Preventing Hospital-Acquired Multidrug-Resistant Organisms With Biosurfactants

Production of Biosurfactants

I need an introduction about why multi-antibiotic resistant organisms (MRO) are important and why they are important in biofilms.
They also will want to methods – saying how you are going to detect the bacteria.

Project 1: Superbugs in hospital environments: understanding and eradicating an important source of nosocomial infection
Introduction

Hospitalised patients develop nosocomial infections, which cause increased morbidity and mortality and result in increased healthcare costs. The environment is one important source of cross-infection and we have recently shown that most surfaces in hospitals are contaminated with biofilms which can contain multi-antibiotic resistant organisms (MRO). The risk of obtaining a nosocomial infection is increased by an average of 73% if the previous patient occupying that room had a MRO. The role of biofilms in maintaining MRO viability and transmission of infection is unknown and will be explored in this study.

Hypothesis:

Persistent exposure to antibiotics and disinfectants in the ICU induces biofilm growth, which subsequently promotes maintenance of MROs;
Infection of multiple patients, over time, with the same strain of MRO is due to the periodic release of free swimming bacteria from these biofilms;
Current cleaning methods and auditing do not deal with dry surface biofilms
Aims:

Determine if MRO persist over time as biofilms and contribute to nosocomial infection.
Compare environmental biofilm formation in the face of detergent cleaning compared to detergent cleaning plus chlorine disinfection with respect to MRO persistence.
Show that quadruple monitoring of environmental surfaces with real-time feedback to cleaners, combined with new generation cleaning, will decrease environmental contamination, remove demonstrable MROs and biofilms from the hospital environment and decrease patient acquisition of MROs.
Research Plan:

Aims 1 and 2. Patients admitted to intensive care units in Australia and Scotland will be screened on admission, weekly and on ICU discharge for MRSA, vancomycin resistant enterococci (VRE) and multidrug resistant gram-negative bacilli (MDRG). Line-associated bacteraemia, S. aureus bacteraemia and MRO data will be collected. All MRO patient strains will be collected and stored at -80oC. Patient demographics, date and bed number(s) will be entered into a database. Those patients with no prior history of MRSA, who had been on the ward for >48 hours and have laboratory isolated MRSA from their clinical samples will be regarded as a new MRO acquisition. Collection of environmental samples will be conducted after routine cleaning and disinfection. Ten high "hand-touch" sites will be screened weekly using chromogenic dip slides. 5 environmental samples for biofilm analysis will be collected monthly for 6 mths from 10 patient bed areas and the nurses' station before being divided into sections and processed by culture, quantitative real-time PCR, next generation sequencing, confocal laser scanning microscopy and scanning electron microscopy. Patient, environmental planktonic and biofilm bacterial strains will be subjected to molecular characterisation by whole genome sequencing. We will correlate geographic and time relationships between environmental, biofilm and patient strains to determine the contribution of the environment, in particular biofilms, strains to nosocomial infection.

Aim 3. Validation that cleaning has removed pathogenic organisms from the environment is important for infection control. Cleaning validation tools include visual inspection, fluorescence marker testing, ATP sampling, and microbial sampling for culture and biofilm demonstration. We will use failure mode and effects analysis to identify and grade each failure point in cleaning and the audit process, the frequency of this failure occurring, the effect of this failure, and whether this failure can be detected during normal protocols.

Multi drug Multi-drug resistant organisms are bacteria which have developed resistance to selected antibiotics such that these antibiotics cannot be used for killing or controlling the spread of these organisms (Ciofu et al., 2015). This becomes a public health issue because antibiotics are designed to control and fight bacteria related infections. Moreover, it takes a long period of time for a drug to be prepared, clinically tested and approved for use in human treatment. Thus the development of resistance against many antibiotics indicates that the health pf the people is compromised. The multidrug-resistant organisms are common in long terms health care facilities like hospitals. These can develop when antibiotics are taken for longer period of time than is required or unnecessary use of these drugs (Malik et al., 2013). At first, these drugs are able to kill a few of these bacteria but with time as the same drugs are used, multidrug resistance bacteria develops. The rise in pathogenic infections in the patients at the hospitals is caused by the environment. In most cases, the biofilms contain pathogens and end up contaminating dry surfaces of the hospitals, prolongs their survival rates and they become tolerant to destruction by the routine disinfection and cleaning activities. However, for a long period of time, there have not existed any methodologies for testing the effectiveness of detergents in destroying the biofilms. It is thus important to ensure that there is a proper method of prevention of hospital acquired multidrug resistant bacteria in order to have the patients get healed. Some of the methods used for prevention of infections involves putting protection barriers and practicing hand hygiene. Another method which is also used involves the environmental contamination but this too is not able to destroy the biofilms. This is because it has been found that the biofilms contain a thick extracellular and polymeric coating. Further testing has found that these biofilms harbor live and multiple resistance strains of bacteria (Penesyan et al., 2015). Thus the major importance of the biofilms is that they are resistant to destruction by the detergents and antiseptics in the control of infections. An example of a microorganism which causes hospital-acquired infections through incorporation into the biofilms is the Staphylococcus aureus. An example of the negative effect of biofilm in patients is that it lowers the rate of healing by a patient such that it creates a high level of inflammation for instance in patients who have wounds like diabetic patients (Malone et al., 2017). The Scientific evidence thus suggests that the antimicrobials used for treatment purposes result in poor outcomes in bacteria which are in the phenotypes of biofilms in dry surfaces.

