Introduction: This is a perspective analytical study. The objectives of which were to identify the different Staphylococcus species that infects Human and to study their sensitivity to Alcohol. The study identified the best type and concentration of Alcohol to be used for cleaning the infected wounds using different isolates of Staphylococci known for its higher resistance to both antibiotics and alcohol.

Materials and methods: Isolates were sub cultured into Nutrient broth and incubated for 3-5hours. Then, 200µl of sub cultured Nutrient broth was added to 2ml Alcohol each time with different concentrations starting from 100% to 50% and a lope full of the mix was streaked at 10seconds, 30seconds, 1minute, and 5minutes.

Results: This study showed that S. aureus was the predominant as the most pathogenic species accounting for 50% of all isolates. This followed by S. epidermidis which accounted for 26.7% of isolates. Other species that were isolated included: S. intermedius (6.7 %), S. schleiferi (6.7 %), S .saprophyticus (3.3 %), S. sciuri (3.3 %), and S. lugdunensis (3.3 %). Ethanol effectiveness on Staphylococcus species showed higher effectiveness at higher concentrations (100%, 90% and 80%) and the effectiveness become as lower with lower concentrations (70%, 60%, and 50%) especially with shorter exposure times. Methanol effectiveness on Staphylococcus species showed also higher bactericidal effects of the absolute (100%) and the 90% concentrations while lower concentrations (80%, 70%, 60%, and 50%) gave higher resistant rates of Staphylococci especially with shorter exposure times.

Conclusion: The best concentrations found to be bactericidal for both Ethanol and Methanol were the Absolute and 90% with 100% and 90% effectiveness rate respectively. The results also showed that Ethanol is much better a disinfectant than Methanol in all concentrations and especially at the lower concentrations.

Keywords: staphylococci, Alcohol, Ethanol, methanol, bactericidal

Staphylococcus species are Gram-positive, non-motile, non-sporing cocci of varying size occurring singly, in pairs and in irregular clusters. They are facultative anaerobes with fermentative metabolism. Their optimum growth temperature is 30ºC-37ºC.¹ Staphylococci are classified mainly by their production of coagulase enzyme that clots plasma into Coagulase positive Staphylococci and Coagulase negative Staphylococci.² Alcohol refers to two water-soluble chemical compounds, ethyl alcohol and isopropyl alcohol, which have germicidal characteristics. These Alcohols are rapidly bactericidal rather than bacteriostatic against vegetative forms of bacteria; they also are tuberculocidal, fungicidal, and virucidal but do not destroy bacterial spores. Their cidal activity drops sharply when diluted below 50% concentration, and the optimum bactericidal concentration is 60%–90% solutions in water. Alcohols are not recommended for sterilizing medical and surgical materials principally because they lack sporicidal action and they cannot penetrate protein-rich materials.³

Study design

This is perspective analytical case control study that used to identify the different Staphylococcus species that infects Human and to study their sensitivity to Alcohol. The study was carried out in the period between February 2015 and September 2015. The study was carried out at Tropical Medicine Research Institute, Khartoum, Sudan.

Collections of specimens

Samples were obtained according to site of infection. Samples from wounds were collected using sterile swabs and also biopsy was taken. Urine samples had been collected in sterile urine containers. Stool samples had been collected in stool containers.

Cultivation of specimens

Wound swaps were cultivated on Blood agar and MacConkey agar, and then sub cultured on Mannitol salt agar. Urine specimens were cultivated on CLED medium and then sub cultured on Mannitol salt agar. Stool specimens were firstly cultivated on Selenite-F-Broth and then sub cultured on XLD media Mannitol salt agar. Staphylococcus isolates were then tested for their Coagulase production that categorized them into two groups. Then Staphylococcus species were identified according to biochemical tests and sugars assimilation tests. Sugars used for the identification of Staphylococcus species included: Lactose, Maltose, Sucrose, Mannitol, Raffinose, and Fructose.

Methodology for alcohol sensitivity

Isolates were sub cultured into Nutrient broth and incubated for 3-5hours. Then, 200µl of sub cultured Nutrient broth was added to 2ml Alcohol each time with different concentrations starting from 100% to 50% and a lope full of the mix was streaked at 10seconds, 30seconds, 1minute, and 5minutes. The media were incubated at 37ºC for 24 hours aerobically and then results were recorded.

Statistical analysis

Results were analyzed by computer using Microsoft office excel program for the tables and the charts presentation.

Staphyloccocal isolates

In this Study 30 strains were isolated. As shown in (Figure 1), S. aureus was the predominant as the most pathogenic species accounting for 50% of isolates, followed by S. epidermidis which accounted for 26.7% of isolates. Other species that were isolated include: S. intermedius (6.7 %), S. schleiferi (6.7 %), S .saprophyticus (3.3 %), S. sciuri (3.3 %), and S. lugdunensis (3.3 %).

Figure 1 Shows the percentage of isolates from the 30samples in a columnar chart.

