Prevalence of Antiseptic-Resistance Genes in Staphylococci

Published Date: 09 Oct 2017

• Shi Guangsen

1. Project Title

Prevalence of antiseptic-resistance genes in staphylococci isolated from the conjunctival sac of orthokeratology (Ortho-k) lens and spectacle wearers in Hong Kong

2. Abstract

1. Background

With the widespread use of disinfectants, there is a growing concern about the increasing incidence of disinfectant-resistant microorganisms and the genes (qacA, qacB, smr, qacG, qacH and qacJ) that encode the loss of susceptibility to QAC worldwide. Most multi-purpose solutions (MPS) contain quaternary ammonium compounds (QACs). It is unknown whether long term use of these solutions will select for ocular pathogens with increased resistance to antiseptics including those used in MPS.

2. Purpose, Methods

In this study the disinfectant-resistance genes in staphylococci isolated from the conjunctival sac will be compared between Ortho-k lens wearers with spectacle wearers by Polymerase Chain Reaction(PCR).

3. Significance of Study

The finding will bring great significance to evaluate the safety of MPS, especially to the children. If long term use of multi-purpose solutions containing quaternary ammonium compounds will select the carriage of organisms harbouring antiseptic-resistance genes. The manufacturer and the food and drug administration need to retest the effect of MPS to the bacteria containing disinfectant-resistant genes, even revise the standard of MPS.

3. Introduction

Disinfectants based on quaternary ammonium compounds (QACs) have been extensively used in hospitals and other health care settings for a variety of clinical purposes and play an important role in the control of infectious diseases due to its wide range of effectiveness and low toxicity.[1,2]It is very important to understand the mode of action of QAC-based disinfectants and the mechanisms of bacterial resistance against such compounds, particularly in the light of the pending post-antibiotic era that the poultry industry is facing so as not to repeat mistakes made with antibiotics.Many researches related tothe mechanism of action of QACs wereconducted in the past 50 years. [2-4]

Gilbert &Moore (2005) proposed the process ofa QAC on a microorganism: progressive adsorption of the quaternary headgroup to acidic phospholipids in the membrane of the microorganism with increasing exposure time and concentration of the QAC. Following adsorption, the QAC penetrates into the cell wall and reacts with the cytoplasmic membranewhich leads to decreaseof hydrophilic voids in the membrane. Proteins and nucleic acids are degraded followed by an eventuallysis of the cell. [5]

With the widespread use of these products, there is a growing concern about the increasing incidence of disinfectant-resistant microorganisms worldwide. [6]Micro-organisms can evolve in a number of different ways to circumvent the toxic effects ofantibiotics, crop protection chemicals, and disinfectants. [1]Reports on resistance to QACs have been reviewed by several authors.[1, 4, 6] The widespread use, and to a degree, the misuse of QACs can cause selective pressure on bacteria and is one of the main reason for the development of resistance to QACs. [6] Bacteria have always been capable of acquiring genes that enable them to survive harsh environments.[7]Acquired resistance to QACs has been observed, notably in staphylococci and the genes that encode the loss of susceptibility to QAC by efflux found in many bacteria, qacA, qacB, smr, qacG, qacH and qacJ, are, in general, plasmid-borne and confer reduced susceptibility to cationic antiseptic agents including dyes (acriflavine, ethidium bromide), QACs (benzalkonium chloride) and biguanides (chlorhexidinedigluconate). [8, 9]

In recent years, considerable research progress has been achievedin the understanding more fully the actions of several types of bacteria to QACs. [10] Most disinfectant-resistant bacteria, such as Staphylococci, Escherichia, Acinetobacter and Pseudomonas, are opportunistic ocular pathogens found in the normal skin flora, commonly isolated from the axillae, perineum, inguinal areas and conjunctival sacs of humans. In the course of colonization, these bacteria may adapt to changing environmental conditions leading to the production of molecular determinants for colonization and virulence, and are able to infect a broad range of animal and plant hosts, including humans.[11, 12]