Cytotoxic Tests

Production of biosurfactants

Studies indicate that biosurfactants can be used in the destruction of most of the bacteria infections including biofilms and multidrug-resistant bacteria (Almatroudi et al., 2015). However, these biosurfactants are only those produced by Lactobacillus jensii as well as Lactobacillus rhamnosus and their effects will be determined by use of clinical sample isolates. The purchased L. jensii and rhamnosus will be inoculated culture on a mechanical shaker in order to produce biosurfactants. The pellets will be collected trough centrifugation and washing done in order to suspend the biosurfactants in the solution. The bacteria will then be collected by filtration leaving the biosurfacants in solution.

Cytotoxic tests

Then albino mice epithelial cells of the lungs will be used for the cytotoxicity test of the biosurfactants. The test was confirmed by observing the production of lactate dehydrogenase at different biosurfactants concentrations (Sambanthamoorthy et al., 2014). The cytotoxic effects of biosurfactants will be measured as a fraction of the lactate dehydrogenase released to the number of untreated epithelial cells.

The bacterial biofilms aseptically collected in the hospital dry surfaces will be exposed to various concentrations of the biosurfactants in microtitre plates containing fresh growth media. The adherent biofilm will be fixed using about 190 microliters of 100% ethanol before staining with crystal violet dye. To determine the possible formation of biofilms, pegs will be immersed into polystyrene microtitre plates containing 190 microliters of 100% ethanol and absorbance measured at 595nm by use of a SpectraMax M5 spectrophotometer machine. The results will be interpreted by making a comparison of the biofilms treated with biosurfactants against those not treated with biosurfactants. These experiments will be carried out in triplicates to enhance accuracy and reproducibility.  

In order to determine the composition of the biofilm obtained from the hospital surfaces, the biofilms will be stained by use of a lectin dye for about 30 minutes, while the proteins will be stained using sypro Orange dye. The DNA of the biofilm will also be stained using Syto 60 dye, all these structures fixed with and the composition of each determined by use of an appropriate imaging software.

Reference List

Almatroudi, A., Hu, H., Deva, A., Gosbell, I.B., Jacombs, A., Jensen, S.O., Whiteley, G., Glasbey, T. and Vickery, K., 2015. A new dry-surface biofilm model: an essential tool for efficacy testing of hospital surface decontamination procedures. Journal of microbiological methods, 117, pp.171-176.

Ciofu, O., Tolker-Nielsen, T., Jensen, P.Ø., Wang, H. and Høiby, N., 2015. Antimicrobial resistance, respiratory tract infections and role of biofilms in lung infections in cystic fibrosis patients. Advanced drug delivery reviews, 85, pp.7-23.

Malik, A., Mohammad, Z. and Ahmad, J., 2013. The diabetic foot infections: biofilms and antimicrobial resistance. Diabetes & Metabolic Syndrome: Clinical Research & Reviews, 7(2), pp.101-107.

Malone, M., Johani, K., Jensen, S.O., Gosbell, I.B., Dickson, H.G., McLennan, S., Hu, H. and Vickery, K., 2017. Effect of cadexomer iodine on the microbial load and diversity of chronic non-healing diabetic foot ulcers complicated by biofilm in vivo. Journal of Antimicrobial Chemotherapy.

Penesyan, A., Gillings, M. and Paulsen, I.T., 2015. Antibiotic discovery: combatting bacterial resistance in cells and in biofilm communities. Molecules, 20(4), pp.5286-5298.

Sambanthamoorthy, K., Feng, X., Patel, R., Patel, S. and Paranavitana, C., 2014. Antimicrobial and antibiofilm potential of biosurfactants isolated from lactobacilli against multi-drug-resistant pathogens. BMC microbiology, 14(1), p.197.