ASIAN J. EXP. BIOL. SCI.

VOl 1 (1) 2010:128-135

Society of Applied Sciences

ORIGINAL ARTICLE

Extramural Aero-bacteriological Quality of Hospital Environment

Apurva K.Pathak* and Karuna S. Verma #
*Deptt. of Pathology & Microbiology, Modern Dental College & Research Center, Indore (M.P.)-453112, India, e-mail: pathak.apurva@gmail.com. # Aeroallergens and Immunology Laboratory Department of Biological Sciences, Rani Durgavati University, Jabalpur (M.P.) India -482001 ABSTRACT An extramural aero-bacteriological study was undertaken to determine typical concentrations of airborne culturable bacteria at the vicinity of a local hospital in the tropical environment of India. Hospital acquired infection is a common phenomenon; however, hospital as a source of infection for the surroundings community is yet to be established. Present aero-bacteriological investigation includes enumeration, identification, and numerical analysis of air borne culturable bacteria in hospital associated environment. Bacteria isolated from the environment during the present study were representative of normal micro flora of the skin, respiratory and gastro-intestinal tracts; it also includes the opportunistic pathogens like Acinetobacter and Flavobacterium species with the environmental nonpathogenic genera. In this study respirable and nonrespirable fraction of bacteria were also quantify. The Pearson correlation study shows that both temperature and humidity have an effect on tenacity and prevalence of the allbacterial type. A regression model with 67.15 % variance is also prepared in order to predict the bio-load for this atmosphere. KEY WORD: Aerosolization, Aero-bacteriological investigation, Enterobacteriaceae and Prediction.

INTRODUCTION A hospital produces waste by giving their service to the patients. This waste can be produced directly in combination with the service (e.g. injection) or in the upstream (e.g. blood or urine cultures in the laboratory) or downstream (vaccine) process. One kind of typical diseases treated in hospitals is infectious diseases. By the services for known or unknown infectious patients, waste can be the source of an infectious agent. If the microorganisms have survived the aerial transport, they can grow and reproduce in the new environment and may spread further. If a living organism inhales the airborne particles, their fate will depend on many factors. Large particles, i.e. larger than 5 m diameters, easily adhere to the mucus membranes of the upper respiratory tracts. Continuously moving cilia guide the particles to the throat, where they removed by coughing or swallowing. Particles smaller than 5 mm inhaled into the deeper parts of the lungs, many bacteria and viruses are thus well adapted by their size to reach the alveoli [1]. The lungs represent one of the largest interfaces between the human organism and its environment, and thus a major site of interaction with microorganisms and its products. Infection of the lung is one of the most frequent causes of morbidity and death. Toxic substances can enter into the lungs either during infection by Gram-negative bacteria (4060% of all cultured-diagnosed pneumonia), or when carried by inhaled airborne particles. Hospital environment consisted high level of potentially hazardous bacteria, fungi and other allergenic and / or immuno-toxic agents. These agents may penetrates into lungs of exposed residences or dwellers and evoke inflammatory reaction leading to respiratory disease, such as asthma, mucous membrane irritation, allergic alveolitis etc. Hospital waste is not necessary a source of infectious agents. Only if the waste suspected to contain infectious agents like bacteria viruses, parasites, or fungi in a sufficient concentration it will be infectious waste. Members of the Klebsiella, Enterobacter and Serratia groups, and Pseudomonas aeruginosa, Ps. cepacia, flavobacteria, and other non-fermentative bacilli can able to grow well in environmental fluids and some enteric organisms (e.g., Escherichia coli and Proteus spp.) that have limited powers of multiplication at environmental sites, are the potential source of infection, once these bacteria are introduced into the

