ respectively. A volume of 2μl of DNA

template (approximately 140-160 ng) was added to a 48 μl master mix solution

which contained 1x-PCR buffer, 2.2 mM MgCl2, 0.2 mM dNTP, 0.4 μM SpaF1,

0.4 μM Spa R1 and 0.02 U/μl of AmpliTaq Gold polymerase. The amplification

took place in a Veriti® 60-well thermal cycler (Applied Biosystem, California,

USA). The AmpliTaq Gold polymerase was first activated at 94℃ for 10 minutes

followed by 30 cycles under the following profile: 94℃ for 30 seconds, 61℃ for

30 seconds, 72℃ for 45 seconds. An extension was performed at 72℃ for 10

minutes. Gel electrophoresis of the amplified PCR product was performed in a 2%

w/v agarose gel with Tris/Borate/EDTA buffer at 120 voltages for 30 minutes. A

molecular size marker (GeneRulerTM100bp Plus DNA Ladder, Thermo Fisher

Scientific Inc., UK) was incorporated parallel during each run. The gel was

stained with SBYR gold for 30 minutes and illuminated under UV light

40
(Appendix II).

3.3.3 Purification of the PCR product

Before performing sequencing, the PCR product was purified by the PCR

quick-spinTM PCR Product Purification Kit (iNtRON Biotechnology, Inc.).

Firstly, PCR product in a membrane of a spin column was purified by adding

binding buffer. Subsequently, washing buffer was added into the column and

centrifuged. After centrifugation, the spin column was centrifuged to dry the spin

membrane. Finally, the column was put into a clean and sterile centrifuge tube.

The purified PCR product was eluted by adding 50 μl PCR grade H2O into the

upper reservoir of the column and centrifuged at 13,000 rpm for 60 seconds, the

purified PCR product was then ready for sequencing.

3.3.4 Spa typing

The purified PCR product underwent cycle sequencing. A volume of 2 μl

purified PCR product was added to 18 μl of master mix solution with BigDye

Terminator Tv3.1, 5X sequencing buffer, 3.2 μM SpaF1 primer. The mixture was

first activated at 96℃ for 1 minute followed by 25 cycles at 96℃ for 10 seconds,

50℃ for 5 seconds and 60℃ for 4 minutes. Afterwards, sequencing reaction

purification was performed by Centri-Sep™ 96-well plate (Applied BioSystem,

California, USA). Finally, DNA sequencing was performed in the 3130xl Genetic

Analyzer (Applied BioSystem, California, USA) and the Spa types were assigned

by the Bionumerics software (Version 6.6, Applied Maths, USA).

41
4. Results

4.1 Patients

From July 2011 to September 2011, a total of 655 (including nurse

workstation) case environmental swabs from 30 patients with MRSA culture

positive and 386 control environmental swabs from 30 MRSA negative patients

were collected for this study. The control patients were found MRSA negative in

their clinical samples during collecting the environmental samples, however, they

did not screen for MRSA carriage. The thirty MRSA positive patients were all

handled with contact precautions. Only one of the patients in medical ward was

isolated in single room while the other 29 patients were cohorted in ward cubicles.

In total, 29 patients (97%) were found to be MRSA positive in their environmental

swabs. Only one patient was found to have no MRSA contamination in his

surrounding environment. Besides, 12 of the 30 (40%) MRSA negative control

patients were found to have MRSA in their environmental samples. The case

patients’ demographics were shown on Table 11.

4.2 Environments

The percentage of MRSA culture positive in case environment, control

environment as well as nurse station were 34.3% (137/400), 9.1% (35/386) and

5.9% (15/255) respectively. Figure 7 shows the numbers of MRSA cultured from

the both case and control environmental surfaces as well as the location of

different surfaces. In the case environment, bed sheets (70%, 21/30) on which

patients sleep were the most frequent site to isolate MRSA, followed by pillows

(55%, 16/29), patient bed frames (52%, 15/29) and patient lockers (52%, 15/29).

Patient folders, oxygen pump knobs and infusion pump adjustment panels also

42
had presence of MRSA by 7% (2/30), 7% (1/15) and 8% (1/12) respectively.

Bathroom outside handles and water tap handles had an absence of MRSA. In the

environment of control patients, cubicle partition ledge was the most frequent area

found to harbour MRSA and accounted for 20% (6/30), followed by bed sheets,

curtains and patient lockers accounting for 17% (5/30). Monkey pulls, patient bed

frames, oxygen pump knobs, patient folders, patient table height adjustment knobs

and infusion pump adjustment panels were found to be MRSA culture negative in

this study.

