An-Najah National University Faculty of Graduate Studies Characterization of Indoor Air Bacterial Isolates from Rafidia Hospital, Nablus-Palestine and their Roles in Nosocomial Infections By Eman Atta Hassan Abedraboo Supervisor Dr. Ghaleb Adwan This Thesis is Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Environmental Sciences, Faculty of Graduate Studies, An-Najah National University, Nablus, Palestine. 2015 iii Dedication I wish to dedicate this thesis to my mother; to my late father whose memory continues to inspire me, husband; to my four children Heba, Sondos, Ahmad and Abederahman; to my sisters and brothers. Finally, to my friends and school team for their continuous love and support. iv Acknowledgements First, I would like to express my deepest gratitude to my thesis supervisor Dr. Ghaleb Adwan who has always been providing kindest support, encouragement and indispensable guidance throughout the accomplishment of this dissertation. I also would like to thank the technicians in the Department of Biology and Biotechnology at An-Najah National University, especial thank for my friend Hiba Bourinee for her help and cooperation. Another thank for the medical staff in Rafidia Hospital for their cooperation. Lastly, I would like to thank my family and to all my friends of the school for their help and cooperation. Thank you all ……….. vi List of Contents No. Subject Page Dedication iii Acknowledgement iv Declaration v List of contents vi List of tables viii List of Figures ix Abbreviations x Abstract xi Chapter One: Introduction 1 1.1 General background 1 1.2 Air sampling methods 3 1.3 Enterobacterial repetitive intergenic consensus (ERIC) PCR 5 1.4 Literature review 6 1.5 Aims of study 10 Chapter Two: Materials and Methods 11 2.1 Study area and site of samples analysis 11 2.2 Media preparation 11 2.2.1 Tryptic soy agar ( TSA ) 11 2.2.2 Mannitol salt agar ( MSA ) 11 2.2.3 Meullar Hinton agar ( MHA ) 12 2.2.4 MacConkey agar 12 2.2.5 Eosin Methylene Blue ( EMB ) 13 2.2.6 Blood agar 13 2.2.7 Simmons citrate agar 13 2.2.8 Triple sugar Iron Agar ( TSI ) 14 2.2.9 Sulfied Indole Motility ( SIM ) Medium 14 2.3 Identification of microorganisms 14 2.3.1 Gram staining 14 2.3.2 Mannitol fermentation 15 2.3.3 Catalase test 15 2.3.4 Slide coagulase test 15 2.3.5 Tube coagulase test 15 2.3.6 Citrate utilization test 16 2.3.7 TSI test 16 2.3.8 Motility test 16 2.4 Air sampling 16 2.4.1 Active air sampling 16 2.4.2 Passive air sampling (sedimentation technique) 17 2.5 Bacterial Identification 18 vii 2.6 Antibiotic susceptibility 18 2.7 DNA Extraction and ERIC PCR 19 2.7.1 DNA Extraction 19 2.7.2 ERIC PCR 19 2.8 Statistical analysis 20 Chapter Three: Results 21 Chapter Four: Discussion 28 References 36 ب الملخص viii List of Tables No. Contents Page 2.1 Categories of microbial indoor air contamination–a concentration criterion of mixed population of bacteria and fungi. 17 3.1 Microbial air load and spectrum of microbial findings in the air samples at surgical operation rooms, intensive care unit and neonatal room at Rafidia Hospital-Nablus by passive air sampling. 23 3.2 Microbial air load and spectrum of microbial findings in the air samples at surgical operation rooms, intensive care unit and neonatal room at Rafidia Hospital-Nablus by active air sampling. 24 3.3 Microbial contamination (Colony Forming Unit) of the air in surgical operation rooms, intensive care unit and neonatal room at Rafidia Hospital-Nablus by active and passive air sampling according to the type of medium. 25 3.4 Antibiotic resistance of bacterial isolates recovered from the air in surgical operation rooms, intensive care unit and neonatal room at Rafidia Hospital-Nablus by active and passive air sampling 26 3.5 T test to differentiate between active and passive sampling methods. 26 ix List of Figures No. Contents Page 3.1. DNA fingerprints generated by ERIC PCR analysis of 10 bacterial isolates (S. aureus and CNS and/or Micrococcus spp.) recovered from clinical and air samples on 1.5% agarose gel. 28 3.2. DNA fingerprints generated by ERIC PCR analysis of 10 bacterial isolates (E. coli and Klebsiella spp.) recovered from clinical and air samples on 1.5% agarose gel. 28 x Abbreviations ERIC PCR enterobacterial repetitive intergenic consensus PCR ICU intensive care unit NR neonatal room SOR surgical operation room CFU colony forming units OT operation theatre ENT ear, nose and throat CoNS coagulase negative Staphylococci S. aureus Staphylococcus aureus P. aeruginosa Pseudomonas aeruginosa E.coli Escherichia coli REP repetitive extragenic palindromic TSA tryptic Soy Agar MSA mannitol salt agar MHA meullar Hinton agar EMB eosin Methylene Blue TSI triple Sugar Iron SIM sulfied Indole Motility NA nutrient agar AHEM acta Hygienica, Epidemiologica et Microbiologica CLSI clinical and Laboratory Standard Institute