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environments from the patients. As a result, large populations built up, and may subsequently survive for a very long time. All the Microorganism of ambient environment must not necessarily be hazardous for humans; they are even a part of the natural micro flora. Changing environmental micro flora can change the human normal micro flora too, as the human being is a part of its larger ecosystem. The immune system ( Innate and acquired) has an ability to maintain the healthy state of the organism, but this system under certain state ( e.g. Immunocompromised, old age, contamination etc.) can be exposed of a massive attack of pathogen agents, protective barriers can break or are broken and the natural balance can be shifted [2].The contribution of airborne microorganisms to the spread of infection is likely to be greater than is currently recognized. This is partly because many airborne microorganisms remain viable while being non-culturable, with the result that they are not detected, and because some infections arising from contact transmission involve the airborne transportation of microorganisms onto inanimate surfaces. [3] The aerosolized spore forming gram positive bacteria are able to survived in air for a long duration and gram negative non-spore forming bacteria can also able to survive in air up to 390 minute as a half life time recorded previously by the workers [4]. Things are gone worse when these microorganism able to multiply in these aerosols [5]. The transportation and forecasting of air borne microorganism is of prime importance to epidemiologist in order to access the chances of outbreak of disease in community environment and to evaluate the degree of pathogenicity of these organism. This comprehensive study has been made to evaluate the quantity, quality, respirable and non-respirable fraction of potentially pathogenic bacteria for this environment. The effects of environmental factors on the total airborne bacterial bio-load also analyzed by using correlation analysis and a regression model are prepared for this environment. Many works previously been done to enumerate and identify the air borne bacteria of nosocomial environment [6-11]. On the contrary, very few of them essentially concerned their study as the hospital as a source of bio-pollutants for the community [12, 13]. This present study deals with the types of bacterial flora originated from hospital or hospital associated waste, which disseminates extramurally posing both occupational, and community related health problem. MATERIALS AND METHODS Jabalpur (Latitude: 23.2; Longitude: 79.95; Altitude: 391.) is the third biggest city of Madhya Pradesh. The city of Jabalpur is set in a most attractive stretch of country. The metropolis itself stands on a rocky stretch of land about 9.6 km. from the River Narmada and 20.8 km. from the marble rocks of Bheraghat. Jabalpur is one of the central districts of India. The city consists of long narrow plains running northeast and southwest, and shut in all sides by highlands farming an offshoot from the great valley of the River Narmada. The hilly tracts in and around Jabalpur are covered by luxuriant vegetations. The climate of Jabalpur is overall pleasant and salubrious, has a year-round tropical climate generally characterized by warm days and cooler evenings. Sampling Site Medical college hospital is 750 bedded; one of the biggest tertiary care hospital of Mahakousal Region. The Medical College surrounded by typical urban mixed type habitation. A large number of people from different region visited here not only for the indoor treatment but also as outdoor patients. The kith and kin of these patients were used to stay outside within the premises of the hospital were also responsible for generating waste of various types. The aero-bacteriological sampling had been done within the premises of medical college hospital, 50 -100 meter apart from the building and at the Devtal area which is situated two kilometer from the hospital area as a control, in duplicate and fortnightly in order to cover all the major season. The metrological data collected from weather station Jabalpur. Apart from temperature and humidity five other metrological parameter (Mean sea level pressure (mb); Precipitation amount (mm), Mean wind speed (Km/h); Maximum sustained wind speed (Km/h); Maximum wind gust (Km/h); Indicator for occurrence of: Rain or Drizzle) were also recorded in order to analyze their effect on airborne bacterial load. Isolation from Air A bioaerosol may need to monitored, not only for mass or number concentration, but also for viability. The amount or proportion of viable microorganisms in the atmosphere can be the critical factor of interest. Thus, the sampling method selected should impose minimum stress on the microorganism and supply nutrients to preserve viability. [14]. Area sampling should always be carried out near the potential sources of bioaerosols such as air supply systems, machinery and at or

