Background: Bacterial contamination in operation theatres (OT’s) and other specialized care units is a major factor for nosocomial infection. Surgical Site Infection (SSI) contributes to 33.1% of nosocomial infection. Microbial contamination of OT’s is a major risk factor for surgical site infection. Aim of study is to identify bacterial colonization of indoor air of OT’s, labor rooms (LR’s) and specialized new born care units (SNCU’s).

Material & methods: Air sampling of 29 (OT’s), 9 (LR’s) and 5 (SNCU’s) was done by settle plate method. Surfaces samples were taken by wet swabs from different sites and equipments in nutrients broth. Samples were then transported to laboratory and processed according to standard operation procedures.

Results: Total of 184 swabs were taken, out of which 134 (72.82%) were found to be positive for bacterial growth. A total of 43 air samples were taken out of which 41 were found to be positive for bacterial contamination. Least CFU/m3 was found in ophthalmology OT (4.4-10 CFU/m3) and highest in gynecology and obstetrics OT (4.4-268.7 CFU/m3.

Conclusion: Surfaces and air in various health facilities of studied hospitals were found contaminated with different types of bacteria including potential pathogens that pose a great risk to patients. Hygiene and sanitation need to be improved in these hospitals to control nosocomial infection and for better management of patients.

Keywords: Kashmir, bacteriological surveillance, operation theaters, settle plate method

SNCU, specialised new born care unit; OT, operation theatre; LR, labour room; SSI, surgical site infection; CFU, colony forming unit; GNB, gram negative bacilli

Microbial contamination of OT’s is a major cause of nosocomial infections.^1–4 Nosocomial infections are an important cause of patient morbidity and death.⁵ Good hospital hygiene is an integral part of infection control programme. Microbial contamination in OT’s is influenced by number of individuals present, ventilation, use of airflow methods, use of adequately sterilized equipment and contaminated surfaces.⁶ Microbiological surveillance provides data about factors contributing to infection. Environmental monitoring means microbiological testing of air, surfaces and equipment to detect changing trends of microbial counts and micro-flora.⁷ Lack of adherence to standard operating procedures can result in adverse patient outcomes in health care settings.⁸

The study was conducted in the Division of Epidemiology and Public Health, Directorate of Health Services Kashmir, J&K, India from January 2019 to June 2019. The permission for the study was taken from Director Health Services Kashmir. There are 10 district hospitals, one in each district of the valley which provides health care services to population of 72 lakh approximately.⁹ Two sampling methods used in the study were:

1. Settle plate method (for monitoring quality of air)
2. Surface swabbing.

Collection and transport of specimen

Air and surface samples were taken from OT’s, LR’s and SNCU’s from district hospitals across Kashmir valley.

Air sampling

Air sampling was done by settle plate method. Blood agar and MacConkey agar plates (four sets, each comprising of one blood agar and one MacConkey agar) were placed at four locations in OT’s, LRs and SNCU’s one meter above ground, one meter away from the wall for one hour (1,1,1). These were then transported to laboratory and incubated at 37^oC for 24 hours under aerobic conditions. After incubation, plates were observed for growth. Isolates were then identified using standard microbiological procedures and CFU/m³ were estimated using Omeliansky formula according to which

 CFU/m³=ax1000

 Pxtx0.2

 a = No. of colonies on Petri plate.

 P = Surface measurement of plate used in cm²

 t = Time of exposure of Petri plate in minutes

Recommended conventional operation theatre values

1. An empty theatre bio load should not exceed 35 CFU/m^3.
2. During surgery bio load should not exceed 180 CFU/m^3.

During air sampling procedure, sterile gloves, surgical masks and protective gowns were used to prevent contamination of agar plates. Plates were checked visually for any bacterial growth before use.