NT not tested MRSA methicillin- resistant S. aureus MDR multi-drug resistant xi Characterization of Indoor Air Bacterial Isolates from Rafidia Hospital, Nablus-Palestine and their Roles in Nosocomial Infections By Eman Atta Hassan Abedraboo Supervisor Dr. Ghaleb Adwan Abstract Air can play a vital role as a reservoir for both pathogenic and non pathogenic living microorganisms. Microbial contamination of hospitals air is considered as a source of hospital-associated infections. The present study aimed to assess microbial profile of air contamination in different wards of Rafidia Hospital, Nablus-Palestine using both active and passive sampling methods, and to ascertain the contribution of some of these microorganisms to infection using cultures from hospitalized patients at the same time using ERIC PCR technique. Results of this research showed that total viable count of Gram-positive bacteria was the most frequent microorganisms cultured from different wards of the hospital. Coagulase negative Staphylococci (CoNS) and Micrococcus spp, were the most predominant among isolates from air samples from all operation rooms, ICU and neonatal room by passive air sampling method. The percentage of CoNS and Micrococcus spp in air of surgical operation rooms, intensive care unit and neonatal room ranged from 61.8%-100% and the average was 5158 CFU/m 2 /h-20187 CFU/m 2 /h. Staphylococcus aureus was the most common microorganisms isolated from neonatal room by active air sampling method, the percentage was 35% and the average was 100 CFU/m 3 . Total bacterial level range was116 xii CFU/ m 3 -1085 CFU/m 3. The percentage of CoNS and Micrococcus spp in air of surgical operation rooms, intensive care unit by active air sampling was 58.8%-100% and the average was 70-1080 CFU/ m 3 . The results showed that most frequent Colony Forming Units were obtained from Blood agar with a range of 4085 CFU/ m 2 /h -8721 CFU/ m 2 /h and Tryptic Soy Agar with a range of 2043 CFU/ m 2 /h-7935 CFU/ m 2 /h by passive air sampling method. Antimicrobial susceptibility pattern of bacterial isolates revealed that the most effective antibiotics were ciprofloxacin , norfloxacin and tetracycline against S. aureus; tetracycline, ciprofloxacin and norfloxacin against CoNS and Micrococcus spp and ciprofloxacin, Trimethoprim/Sulfamethoxazole and tetracycline against Bacillus spp. ERIC PCR profile based on number and size of generated bands revealed that clinical bacterial strains of S. aureus, E. coli and Klebsiella spp were not clonally related to airborne isolates collected at the same time. Data presented in this study may be valuable to develop interventions to improve microbial indoor air quality in various hospital wardsand also for preventing or decreasing the occurrence of the nosocomial infections. 1 Chapter One Introduction 1.1 General Background Indoor air quality is a term which refers to the air quality within and around buildings and structures especially as it relates to the health and comfort of its occupants. Indoor air can be polluted by various compounds such as carbon monoxide, volatile organic compounds, particulate matter and microbial contaminants (molds, bacteria, viruses) and any action that introduces harmful contaminants into the air within the building. The concern for quality indoor air is necessary especially in institutionalized settings that accommodate a large number of people such as hospitals, nursing homes, prisons, schools, family houses, offices, dispensaries, hotels and classes because contaminated air can cause both mild and severely irritating health conditions (Tambeker et al., 2007). The quality of air in hospitals in relation to microbial contamination at a given time period is determined by the quality of air entering into the building, the number of occupants in the building, their physical activities and resultant aerosol generation, human traffic and the efficiency of ventilation (Adebolu and Vhirterhre, 2002). 2 Nosocomial infections acquired during hospitalization for proper management depend on the characteristics of the microorganisms, with a high risk of being acquired when the healthcare environment is contaminated. Some patients acquire other disease other than the one they were admitted to and this results from contact with a carrier of the pathogen directly or indirectly through inanimate objects. Improper/unhygienic ventilation system can continually be a source of nosocomial infection (Ayliffe et al., 1999). Sneezing has been described as the most vigorous mechanisms of generating millions of airborne microbial infections into the hospital environment (Pasquaria et al., 2000). While the larger droplets fall to the ground or nearby surfaces, smaller ones are rapidly evaporated into a non-volatile form where they remain suspended in the air thereby serving