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near the workers position; Duplicate samples in time and place must always be taken [15]. The Andersen 2-stage viable (microbial) particle sampler (2-STG) used for sampling which has been developed for monitoring bioaerosols. It is a multi-orifice, cascade impactor with 400 holes per stage, drawing air at a flow rate of 28.3 L min-1. This sampling rate is comparative to the breathing rate of a person going about their normal work. However, worker respiration rates will rise with increased metabolic rate. In women, respiration rates are lower [16]. The different stages separate the airborne particles in size fractions. The stages have 50% cut-off diameters of 0.6 to 7 m (depending on the orifice used) and the impaction holes arranged in a regular pattern that facilitates counting of colonies [17]. As the air velocity increases across the different impacting surfaces, the smaller particles deposited resulting in the upper stages collecting the larger particles while the lower stages collect the smaller particles [18].The organisms deposited because the air forced to make a 90 degree turn to pass around the edge of the plate. This allows the smaller particles to pass around the growth medium while the larger particles cannot make the turn and impact on the surface [16]. Dividing bioaerosols into size categories is important for epidemiological research [19]. Culture methods were used everywhere for the measurements of airborne microorganisms in the work environment [20]. For this study, air sampling done on Tryptone Glucose Yeast Extract (TGYE) Agar Medium (Hi Media), and Eosin Methylene Blue (EMB) Agar Medium (Hi Media) with the help of modified two stages Andersen Sampler [21, 22]. The sampler placed at one meter height from the ground; operated for two minutes at both the site in duplicate at morning hour before the OPD started. For enumeration and identification of total viable type of bacterial population present in air, the TGYE medium plate were kept on upper stage of the sampler, whereas for enumeration and isolation of respirable fraction of gram- negative bacteria, the EMB media plate were kept on lower stage of the sampler. Isolation from Sources Effective source control is the key issue in all matters concerning hygiene and hospitals are by no means an exception, [23] for this the source must be identified. This sampling was carry out to identify the presence of the bacteria in dust, decayed materials, and debris of hospital residues originated by the community and compare with those present in air. Water samples collected from associated sewage system of hospital, by holding the glass stopper, sterile bottle near its base in the hand and plugging it (necked downward below the surface) and transported to the laboratory in an icebox to avoid unpredictable changes in physiochemical as well as bacteriological characteristics. The dust and top soil of debris sampled in sterile polythene airtight bags. Processing of samples was done by serial dilution technique (10-2 to 10-4) to get only a few cells per ml. One ml of inoculums from each dilution poured onto sterilized Petri plates of respective media (TGYE & EMB) at 45 0C by using Pour plate technique [24] and incubated at 37 2 0C for 24 to 48 hrs. Air Contamination Standards The level of bacterial contamination of air is usually expressed in terms of number of bacteriacarrying particles per m3 (bcp m-3) or the bioload (B). B can be calculated from the below formula:

B=

1000N bcp m RT

Where N is the number of colonies counted on the sample plate, T is the duration of the test in min, and R is the air-sampling rate in liters/min. (25). The recommended threshold value (TLV) for bioload is 50 CFU/m3 (WHO). Identification of Isolates Bacteria can be identified by morphology, gram-staining, growth on specific substances and under special conditions, and production of specific metabolites [17]. After gram staining of bacteria, a study by Krahmer et al. [26] divided the results into four categories as actinomycetes, gram-positive rods, gram-negative rods, and gram positive cocci. In this study, identification of isolates was done by using standard methods and manuals[25, 27-31]. Statistical Analysis The number of samples collected will influence the precision of the exposure estimate and the associated confidence limits [32]. Monitoring environments can be difficult and time consuming, which can lead to a small number of samples being collected [33]. In order to analyze the effect of various environmental factors on the prevalence of airborne bacterial population and degree of its

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effectiveness with other environmental factors, multiple correlation coefficients, and multiple regressions analysis was done. Multiple linear regressions can only be used if each independent variable displays a linear relationship with the dependent variable. If that criterion is met then several assumptions have to be tested for multiple linear regressions to be appropriate [34-40].All the data were presented in the form of table and figures. RESULT Present study reported an average of 2.5 X 103 bcp m-3 of total aerobic viable culturable bacterial bioload in extramural hospital environment and the bioload of the species of family Enterobacteriaceae was reported 35-bcp m-3. The bioload of total aerobic viable culturable bacteria ranged from zero to a maximum of 9012 bcp m-3 and the respirable fraction culturable gram negative bacteria was ranging from zero to 106 bcp m-3 (Figure:1). Filamentous bacteria belong to the groups of Actinomycetes & Arthobacter were predominant in extra mural environment, probably due to its origin from the soil. Grampositive rod and pleomorphic bacteria belonging to the Bacillus, Actinomycetes & related genera account 33 percent of total type of bacteria isolated. (Figure: 2). Source study also reported similar type of organism in the soil, medical waste of extra mural environment and the control environment. The concentrations of these groups recorded higher in winter (39%) but the airborne concentration of total aerobic viable culturable bacteria in extramural hospital environment reported highest during the summer (Figure: 3). The Gram-negative bacteria in the outdoor atmosphere recorded higher during the winter (42%) than the summer and monsoon; but it represents only 1.5% of total viable culturable bacteria, though it shows higher diversity at species level. The fraction of viable culturable gram negative bacteria isolated on lower stage of sampler, which could affect the lower respiratory tract, is recorded lowest in monsoon period (20 bcp m-3) and the average of incidence in summer and winter is identical (30 bcp m-3) even though the consistency occur only in winter season. In winter, Proteus and Salmonella were dominant genera, whereas in summer, Citrobacter was dominant. The type of species isolated from hospital environment during that study were Providencia sp., Proteus sp.( mirabilis), Citrobacter frundii, Serratia plymuthica, Pasteurella spp., Enterobacter gergoviae. Enterobacter cloacae & Escherichia coli were also recovered from the soil and water of the intra-mural environment but the species Providencia sp., Xenorhabdus & Edwardsiella tarda was not reported from soil & water sampled. During the study of sources of these microorganisms, the hospital sewage study revealed that the sewage is highly contaminated with the spp. of Citrobacter, Salmonella, and Enterobacter. The species of Enterobacter were also reported from control environment during the present survey with dissimilar carbon assimilation profile. The effects of environmental factors on the quantity of airborne bacteria studied, by using correlation coefficient and regression analysis. The Pearson correlation is strongly positive between average temperature with the total viable culturable bacteria of hospital environment (+0.709) but it is negative correlated with the humidity (-.663) (Table 1). Whereas, the humidity had, negative and temperature had a positive correlation with the respirable fraction of gram-negative bacteria isolated on Eosin Methylene Blue Agar (EMB).
Table 1: The Quantity Of Airborne Bacteria Studied, By Using Correlations Coefficient Bioload of total type of bacteria isolated on NAM -.663 .709 .000 .000 Bioload of total type of bacteria isolated on EMB -.044 .165 .419 .221 Bioload of total Enterobacteriaceae. .093 -.443 .332 .015

