Susceptibility testing of Pseudomonas aeruginosa and Staphylococcus aureus to some commonly used Hospital disinfectants

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1 International Scholars Journals African Journal of Microbiology ISSN: Vol. 4 (4, pp , April, Available online at International Scholars Journals Author(s retain the copyright of this article. Full Length Research Paper Susceptibility testing of Pseudomonas aeruginosa and Staphylococcus aureus to some commonly used Hospital disinfectants Ayangwa IA 1 and Colman SA 2 * 1 Department of Medical Microbiology, Federal College of Vetenary and Medical Laboratory Technology Vom, Plateau State, Nigeria. 2 National Agency for Food and Drug Administration and Control (NAFDAC, Lagos. Nigeria. Accepted 12 January, 2015 In the hospitals, one of the ways to control microbial contamination is by disinfecting the rooms, furniture and equipments used by patients and the health workers. It was therefore important to ascertain that in the two General Hospitals in plateau state, Nigeria. A total of 260 swab samples were collected from the hospital s beds, tables, floors, sinks, equipments and used disinfectants, were cultured, growths were identified macroscopically, microscopically and through biochemical test. Of the 81.92% bacteria isolated, Staphylococcus aureus had the highest percentage of occurrence (44.13%, Pseudomonas aeruginosa had the least percentage of occurrence (15.02% and other bacteria such as Proteus species, Coagulase negative Staphylococcus and Candida species (40.85%. Six disinfectants were used for the susceptibility testing; P, D, J, I, S, and G. isolates were used for the determination of the Minimum Inhibitory Concentration (MIC and Minimum Bactericidal Concentration (MBC. P (Chloroxyhexidine Gluconate 0.3% and Germicides 3% was found to be most effective in bactericidal of Staphylococcus aureus and Pseudomonas aeruginosa. Disinfectant G and S were bacteriostatic. Working concentrations recommended by the manufacturers were not adequate to completely suppress the growth of Ps. aeruginosa and S. aureus. High isolation of Staphylococcus aureus, Pseudomonas aeruginosa and other organisms encountered in our hospitals could be as a result of improper disinfection of the patient s rooms and furniture. There is need therefore to appropriately disinfect the hospital environment with the appropriate concentration of the disinfectants. Key words: Isolation, bacteria, susceptibility, disinfectants and Hospital environment. INTRODUCTION Most infections acquired in the hospitals are caused by microorganisms that are commonly present in general population in which they caused disease less often and usually milder form than in the hospital (Ayliffe et al., Within the hospital environment in-animate objects can provide opportunities for contact with transmission of microorganisms but hospital disinfectant can help in breaking the epidemiological chain of the infection, however, many in the medical community considered the inanimate objects to be of less importance in the incidence of hospital infection (Scarpitta, *Corresponding author. colman.sunday@yahoo.com Tel: In the hospitals, one of the ways of controlling microbial contamination is by disinfecting the furniture, floors, and walls using chemical disinfectants (Greenwood et al., One of the activities that nurses do daily in the hospitals is to keep the hospital environs biologically safe by disinfecting the patient s units which are basically made up of beds, mattresses, chairs, step stools, bells and tables with items for personal use (Denise et al., A wide variety of chemical agents are used as disinfectants in the healthcare settings, these include glutaraldehyde, sodium hypochlorite, fenolics, quaternary ammonium compounds chlorhexidine. Quaternary ammonium compounds are cationic surfactants that are widely used for the control of bacterial growth in clinical