In the nurse workstation, the ambulatory chair armrests had the highest

MRSA positive rate of 21% (4/19), followed by fax machines which accounted

for 14% (4/29) (Figure 8)

43
Table 11. The demographics of MRSA positive patients

Parameter No. of patients

Sexs
Female 19
Male 11
Mean age ± SD 76.6 ± 14.9
Age group (years)
18-50 1
51-64 3
65-74 5
≧ 75 21
Mean length of bed occupancy ± SD 9.3 days ± 21.8
Specialty (Unit)
Medical 12 (40%)
Geriatrics 6 (20%)
Orthopeadics 4 (13%)
Medical Nephrology 3 (10%)
General Surgical 2 (7%)
Surgical Urology 2 (7%)
Medical Neurology 1 (3%)
Specimen Sources
Respiratory 13 (43%)
Wound 6 (20%)
Nasal Swab 5 (17%)
Urinary 3 (10%)
Blood culture 1 (3%)
Pleural fluid 1 (3%)
Eye swab 1 (3%)

44
Fig. 7 MRSA found on the environmental surfaces of case and control patients
(A) Numbers of MRSA isolates found in near patients environments. A, bed sheet; B, pillows; C, patient bed frame; D, patient locker; E, blanket;
F, cubicle partition ledge; G, bedside table top; H, curtain; I, patient table height adjustment knot; J, bed tilt adjustment panel; K, monkey pull, L,
locker handle bars; M, patient folder; O, oxygen Pump knobs; infusion pump adjustment panel. (B) A picture shows the settings of the
environmental surfaces surrounding the case and control patients; P, case and control patients.

45
 MRSA POSITIVE

0.5
1.5
2.5
3.5
4.5

0
1
2
3
4
Fax machine

Ambulatory chair
armrest

CMS = Clinical Management System

Computer keyboard
and mouse

Barcode Reader

Nurse workstation
Door handle inside and
outside ward

Fig 8. Numbers of MRSA found on the non-near patients’ environmental surface

Nurses station around
CMS (top surface)

Telephone

Toilet/ Bathroom
outside handle
MRSA Positive

Water tap handle

46
4.3 Spa types of the MRSA isolates

In total, 216 MRSA isolates (30 clinical isolates, 151 case environments and

35 control environments) were performed spa typing and eight spa types were

found. The most predominant spa type in this study was t1081 and accounted for

63.3% (135/216) of the total isolates. Then t032 accounted for 17.6% (38/216)

and was the second predominant spa type. The third prevalent spa type was t037

and accounted for 7.4% (16/216). Table 12 shows the distribution of various spa

types in the clinical samples and environment. t091 and t1378 were found in the

control environment and accounted for 20% (7/35) and 2.9% (1/35) of the total

control environmental isolates, respectively. t4677 was found in the nurse

workstation only and accounted for 6.7% (1/15) of the total isolates from this area.

The Spa repeats of each of the Spa types were shown on Table 13.

Table 12. Spa types found in clinical samples and environmental surfaces

Spa No.(%) No. (%) of environmental isolates

types of
clinical Case Control Nurse
isolates Environment Environment Station Subtotal Total
t1081 19(63.3) 93 (68.4) 15 (42.9) 8 (53.3) 116 135
(62.4) (62.5)
t032 6 (20) 17 (12.5) 12 (34.3) 3 (20) 32 (17.2) 38 (17.6)

t037 3 (10) 13 (9.6) - - 13 (7.0) 16 (7.4)

t002 1 (3.3) 7 (5.1) - 3 (20) 10 (5.4) 11 (5.1)

t6508 1 (3.3) 6 (4.4) - - 6 (3.2%) 7 (3.2)

t091 - - 7 (20) - 7 (3.8) 7 (3.2)

t1378 - - 1 (2.9) - 1 (0.54) 1 (0.5)

t4677 - - - 1 (6.7) 1 (0.54) 1 (0.5)

Total 30 (13.9) 136 (63) 35 (16.2) 15 (6.9) 186 216

(86.1)