Pearson Correlation Sig. (1-tailed)

Hum. Avg Temp.Avg Hum. Avg Temp.Avg

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Whether, independent variable had a linear relationship with the dependent variable, multiple regression analysis with residual technique is used. If the errors distributed in any fashion other than normal, this would suggest a nonlinear trend. All residuals distributed normally in the present analysis. The stepwise multiple regression analysis (CRITERIA: Probability-of-F-to-enter <= .050, Probability-of-F-to-remove >= .100, Confidence Limit enter 95%) was used to determine the variation in bacterial bioload in the environment under study that was explained by the estimated sample regression plane, uses environmental factors (i.e. Temperature, Humidity, Wind speed, Precipitation, and fog) as the independent variables and which were best fitted in the criteria. Temperature and humidity account for 67.15 % variation in total type of bacterial bioload in hospital environment (Figure: 4), but it did not affect the total gram-negative bacteria and Enterobacteriaceae bioload of air for the environment; as its coefficient of determinates was only 0.028 and 0.196 respectively, aerosolization of these organism might be due to the mechanical forces. Estimated Multiple Regression Model of Total type of Bacteria in Hospital Environment. = -141.48 - 43.43 (Average Humidity) + 210.58(Average Temperature). Estimated Multiple Regression Model of total isolates on EMB in Hospital Environment = 17.31+ 3.44E-02 (Average Humidity) + 0.91(Average Temperature) Estimated Multiple Regression Model of total Enterobacteriaceae in Hospital Environment. = 57.39 + 7.96E-02 (Average Humidity) -1.02(Average Temperature) DISCUSSION The previous study reported the total viable bacterial counts in intramural hospital environment were as low as 20-cfu m-3 to as high as 539-cfu m-3 by many workers [41-45]. That made it clear that the viable cultrable bacterial count of extramural environment is independent of intramural environment. Later studies revealed that the gram-negative bacteria generally derived from the intramural environment of hospital, soil, and vegetation [46, 47]. In this, present studies based on carbon assimilation profile and biochemical characteristics of isolated bacteria shows that not all but some of the bacterial source is intramural environment of the hospital. Gram-negative bacteria have been described to persist longer than gram-positive bacteria [25, 24]. Humid conditions improved persistence for most types of bacteria. The noninfectious hospital waste compared with household waste shows general hospital waste is not necessary more dangerous then household waste, but the hospital waste contained a greater variety of microorganisms, whereas enteric bacteria [48, 49, 15] dominate the household waste. Quantitatively only 1.5% of total viable bacterial bioload were belonging to the members of Enterobacteriaceae, whereas, qualitatively it represents 28% of total identifiable species, similar concentrations known from other investigations [47]. The concentration of gram-negative bacteria reported highest during the winter, it is only due to the fact that; rate of desiccation of gram-negative bacteria are high in higher temperature. Gram-positive cocci were predominant airborne bacteria reported by many workers [50, 51, 52], but these findings is contrasting with the present survey is probably due to the extra mural condition and comparatively slow growth on the TGYE of these bacteria than other bacteria [53]. The species of Proteus and Pseudomonas were isolated from associated sewage water of medical college hospital. Tambekar (13) reported Proteus mirabilis as a dominant species among gramnegative bacteria Pseudomonas spp. is dominant which is isolated in the entire sample collected from these extra mural environment during the present work. These findings are similar to the other workers [54, 55, 13] representing that the nosocomial environment is the major source of pseudomonads dissemination to the surrounding environment. The isolated species Klebsiella and Enterobacter were also reported by the previous workers in nosocomial environment [56, 40]. Masaki, et al. [56] by using settled plate method in intramural environment found that, Citrobacter and Enterobacter are predominant spp., that study reiterated the present findings. The source of Enterobacter spp. could also be the outer environment as it is isolated from control environment i.e. mostly vegetation and human activity. According to Krishna Prakash [57] infections with Clostridium tetani, Pseudomonas cepacia, Flavobacterium meningosepticum are nearly always and infections byof Morgenella, Pseudomonas aeruginosa and members of the Klebsiella-Enterobacter - Serratia group are often, acquired form independent environmental sources (exogenous), and reiterated the present findings. The effect of environmental factors on viable airborne bacteria revealed that, the

