How Do We Clean Up This Mess?-A Review of The Testing Methodologies Used for Detection of Live Bacteria in Healthcare Environments

We have been aware of microorganisms and their impact on human health for over 150 years. Anton Van Leeuwenhoek was the first person to document microscopic observations of bacteria in 1676 (although a Jesuit priest, Athanasius Kircher is also credited by some as the first to observe microorganisms at around the same time). In 1878 Robert Koch discovered how to grow bacteria in a Petri dish (named after his assistant Julius Petri). Circa 1900 Martinus Beijernick extended the work of both Koch and Pasteur to develop what is now the enrichment culture techniques used today. However, in the present day, it is generally believed that the vast majority of microorganisms present in common environments cannot be successfully cultured and characterised using current standard testing methods [1-5]. Ever since the discoveries of Semmelweis in 1847, and in spite of our industrial, academic and clinical efforts to reduce HAI’s (Hospital Acquired Infections), we have had varying degrees of success. There have been longstanding theories and hypotheses surrounding the contributing factors to HAIs; for example, effectiveness of hand hygiene and the impact of airborne contaminates amongst many others. Although there has been a significant amount of research done in the food preparation sector, until recently there has been limited evidence focused on surface bio burden, and its relevance in specialised critical areas of healthcare [6-8].

However, in the present day, it is generally believed that the vast majority of microorganisms present in common environments cannot be successfully cultured and characterised using current standard testing methods [1][2][3][4][5]. Ever since the discoveries of Semmelweis in 1847, and in spite of our industrial, academic and clinical efforts to reduce HAI's (Hospital Acquired Infections), we have had varying degrees of success. There have been longstanding theories and hypotheses surrounding the contributing factors to HAIs; for example, effectiveness of hand hygiene and the impact of airborne contaminates amongst many others. Although there has been a significant amount of research done in the food preparation sector, until recently there has been limited evidence focused on surface bio burden, and its relevance in specialised critical areas of healthcare [6][7][8].
The root cause of this is multifactorial, although one main reason can be associated with the technological advances, or lack thereof in the standard testing methodology used to confirm successful cleaning of clinical areas. In 2004, when Dancer proposed a standard for microbiological assessment of surfaces, many routine molecular techniques were not available outside either academic research or the military [5]. It took over a decade more, before the creation of the UK Environmental Special Interest Group (ESIG) used predominantly to initially detect the presence or absence of microorganisms and to subsequently identify ideally to a species level. However, techniques such as culture have not changed significantly in many years and continue to have significant limitations in both sensitivity and specificity. Although a pure bacterial culture remains important for the study of virulence, antibiotic susceptibility, and genome profiling in order to facilitate the understanding and treatment of caused diseases, there continues to be little correlation between in-vitro evidence and its relevance in-situ.
The recent availability of molecular diagnostic technology has provided greater sensitivity and specificity in the detection of microorganisms. Evidence would suggest improvement in detection methods provides greater insight into the efficiency of current cleaning and decontamination of clinical environments [10]. Improved detection methods may also help us to understand the true extent of bacterial resistance to disinfectants. It is now considered "routine" for molecular diagnostics to deliver results in hours and minutes compared to traditional culture results taking days and in some cases weeks.

Visual inspection
Unfortunately, due to many factors including the lack of a fast, accurate test, visual inspection is still used by the vast majority of healthcare institutions, even though the evidence is clear that it is not an accurate test of cleanliness, let alone bioburden [11,12].

Total ATP
Adenosine Tri Phosphate is present in all living cells except viruses. It is measured by the inexpensive test kits, in relative light units (RLU), and is a fast, simple way to measure extra cellular and some intra cellular ATP. Total ATP testing has been undertaken for more than a decade in the food industry, yet no statistically relevant direct relationship has been found between RLU and Colony Forming Units (CFU) [13,14]. As there is no certainty that the test equipment is able to measure intra cellular ATP, even a zero count cannot be considered relevant.