as a source of infection when inhaled by other occupants of the hospital including staff. Drug resistant pathogens and fungi strains are of interest in hospitals. Hospitals have the potential for pathogen spread because they have contact with different medical furniture, instruments, air, medical staff and others, although the infections caused by such nosocomial pathogens involves a contaminated environment which should have applied strict safety biosecurity procedures (Mazzali et al., 2003). In addition, it seems that the role of airborne microorganisms in development of hospital-acquired infections has been underestimated because many of these airborne microorganisms cannot be cultured easily (Heidelberg et al., 1997). Moreover, some of the infections resulting from contact rout have resulted from airborne transportation of microorganisms onto surfaces 3 (Poirot et al., 2007; Bergeron et al., 2007; Abdollahi and Mahmoudzadeh, 2012). Although the cause-and-effect relationship between airborne pathogen levels and nosocomial infections is not known yet, it could be hypothesized that lowering the level of these pathogens in the air would result in providing an environment that would help decrease the risk of nosocomial infection (Poirot et al., 2007; Guriz et al., 2008; Wood et al., 2010). Hospital acquired infection rate varies from 5-10% in the developed countries to 25% or more in developing countries. These infections are mainly caused by microorganisms or surfaces contaminated by the microorganisms or air contaminated with microbial infections nuclei (Odimayo et al., 2008). Nosocomial infections can cause severe pneumonia, infections of the urinary tract, bloodstream and other parts of the body. The microorganisms implicated can enter the body through wounds, catheters as well as by inhalation (Prescott et al., 1999). 1.2. Air sampling methods At the moment, the only effective means of quantifying airborne microbes is limited to the count of colony forming units (CFU). The CFU count is the most important parameter, as it measures the live micro-organisms which can multiply. Air samples can be collected in two ways: by active air samplers or by passive air sampling (the settle plates). Both methods are widely used (Pasquarella et al., 2000). In active monitoring a microbiological air sampler physically draws a specific volume of air 4 through or over a particle collection device which can be a liquid or a solid culture media or a nitrocellulose membrane and the quantity of microorganisms present is measured in CFU/m 3 of air. This system is applicable when the concentration of microorganisms is not very high, such as in an operating theatre and other hospital controlled environments. There are many different types of active samplers on the market, each based on a different design. Unfortunately, there are many drawbacks that make it difficult to interpret correctly the results obtained by these devices. Different active samplers give different results in the same place at the same time (Pasquarella et al., 2000). Passive monitoring uses “settle plates”, which are standard Petri dishes containing culture media, which are exposed to the air for a given time in order to collect biological particles which “sediment” out and are then incubated. Results are expressed in CFU/plate/time or in CFU/m 2 /hour (Napoli et al., 2012). The main criticism of settle plates is that the measured microbial fallout is not at all or is only weakly correlated with the counts determined by other quantitative methods and with a defined volume of the surrounding atmosphere. The settle plate method is still widely used as a simple and inexpensive way to qualitatively assess the environments over prolonged exposure times. Settle plates are not to be used for quantitative estimations of the microbial contamination levels of critical environments. Settle plates are sterile, economical and readily available. The results obtained by settle plates are reproducible and reliable. Many places in an environment can be checked at the same time. 5 Data collected on settle plates set in different places, by different operators, can be compared and understood (Pasquarella et al., 2000). According to some authors, passive sampling provides a valid risk assessment as it measures the harmful part of the airborne population which falls onto a critical surface, such as in the surgical cut or on the instruments in operating theatres (French et al., 1980). 