MRSA is a Gram positive, non-motile, facultatively anaerobic coccus which does not form spores. It is both catalase positive and coagulase positive, and forms golden yellow colonies when growing on standard nutritive media and a rose--mauve colony on CHROMagar. MRSA is found in the normal human flora on nasal passages, skin, and mucous-membranes and is an opportunistic pathogen, causing death, notably in old and immunocompromised patients. By definition, MRSA is resistant to the antibiotic, methicillin. MRSA is widely distributed in hospitals, where it can survive on walls and other surfaces.
Members of the genus Staphylococcus are environmental organisms and are not restricted to humans or other animals. Methicillin resistant S.aureus are known to be able to survive for long periods in the hospital environment, on walls and floors and in bedding. As yet however, there seems to be no reports of these organisms being found in soils. Such a finding would be important as it would provide an obvious mechanism by which MRSA could be carried from the external environment into hospitals.
The aim of the work reported here was therefore, to determine if MRSA are present in soils and if there is a correlation between the incidence of these organisms in soils and the proximity of the sampling sites to hospitals in both the UK and Saudi Arabia.
Key words: Pathogenic bacteria, environmental bacteriology, antibiotic resistance, resistant pathogens, Staphylococcus aureus, hospitals contamination, MRSA.
Introduction
The increasing development of antibiotic resistance is a problem worldwide. As early as the late of 1940's, approximately 60% of hospital-acquired strains were resistant to penicillin (Cookson et al., 1989; Bradley et al., 1991; Mulligan et al., 1993; Cormican et al., 1996), while less than 10% are susceptible today (Murray et al., 1998). In order to combat this resistance semi-synthetic penicillins which are not susceptible to beta-lactamase hydrolysis (such as methicillin) were introduced in the 1960's. Unfortunately bacterial resistance to these antibiotics has also developed, and methicillin-resistant Staphylococcus aureus (MRSA) currently account for a large proportion of clinical isolates of this pathogen, worldwide Methicillin resistance is due primarily to a novel form of a penicillin binding protein (Hartman and Tomasz, 1984). Until recently, vancomycin was the only uniformly effective treatment for Staphylococcal infections and, as a result, it has been used as the last line of defense against multi-resistant S.aureus. By 1996, however, the first clinical isolate of S.aureus with reduced susceptibility to vancomycin was reported. A year later it was reported that up to20 % of MRSA isolated from hospitals throughout Japan were heterogeneously resistant to vancomycin, and that this resistance develops into VRSA upon exposure to vancomycin (Hiramatsu et al., 1997); the spread of resistance to this antibiotic is now being seen in a number of countries.
In the hospital environment, clinical isolates may be less susceptible to many commonly-used disinfectants than are standard, culture collection strains. There has been concern in recent years that, as with antibiotics, intensive exposure of hospital pathogens to disinfectants and antiseptics (biocides) may result in the emergence of resistance to these agents. Evidence for reduced susceptibility to biocides from exposure to these agents has been both laboratory-and field-based. It is reasonable to assume that in a clinical environment, microorganisms will be exposed to some extent to sub-inhibitory amounts of biocides remaining on environmental surfaces, or even on mucosae or skin.
Rationale behind the use of CHROMmagar to isolate MRSA
A number of isolation media are currently available which claim to specifically isolate selected groups, as wells as individual species, of bacteria and yeasts. However, such claims for specificity, although based on extensive trials, should never be accepted at face value. For example, we have isolated a species of Bacillus which produced the colour change on CHROMagar, a medium which is claimed by the manufacturers to be specific for methicillin-resistant Staphylococcus; clearly then, confirmatory checks on any bacteria assumed to be MRSA (i.e.presumptive MRSA) have to be made (Flayhart et al., 2005; Gurran et al., 2002).
Materials and methods
A range of surface soil samples were collected from the vicinity of hospitals in Riyadh, Saudi Arabia and Sheffield, UK. Samples (1g) were added to sterile distilled water (99ml) and shaken at 70 rpm for 15 mins. The suspension was then diluted from 10-3to 10-6 and 0.1 ml was spread onto the surface of CHROMmagar MRSA medium (prepared as per manufacturer's instruction); plates were incubated in triplicate at 30°C for 18-24 h.
Colonies of MRSA appear rose-mauve, while the other bacteria isolated are colourless or blue; any MRSA sensitive bacteria were inhibited and prevented form growing on the medium. In order to confirm methicillin resistance, presumptive MRSA colonies were picked off and transferred to fresh CHROMagar to select single colonies. These were the transferred to produce a lawn on Mueller Hinton agar. In order to test for the antibiotic sensitivity of the isolates, antibiotic test discs (Oxoid) were placed at the centre of the plates and these were incubated at 37°C. Any presumptive MRSA were checked to determine if they were acid fast and coagulase positive, using standard methods (Flayhart et al., 2005; Gurran et al., 2002).