2 Ayangwa & Colman 110 environment (McBain et al., Despite the diversity of pathogens involved in hospital infections, Staphylococcus aureus and Pseudomonas aeruginosa play important roles in the incidence of hospital infections, these two organisms appear as contagious agents harboured by health professionals and by medical articles (Denise et al., The use of antimicrobial agents has significantly reduced Staphylococcus infections, however, pathogenic Staphylococci have become increase resistance to the common used anti microbial agents worldwide (Yohannes et al., This work is to isolate the possible bacteria (Staphylococcus aureus and Pseudomonas aeruginosa in the hospital environment, and to determine the susceptibility of these organisms to some commonly used disinfectants in the hospital by determining its MIC and MBC. MATERIALS AND METHODS Sample Collection A total of 260 samples were collected from the beds, sink/washing hand basins, floors, tables, cupboards, and bedsides of the patients in the wards, theatre rooms, maternities, amenities, OPD, and emergency units using sterile swap sticks. It was transported to the Microbiology Laboratory, Federal College of Veterinary and Medical Laboratory Technology Vom, Plateau State for analysis. Two Hospitals were used for the collection of the samples; Jos General Hospital and Vom General Hospital, all in Plateau state, Nigeria. Cultural Technique The swaps were inoculated aseptically into nutrient broths and incubated aerobically at 37 oc for 24hrs, it was observed for turbidity of the broths and was then subcultured into Blood agar, McConkey Agar, Mannitol salt agar and cetrimide, was incubated for 24hrs at 37 oc. The plates were then examined macroscopically for colonial characteristics. Gram staining technique was carried out as described by Ochei and Kolhatar, Further test (Catalase test, Coagulase, Indole test, Urease test, Oxidase test, Citrate utilization test, methyl blue, Voges-proskauer test and Sugar fermentation test was carried out to identify the specie level (Kloos et al., 1997; Murray et al., Susceptibility Testing The disinfectants were collected from the same hospitals where samples were collected, its exact concentration as described by the manufacturers were also noted. The disinfectants were prepared as instructed by the manufacturer s dilutions and dilution higher than that required by the manufacturer. Colonies from the cultured plates were inoculated into nutrient broth and was reincubated. It was dissolved 1:1000 with sterile distilled water to obtain the standard inoculums size as documented by National Committee for Clinical Laboratory Standards (1997 which was compared to give equal turbidity with 0.5 McFarland standard. Test Procedure to Determine the Minimum Inhibitory Concentration (MIC Described by Giovanni Et Al., (1995; 1999 and Loren ( sterile capped test tubes were placed in a rack numbered ml of nutrient broth was distributed into tubes using sterile pipette. Each disinfectant was diluted according to the manufacturer s instructions. The two fold dilution of the disinfectants was carried out. Drop of 1 / 1000 dilution of the overnight broth culture of the organism was inoculated in to all tubes using sterile pasture pipettes and inoculated at 37 oc for 24hrs. The following day the tubes were read and the MICs was read as the highest dilution that inhibits the growth of the test organisms. Determination of Minimum Bactericidal Concentrations (MBC MBCs were determined by first selecting the tubes that showed no growth during the MICs determination. A loopful from each tube was sub-cultured into fresh nutrient agar plates. They were incubated at 37oc for 24hrs. The highest dilution at which no growth was observed was noted as the MBCs. RESULT Out of the two hundred and sixty (260 swab samples collected from the hospital environment, two hundred and thirteen (213 growths were noted. Of the 81.92% growth, 59.15% (Pseudomonas aeruginosa and Staphylococcus aureus are of the research interest and 40.85% (other bacteria which are not of the research interest. The sources of the isolates were beds (40.40%, tables (24.41%, floor (.55%, sink (13.%, equipments used in the hospital (5.16% and the least is the disinfectant used (2.34% showed in Figure 1.