Prevalence of myopia varies in different parts of the world. In some East Asian countries, such asSingapore, China, Japan, Korea, and Hong Kong, children develop myopia earlier than in most other parts of the world. The prevalence of myopia among Hong Kong children has risen significantly over the past 30 years.According to published reports, about 80% of children developed myopia by the time they left school. [13]

Therefore, the number of contact lens wearers is increasing significantly. In 2005, Barr reported that over 125 million people were using contact lenses. [14]The contact lens market is expanding to include children and teenagers. The number of children wearing contact lenses is increasing due to the success of newer lens designs for myopic control. [15] Since the development of myopia often begins at or below the age of six yearsand becomes progressively worse during school-age years,the studies on myopiccontrol are always conducted on school children. [16]Cho’s report showed about 50% less increase in axial length and vitreous chamber depth of the Ortho-k groupcompared to that of the control group after 24 months of Ortho-k lens wearing.With theimprovement of materials and designsand fitting of lenses, Ortho-k lenses are considered by many practitioners to besafe enough forwear during sleep.Overnight Ortho-k wearing has been becoming increasingly popular to control the development of myopia, particularly in Asian countries. [15]

Contact lens wear is generally safe.However, mildcomplications can occur and if not handled properly, a mild complication can develop into a serious one. Almost all seriouscomplications were associated with poor hygiene, improper usage, and poor contact lens maintenance. [17]

Contact lens wear, especially extended wear and overnight wear, has been recognized as a significant risk factor for thedevelopment of microbial keratitis.Keratitis inducing pathogens were not only isolated from the corneas but also fromcontact lenses and lens accessories used by patients. [18, 19]Contact lenses and lens accessories are susceptible to contamination with microorganisms.In published reports, the contamination rate of contact lenses and lens accessories varied greatly. [20-22]

To reduce the risk of contamination (and subsequent infection of the eye), disinfecting solutions are used to inactivate microorganisms on the lenses and lens accessories. Most multi-purpose solutions (MPS) contain QACs, which have a high level of activity against a wide range of common ocular pathogens (Richardson et al 1993; Miller et al 2001). [23, 24]

However, it is unknown whether long term use of these solutions, especially among children and young patients,will lead to a selection for ocular pathogens with increased resistance to antiseptics including those used in MPS. It is important to clarify as disinfectant-resistant microorganisms will greatly decrease the effectiveness of MPS and increase the risk of microbial corneal infection in contact lens wear.

The objectives of this study is:

To compare the disinfectant-resistance genes in staphylococci isolated from the conjunctival sac of Ortho-k lens wearers with those isolated from non-contact lens (spectacle) wearers.

4. Methodology

Study design

Twenty to thirty children (6–12years) who are currently participating in an Ortho-k project (myopic control study) at The Hong Kong Polytechnic University and have been wearing Ortho-k lenses for at least 1 year will be recruited. Each subject will be required to come to the Optometry Clinic only once.

A control group of 20-30 children wearing glasses for myopic correction will also be recruited from the myopic control study.

The ocular health of each subject will be assessed usingslitlampbiomicroscopy at the first data collection visit. All subjects should be in good general and ocular health. All subjects/parents have to sign the consent form at this meeting.

After signing the consent form, samples from each subject in control group will be collected from the left conjunctival sac and be sent to the laboratory for microbiological culture within 2 hoursof collection.

Methods for sample collection

A sterile cotton swab soaked in sterile phosphate-buffered saline(PBS) will be used to swab the lower conjunctiva of the left eye with a gently rolling motion twice. Then the swab will be broken off in a labeled bijou bottle containing 10ml sterile Brain-heart infusion (BHI) broth. The bottles will be vortexed for 1 min to release any micro-organisms adhering to the swabs.

Microbial Assessment

The extracted samples will be transferred to the laboratory for microbiological examination within 2 hours of collection.BHI bottles will be incubated at 35°C for 24 hours.