Culture
The first culture conditions used various parameters such as incubation time, nutrients, atmosphere, and temperature. Refinement around such parameters has continued since its conception over 100 years ago. The use of culture in clinical microbiology was prompted by microbiologists initially specializing in intracellular bacteria [15,16]. The shell vial procedure allowed the culture of terest is the discovery that many organisms cannot survive in the high-nutrient conditions favoured by standard cultural practices [1,2,20,21]. The development of low-nutrient media has greatly increased the number of organisms that have been successfully cultured. Many soil organisms are incapable of surviving under high-oxygen conditions. These obligate anaerobes would naturally not survive standard culturing approaches, but there have been some successes with growing them in anaerobic chambers [22].
However, in respect to cultures usefulness in accurately testing the efficacy of disinfectants, the US CDC states, "Attempts to substantiate the bactericidal label claims of phenolics using the AOAC Use-Dilution Method occasionally have failed" [23]. However, results from these same studies have varied dramatically among laboratories testing identical products (figure1) [24].

Polymerase Chain Reaction (PCR)
PCR is a technique used to amplify a segment of DNA across several orders of magnitude [25]. This then generates thousands to millions of copies of a particular DNA sequence. Developed in 1983 by the Cetus Corporation [26], it is a reliable way to repeatedly replicate a focused segment of DNA. This technique is used in biomedical research, criminal forensics, and molecular archaeology [27]. PCR is now a common and often indispensable technique used

Bacteria Specific Rapid Metabolic Assay (BSRMA)
This test allows surface, water and air testing for estimation of bacterial bio burden/ load with increased sensitivity and specificity compared to culture techniques [31][32][33]. Within a few minutes of sampling, the assay measures only the live bacteria present and requires no culture procedures. This type of test should not be confused with the standard total ATP (Adenosine Tri Phosphate) tests used in some food manufacturing facilities, which due to the lack of any relationship between total ATP and bacterial ATP, have no diagnostic value. There are multiple recent studies regarding the efficacy of bacteriophage-related lytic enzymes (BPLE) or viruses that infect bacteria. By degrading the cell wall of the targeted bacteria, these lytic enzymes have been shown to efficiently lyse Gram-positive and Gram-negative bacteria [34]. Bacteriophages are viruses that kill bacteria. They do not contribute to antimicrobial resistance, are safe for humans, animals, and the environment.
The current focus on BPLE's has been on their use as anti-infectives in humans and more recently in agricultural research models. The initial translational application of lytic enzymes, however, was not associated with treating or preventing a specific disease but rather as an extraction method to be incorporated in a rapid bacterial detection assay [35].
We can trace the translational history of BPLE's from their initial discovery in 1986 for the rapid detection of group A streptococcus in clinical specimens to evolving applications in the detection and prevention of disease in humans and in agriculture [30,31] to Colony Forming Unit count [31,32]. The BSRMA was compared with the gold standard culture techniques as part of a standard of care orthopaedic pathway within an NHS hospital. The difference in sensitivity of the two tests using the same samples was stark. It is clear from this, and other studies [36,37], that the BSRMA test method is far more sensitive than standard culture techniques and, increases the users' ability to see the numbers of live bacteria, in areas previously determined to be bacteria free. Evidence of this is seen in a recent study [10], using the BSRMA test, at multiple time points over a 4-day period.
One of the most interesting findings in the study was the levels of contamination seen immediately after the finish of the operating list, after standard night-time cleaning with Sodium Hypochlorite, and then again immediately before the first patient entered the operating theatre the next morning. Importantly, this study showed that bacterial bioburden on surfaces had returned to approximately the same levels as at the end of the previous days operating lists.
Had just culture been used, levels of bacteria on surfaces at all three time points would have remained unseen.

Summary
In environmental cleaning and contamination there are two main factors associated with HAI's, namely, the types of microorgan-

Final Comments
The current methods of environmental hygiene audit in hospitals and other healthcare facilities need to be supported by microbiological sampling, and further developed in line with the sample pathway previously described. The focus on process has been well described, as is the focus on the people who support it. The potential for improved changes in the behaviour of staff who know they are being observed is well understood. Goodheart's law, sometimes confused with Heisenberg's uncertainty principle, but which is in fact a social analogue of Heisenberg's uncertainty principle, states "Measuring the system usually disturbs it". In reality, the Hawthorne effect, describes a type of reactivity in which individuals modify or improve an aspect of their behaviour in response to awareness of being observed. Each variation of the theme has a common core of understanding, "If people know they are being observed, they change their behaviour". This idea has been described many times as the "observer expectancy effect" and has been used in industry and academic institutions over many years. It may significantly underutilise as a method of achieving improvements in healthcare staff behaviour [48][49][50][51]. It could therefore have a significantly positive impact in helping to reduce the now visualisable surface bioburden that is almost certainly endemic in our healthcare facilities.