Phenotypic Detection and Quality Assessment of Indoor Air-Borne Microorganisms Using Passive Air Sampling Technique (Settle Plate) at A Tertiary Care Teaching Hospital in Puducherry

Microbiological quality of an indoor air is one of the indicators of proper hygienic conditions in a health care hospital. This study intends to assess the bacteriological profile of an indoor air by settle plate method. About 100 air samples were collected from various locations in the hospital by using 5% Sheep blood agar and Sabouraud dextrose agarculture plates. Frequent isolation of common aerobic bacterial flora like Staphylococcus aureus (27.72%) followed by Diptheroids (24.75%), Bacillus spp. (24.09%), Micrococcus (23.43%) were observed. Among fungal isolates Aspergillus niger (25.00%) were more prevalent followed by Zygomyces (23.07%), Fusarium spp. (21.15%), Aspergillus flavus (17.30%) and Aspergillus fumigatus (13.46%) in the hospital environment. Most of the isolates identified were a part of the normal aerobic microbial flora; however, a minimum degree of bacterial and fungal load were observed in the casualty and in the wards due to the constant patient traffic and unrestricted


INTRODUCTION
Quality of Indoor air plays a major role in the quality of health care facilities in hospitals. Many hospitals from various countries had reported that the important risk factors of nosocomial infections is the microbial contamination of indoor air in their hospital environment. There is an increase in contamination by both bacteria as well as fungus due to indoor humidity and the varied temperature. There are two types of air sampling, passive and active air sampling techniques [1][2][3][4][5] . In earlier days, Louis Pasteur showed significance of air borne microbes and the infections 4 . Many methods have been developed for isolation of bacterial and fungal in air. The microbes are collected from air through porous solid filters or aero scope 3 , Settle plate method 4,6-10 , Bio Sampler (Gelatin filters) 11 and centrifugal air sampler to assess the quality of air in the hospitals 12 . These methods shows highly sensitive than the conventional one. However, settle plate is cheaper, cost-effective, and easier, allow the bacteria or fungus to sediment within a short-term duration 8 .
Most of the hospital infections (90%) are bacterial related infections [14][15][16] . The hospital environment are contaminated with bacteria from various sources like overcrowding of visitors to the emergency, Intensive care units and wards during the visitor's time 17 . Usually, a nosocomial infection disseminates through air from outdoor to Indoor and this could be prevented by an immediate action of infection control 18 . The microbiological indoor air quality is an indicator for identification of proper sanitary conditions in the hospital, including restricted aeration 11,13 . Primarily an overall contamination can occurs in the hospital environment, due to the poor quality of Indoor air in the OTs, ICUs and wards. Other possibilities of nosocomial infections like improper operative techniques of surgeons, sensitivity of the patients and incorporation of surgical implants and antibiotics prophylaxis 19 . The principle objective of this study is to estimate and assess the prevalence rate of various microorganisms present in the indoor air by passive air sampling technique at various locations in the hospital.

MAteRiAls AND MethODs
This study was carried after obtaining approval from their respective Heads' of various clinical departments like General Medicine, General Surgery, Obstetrics and Gynecology and Orthopedics of 1200 bedded tertiary care hospital, Puducherry during the period of three months(August -October 2017).

Sample Collection
A total of 100Air samplings were done (in duplicates) by using conventional passive air sampling technique -Settle plate method or Sedimentation method. These samples were obtained from various places of the hospital viz., Casualty, General Medical and Surgical wards (Male/Female), Intensive Care Units (Medical, Neonatal and Pediatrics), Obstetrics and Gynecology (Labor ward), Ortho Ward (Male/ Female) and various Operation theatres. Air Samplings were done on the third working day of every week for three months.

Passive Air sampling method (Settle Plate method)
Culture plates of 5% Sheep blood agar (SBA) for bacterial isolation and Sabouraud Dextrose Agar (SDA) for fungal isolation were used. These plates were kept exposed in the air, at their respective locations in the hospital wards, Operation theatres, casualty and ICUs for 1 hr, at 1m above the floor and 1m from the wall [11] .The procedure was done twice, at 8.00 AM before the routine work started and then at 5:00 PM, after the routine active working hours 4 . The edges of the plates covered with paraffin film to prevent the contamination. For bacterial isolation, SBA was incubated at 37°C for 24 hours and for fungal SDA was incubated at 25°C for up to 10 days. The entire process was repeated every week at an interval of seven days for three months at the same locations. Bacteria were identified by using colony morphology, preliminary tests (Gram stain, Catalase, Oxidase and Motility test) and biochemical tests like Indole production, Methyl Red, Voges-Proskauer, Citrate utilization, Urease test and Triple sugar iron agar (TSI) test). In case of filamentous fungal colonies, Lacto Phenol Cotton Blue (LPCB) mounts were performed.