Surface sampling

Moistened sterile cotton swabs in nutrient broth were used to collect samples from different sites like operation table, autoclaved instruments, drug trolley, walls, floor and light. All samples were properly labeled and immediately transported to microbiology laboratory. Swabs taken from different sites were inoculated on blood agar & MacConkey agar plates. These were incubated at 37^oC for 18-24hrs under aerobic conditions. After incubation the isolates were identified by colony characteristics, gram reaction and standard biochemical tests.¹⁰

Total of 184 swabs were taken from different critical care facilities. Out of 184 swabs, 134(72.82%) were found to be positive for bacterial growth and 50(27.17%) swabs were found to be sterile as shown in Table 1; Bacillus spp. accounts for (n=50, 26.17%), followed by Coagulase Negative Staphylococcus (CONS) (n=38,19.8%); Acinetobacter spp. (n=36 18.8%); S. aureus (n=29,18.8%); Pseudomonas spp. (n=28 14.65%); Klebsiella spp. (n=6, 3.14%); E. coli (n=3,1.5%) and Enterobacter spp. (n=1, 0.5%). Most common organism isolated from general surgery OT was Acinetobacter spp. (n=11, 26.19%); followed by Enterobacter spp. and Klebsiella spp. (n=1, 2.3%) each. Bacillus spp. (n=18, 33.9%) was the most common isolated organism from gynecology and obstetrics OT followed by CONS (n=9,16.9%); S. aureus (n=8,15.09%); Acinetobacter spp. (n=8,15.09%); Pseudomonas spp (n=7,13.2%) and Klebsiella spp (n=2, 3.7%). Most common organism isolated from ophthalmology was Bacillus spp. (n=6, 66.6%) followed by CONS (n=2, 22.22%), E. coli (n=1,11.11%). Most common organism isolated from orthopedics was S. aureus (n=3,37.5%) and Acinetobacter spp. (n=3,37.5%) followed by CONS (n=3,12.5%) and Bacillus spp.(n=3,12.5%). Most common organism isolated from LR was Bacillus spp. (n=10,22.7%) and Pseudomonas spp. (n=10,22.7%) followed by CONS (n=9,20.45%) and Acinetobacter spp. (n=9,20.45%); S. aureus (n=3,6.8%); Klebsiella spp. (n=2,4.54%) and E. coli (n=1,2.27%). Most common organism isolated from SNCU was Bacillus spp. (n=9,30%) followed by CONS (n=6,20%); S. aureus (n=5,16.66%); Acinetobacter spp. & Pseudomonas spp. (n=4, 13.33%), each, followed by Klebsiella spp. (n=1,3.33%). Distribution of pathogens and their percentage from swabs, obtained from different operation theatres, labor rooms and specialised newborn care units is shown in Table 2. Bacterial CFU/m³ of air were in range of 4.4 CFU/m³ to 268.7CFU/m³ with ophthalmology theatre having lowest count and gynecology theatre, the highest count. Bacterial count (CFU/m³) of air from various operation theaters and critical care facilities (air sampling) is shown in Table 3.