Results and Discussion
The mauve colonies isolated on CHROMagar were considered to be presumptive for MRSA. However, confirmative tests are required to avoid false negatives. As a result, the following confirmative tests on the soil isolates were made: 1) the isolates were seen under the light microscope to be cocci in grape-like clusters, indicating they were members of the genus Staphylococcus; 2) the isolates were then shown to be both Gram positive and coagulase positive, but non- acid fast (as determined using Ziel-Neelsen's method), thereby confirming that they are S.aureus and finally, since the colonies were found to be resistant to methicillin and penicillin G, it can be concluded that they are, as indicated by CHROMagar, methicillin resistant S.aureus. In order to make certain that MRSA were in fact counted, occasional microscope analysis of individual colonies were made to confirm that no false positive bacilli, or bacteria which were not cocci, were isolated
.
Table 1. Distribution of MRSA in soils adjacent to hospitals in Riyadh (distance from hospital in meters).
Riyadh Hospitals
10 m
500 m
2000 m
Riyadh Medical Complex
(Centre)
15.9 x 105
± 6.8 x 102
10.6 x 105
± 1.7 x 102
26.6 x 105
± 7 x 102
Aleyman Hospital
(South)
0.1 x 105
± 0.5 x 102
4.8 x 105
± 4.6 x 102
0.1 x 105
± 1.8 x 102
Alyamama Hospital
(East)
10.6 x 105
± 2.6 x 102
0.2 x 105
± 1.1 x 102
0.1 x 105
± 0.2 x 102
King Khalid University Hospital
(West)
0.8 x 105
± 0.3 x 102
1.1 x 105
± 0.15 x 102
2.2 x 105
± 0.4 x 102
Kingdom Hospital
(North)
14.6 x 105
± 2.6 x 102
1.1 x 105
± 0.1 x 102
1.1 x 105
± 0.14 x 102
Tables 1 and 2 show that MRSA were isolated from all of the soils sampled around the hospitals in Saudi Arabia and the UK. Table 1 shows the isolation of MRSA for hospitals in Riyadh. It is clear that there is a wide variation in numbers of bacteria isolated from all soils at individual distances from all of the hospitals. No obvious trend such as a decrease in numbers with distance from an individual hospital, was universally found, although this trend is seen in the case of distance from the Alyamama hospital. In the case of the Riyadh Medical Complex and the King Khalid University Hospital very large numbers of MRSA are found in soils sampled at a distance of 200 meters. The large numbers of MRSA in the first case could be explained by the presence of a high population of people with poor access to medial facilities, while the large population of MRSA in the sample obtained 200 metres from the latter hospital may be explained by the fact that this sampling has a high population density and a relatively large number of medical centres.
Table 2. Distribution of MRSA in soils adjacent to hospitals in Sheffield (distance from hospital in meters).
Sheffield Hospitals
10 m
500 m
2000 m
Thornbury Hospital
9.4 x 102
± 5.8
20.5 x 102
± 10.7
2.4 x 102
± 13.4
Children's Hospital
21.3 x 102
± 1.8
5.1 x 102
± 6.9
2.2 x 102
± 3.8
Methicillin resistant bacteria were also isolated from soils in the neighborhood of two hospitals in Sheffield. Here however, much smaller populations of these antibiotic resistant bacteria were found compared to Saudi soils, possibly because some surface bacteria are likely to washed to lower levels in the UK soils due to the higher incidence of rainfall here when compared with surface soils from Riyadh.
The variation in MRSA populations in both soils can be explained here as the result by the fact that the sampling points varied in relation to soil type, horticultural treatment, and vegetation cover (all factors which will influence bacterial population numbers). As a result, it is not surprising then that bacterial numbers in general, and MRSA in particular should vary widely. Similarly, there is no obvious correlation between proximity to the hospital and the number of MRSA isolated.
An isolate representative isolate of S aureus from soil adjacent to the Hallamshire hospital was found to not to be inhibited by penicillin G or ampicillin. These beta lactam antibiotics act as proxies for methicillin which is only available clinically. Although it is worrying that antibiotic resistant bacteria occur in soils adjacent to Sheffield hospitals, it is reassuring to note that vancomycin (which is generally regarded as the last useful antibiotic where generalized antibiotic resistance occurs) resistance was not found.
The results of this study show that MRSA can be readily isolated from soils both in Saudi Arabia and the UK. Although there is no correction between the distance from the hospitals studied, soils adjacent to these hospitals clearly contain MRSA.
Conclusion
The implications of the findings reported here are obvious, MRSA occur in soils in the neighborhood of hospitals, both in Saudi Arabia and the UK and can therefore be carried into medical facilities on the shoes of staff or visitors. The frequency with which MRSA can be isolated from all of the soils tested, particularly the Saudi soils, is perhaps surprising. At first sight, this could be explained by assuming that the CHROMagar, which is sold as a medium selective for the isolation of these bacteria, is not in fact specific to MRSA. While species of Bacillus were occasionally isolated form soils which gave the indicator colour change on this medium it was nevertheless confirmed that most of the mauve colonies on the isolation plates were cocci; a finding which clearly shows that MRSA are remarkably widely distributed in urban soils in these two countries and presumably also elsewhere.
Acknowledgement
Thanks are due to Prof. Milton Wainwright for her contribution to this study. The study was supported by the College of Science Research Center, King Saud University, Saudi Arabia, for support.