Bactericidal effect of ethanol on staphylococcus species

Result shows that higher concentrations of ethanol are more effective than lower concentrations in the disinfection process. It also shows that more time of exposure results in more effective disinfection by Ethanol. As shown in Table 1 & Figure 2 Bactericidal affect of different concentrations of ethanol (100%, 90%, 80%, 70%, 60%, and 50%) on the different Staphylococcus species at different exposure times (10seconds, 30seconds, 1minute, and 5minutes) by percentage.

Figure 2 The Bactericidal effect of Methanol on Staphylococcus species

Bactericidal effect of methanol on staphylococcus species

Result shows that higher concentrations of Methanol are more effective than lower concentrations in the disinfection process. It also shows that more time of exposure results in more effective disinfection by Methanol. As shown in Table 2 & Figure 3 Bactericidal effect of different concentrations of Methanol (100%, 90%, 80%, 70%, 60%, and 50%) on the different Staphylococcus species at different exposure times (10seconds, 30seconds, 1minute, and 5minutes) by percentage.

Figure 3 The Bactericidal effect of Methanol on Staphylococcus species

Our study showed that S. aureus was the most predominant pathogenic species accounting for 50% of isolates, followed by S. epidermidis which accounted 26.7% of isolates. Other species that were isolated include: S. intermedius (6.7 %), S. schleiferi (6.7 %), S .saprophyticus (3.3%), S. sciuri (3.3%), and S. lugdunensis (3.3%). These results are similar to the finding of Seif Eldin et al., (2000)who found in 92% of all pus cultures from patient with abscesses, microbial agents was identified, the large majority being S. aureus (69%). These results also agree partially with Tanusri et al.,⁴ who found among the Staphylococci isolates 116(86.57%) were coagulase positive Staphylococcus aureus. And a total of 18(13.43%) Coagulase-negative Staphylococci were isolated. Sixteen isolates (11.94%) out of 134 were identified as Staphylococcus epidemidis and two isolates (1.49%) were Staphylococcus haemolyticus. In Sudan, there was no relevant published data on the bactericidal effects of Ethanol and Methanol on different species of Staphylococci. The study proved that higher concentrations of both Ethanol and Methanol are more effective than the lower concentrations, and this finding does not agree with the CDC that proved that 70% (commercial spirit) Ethanol is the most effective bactericidal agent. Their explanation for this is that the mechanism of alcohol killing is by denaturation of proteins and absolute ethyl alcohol, a dehydrating agent, is less bactericidal than mixtures of alcohol and water because proteins are denatured more quickly in the presence of water (CDC, 2008). The study showed that 70% Ethanol is only 50% effective in 10seconds exposure time and 70% Methanol is 30% effective in 10seconds of exposure. The results of this study showed resistance of Staphylococci to lower concentrations of Ethanol and Methanol including the commercial spirit (70%). In a study done by the CDC.³ the bactericidal activity of various concentrations of ethyl alcohol (ethanol) was examined against a variety of microorganisms in exposure periods ranging from 10seconds to 1hour. Pseudomonas aeruginosa was killed in 10seconds by all concentrations of ethanol from 30% to 100% (v/v), and Serratia marcescens, E. coli and Salmonella typhosa were killed in 10seconds by concentrations of ethanol from 40% to 100%. The Gram-positive organisms S. aureus and Streptococcus pyogenes were slightly more resistant, being killed in 10seconds by ethyl alcohol concentrations of 60%–95%.

The Results of this study showed that Ethanol is more effective than Methanol as a Bactericidal agent, this finding also agrees with the study done by the CDC that proved Methyl alcohol (methanol) has the weakest bactericidal action of the alcohols and thus is seldom used in healthcare.³

The results showed that the most frequent isolates among the staphylococcus species was S. aureus as the major human cause of disease followed by S. epidermides and then other Staphylococci. The results of ethanol effectiveness on Staphylococcus species showed higher effectiveness of higher concentrations (absolute, 90% and 80%) and the effectiveness becomes lower with lower concentrations (70%, 60%, and 50%) especially with shorter exposure times. The results of Methanol effectiveness on Staphylococcus species showed also higher bactericidal effects of the absolute and the 90% concentrations while lower concentrations (80%, 70%, 60%, and 50%) showed higher resistant rates of Staphylococci especially with shorter exposure times. The best concentrations found to be bactericidal for both ethanol and Methanol are the higher Absolute and 90% with 100% and 90% effectiveness respectively. The results also showed that Ethanol is much better disinfectant than Methanol in all concentrations.

From results of this study it's recommended that:

1. Ethanol 80% and above (90% and absolute) are to be used for disinfection of surfaces and for wound cleaning instead of 70% (commercial spirit).
2. Methanol must not be used as a disinfectant except in its very high concentrations (Absolute and 90%).
3. Future studies are recommended with bacteria other than staphylococci especially that infect the wounds.
4. Future studies on the surfaces disinfected with different concentrations of ethanol and other alcohols.
5. Future research is needed to study other different disinfectants and their effectiveness on Staphylococci and also on other bacteria.

None.

The authors declare no conflict of interest.

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