RESULTS
Tabel 1 shows the details on the percentages of micro-organisms isolated from air samples collected from various locations of the hospital. Nearly, 120 bacterial and 18 fungal isolates were identified during 8 am and 192 bacterial and 31fungal isolates were identified at 5 pm sampling. In this study, the microbial contamination was found higher in wards (122 isolates) and low in operation theatres (8 isolates) which is shown in Table 2. Different microbial flora were found contaminating indoor air, which range from higher prevalence of Staphylococcus aureus (27.72%) followed by Diptheroids (24.75%), Bacillus spp. (24.09%) and Micrococcus (23.43%). Among fungal isolates, Aspergillus niger (25.00%) was more prevalent followed by Zygomyces (23.07%), Fusariumspp. (21.15%), Aspergillus flavus (17.30%) and Aspergillus fumigatus (13.46%)in the hospital environment. There was no significant statistical difference between the pre-work and post work of microbial load of air in different locations of the hospital. Recently, one of the study found that an anaerobic bacterium, Clostridium difficile, were disseminated through aerosols and it was identified by slit sampling technique 21 . Nearly 4-11% of total microorganisms were isolated in urban air 13 . Two organisms were identified with human related bacteria viz., Allocococcus otitis in government hospital and Bacillus licheniformis in private health care centers by using Impact samples 22 . Using conventional method like settle plate technique, 57% for S. aureus and 7% for Aspergillus niger were detected 23 . Similarly, in the present study, 27.72% for S. aureus and 25.00%Aspergillus niger was found higher, when compared to the other bacterial as well as fungal isolates from the hospital regions. There was no statistical significance found on microbial load between the pre-work and post work from different locations of the hospital. The major impact on microbiological indoor air quality is to prevent the risk and critical to the patients as well as healthy concern 1 . According to Huanget al., surface samples are highly contaminated when compared to the air samples. Nearly, 19.3% P. aeruginosa was found in the surface area and but higher percentage was found 39.1% in air samples 3 . However, Pseudomonas isolate is completely absent in the present study. Both surface air system and settle plate methods for air sampling procedures particularly in the operation theaters and found total viable count at rest was 12.4 CFU/m 3 and during surgical time the count was higher 722.5 CFU/m 2 /hr 4 . Hence, we collected the samples at theatres and yielded a low prevalence of microbial contamination this may be due to absence of surgical procedures in the theaters. Schulsteret al highly recom-mended CDC guidelines for environmental infection control in health-care facilities 18 . Postoperative wound infections occur due to the lack of guidelines, improper sanitation and use of surgical aprons. This could be one of the reasons for spreading of nosocomial infections and emerging antibiotic resistance to the patients [14][15][16] . Fitting of laminarair flow systems with High-Efficiency Particulate Air (HEPA) Filters in operation theatres, could help to reduce the infections for the patients 16 .
In OT samples, found highest prevalence is CONS (53.5%), S. aureus (33.1%) and MRSA (7.7%) 6 . In contrast to our study, S. aureus about 27.72% had obtained in various locations of our hospital. Cleaning the OTs and other wards regularly, this can reduce the microbial burden of the patients 6 . In some studies, the authors down regulated the settle plate method for trapping the air-borne microorganisms in comparison with nitrocellulose membrane 24 . Lacunae of this method are the exposure time, because of dryness and wrinkles in the agar surface. Nearly, 29.7% grampositive cocci and 70.3% gram-negative bacilli were identified from the ICUs 19 . In the present study, 51.15%, 24.75% and 24.09% of grampositive cocci, gram-negative bacilli and grampositive bacilli were isolated respectively. Highest microbial air contamination were identified in the wards (34.36%) and the lowest contamination in operation theatres (2.25%).

CONCLUSION
We concluded that our short-term study is highly limited only to the hospital environment and the bacteria and fungus that we isolated in various locations of the hospital were identified up to the genus level. However, we could not be able to perform antibiotic sensitivity testing as well as molecular identification for these isolates to predict the resistance and sensitive genes. Based on this study we highly recommend, to restrict the entry of visitors to ICU patients and wards during visitors time and also to avoid overcrowding. Performance of molecular diag-nosis for bacterial and fungal isolates to know the true picture of hospital acquired infections of this study. The main stay of this study is to make awareness to the physicians or surgeons and other health care committees in the hospital that intend to prevent the spreading of nosocomial infections among the patients as well as health care workers.

ACKNOWleDGeMeNt
Author is grateful to the Chairman, Vice-Chancellor, Dean-Academic and Dean -Research of Mahatma Gandhi Medical College and Research Institute, Puducherry (Sri Balaji Vidyapeeth, Deemed-to-be-University) for providing facilities for this Research Project.