3 % of growth 111 Afr. J. Microbiol Beds Floors Tables Sinks Equipments Disinfectants Source FIGURE 1. Distribution of Isolates from the Hospital Environment and their Source. Table 1. Distribution of bacteria isolated from the hospital environs and wards. Source S. aureus (% P. aeruginosa (% Other Bacteria (% Total (% Casualty 23 ( ( ( (20.66 Theatre 19 ( ( ( (23.47 Maternity 13 ( ( ( (12.21 A & E 11 ( ( ( (.04 Psychiatric 10 ( ( ( (7.51 Paediatric 7 ( ( ( (9.86 OPD 6 ( ( ( (8.45 Amenity 5 ( ( ( (3.76 Total 94 ( ( ( (81.92 N = 260 sampled, p value < The distribution of the isolates in the hospital environment showed that S. aureus had the highest percentage of occurrence (44.13% higher than that of Ps. aeruginosa (15.02%. Other bacteria of not interest were 40.85% as seen in Table 1. The Minimum Inhibition Concentrations (MIC of the six hospital disinfectants (P, S, D, G, J and I used to detect its potency on S. aureus and Ps. aeruginosa isolated in the hospital showed that, all the disinfectants inhibit the growths of both Ps. aeruginosa and S. aureus of the isolates in tube one (1, tube two ( 1 / 2 all the disinfectants inhibits the growth of both bacteria except in S were only 11(75% of the Ps. aeruginosa isolates were inhibited. In tube three ( 1 / 4 all the disinfectants inhibits the growth of S. aureus except in I which inhibited only 12(85.71% of the isolates and Ps. aeruginosa was inhibited by P and J (all the isolates. In tube four (1/8 P, J and I inhibited 7(50.00%, 7(50.00% and 3(21.43% respectively of Ps. aeruginosa, at same tube

4 Ayangwa & Colman 112 Table 2. Minimum Inhibitory Concentration (MIC of the six hospital disinfectants for isolates of Pseudomonas aeruginosa and Isolates of Staphylococcus aureus. DISINFECTANTS IN-USE DILUTION USED DILUTION Number percentage (% a giving dilution Pseudomonas aeruginosa Staphylococcus aureus 1(% ½(% ¼(% 1 / 8(% 1 / 16(% 1 / 32(% 1(% ½(% ¼(% 1 / 8(% 1 / 16(% 1 / 32(% 1 / 64(% 1 / 128(% 1 / 256 P (50 0 (0 0 (0 10( ( (0 S ( (21.43 D (50.00 G (64.29 J I ,2 8 (57. 0 (0 0 (0 0 (0 0 (0 0 (0 0 (0 0 (0 0 (0 0 (0 7 0 (0 0 (0 (50 3(21. 1(7.1 0( ( ( ( (0 0 (0 0 (0 0 (0 0 (0 12( (85.7 3( ( (0 0 (0 0 (0 7 0 (0 0 (0 0 (0 ( (0 0 (0 0 (0 ( (0 0 (0 0 (0 0 (0 Table 3. Minimum Bactericidal Concentration (MBC of the six hospital disinfectants of isolates of Pseudomonas aeruginosa and isolates of Staphylococcus aureus. Number percentage (% a giving dilution DISINFECTANTS Pseudomonas aeruginosa Staphylococcus aureus 1(% ½(% ¼(% 1 / 8(% P 8(57. 2( / 16(% 1(% ½(% ¼(% 0 (0 1 / 8(% 1 / 16(% 1 / 32(% 1 / 64(% 12( ( / 128(% 1(7. 1 / 256(% 0 (0 S 8(57. 1(7. 0(0 0 (0 D 10( ( (0 0 (0 G 13( ( (0 0 (0 7( (7. 0(0 0(0 0 (0 0 (0 0 (0 12( (57. 1(7. 0 (0 0 (0 0 (0 9( (.29 0 (0 0 (0 0 (0 J 10( ( ( (0 12( ( (7. 0 (0 0 (0 0 (0 I 7( (.29 1(7.1 1 (0 (10 9( ( ( (0 0 (0 0 (0 0 (0

5 113 Afr. J. Microbiol. Table 4. The active ingredients in the disinfectants. Disinfectants Active ingredients P Chlorhexidine Gluconate 0.3% and Germicides 3% S Hibitane (Chlorhexidine Gluconate 1.5% and Ceta Vion (Cetrimide 15% w/v D Chloroxylenol 4.8% w/v, Oleumpini Aromaticum 9.0% V/V, Denatured spirits 11.3% w/v, Sapo Vegetalis 5.8% w/v Sacharum G J I Dicloroxylenol and Chlorophenol 32% V/V Phenols Sodium Hypochloride 3.5% w/v Chloride Povidone Iodine 5% w/v all the disinfectants were effective on isolates of Staph. aureus except in the case of S and I 