Aerobic cultures on non-selective media should not exceed 35CFU/m³ of air in an empty room and 180CFU/m³ of air during surgery for conventional theatre. Total of 43 air and 184 swab samples from different surfaces of operation theaters and other critical care facilities were taken during study period. Out of 43 air samples, 41 were found to be contaminated, but only 2 had CFU/m³ beyond acceptable level. These two critical care facilities were gynecology & obstetrics OT’s. This is due to poor ventilation and cleaning practices and high traffic density of people to these OT’s. Surface disinfection in these OT’s is not done according to recommendations because of presence of heavy patient load. Duguid and Wallace, (1948) reported that increased activity enhanced the dispersion of bacteria and movement can shed upto 10⁴ skin scales per minute of which,10% contain cluster of micro-organisms.¹¹ Ophthalmologic OT’s have lowest level of CFU/m³ (4.4-10 CFU/ m³); Anjali et al.¹² & Mir RF eta^l3 & Kiranmai et al.¹⁴ &  Javid et al.¹⁵ also have reported lowest level of contamination in ophthalmologic OT.^12–15 It has been observed that counts in the range of 700 – 1800/m³ were related to significant risk of infection and risk is slight when they are below 180 CFU/ m³.¹⁶ Settle plate method for air sampling showed highest percentage of CONS (n=22), followed by Bacillus spp (n=22). This is in line with other studies from India and abroad.^{4,12,15,17,18} CONS is considered as an exogenous organism. Source of CONS in the study can be normal skin flora of medical personnel, patients and fabrics.¹⁹ Other species isolated are S. aureus (n=18), Acinetobacter spp. (n=12), Pseudomonas spp. (n=9), E . coli (n=2) and Klebsiella spp. (n = 1). S. aureus was isolated in a study done by Qudiesat et al, Desai SN, and Yadav M et al.^17,19,20 In addition Yadav M et al.²¹ also showed isolation of Acinetobacter spp. in their study. In our study E . Coli, Klebsiella spp. and Pseudomonas spp. are also isolated, Kiramai et al.¹⁴ has also showed isolation of same species.¹⁴ S. aureus and CONS are an important cause of SSI.^22,23 Source of S. aureus may be anterior nares, axilla or groin of either health care personal or patient. This bacteria can survive for several month on dust in hospital environment. SNCU serves as nursery for sick neonates, so high level of sterility should be maintained there. In our study SNCU shows higher level of contamination with Bacillus spp and CONS followed by S. aureus & Acinetobacter spp. Other gram negative organisms isolated are Pseudomonas spp., E . coli and Klebsiella spp. This is in accordance to study done by Okon et al.⁴ CONS is an important cause of sepsis in premature newborns. High level of bacterial contamination may be attributed to high movement of medical staff and mother of babies in the neonatal units. Transmission of these bacterial species can occur during transportation of patients from ward to OT or from other specialized care units. A significant percentage of Acinetobacter spp. has been isolated in our study. Infected patients spread Acinetobacter spp. to air of health care facilities. Strain can remain in the air for about four weeks. Extensive air borne infection control measures must be taken to prevent infection. A total of 184 swabs from different surfaces were taken. After 24 hours of aerobic incubation at 37^oC, 134 showed growth on culture media (72.82%). A total of 191 isolates were obtained from positive samples. Highest number of organisms obtained were Bacillus spp (n = 50, 26.17%), followed by CONS (n = 38, 19.8%), Acinetobacter spp. (n=36, 18.8%), S. aureus (n=29, 15.1%) and GNB (n=10, 5.14%). Such a high level of contamination is also reported by Okon etal.⁴ This high level of contamination may be again attributed to low adherence and compliance to infection control practices. Other factors contributing to such high level of contamination are a lack of hand hygeine practices, education level and awareness of health care professionals.

Microbial quality of air and surfaces in OT’s may be considered a mirror image of hygienic conditions of hospital. Our study has shown that air and surfaces in different health care facilities of studied hospitals is contaminated with different types of bacteria. Presence of potential pathogens in OT’s, LR’s and SNCU’s can pose a great risk to patients. Surveillance of OT’s and other critical care facilities along with infection control measures is helpful in controlling nosocomial infection.

Epidemiologist Kashmir wishes to thank Director Health Services Kashmir for her continuous support and motivation. Thanks to Chief Medical Officers, Medical Superintendents and District Health Officers of all the districts of Kashmir Division who contributed to this study. Special thanks to staff of Epidemiology, Public Health Division Barzulla, Srinagar. Again Special thanks to Mr. Mustafa Khan (DEO), Mudasir Ahmad Yatoo (DEO) and Irfana Bhat (DEO) for compilation of study.

The author declares that there is no conflict of interest.