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Figure : 1 Respirable & Non-respirable fraction of Bacteria in Hospital Environment

10000 9000 8000

Bioload of total bacteria (Upper airway) Non respirable Fraction of B Respirable Fraction of B
6467

9012

106 7280

106

106

120
Bioloadof Enterobacteriacieae (Lower airway)

100
88

4064

4000
1573 1424 1378

3234

3711

5000 3000 2000 1000 0

53

53

53

53

53

53

4947

6000

5690

7000

80 60
35 2120 35

2403

35

35

35 1484 1272

40
18 230
D ec

1202

919

812

639

583

0
M ay M ay Ja n Ja n F eb F eb M ar M ar A pr 1 15 A pr 15 1 15 1 15 1 15

0
1

548

15

18

18

18

18

20 0

Ju n 15 e Ju ne 1 Ju ly 15 Ju ly 1 A ug 15 A ug 1 S ep 15 Se p

O c 15 t O ct 1 N ov 15 N ov

Date of Sampling

Figure : 2 Total type of bacteria encountered hospital environment G ve Coccobacili 3% G +ve Rod 21% Filamentous 35% Monsoon 21% G +ve Pleolomorph 12% Vibrio & Rod 1%

Figure : 3 Seasonal Bioload of Bacteria Winter 19%

15

D ec

Summer 60% G -ve Rod 28% Estimated vs. actual total Bioload. Shown as the dependent variable (Bioload) versus the model predicted cells per m3. Bioload of Total aerobic viable culturable bacteria

10000 8000 6000 4000 2000 0 1 0 1 2

Regression Standardized Predicted Value of Total aerobic viable cuturable bacteria

the significant of relation between environmental factors with viable airborne bacteria is higher with humidity i.e. higher temperature and lower humidity favours the dissemination of gram-negative bacteria to the adjacent areas of hospital environment. As for as members of family Enterobacteriaceae is concerned, the lower temperature and moderate humidity favours its tenacity in air.The Bioload of viable culturable bacteria taken from extramural environment associated with a tertiary care hospital have successfully been applied to a mathematical equation that has made it possible to estimate the extramural bacterial bioaerosol concentrations. This model shows to be accurate to 67% for the sample size. Though the model which could not be able to explain >90% of variation accounted for by the independent variable is inappropriate (58), yet this model shows that the humidity and temperature is the major factor which govern the bioaerosol dissemination for this environment. Finally, it should be noted that most of the pathogenic bacteria, which are not represented in the control environment and of which biochemical profile is similar to the isolated species from sources associated with hospital environment are might be derived from the hospital environment. According to Kramer et al. (26), the most common nosocomial pathogens may well survive or persist on surfaces for months and as unicellular organisms like virus and bacteria have no possibilities to become airborne by their own force (1) can thereby be a continuous source of transmission. Thus, the hospital managements must adopt for a stringent practices to avoid bioaerosol generation. ACKNOWLEDGEMENT We are greatly thankful to the Dr. S.M. Paul Khurana Honble Vice- Chancellor Rani Durgavati Vishwavidyalaya, Jabalpur for providing academic and materialistic supports without which it is hard to perform this work. REFERENCES
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*CORRESPONDING AUTHOR: DR. APURVA K. PATHAK (ASST. PROFESSOR). Deptt. Of

Pathology & Microbiology, Modern Dental College & Research Center, Indore (M.P.)-453112, India, e-mail: pathak.apurva@gmail.com

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