47
Table 13. The Spa repeats succession of isolated Spa types

Spa types Spa type repeats succession

t1081 08-16-02-43-34-17-34

t032 26-23-23-13-23-31-29-17-31-29-17-25-17-25-16-28

t037 15-12-16-02-25-17-24

t002 26-23-17-34-17-20-17-12-17-16

t6508 08-17-34

t091 07-23-21-17-34-12-23-02-12-23

t1378 26-23-23-13-23-31-29-17-31-29-17-25-17-25-16-16-28

t4677 08-16-02-25-02-24-25

4.4 Relationship of the spa types between patients and the environment

For 29 case patients, five spa types were discovered; 27 patients were found

to have the MRSA isolates with same spa type in the clinical samples and their

surrounding environments. This revealed a 93.1% (27/29) agreement (Figure 9).

For the two out of 29 patients (6.9%), the spa type found in the case environment

was different from that in patients. The length of bed occupancy of these two

patients was less than 48 hours. In addition, nine out of the 27 patients (33.3%)

were found to have more than one spa types in the case environment.

4.5 Antimicrobial resistance profiles of different spa types

Overall, the erythromycin resistance rate was 74.1% (160/216). Among

erythromycin-resistant isolates, 4.4% (7/160) and 95.6% (153/160) had

constitutive (cMLS phenotype) and inducible (iMLS phenotype) resistance to

clindamycin, respectively (Table 14). Among the three predominant spa types,

48
t1081, t032 and t037, >70% of the isolates were shown to have resistance to

erythromycin, clindamycin, ciprofloxacin. All the t002 isolates were shown to

have 100% (11/11) resistance to clindamycin, erythromycin, tetracycline and

ciprofloxacin. Only t1081 showed 9.6% (13/135) resistance to mupirocin and t032

showed 7.9% (3/38) resistance to rifampicin; all others isolates were shown to be

sensitive to these two drugs.

49
 Spa type different from the patient
Spa type same as patient
12
No. of sites MRSA positive in case environment

t1081 t032
10

t1081 t002
8
t1081t1081 t032
t1081
t1081 t037
6
t1081 6508 t1081 t1081
t1081 t002
4
t1081 t1081 t1081
t1081
t1081 t002 t037
2 t1081
t1081 t032 t1081 t032 t032 t037 t6508
t1081
t1081 t1081 t1081 t032 t032 t037
0

M035 (t032)

M026 (t032)

M030 (t032)

M028 (t032)

M015 (t032)

M021 (t037)

M019 (t037)

M018 (t037)

M014 (t002)
M005 (t1081)

M013 (t1081)

M002 (t1081)

M025 (t1081)

M017 (t1081)

M020 (t1081)

M022 (t1081)

M031 (t1081)

M023 (t1081)

M034 (t1081)

M011 (t1081)

M023 (t1081)

M012 (t1081)

M003 (t1081)

M016 (t1081)

M027 (t1081)

M033 (t1081)

M029 (t1081)

M024 (t1081)

M004 (t6508)
M001 (t032)
MRSA patients

(M024 was the only patient found no MRSA contamination in the environment and isolated in single room. Other patients were placed in
cohorted cubicles.)

Fig 9. Correlation of the spa types between the patients and their surrounding environment

50
Table 14. The antimicrobial profiles of the 5 spa types isolated from patients

Erythromycin(%)

Chloramphenical
Co-trimoxazole
SPA

Ciprofloxacin
D zone + (%)
Clindamycin

Tetracycline
Minocycline
Gentamycin
TYPE

Mupirocin
Rifampcin
Acid (%)
(total

Fusidic
no)

(%)

(%)

(%)

(%)

(%)

(%)

(%)

(%)
t1081 97 12 99 42 1 105 135 13
- - -
(135) (72.6) (8.8) (73.3) (31.6) (0.7) (77.8) (100) (9.6)

t032 30 30 1 3 1 37
- - - - -
(38) (78.9) (78.9) (2.6) (7.9) (2.6) (97.4)

t037 12 5 16 10 5 1 15 16
- - -
(16) (75) (31.2) (100) (62.5) (31.3) (6.3) (93.8) (100)

t002 11 11 7 11 11
- - - - - -
(11) (100) (100) (63.6) (100) (100)

t6508 3 3 1 7
- - - - - - -
(7) (42.9) (42.9) (14.3) (100)

Total
71.9% 7.8% 73.7% 29% 2.8% 0.5% - 1.4% 61.3% 95.9% 6%
(216)

51
5. Discussion

5.1 Environmental contamination by patients

In this study, widespread contamination of the environment with MRSA was

found in patients with MRSA infection or colonization and the contamination rate

is higher than some previous studies (Eibicht and Vogel, 2011; Wang et al., 2011).