The broth will be subcultured onto SA select agar which will be incubated at 35°C for 24 hours. All culture media will be purchased from Oxoid Ltd, Basingstoke, UK. Positive cultures will be isolated, Gram-stained and identified to the genus level, using standard biochemical tests for microbiological identification. After identification, all bacteria belonging to Staphylococcus will be stored at -20°C for further study.

DNA extraction

Two colonies of each strain will be inoculated into the eppendorf tube containing 200µllysis solution which included 10µllysostaphin (500U/ml), 10µl lysozyme (5000U/ml), 4µl EDTA (0.5M), 2µl Tris-Hcl (1M), and 174µl MiliQ water. Then eppendorf tubes will be votexed to homogenize bacterial suspension and incubated with agitation at 37°C for 1 hour. 0.5ml 1M Tris, 0.5ml 0.1M EDTA, 5ml 1M NaOH, 2.5ml 10% SDS and 41.5ml distilled water are added into a sterile bottle to prepare TENS solution. 300µl TENS will be added into each eppendorf tube, and gently mixed (up and down for 8-10 times),after that 150µl KOAC will be added into each tube and mixed gently (up and down for 8-10 times). The tube will be placed on ice for 20 minutes and centrifuged at 15000rpm for 20minutes and the supernatant will be transferred to a new eppendorf tube.

Each new tube will be added 1ml 100% EtOH (cold from frig) and mixed gently and placed on ice for 30 minutes, followed by centrifuging at 15000rpm for 30minutes. The supernatant in each tube will be discarded.To each tube, 1ml 70% EtOH (room temperature)will be added and mixed gently and centrifuged at 15000rpm for 5minutes. The supernatant in each tube will be discarded and the precipitate will be aired dry at room temperature for at least 10 minutes. To each tube, 50µl MiliQ of water will be added and the content containing the bacterial DNA will be stored at 20°C and used in the PCR.

PCR of qac genes

PCR will be carried out for detection of smr, qacA/B, G, H and J using primer sequences published previously (Table 1). [8, 9] The volume of each reaction in the PCR will be 50µl. Each reaction tube will contain 5µl of 10x Expanded High Fidelity buffer (without MgCl2) (Roche, Lewes, UK), 4µl of MgCl2 at a final concentration of 2mM (Roche, Lewes, UK), 8µl of dNTPs (1.25mM), 1 µl (50pmol) of forward primer, 1µl (50pmol) of reverse primer and 20µl of sterile H₂O. Template bacterial DNA (10µl) will be added, and a negative control will be included containing 10 µl of sterile water instead of template DNA. Finally, 1U of ExpandedHigh Fidelity Enzyme mix (Roche, Lewes, UK), containing thermostableTaq DNA polymerase and Tag DNA polymerase with proof-reading activity, will be added to each reaction. The cycling conditions will be as follows: DNA denaturation at 94°C for 3 min, followed by 25 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min. This will be completed with an extension step of 72°C for 5 min. PCR products will be analyzed by electrophoresis on a 2% (w/v) agarose/1x TBE gel.

Table 1. Primers used for polymerase chain reaction (PCR)

Statistical analysis

Statistical analyses were performed using SPSS system for Windows version 16.0 (SPSS Inc., Chicago, IL, USA).

5. Timetable of work

6. Budget with justification

7. Summary:

Currently, over 125 million peopleuse contact lenses and lens accessories. Contact lenses are susceptible to contamination with microorganisms. To reduce the risk of contamination (and subsequent infection of the eye), disinfecting solutions are used to inactivate microorganisms on the lenses and lens accessories. Most multi-purpose solutions (MPS) contain QACs, which have a high level of activity against a wide range of common ocular pathogens. However, it is unknown whether long term use of these solutions will select for ocular pathogens with increased resistance to antiseptics including those used in MPS.

The finding will bring great significance to evaluate the safety of MPS, especially to the children. If long term use of multi-purpose solutions containing quaternary ammonium compounds will select the carriage of organisms harbouring antiseptic-resistance genes. The manufacturer and the food and drug administration need to retest the effect of MPS to the bacteria containing disinfectant-resistant genes, even revise the standard of MPS.