1. Al-Benna S. Infection control in operating theatres. J Perioper Pract. 2012;22:318–22.
2. Fleischer M, Bober-Gheek B, Bort Kiewicz O, et al. Microbiological control of airborne contamination in hospitals. Indoor Build Environment. 2006;15:53–56.
3. Ensayef S. Al-Shalchi S, Sabbar M. Microbial contamination in the operating theatre . A study in a hospital in Baghdad. East mediterr Health J. 2009;15:219–223.
4. Okon KO, Osundi S, Dibal J, et al. Bacterial contamination of operating theatre and other Specialized Care Unit in a tertiary hospital in Northeastern Nigeria. Afr J Microbiol Res. 2012;6:3092–3096.
5.  Landrien A, Bissery A, Kac G. Monitoring air sampling in operating theatres: Can particle counting replace microbiological sampling. J Hosp infect. 2005;61(1):27–29.
6. Griffith CJ, Cooper RA, Gilmore J, et al. An evaluation of hospital cleaning regimes and Standards. The hospital infection Society. J Hosp Infect. 2002;51(2):79–84.
7. Sandle T. Environmental monitoring risk assessment. Journal GXP compliance. 2006;10(2):54–74.
8. Sehulster L, Chin RY, Arduino MJ, et al. Guidelines for environmental infection control in health care facilities. Morbidity and Mortality weekly Report Recommendations and Reports RR. 2003;52(10).
9. Census of India website. Office of the Registrar General and Census Commissioner, India. 2019.
10. Collee JG, Fraser AG, Marmion BP, et al. Mackie and MC Cartney Practical Medical microbiology. 14th edn. India. Elsevier; 2007:131–148.
11. Hughes SPF, Anderson FM. Infection in operating room. J Bone Joint Surg. 1999;81:754–755.
12. Anjali K. Environmental microbiological surveillance of operation theatres in a tertiary care hospital. Int J Cur Res. 2015;7(03):139;77–80.
13. Mir RF, Singh VA, Shinu P. Pre and post fumigation bacteriological profile of various operation theatres, in MMIMSR-a three Years retrospective Study. J Pharmacies Biomed Sci. 2013;36(36):1887–1891.
14. Kiranmal S, Madhavi K. Microbiological surveillance of operation theatres , intensive care units and labor room of a teaching hospital in Telangana, India. Int J Res Med Sci. 2016;4(12):525660.
15. Javed I, Hafeez R, Zubair M, et al. Microbiological Surveillance of operation theaters and ICU’s of a teaching hospital, Lahore. Biomedica.2008;24:99–102.
16. Parker MT. Hospital acquired infections: guidelines to laboratory methods. In hospital – acquired infections: guidelines to laboratory methods.1978;28–32.
17. Desai SN, Kikani KM, Mehta SJ. Microbiological Surveillance of operation theaters and intensive care unit of Teaching hospital in Surendranagar, Gujarat. Gujarat Med J. 2012;67:95:7.
18. Najotra DK, Malhotra AS, Slathia P, et al. Microbiological Surveillance of operation theaters : Five year retrospective analysis from tertiary care hospital care hospital in North India.
19. Chacko L, Jose S, Isac A, et al. Survival of nosocomial bacteria on hospital fabrics. Indian J-Med. Microbiology. 2003;21:291.
20. Qudiesat K, Abu-Elteen K, Elkarmi A, et al. Assessment of airborne Pathogens in health care settings. Af J Microbial Res. 2009;3(2):66–76.
21. Yadav M Pal, Sharma SH, Khumanthem ST. Microbiological surveillance of operation theatres in a tertiary care hospital in North East India. Int J Res Med Sci. 2017;5(8):3448–3453.
22. Owens CD, Stoessel K. Surgical Site Infections: Epidemiology, microbiology and Prevention. J Hospital infect. 2008;2:3–10.
23. Najotra DK, Kakru DK. Bacteriology and antibiogram of skin and soft tissue infections from a tertiary care hospital. Indian J med Spec. 2012;3:26–30.