This may be due to the crowded cubicle with the same kind of infectious organism.

In our hospital setting, the patients diagnosed with the same infectious disease

organisms, e.g. MRSA will be cohorted in the same cubicle. If isolation is not

possible, placing patients close to other patients at high risk of being infected will

be avoided, e.g. immunocomprised patients, patients with open wounds or with

long bed occupancy. Designated medical devices, e.g. blood pressure cuff and

stethoscope are provided to them. Our hospital will consider patients with known

MRSA carriage to be “colonized for life” and place them under contact

precautions whenever they are readmitted. For cubicles cohorted with MRSA

patients, the floor and walls are cleaned as routine practice by using neutral

detergent to remove organisms and dust; however, designated mops and buckets

should be used. After cleaning, items should be disinfected by 1,000 ppm sodium

hypochloride. MRSA positive patients should be managed under the infection

control guideline published by the CCIDER of Hospital Authority (HA) to limit

the risk of environmental contamination. If cleaning practices are not followed

properly, it is inevitable that the cubicle will have a large amount of MRSA

contamination.

To lower the contamination rate, the cleaning practice in the cohorted

cubicles should be revised by the ICT, twice daily as usual practice may not be

enough to eliminate the MRSA which has survived in a cubicle. Study shows a

52
reduction in levels of microbial contamination when wards received enhanced

cleaning (Dancer et al., 2009b). Besides, the cleaning tools can be one of the

factors contributing to contamination of the environment. A study recognizes that

accumulation of bacteria on wipes is occurs and increases the chance of cross

transmission of MRSA if rinsing not conducted thoroughly and between wiping

(Cheng et al., 2011a). Instead of conventional cloths, a study used ultramicrofibre

cloths to clean the environmental surfaces and found that their decontamination

ability was outperformed consistently (Wren et al., 2008).

The enrichment step before culturing the swabs can recover more MRSA and

may be another reason for the higher isolation rate of MRSA in this study (Van

Heirstraeten et al., 2009). The only one case patient showing MRSA negative in

his surrounding environment was located in the infectious disease block with

single room isolation and special ventilation. HCWs should wear masks, gloves

and personal protective equipment (PPE) before entering the room and then take

off the PPE, gloves and masks after leaving the room and wash hands thoroughly.

A good practice in contact precautions can be one of the factors behind reducing

the environmental contamination.

Contamination of environmental surfaces of control patients raises several

issues in terms of infection control policies. Currently, the guidelines recommend

terminal cleaning after discharge of MRSA patients. So, the contamination can be

due to continued viability of MRSA shed by previous occupants if housekeeping

was not performed thoroughly (French et al., 2004; Hardy et al., 2006). In general,

the cleaning practice in our hospital is cleaning the wards twice daily with 1,000

ppm sodium hypochloride according to the guideline of our ICT. The

responsibility for cleaning usually rests with the healthcare assistants, who are

53
often very busy and are almost permanently understaffed in our hospital. So, the

practice cannot be followed sometimes or the ward supervisor cannot monitor the

cleaning procedure which results in a high risk of transmitting MRSA. Studies

have shown an increased risk of infection after periods of insufficient nurse

staffing or excessive workload (Dancer et al., 2006; Hugonnet et al., 2007).

Sometimes, the HCWs may pay less attention to the cubicles without cohorting

infectious disease patients as they think that such areas are much cleaner when

compared with cubicles cohorting patients with infectious diseases. Our hospital

does not have an active surveillance program, so the negative control patients

cannot be excluded as MRSA carriages. Also, the hands of HCWs can be one of

the routes to carry MRSA from positive patients to control patients’ surrounding

environment if hand hygiene is not performed well. In addition, the HCWs do not

screen for MRSA carriages so they can be the carrier to shed MRSA to the

controls’ environment.

The MRSA contamination rate in the nurse workstation (5.9%) is

comparatively lower than the near patients’ environment (34.3%). The hygiene

level is better in the working area of the HCWs because these areas are seldom

touched by patients. Besides, these data indicate that the HCWs have good

compliance with hand hygiene regimens after caring for patients. In one study, the

MRSA contamination rate on the surfaces of working tables in ward staff centres

was 30.4% which was relatively higher than in the present study. This suggests

that regular disinfection is necessary (Oomaki et al., 2006).