6(42.86% each. Tube five ( 1 /16 and six ( 1 / 32 were both turbid. Staph aureus was inhibited at tube five ( 1/ 17 by P (100% and G, while S (0.00%, D and J inhibits 12(85.71% each, while I had 3(21.43%. At tube six ( 1 / 32 only Purit cleared the whole isolates of Staph. aureus, S and I did not clear any of the isolates. At tube seven ( 1 / 64 only P inhibited 10(71.43% of the Staph. isolates while all other disinfectants could not inhibit any. Purit inhibits 5(35.75% at tube eight ( 1 / 128 while others do not inhibits. At tube nine ( 1 / 256, none of the disinfectants were able to clear the growth (Table 2. Minimum Bactericidal Concentration (MBC of the six commonly used hospital disinfectants on both Ps. aeruginosa and Staph. aureus reviled that all the disinfectants inhibits the growths of both Ps. aeruginosa and Staph. aureus in tube one (1, in tube two ( 1 / 2 all the disinfectants inhibits the isolates of Staph. aureus, while only Purit inhibits the growth of Ps. aeruginosa isolates. Tube three ( 1 / 4 reviled that P, D, J and G inhibits the growth the isolates of Staphylococcus aureus while P inhibited 57.% of Pseudomonas aeruginosa, tube four ( 1 / 8 shows that P and G were the only disinfectants that inhibits the isolates of Staph., P still inhibits the growth of Staph. aureus at 1 / 16 dilution (tube 5. At tube six ( 1 / 32 dilution, P cleared 50% of the isolates of Staph. aureus. At 1 / 128 dilution of tube eight, P cleared 1(7.10% of Staphylococcus aureus isolates. At tube nine ( 1 / 256 none of the six disinfectants inhibited the growths of the isolates as seen in Table 3. Table 4 showed the active ingredients of the six (6 hospital disinfectants that was used against the two (2 isolates (Pseudomonas aeruginosa and Staphylococcus aureus. DISCUSSION Because of the diversity of microorganisms that can be isolated from the hospital, we decided to focus our research on Staphylococcus aureus and Pseudomonas aeruginosa. The study reviled that, Staphylococcus aureus had the highest percentage of occurrence (44.13% which means Staph. aureus remain the major cause of the nosocomial infection as reported by Yohannes et al, (1999. Pseudomonas aeruginosa (15.02% is another important agent of nosocomial infection isolated in this research. Other microorganisms (40.85% were also encountered such as; Proteus species, Coagulase negative Staphylococcus and Candida species as was observed by Pannati (1997. The highest percentage of S. aureus was isolated from the casualty ward (10.80% where it should not be found. Likewise the highest occurrence of Pseudomonas aeruginosa (11.00% was isolated in the theatre room which means patients can be exposed to infection easily as a result. Ps. aeruginosa developed resistance to most of the commonly used Hospital disinfectants following the manufacturer s instruction. But at higher concentration, some isolates of the Ps. aeruginosa were inhibited by P (100.00%, (50.00%, J (29. 00%, and I (21.00% while S has the ability to inhibit the growth of the organisms without necessary killing them as shown in the MBC carried out on those inhibited in the MIC. P disinfectant was found to be very effective on the isolates most especially on Staphylococcus aureus followed by G which had its MBC at tube 1-4 cleared (100% each, tube 5 and 6 had (64.29% and (.29% respectively. S had the least efficiency on the isolates of Staphylococcus.