1.3 Enterobacterial repetitive intergenic consensus (ERIC) PCR Airborne pathogen levels and their role in nosocomial infections will be evaluated by comparing certain bacterial organisms with cultures from hospitalized patients collected at the same time using Enterobacterial repetitive intergenic consensus PCR (ERIC PCR). ERIC PCR is a PCR- fingerprinting technique but it is not arbitrary because the primer was designed to known target sequence. The primers used in ERIC PCR are complementary to repetitive sequence that highly conserved in bacterial genomic DNA. There are few repetitive sequences have been reported in bacterial genome include enterobacterial repetitive intergenic consensus (ERIC) sequences, repetitive extragenic palindromic (REP) sequences, and BOX elements. The enterobacterial repetitive intergenic consensus (ERIC) sequences are present in many copies in the genomes of different enterobacteria such as Escherichia coli, Salmonella typhimurium (Hulton et al., 1991; Zulkifli et al., 2009). These elements are highly conserved at the nucleotide level, 126 bp long, and include a central core inverted repeat. The position of ERIC elements in enterobacterial genomes varies between 6 different species and has been used as a genetic marker to characterize isolates within a bacterial species (Versalovic et al., 1991; Son et al., 2002). In ERIC-PCR a band pattern is obtained by amplification of genomic DNA located between ERIC elements or between ERIC elements and other repetitive DNA sequences. This technique uses consensus primers in the PCR to amplify DNA sequences located between successive repetitive elements such as RRIC sequence for subtyping Gram-negative enteric bacteria (Hulton et al., 1991; Zulkifli et al., 2009) 1.4 Literature review Approximately 10% of the nosocomial infections in both immune- compromised and healthy people are caused by airborne bacteria (Gioffre et al., 2007). It has been suggested that many pathogens can survive as bioaerosol, spread considerable distances, and result in infections (Moletta- Denat et al., 2010). Thus, recognition of microbial profile and control of microbial contamination of hospital air wards has great importance especially for those infections that an airborne transmission is postulated (Perdelli et al., 2006). The quality of indoor air depends on external and internal sources, such as ventilation, cleaning procedures, the surgical team and their activity (Fleischer et al., 2005). In one study, air samples from ten conventionally ventilated operating rooms were taken simultaneously by the sedimentation method and by the air sampler. The most commonly isolated pathogenic species of bacteria were: Staphylococcus aureus (S. aureus), Enterococcus spp., Streptococcus 7 spp., Pseudomonas aeruginosa (P. aeruginosa), Acinetobacter lwoffii and Alcaligenes faecalis. The dominant fungal species were Penicillium spp. and Cladosporium spp (Fleischer et al., 2005). Air samples from seven different operation theatres were processed and the isolates were S. aureus, Coagulase negative Staphylococci (CoNS), Acinetobacter spp. and Klebsiella spp. The dominant bacterial species identified were CoNS (Kaur and Hans, 2007). Javed et al., (2008) showed that S. aureus was isolated from all the air samples obtained from the various operation theatres (OTs) except ENT (ear, nose and throat). Coagulase negative staphylococci were isolated from air samples from all the OT with the lowest prevalence in eye (50%) and urology (48%). Other pathogens were also isolated such as Aspergillus spp., Bacillus spp., and Streptococcus spp. In recent study, higher concentration of microorganisms was detected when medical staff was present in the room and investigation or treatment was carried out. The majority of microbial findings in the air were Gram- positive cocci (CoNS, Micrococcus spp., Sarcina spp.). Findings of Gram- negative stems were sporadic (Pseudomonas aeruginosa) as well as the incidence of microscopic fungi (Cladosporium spp., Penicillium spp.) (Vackova et al. 2011). Male medical ward and male surgical general ward showed the highest bacterial and fungal growth while the operating theatre was almost free of microbial burden. The bacteria isolates were S.aureus, Klebsiella spp., Bacillus cereus (B. cereus), B. subtilis, Streptococcus pyogenes and Serratia marscences while the fungi isolates included Aspergillus flavus, Penicillium spp., Fusarium spp., Candida albicans and 8 Alternaria spp. Staphylococcus aureus was the predominantly isolated bacterium while Penicillium sp. was the most isolated fungus (Awosika et al., 2012). In other study, the microbial profile of air samples showed that P. aeruginosa was the predominantly isolated bacteria from thoracic surgery ward; S. epidermidis from bone marrow transplantation ward and neonatal ward; Enterococcus from ICU and Acinetobacter from operating room. Other microorganisms were also isolated from these wards such as Proteus, Stenotrophomonas maltophilia, Enterobacter, S. aureus, Streptococcus group D, E. coli, Klebsiella and Candida albicans. Cladosporium was the most frequent fungi found (Abdollahi and Mahmoudzadeh, 2012). Furthermore, Qudiesat et al. (2009) noted that, from their studies in two selected hospitals (a private and a public) in Jordan, the air quality in terms of biological contamination in the governmental hospital was worse than that of the private hospital in all units. In both hospitals, S. aureus, Micrococcus luteus and CoNS were among the most common bacteria identified whereas fungal species Aspergillus spp., Penicillium spp., Rhizopus spp. and Alternaria spp. were isolated in both hospitals. Recently, in cross sectional research from 30 wards in five educational hospitals, the highest fungal populations were Penicelium spp. (32.06%), Cladosporium spp. (20.5%), Aspergillus fumigatus (14.61%) and A. niger (7.43%), respectively. The highest bacterial population was coagulase- negative staphylococci (32.49%), Bacillus spp. (14.74%), Micrococcus spp. 9 (13.68%) and Staphylococcus aureus (11.34%), respectively (Hoseinzadeh et al., (2013). It was reported that the highest bacterial population was recorded in the evening between time 5pm and 6pm compared to the morning and afternoon, ranging from 15cfu/m 3 to 47cfu/m 3 in the Faith Medical Hospital and 17cfu/m 3 to 52cfu/m 3 in the Central Hospital, with the children ward recording the highest bacterial counts of 47cfu/m 3 and 52cfu/m 3 in the Faith Medical Center and Central Hospital, respectively. The concentration of fungal population in air of the five different wards in the two hospitals studied was recorded high in the evening, with values ranging from10 cfu/m 3 to 53 cfu/m 3 . Six bacterial species and four fungal genera were identified, among which the bacterial isolates were: S.aureus, S. epidermidis, E. coli, P. aeruginosa, Proteus mirabilis and Klebsiella aerogenes and the fungal isolates included Aspergillus, Penicillum, Mucor,and Fusarium. The degree of frequency of microbial distribution was high in the bacteriological laboratory and female ward and lowest in the operating room (Ekhaise et al., 2008). It was reported from autopsy room air 14 bacterial and 26 fungal species were cultured. Most frequently isolated bacteria were CoNS, Micrococcus spp., Bacillus spp., and diphtheroid bacillus for the Gram- positive, and Acinetobacter spp., Proteus mirabilis, and E. coli for the Gram-negative groups. Most frequently isolated fungi were Penicillium spp., Alternaria spp., and Aspergillus flavus (Sonmez et al., 2011). 10 1.5 Aims of the study In Palestinian hospitals there are no previous studies on the prevalence airborne microorganisms. The present study aimed to assess microbial profile of hospital air contamination in different wards of Rafidia Hospital- City of Nablus using both active and passive sampling methods and to further assess the correlation between the results of the different sampling methods. In addition, to ascertain the role of some of these airborne microorganisms in nosocomial infections in cultures collected from hospitalized patients at the same time using ERIC PCR technique. 11 Chapter Two Materials and Methods 2.1 Study area and site of samples analysis Hospital air samples were collected between September and October 2014. The collected samples were cultured, identified and analyzed in Department of Biology and biotechnology, Science College, An-Najah National University, Nablus-Palestine. 2.2 Media preparation 2.2.1Tryptic Soy Agar (TSA) Tryptic Soy Agar (Acumadia,USA) was prepared according to manufacturer's instructions. In a 1L bottle, 500 ml of deionized water was mixed with 20g TSA, heated and stirred until the agar dissolved. The solution was then autoclaved at 121°C for 15 minutes and allowed to cool to about 45 °C to 50 °C. A 20-25 ml agar was then poured into Petri dishes and the dishes were covered and left overnight at room temperature. After that, Petri dishes were stored at 4°C. 2.2.2 Mannitol salt agar (MSA) BBL TM Mannitol agar (BD,USA) was prepared according to the manufacturer's instructions. In a 1 L bottle, 500 ml of deionized water was mixed with 55.5g MSA, heated and stirred until the agar dissolved. The solution then autoclaved at 121°C for 15 minutes and allowed to cool to 12 about 45 °C to 50 °C. A 20-25 ml agar was then poured into Petri dishes and the dishes were covered and left overnight at room temperature. After that, Petri dishes were stored at 4°C. 2.2.3 Meullar Hinton agar (MHA) Meullar agar (BD,USA) was prepared according to manufacturer's instructions. In a 1 L bottle, 500 ml of deionized water was mixed with 19g MHA and 10g NaCl, heated and stirred until the agar dissolved. The solution then autoclaved at 121°C for 15 minutes and allowed to cool to about 45 °C to 50 °C. A 20-25 ml agar was then poured into Petri dishes and the dishes were covered and left overnight at room temperature. After that, Petri dishes were stored at 4°C. 2.2.4 MacConkey Agar: MacConkey agar (HIMEDIA,India) was prepared according to the manufacturer's instructions. In a 2 L bottle, 1 L of deionized water was mixed with 49.53g MacConkey, heated and stirred until the agar dissolved. The solution then autoclaved at 121°C for 15 minutes and allowed to cool to about 45 °C to 50 °C. A 20-25 ml agar was then poured into Petri dishes and the dishes were covered and left overnight at room temperature. After that, Petri dishes were stored at 4°C. 13 2.2.5 Eosin Methylene Blue (EMB) Agar EMB medium (HIMEDIA,India) was prepared according to the manufacturer's instructions. In a 2 L bottle, 1 L of deionized water was mixed with 35.96g of EMB agar, heated and stirred until the agar dissolved. The solution then autoclaved at 121°C for 15 minutes and allowed to cool to about 45°C to 50°C. A 20-25 ml agar was then poured into Petri dishes and the dishes were covered and left overnight at room temperature. After that, Petri dishes were stored at 4°C. 2.2.6 Blood agar A 1L bottle containing deionized water (475mL) and 11.6 g Nutrient Agar were heated and stirred until the agar dissolved. Then, the solution was autoclaved at 121°C for 15 minutes and allowed to cool to about 45°C to 50°C. After that, 25 ml of sterile defibrinated blood was added aseptically and mixed thoroughly. A 20-25 ml agar was then poured into Petri dishes and the dishes were covered and left overnight at room temperature. After that, Petri dishes were stored at 4°C. 2.2.7 Simmons Citrate Agar Simmons citrate agar (HIMEDIA, India) was prepared according to the manufacturer's instructions. In a 2 L bottle, 1 L of deionized water was mixed with 24.28 g of Simmons citrate, heated and stirred until the agar dissolved. Ten ml of Simmons citrate agar was dispensed into tubes, 14 autoclaved at 121°C for 15 min. The medium was prepared as slant agar tubes and then stored at 4°C. 2.2.8 Triple Sugar Iron (TSI) Agar TSI agar (Acumedia, USA) was prepared according to the manufacturer's instructions. In a 2L bottle, 1000ml deionized water was mixed thoroughly with 60 g of Triple sugar iron agar, heated to dissolve the agar. Ten ml of Triple sugar Iron medium was dispensed into tubes, autoclaved at 121°C for 15min. The medium was prepared as slant agar tubes and then stored at 4°C. 2.2.9 Sulfied Indole Motility (SIM) Medium SIM medium (Acumedia, USA) was prepared according to the manufacturer's instructions. In a 1L bottle, 500ml deionized water was mixed with 15g of SIM agar, heated and stirred until agar dissolved. Medium was dispensed into tubes to a give depth of about 4-5 cm. Then the medium was autoclaved at 121°C for 15min, allowed to cool and then stored at 4°C. 2.3 Identification of microorganisms 2.3.1 Gram staining Gram staining of bacteria was performed as described previously (Cappiccino and Sherman, 1996). 15 2.3.2 Mannitol fermentation Aseptically, a single line of inoculation of test organism was cultured on MSA plate. The plates were then incubated for 24hours at 37ºC (Cappiccino and Sherman, 1996). 2.3.3 Catalase test Catalase test was carried out by the addition of 40μl of 3% hydrogen peroxide (3 ml of 30% stock hydrogen peroxide concentration with 97 ml sterile water) on bacterial colony cultured on NA or on slide (Cappiccino and Sherman 1996). 2.3.4 Slide coagulase test One Staphylococcal colony from NA and 100μl of diluted citrated plasma (1 ml citrated plasma with 3 ml sterile normal saline) were mixed on a slide. Agglutination or clumping of cocci within 1minute was considered as positive (Cappiccino and Sherman 1996). Negative samples were further tested by tube coagulase test. 2.3.5 Tube coagulase test This test was done by inoculating 1ml of diluted (1:4) fresh citrated human plasma with a catalase positive colony from NA in a tube. The tube was then incubated for 18-24 hours at 37ºC and inspected from time to time for the presence of clumping or agglutination (Cappiccino and Sherman 1996). 16 2.3.6 Citrate utilization test Citrate utilization test was carried out by inoculation of Simmons citrate agar by means of stab-and streak (Cappiccino and Sherman 1996). 2.3.7 TSI test TSI test was carried out by inoculation the TSI agar slants by means of stab-and-streak (Cappiccino and Sherman 1996). 2.3.8 Motility test A well-isolated colony was picked with a sterile needle, and then the SIM medium was stabbed to within 1-2cm of the bottom of the tube. The needle was kept in the same line it entered as it is removed from the medium. The tube was incubated at 35°C for 18-24h (Cappiccino and Sherman 1996). 2.4 Air sampling 2.4.1 Active air sampling Air was sampled using Air Sampler based on manufacturer instructions. Air was blown on a Tryptic Soy Agar plate as a standard growth medium for bacteria. The flow rate was calibrated at 11.0 L/min and samples of 50 L were collected. Active air sampling was repeated twice in each location and was carried out 2 times weekly for 3 weeks. Microbial air pollution was evaluated according to the recommendation published in AHEM (Acta Hygienica, Epidemiologica et Microbiologica) 17 No. 1/2002, State Health Institute, Prague (Vackova et al., 2011). The evaluation was carried out in one of the five categories as shown in Table 2.1. Table 2.1 Categories of microbial indoor air contamination–a concentration criterion of mixed population of bacteria and fungi Microbial contamination Bacteria (CFU/ m 3 ) Fungi (CFU/ m 3 ) very low < 50 < 25 Low < 100 < 100 Middle < 500 < 500 High < 2000 < 2000 Very High >2000 >2000 2.4.2 Passive air sampling (sedimentation technique) Sedimentation technique was carried out as described previously (Awosika et al., 2012). This technique was done using open Petri dishes containing different culture media. Duplicate set of plates of each medium (Tryptic Soy Agar, blood agar, MacConkey agar and Mannitol salt agar) were distributed at different sites of wards/units examined. The plates were labeled with sample number, site within the ward and date of sample collection. The plates were placed at 2 chosen sites in the concerned wards at about 1 meter above the ground level. The samplings were done at the morning hours (8.00–12.00 am) two times weekly for 3 weeks. All samples were collected withclosed windows and doors. Samples from operating 18 room were carried out at different critical sites (near the surgeon, near the surgical instruments) and were left open to the air for one hour during operation times. The plates were covered and transferred immediately to the Microbiology Laboratory- An-Najah N. University for incubation. Plates were incubated at 37 °C for 48 hours; the total numbers of colony forming units (CFU) were enumerated. The identification of the isolates was carried out according to standard procedures. 2.5 Bacterial Identification Media used by passive and active air sampling were transferred to the laboratory and kept in incubators for 48h at 37 o C. The number of CFU/m 2 was then calculated.. Bacterial colonies were characterized by cultural, morphological and microscopic examination, and further identification was carried out by biochemical tests including: catalase activity, lactose fermentation, mannitol fermentation, blood haemolysis, coagulase test, motility test, TSIand citrate utilization test. Colonies of fungal growth were identified based on colony appearance and microscopic examination. 2.6 Antibiotic susceptibility test Antimicrobial susceptibility for some bacterial strains was determined according to the Clinical and Laboratory Standard Institute (CLSI) using the disk diffusion method (CLSI, 2011). Some bacterial isolates was examined for resistance using the following antibiotic disks (Oxoid): ciprofloxacin (5 µg), norfloxacin (10µg), Trimethoprim/ Sulfamethoxazole 19 (1.25/23.75µg), tetracycline (30µg), Cefotaxime (30µg), Oxacillin (1µg), ceftriaxone (30µg), Aztreonam (30µg) and nalidixic acid (30µg). All chemicals or powders /discs were purchased from USA. Zones of inhibition were determined in accordance with procedures of the Clinical and Laboratory Standard Institute (CLSI, 2011). 2.7 DNA extraction and ERIC PCR 2.7.1. DNA Extraction Total genomic bacterial DNA for certain airborne isolated strains and fromclinical samples collected from inpatients by hospital lab was extracted for PCR as previously described by Adwan et al., (2013). Briefly, a loop full of bacterial cells were scraped off an overnight nutrient agar plates, washed twice with 1 ml of 1X Tris-EDTA buffer (10 mM Tris- HCl, 1 mM EDTA, pH 8). The pellets were then re-suspended in 500μl of sterile distilled water and boiled for 10-15 min. After that, the suspension was incubated on ice for 5-10 min. Debris were pelleted by centrifugation at 11,500 X g for 5 min. DNA concentration was determined using spectrophotometer and the DNA samples stored at -20ºC until further use for ERIC PCR analysis. 2.7.2 ERIC PCR ERIC (Enterobacterial repetitive intergenic consensus) PCR was performed using Primer ERIC1: 5`-ATG TAA GCT CCT GGG GAT TCA C-3 and Primer ERIC2: 5-AAG TAA GTG ACT GGG GTG AGC G-3. Each PCR 20 reaction mix (25μL) was carried out using 12.5μL of PCR premix with MgCl2 (ReadyMix TM Taq PCR Reaction Mix with MgCl2, Sigma), 1μM of each primer, 3μL DNA template. In addition, the master mix was modified by increasing the concentration of dNTPs to 400μM, MgCl2 to 3mM and Taq DNA polymerase to 2U. DNA amplification was carried out using the thermal cycler (Mastercycler personal, Eppendorf, Germany) according to the following thermal conditions: initial denaturation for 2 min at 94ºC will be followed by 30 cycles of initial denaturation 94ºC for 60 s, 40ºC for 120s and 72ºC for 90s, with a final extension step at 72ºC for 5 min. The PCR products were analyzed by gel electrophoresis on 1.7% agarose gel, after that, the gel was stained with ethidium bromide (0.5μg/ml), and then the gel was photographed for further analysis. Fingerprints were compared visually. 