The degree of environmental contamination can be a reflection of hygiene

practices. However, it could not be evaluated in our study design because we did

not audit the cleaning practice of wards. The length of the bed occupancy was

54
greater than 48 hours in 16 of the 29 patients while 13 of the 29 patients it was

less than 48 hours. These data cannot show the apparent influence of the length of

bed occupancy on environmental contamination.

5.2 Contamination of environmental surfaces

Bed sheets, pillows and patient bed frames are the surfaces highly

contaminated by MRSA patients. These are the high-touch sites by the patients. In

our hospital, the high-touch surfaces, e.g. bedside tables and bedrails of MRSA

patients are recommended to be cleaned at least twice daily with 1,000 ppm

sodium hypochlorite and 70% alcohol is used to disinfect metal surfaces, e.g.

patient lockers. The high contamination rate in the high-touch surfaces shows that

the current cleaning practice and barrier strategies in our hospital may be

insufficient and ineffective to control the spread of MRSA. Studies show that

these environmental surfaces are not cleaned well after patient-discharge (terminal)

cleaning (Carling et al., 2008a, Carling et al., 2008b). The environmental surfaces,

including bed rails, bedside tables and patient furniture fall into Spaulding’s

noncritical category according to the CDC guideline for Disinfection and

Sterilization of healthcare facilities (2008) (Kohn et al., 2003; Rutala et al., 2008).

The guidelines note that these objects could potentially contribute to secondary

transmission by contaminating the hands of HCWs (Bhalla et al., 2004). These

near patient surfaces are easily contaminated by patients and a study show that

patient-occupied bed rails were re-contaminated within 2 to 6 hours after

disinfection (Schmidt et al., 2012). Recently, copper-based surfaces have been

advanced as a complement to standard cleaning methods to control the bacterial

burden associated with high-touch surfaces within healthcare environments

55
(Sharpe and Schmidt, 2011). A range of alloys containing more than 60% copper

have been registered by the U.S. Environmental Protection Agency (EPA) due to

their ability to kill disease-causing bacteria which include MRSA and demonstrate

antimicrobial efficacy as a sanitizer. Study show that copper alloys have a lower

recolonization rate when compared with traditional environmental surfaces made

of plastic and aluminium (Sharpe et al., 2011). Implementation of new surfaces

material in healthcare settings, e.g. bedside tables or patients’ lockers can be one

of the strategies to reduce the contamination rate of high touch surfaces.

Patients’ folders, oxygen pump knobs and infusion pump adjustment panels

are found to have MRSA contamination in case patients only. These areas are

usually touched by HCWs than patients. If the HCWs do not have good

compliance with hand hygiene protocols, their hands can be a source to

contaminate those surfaces after patient care. Moreover, these areas are not

high-touch surfaces so they can be easily ignored by cleaners.

Cubicle partition ledge is the most frequent area in which to find MRSA in

the control environment. It is a surface that easily accumulates dust particles and

is rarely cleaned. MRSA carried on dust particles is capable of being aerosolized

and survive for a long time. During bed making, MRSA air contamination can

increase 10-fold (Shiomori et al., 2002).

MRSA is found on bed sheets, curtains and the patient lockers of the negative

control. It was probably contaminated by the previous patient followed by

inappropriate cleaning practice which cannot eliminate the MRSA. Terminal

cleansing and changing of curtains are the usual practice in our hospital when a

patient is discharged from a room or isolation is discontinued. Sometimes, the

cleaners may not completely follow the procedure in the absence of supervision,

56
especially with regard to changing curtains, as it is labour intensive to renew

curtains once the MRSA positive patients are discharged.

Fax machines, computer keyboards and barcode readers on the nurse

workstation are touched by HCWs frequently. Contamination of these areas may

be due to the incompliance with hand hygiene of HCWs. In our hospital, the

HCWs are recommended to perform the five moments of hand hygiene published

by the WHO. After patient care of MRSA patients, if HCWs do not use hand rub

to clean their hands will result in contamination of the nurse station when they

proceed to the next job at the nurse workstation.

Most of the time, the healthcare assistants in ward only clean the high-touch

surfaces, but non near-patient hand-touch sites, e.g. infusion pumps, door handles

or monkey pulls are rarely prompted to attention. To eradicate this persistent

organism, hydrogen peroxide vapour or ultraviolet light can be used as a routine

approach. They have a high penetrating ability to the areas that the cleaners are

rarely aware of and are difficult to reach, e.g. cubicle partition ledge. In our

hospital, hydrogen peroxide vapour is used in the cubicles with highly contagious

patients or multidrug resistant organism carriers after they are discharged.

Although these devices may have merit, they can be conducted only when the

cubicles or rooms are empty.

5.3 Molecular characteristics of the MRSA isolates

The most predominant spa type isolated in this study was t1081, which

accounted for 62.5% of the total MRSA isolates. It was correlated with a previous

study that spa type t1081 (CC45/agr IV-MRSA-IV and –V) have been commonly

found in many residential care homes for the elderly in Hong Kong (Ho et al.,

57
2008b). There was no significant correlation with the specimen type, length of bed

occupancy, sex or age of the patients. t1081 is belonged to clonal complex

CC45/agr IV and ST45 clone, it has higher transmissibility than the other less

common spa types which can explain the high prevalence of it in healthcare

settings (Cheng et al., 2011). In Belgium, CC45/agr IV MRSA isolates were

found to account for 47% isolates from 100 hospitals in 2001, suggesting the

ability of this clone to disseminate rapidly and widely (Denis et al., 2004). The

second and third prevalent spa types were t032 and t037 which accounted for

17.6% and 7.4% of the total isolates respectively. t032 is the main cause of

bacteraemia within the hospital at the Scottish MRSA Reference Laboratory

(Murchan et al., 2004) and was the second most common spa type deposited on

the Spa Server, accounting for 9.83 % of all the isolates. It is widely spread

throughout Europe (Ghebremedhin et al., 2007). t037 is related to the epidemic

clone of the Brazilian/Hungarian strain (Oliveira, et al., 2001; Chung, et al., 2004).

Recently, it has been reported in Malaysia and several other cities in China (Sun

et al., 2009; Neela et al., 2010). One study found that it is the most frequent spa

type found in the HA-MRSA isolates, SCCmec type III, in Taiwan, Hong Kong,

Thailand, Vietnam and India (Song et al., 2011). In addition, t1081 and t032 are

predominant spa types found in the control environment which could act as a

reservoir for MRSA transmission through the hands of HCWs.

5.3.1 Molecular characteristics of the MRSA isolates between patients and

their environments

MRSA isolates were found to be indistinguishable between the clinical

isolates of the patients and their environment. Our finding is consistent with some

studies shown in Table 1 that reports similarly high correlations between patients
58
and environmental MRSA sources. This shows that the environment is likely

contaminated by patients’ MRSA isolates and acts as a reservoir for MRSA

transmission if infection control is implemented ineffectively. Two patients were

found to have the spa type of MRSA strain distinguishable between the clinical

isolates and the environmental isolates. We found that the length of bed

occupancy in these two patients was less than 48 hours when environmental

samples were taken. So lack of time to contaminate the environmental surfaces

could be one of the reasons. Besides, the distinguishable strains can be shed by

previous bed occupants with unrecognized MRSA colonization and become the

source if terminal cleaning was not performed thoroughly. HCWs or visitors can

be the source to import MRSA strains that were not eradicated by daily cleaning

practices. Nevertheless, this does not dispute the possibility that the different

strains came from the patients themselves, since patients can be colonized or

infected by several distinct strains (Cespedes et al., 2005; Lim et al., 2006). This

can also explain in some cases the patients found to have two spa types in their

surrounding environments.

6. Conclusion

HAIs represent a substantial cost in lives lost, prolonged rehabilitation and

dollars spent on healthcare. Patients colonized or infected with MRSA have a high

risk of contaminating their surrounding environments which play a significant role

in spreading MRSA. The findings in this study should be considered with the

understanding in standard hospital hygienic care, e.g. cleaning, disinfection and

hand washing. The failure to control the spread of MRSA in our hospital is likely

due to several factors, such as incompliance with hand hygiene protocols of

59
HCWs, poor adherence of HCWs to barrier contact precaution guidelines, lack of

active surveillance to find out the MRSA carriage and screening the carriage state

of HCWs.

Several new strategies can be applied in our hospital setting to complement

the present infection control practice. These include the promotion of better

cleaning through education, training, monitoring, using checklists or fluorescent

dye indicator systems as part of behavioural modification programs. More

frequent cleaning, the addition or better placement of sinks and alcohol dispensers

with appropriate educational and monitoring programs can also help to increase

compliance and effectiveness. The incorporation of surface materials with

inherent bactericidal properties used in synergy with current hygiene practices

offers a new paradigm for healthcare design that will lead to better outcomes.

Active surveillance of MRSA carriers can be implemented in some high risk

wards, e.g. burns unit, surgical ward or intensive care unit. A study shows success

in this “Search and Destroy” policy (Vos et al., 2009). After identifying the MRSA

carriage, decolonization therapy is preformed to remove MRSA and reduce the

chance of MRSA transmission.

In conclusion, we have shown that the environment of isolation rooms with

patients who are infected and colonized with MRSA is often positive for MRSA,

and that patient and environmental isolates are usually indistinguishable.

Environmental reservoirs may be a significant contribution to endemic MRSA,

but consecutive prospective studies are needed to assess the correlation between

environmental MRSA and the acquisition of MRSA by patients which result in

development of effective interventions in control transmission of MRSA.

60
7. Limitations of the study

There are several limitations in this study which include the relatively small

sample size. The degree of environmental contamination may be a reflection of

hygiene practice; however, the potential confounders of hygiene cannot be

identified in our study design because we did not audit the cleaning practices to

confirm that this was done appropriately.

In addition, we did not screen the HCWs and control patients before

commencing the study. So we cannot exclude the possibility of transmission of

MRSA from colonized HCWs and patients to the environment. Multi-locus

sequence typing (MLST) and SCCmec typing by multiplex polymerase chain

reaction was not performed in this study which may show more diversity in the

molecular pattern of the MRSA isolates. However, good correlation was

documented between spa typing and MLST in some previous molecular

epidemiological investigations (Ho et al., 2008a; Ho et al., 2008b; Cheng et al.,

2011b).

61
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9. Appendix I

Result of lowest limit of detection (LLD) of the laboratory culture

method. Tables showing the actual CFU of MRSA isolated in various
dilutions

1st trial 2 x 104 1 x 104 5 x 103 2 x103 1 x 103 1 x 102

CFUmL-1 CFUmL-1 CFUmL-1 CFUmL-1 CFUmL-1 CFUmL-1

Actual
inoculums 198cfu 97cfu 55cfu 24cfu 3cfu 1cfu
(By back
titration)
OX-MSA 8cfu 5cfu 2cfu Neg Neg Neg
Direct
plating

OX-MSA Pos Pos Pos Pos Pos Neg

24h
enrichment

OX-MSA Pos Pos Pos Pos Pos Pos

48h
enrichment

2nd trial 2 x 104 1 x 104 5 x 103 2 x103 1 x 103 1 x 102

CFUmL-1 CFUmL-1 CFUmL-1 CFUmL-1 CFUmL-1 CFUmL-1

Actual
inoculums 202cfu 105cfu 56cfu 28cfu 8cfu 2cfu
(by back
titration)
OX-MSA 4cfu 3cfu 1cfu Neg Neg Neg
Direct
plating

OX-MSA Pos Pos Neg Neg Neg Neg

24h
enrichment

OX-MSA Pos Pos Pos Neg Neg Neg

48h
enrichment

81
3rd trial 2 x 104 1 x 104 5 x 103 2 x103 1 x 103 1 x 102
CFUmL-1 CFUmL-1 CFUmL-1 CFUmL-1 CFUmL-1 CFUmL-1
Actual
inoculums 210cfu 112cfu 61cfu 24cfu 4cfu 1cfu
(by back
titration)
OX-MSA 5cfu 4cfu 1cfu Neg Neg Neg
Direct
plating
OX-MSA Pos Pos Pos Pos Neg Neg
24h
enrichmen
OX-MSA Pos Pos Pos Pos Pos Neg
48h
enrichment

82
10. Appendix II.

Gel Electrophoresis of the PCR product under UV illumination

DNA markers (lane 1, 19), MRSA isolates (lane 2-16), Positive control,
MRSA strain with spa type t1083 (lane 17), Negative control, SCON ATCC
11490 (lane 18)

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11. Appendix III

Photo guideline for environmental sampling

(Near patient surfaces)

84
85
86
87
88
89
90
91
(Non-near patient surfaces)

92
93
94
95