6 Ayangwa & Colman 1 P was the disinfectant that was very effective in the elimination of Ps. aeruginosa killing the bacteria at tube 1,2,3 and 4 as 100%, 100%, 57.% and.29% respectively, followed by G that was bactericidal in tube 1,2 and 3 as 100%, 92.86% and 50.00% respectively. The least of the disinfectants to be bactericidal on Pseudomonas aeruginosa was S with 100%, 57.% and 7.% in tube 1, 2 and 3 respectively. Because of the extensive and long lasting use of different disinfectants in the hospital wards from which these isolates originated, it validate our epidemiological observations. Some of the disinfectants may not be active because of their ingredients as stated by Giovanni et al, (1995. CONCLUSION It was observed that attentions were made more on the disinfection of the floor in the hospital than the other areas (Beds, Tables, Sink Chairs, etc. without bearing in mind that the ignored areas harboured high levels of bacteria. There was high isolation of pathogenic Bacteria in these study locations. Staphylococcus aureus and pseudomonas aeruginosa were isolated in the study locations. Other pathogenic bacteria were also encountered. Chlorhexidine Gluconate 0.3% and Germicides 3% was most effective than other disinfectants on S. aureus and Ps. aeruginosa. Some disinfectants eliminate the isolates at concentration higher than that of the manufacturers. It is therefore important to assess the disinfectants frequently before making use of them. As part of the good hospital practice, an utmost care should be taken to use the optimal concentrations of the disinfectants to reduce the incidence of the hospital acquired infections for the better patients management and survival. Kloos WE, Lambe DW (1997. Staphylococcus. In. Balows Hauserman, K.L., Isenberg, H.D. shadowy, H.J (ed. Manual of Clin. Microbiol., 18: e Based Paediatrics Chapter. Www hawaaii.edu/medicine/peditrics/pedtext/s06c04.html Loren GY (2003. Inhibitory and Bactericidal Principles (MIC and MBC. cas Mac Bain AJ, Ledder RG, Moore LE, Catrenich CA, Gilbert P (2004. Effects of quaternary-ammoniumbased formulation on bacterial community dynamics and antibacterial susceptibility. Applied Environmental Microbiology 70: Murray PR, Baron EJ, Pfaller A, Tenover FG, Yolken RH (1995. Manual of Clinical Microbiology 6 th ed. American Society of Microbiology Washington DC. 227,282, NCCLS- National Committee for Clinical Laboratory Standards (1997. Methods for dilution of antimicrobial susceptibility tests for bacteria that grows aerobically. 4 th ed Approved Standard. M7- A4 NCCLS Wayne, pa. Ochei J, Kolhartkar AA (2000. Medical Laboratory Science, theory and practical. Tata McGraw Hill. 7: , 9:615. Pannuti CS (1997. An importancia do meio ambiente hospitalar. In: Rodrigues EAC et al. Infeccoes hospitalares prevencaoe controle. Sao Paulo: Sarvier: Scarpitta, CRM., Limpeza e desinfecccao de artigos hospitalares. Limpeza e desinfeccao de areas hospitalares In: Rodrigues EA et al., Infeccoes hospital areas Prevencao e controle. Sao Paulo Yohannes Mengistu, Worku Ergie and Bahrie Bellete (1999. In Vitro Susceptibility of Staphylococcus to Chlorhexidine and antibiosis. Ethiop. J. Health Deve., 13 (3: REFERENCES Ayliffe GA (1987. Hospital disinfection and antibiotic policies. Chemotherapia 6: Denise de A, Emilia LS, Carlos RP (2000. A Bacteriological study of hospital beds before and after disinfection with phenolic disinfectant. R3ev. Panem Seiud Public 7:3. arvier Giovanni BO, Paola T, Paola V (1995. In vitro activity of commercial manufactured disinfectants against P. aeruginosa. European J. Epidemiol., 2 (4: Greenwood D, Richard CBS, John FP (1997. Medical Microbiology 15 th edition. 7:76-78, 15: , 29: , 67: