Have you wondered if Staphylococcus epidermidis cause diseases? This Microbiology research report was produced as part of a class assessment. It is shorter than the usual reports submitted to peer-reviewed journals and it is not a thesis. However, it is a stepping-stone to publishing a thesis of year-length research quality and it is within an area of my research interests.
Microbes comprising the normal microflora are potentially pathogenic and their identification with the appropriate characterisation tests for pathogenicity factors is important to establish if they are the causative agent to a disease. We identified and characterised four bacterial species: Staphylococcus aureus and Enterococcus faecalis that were provided as a class laboratory exercise, and Streptococcus sanguis and Staphylococcus epidermidis isolated from our throat, nasal cavity and forehead. The isolation and identification of these species by utilising universal microbiology laboratory resources was uncomplicated and yielded no peculiar results. In spite of this, the characterisation of the microflora species for bacteriocin-like inhibitory substance (BLIS), proteolytic and DNAse enzyme production gave negative results. While S. aureus presented results that demonstrated a pathogenicity greater than the microflora species. From the information gathered from characterising and determining the isolated bacteria pathogenicity factors, we can utilise the knowledge to assist in clinical diagnosis and treatment.
INTRODUCTION
A diverse population of microorganisms reside on the external surfaces of our body that includes the mouth, gut, nasal passages and skin. The normal microflora is regarded as non-pathogenic such as Streptococcus sanguis which dominates the microflora at the back of the throat and Staphylococcus epidermidis which dominates the surface of the skin (1). However some of the bacteria that are part of the normal microflora are potentially pathogenic when the host is immunosuppressed, the bacteria resides in a different part of the body or a strain of the bacteria acquires new pathogenic abilities (2). In other words, the healthy host-microflora relationship is broken and this leads to the subsequent injury of the host (3). S. epidermidis and S. sanguis can both cause endocarditis since its hydrophobic nature of its cell surface allows it to colonise damaged heart valves (4). Pathogens such as Streptococcus pyogenes, Streptococcus pneumoniae, Escherichia coli, and Staphylococcus aureus can be found growing as part of the normal microflora, however their numbers are not significant to cause disease. This is mainly due to other competing for resources and the host’s ability to keep the pathogen in restraint owing to the immune system (5).
The diagnosis of many microbiological diseases relies primarily on the symptoms of the patient and in most cases the disease is quickly determined (6). However many different diseases demonstrate similar symptoms, thus the clinician may not provide a defined diagnosis. Many diseases cannot be determined by the symptoms alone, so more precise tests are required. Many tests involve the bacterial analysis of the patient. In a bacterial disease, it is important to isolate and identify the causative pathogen so that effective medical treatment can be administered effectively. The tests, however, will identify numerous bacterial species so it is important to characterise the pathogenic or virulence factors associated with each isolate. It is also important to realise the pathological effects of these factors. This is so that the microbiologist can deduce which species is causing the injury; otherwise the microbiologist could blame the disease on a harmless commensal.
We wanted to characterise the dominant bacterial species residing in a few of the areas of a healthy adult male person. As a means of comparative analysis, two established pathogens – S. aureus and Enterococcus faecalis were characterised as well (7). Extraction, isolation, followed by identification of the bacteria provided the base to characterise the pathogenicity factors known to that species. Different pathogenicity factors, such as antibiotic resistance, are associated with bacteria including those that are microflora. By characterising and determining the isolated bacteria pathogenicity factors, we can utilise the knowledge to assist in clinical diagnosis and treatment.
MATERIALS AND METHODS
Two unknown species, isolates #83 and #97, were originally grown on separate blood agar plates by Otago University teaching staff. Throat, nasal, teeth and forehead samples from Samuel Fung, the author, were obtained using the techniques described in the Laboratory Manual (8, p14), and these were grown on blood agar plates at 37˚C for 24 h in 5% CO2 except for the forehead sample which was incubated in full aerobic environment.
Isolation and Identification of samples
As isolates #83 and #97 were already single colonies on their blood agar plates, there was no further need for isolation and enrichment. Only one colony type from each of the throat, nasal, teeth and forehead samples were isolated. White colony-types from the nasal and forehead samples and yellow colony-types from the teeth and throat samples were isolated and enriched on blood agar at 37ºC for 24 h in 5% CO2 except for the forehead sample which was incubated in full aerobic environment. The isolates’ colony characteristics were recorded and they were Gram stained to examine their cellular morphology characteristics under light microscope. The isolates were tested for catalase production with hydrogen peroxide (8, p16). They were inoculated in Todd Hewitt broth (THB) with supplemented bile salts and incubated on mutans selective agar for 24 h at 37ºC. The isolates from the nasal and forehead samples were suspected to be staphylococcal and they were plated on mannitol sodium chloride agar for 24 h at 37ºC.
Confirmation and Characterisation of Isolates
Isolate #83, namely S. aureus, was confirmed by growing it on mannitol sodium chloride agar for 24 h at 37ºC. Characterisation of S. aureus involved measuring proteolytic enzyme production using skim-milk agar (8, p28), DNAse production using DNAse agar (8, p29) and β-lactamase production using Nitrocefin discs (8, p30).
Isolate #97, namely Enterococcus faecalis, was confirmed by growing it on bile esculin azide agar for 24 h at 37ºC. Characterisation of E. faecalis involved testing for bacteriocin-like inhibitory substances (BLIS) production by deferred antagonism (8, p23) utilising the indicator strains: Micrococcus luteus T-18 (labelled as I1), S. pyogenes FF-22 (I2), S. anginosus T-29 (I3), S. uberis ATCC27958 (I4), S. pyogenes 71-698 (I5), Lactococcus lactis subsp. lactis T-21 (I6), S. pyogenes 71-698 (I7), S. pyogenes W-1 (I8) and S. equisimilis T-148 (I9). Inhibition of the indicator strains through the E. faecalis band was measured. We also tested the degree of resistance of our sample to the antibiotics ampicillin and gentamycin using E test antibiotic-strips (8, p31-2).
The throat isolate, namely S. sanguis, was confirmed by growing it on mitis salivarius agar at 37ºC for 24 h. It was also tested for bacitracin and optochin sensitivity with blood agar as the medium (8, p17). We characterised S. sanguis for BLIS production by deferred antagonism, proteolytic and DNAse enzyme production.
The nasal isolate, namely S. epidermidis, was confirmed by testing for coagulase production (8, p16) and growth in 6.5% (w/v) sodium chloride broth over 72 h at 37ºC. It was characterised for BLIS production by deferred antagonism, proteolytic and DNAse enzyme production.
The forehead isolate was regarded to be the same species as the nasal isolate. All the confirmation and characterisation tests performed on the forehead’s S. epidermidis were the same as the nasal isolates tests.
We repeated all the identification tests on the teeth isolate such as the catalase production, growth in THB and bile, and conducted a second Gram stain with cell morphological inspection.
RESULTS
Table 1. Identification test observations of samples
| | #83 S. aureus | #97 E. faecalis | Throat S. sanguis | Nasal S. epidermidis | Forehead S. epidermidis | Teeth |
| Haemolysis type | β | γ | α | γ | γ | α |
| Colony characteristic | Grey | White | Grey | White | White | Grey |
| Microscopic observation | Cocci, Cluster, G+ | Cocci, Chain, G+ | Cocci, Chain, G+ | Cocci, Cluster, G+ | Cocci, Cluster, G+ | Rod, 1x3 length, G+ |
| Catalase test | +ve | -ve | -ve | +ve | +ve | -ve |
| THB + bile | NN | +ve | -ve | NN | NN | -ve |
| Mutans selective agar | NA | NA | -ve | NA | NA | -ve |
NN Test not necessary on this sample isolate
NA Test not applicable to this isolate species
G+ Gram-positive
G- Gram-negative
We could not identify the teeth isolate because the cellular morphology was a Gram-positive rod of three times the length that its width. It was not an identifiable isolate as it was not described in the Laboratory Manual (8, p18), therefore we did not have the foundation to use the tools to identify it. With no direction to help us to appropriately characterise it, we did not continue examining the teeth sample isolate.
Isolate #83 was confirmed to be S. aureus with the growth of yellow colonies as well as the colour change of the mannitol sodium chloride from pink to yellow. The observations in Table 1 of S. aureus are consistent with standard observations of S. aureus (7). S. aureus gave positive results when tested for β-lactamase, proteolytic and DNAse enzyme production. It showed 5 mm clear radius zone for the proteolytic enzyme on the agar and 0.7 mm clear radius zone for the DNAse enzyme.
Isolate #97 was confirmed to be E. faecalis as it managed to grow on the bile esculin azide and blacken the agar due to esculin hydrolysis. It was tested for its minimum inhibitory concentration (MIC) to ampicillin and gentamycin, and we found that the MIC was 0.25 mg/L and 3 mg/L respectively. When tested for any BLIS production by measurement of deferred antagonism of indicator strains, E. faecalis did not inhibit any of the indicator strains except I6 by up to 3 mm from the edge of the isolate’s streak.
We confirmed the throat isolate was S. sanguis as it was not sensitive to bacitracin or optochin, however this alone does not fully determine the isolate as S. sanguis. Our mitis salivarius agar produced only small circular colonies, and we used this to verify it from other bacteria expressing similar characteristics such as Streptococcus salivarius which produces large mucoid colonies. Another bacteria with similar characteristics to S. sanguis is Streptococcus mutansS. sanguis for BLIS, proteolytic and DNAse enzymes gave negative results. but our isolate did not grow on mutans selective agar. Characterisation of
Both nasal and forehead isolates produced virtually identical results in all the tests performed on it. Due to the positive catalase test and the cluster-like cell appearance (Table 1), the isolates could still have been S. aureus. The negative coagulase test ruled out that it was S. aureus and the growth in the NaCl broth meant it was not Staphylococcus saprophyticus. Thus the isolates were most likely to be S. epidermidis. We characterised the nasal and forehead S. epidermidis and found no production of BLIS, proteolytic and DNA enzymes.
DISCUSSION
The S. sanguis and S. epidermidis we isolated from the throat, nasal passage and forehead, are also the scientifically established normal microflora of those areas (10,11,12). The results showed that the S. sanguis and S. epidermidis isolates produced no BLIS, proteolytic or DNAse enzymes however we expected some positives even if they were harmless microflora (1). S. sanguis was α-haemolytic, so we hypothesised that it released proteolytic enzymes to hydrolyse the blood proteins for soluble amino acid nutrients, thus contributing to some of the haemolysis. It was also presumed that the two species could produce BLIS (13), because they exerted such dominance in their area – BLIS was probably a factor of antagonism to other bacteria. However as no BLIS was produced from S. sanguis and S. epidermidis, BLIS cannot be a factor for their population majority. Other factors, such as an efficient metabolic system, could be the cause.
We reason that S. sanguis and S. epidermidis does not produce proteolytic or DNAse enzymes maybe because it was not pathogenic within their respective environment. If they were to produce those enzymes, then it could harm the host and cause serious skin and mucosa infection. But given that the host was healthy, the two isolates did not or could not produce the enzymes.
The S. aureus isolate we tested appears to be the most pathogenic microbe of the four species. It possessed the ability to destroy β-lactams and it could actually produce and release proteolytic and DNAse enzymes. Compared to the isolates from the healthy human individual, this species could cause disease at much lower numbers and it could survive treatment from a spell of β-lactam antibiotics (6). It was unfortunate that we could not measure the amount of β-lactams to consider the MIC or whether it was resistant to other β-lactam forms such as ampicillin. S. aureus is well known to contribute to many diseases like food poisoning, toxic shock syndrome, and skin infection (6). The symptoms associated to these diseases are caused from its many toxins it produces. So in a medical microbiology diagnosis, many specimens from the body can be analysed such as the pus, vomit or blood (6). Careful treatment regimes should be followed so that antibiotic-resistant bacteria are selected for.
E. faecalis can grow in the gut hence it can survive the bile salts that kill most other microorganisms, including S. sanguis and S. epidermidis (3). It also produced a BLIS that inhibited the indicator strain I6, whereas the other isolates tested could not inhibit any indicator strains. E. faecalis may possess a pathogenicity factor as it was not completely inhibited by the antibiotics ampicillin and gentamycin. In spite of this, there was no basis for comparison to other microorganisms, so the MIC values for E. faecalis do not convey whether this was a high MIC or low MIC.
It would have been interesting to characterise the S. sanguis and E. faecalis for its production of surface antigen by Lancefield typing. Also, it would have been useful to test every isolate for their antibiotic sensitivity, and not just E. faecalis. The importance of this is to assist the clinician in administrating the correct amount of antibiotics to infected people, while lessening the unwanted problem of selecting for antibiotic-resistant pathogens (7).
The four identified isolates are medically important and the knowledge of ‘what they are like,’ has led us to find a common medical condition they all cause. They all individually contribute to infective endocarditis (4,1). Hospitals, especially surgeons, should have preventative measures to ensure that those bacteria do not cause post-operative infection. However, if endocarditis does develop, then testing for the causative agent will be timelier with this information.
ACKNOWLEDGMENTS
I thank the Department of Microbiology especially Professor John Tagg and his demonstrators for their assistance and guidance.
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