2.8 Statistical analysis Generated data was analyzed by simple mean value and percentages.T-test to differentiate between two sampling methods ( A p value of < 0.05 was considered to be dependent) 21 Chapter Three Results Microbial profiles of simultaneous cultures obtained from hospital air samples by active and passive sampling in different wards are presented in Tables 1 and 2. The results indicated that all air samples collected from surgical operation rooms, intensive care unit and neonatal room were contaminated to some extent with different types of microorganisms. The total viable count of Gram-positive bacteria was the most frequent microorganisms cultured from different wards. It was also found that CoNS and Micrococcus spp, were the most predominant among isolated bacteria from air samples collected from various wards by passive air sampling method. The percentage of CoNS and Micrococcus spp in air of surgical operation rooms, intensive care unit and neonatal room by passive air sampling has ranged from 61.8%-100% and the average was 5158 CFU/ m 2 /h-20187 CFU/ m 2 /h (Table 1). Staphylococcus aureus was the most common microorganisms isolated from neonatal room by active air sampling method, the percentage was 35% and the average was 100 CFU/ m 3 . Total bacterial level in these rooms had a range 116CFU/ m 3 - 1085CFU/m 3. The percentage of CoNS and Micrococcus spp in air of surgical operation rooms, intensive care unit by active air sampling was 58.8%-100% and the average was 70-1080 CFU/m 3 (Table 2). The findings of this research showed that most frequent Colony Forming Units were obtained from Blood agar with a range of 4085 CFU/ m 2 /h - 22 8721 CFU/ m 2 /h and Tryptic Soy Agar with a range of 2043 CFU/ m 2 /h- 7935 CFU/ m 2 /h by passive air sampling method (Table 3). The antimicrobial susceptibility pattern of bacteria isolates revealed that the most effective antibiotics were ciprofloxacin , norfloxacin and tetracycline against S. aureus; tetracycline, ciprofloxacin and norfloxacin against CoNS and Micrococcus spp and ciprofloxacin, Trimethoprim/Sulfamethoxazole and tetracycline against Bacillus spp. The antibiotic resistance profile of the different microorganisms isolated from hospital air in surgical operation rooms, intensive care unit and neonatal room by active and passive air sampling is presented in Table 4. Results in table 5 indicate the presence of statistically significant difference at (α= 0.05) between the two sampling methods in favor of passive sampling method. ERIC PCR profile revealed that clinical bacterial strains S. aureus, E. coli and Klebsiella spp. and those isolated from air samples collected at the same time were not clonally related (Figures 1 and 2). 23 Table 3.1: Microbial air load and spectrum of microbial findings in air samples collected from various hospital wards by passive air sampling. Room Average (range) of CFU/m 2 % S. aureus CoNS and Micrococcus spp. Bacillus spp. Corynobacteri um spp. E. coli Klebsiella spp. Fungi and yeast SOR1 668 (0-1335) 6.8% 9152 (5655-12647) 93.2% 0 0.0% 0 0.0% 0 0.0% 0 0.0% 0 0.0% SOR2 0 0.0% 17753 100% 0 0.0% 0 0.0% 0 0.0% 0 0.0% 0 0.0% SOR3 1964 (0-5892) 23.5% 5158 (9898-18381) 61.8% 26 (0-79) 0.3% 53 (0-178) 0.6% 0 0.0% 0 0.0% 1152 (786-1335) 13.8% SOR4 0 0.0% 11076 86.5% 0 0.0% 0 0.0% 79 0.6% 0 0.0% 1650 12.9% SOR5 4438 (2121-6756) 17.7% 20187 (17593-22781) 80.4% 118 (79-157) 0.5% 0 0.0% 0 0.0% 0 0.0% 354 (79-628) 1.4% SOR6 864 (0-1728) 8.6% 8916 (4713-13119) 89.4% 39 (0-79) 0.4% 0 0.0% 0 0.0% 0 0.0% 157 (97-236) 1.6% ICU 1126 (0-2514) 7.9% 11076 (6520-15004) 78.8% 79 (0-236) 0.6% 681 (0-1728) 4.8% 0 0.0% 26(0-79) 0.2% 1047 (0-2749) 7.3% NR 1453 (550-2357) 15.4% 6913 (5185-7934) 73.2% 79 (0-157) 0.8% 20 (0-79) 0.2% 20 (0-79) 0.2% 0 0.0% 962 (0-2749) 10.2% SOR: Surgical operation room; ICU: Intensive care Unit; NR: Neonatal Room; CFU: Colony Forming Unit 24 Table 3. 2: Microbial air load and spectrum of microbial findings in air samples collected from various hospital wards by active air sampling. Room Average (range) of CFU/m 3 % Total bacterial CFU/ m 3 S. aureus CoNS and/or Micrococcus spp. Bacillus spp. Corynobacteria spp. Fungi and yeast SOR1 570 0 0.0% 570 (50-1090) 100% 0 0.0% 0 0.0% 0 0.0% SOR2 250 0 0.0% 250 100% 0 0.0% 0 0.0% 0 0.0% SOR3 116 3 (0-10) 2.3% 103 (20-200) 79.8% 10 (0-20) 7.6% 0 13 (0-40) 10.1% SOR4 100 30 30% 70 70% 0 0.0% 0 0.0% 0 0.0% SOR5 1085 0 0.0% 1080 (660-1500) 92.3% 0 0.0% 5 (0-10) 0.43% 85 (30-140) 7.3% SOR6 100 0 0.0% 100 (0-200) 58.8% 0 0.0% 0 0.0% 70 (0-140) 41.2% ICU 533 3 (0-10) 0.6% 523 (10-1000) 97.6% 7 (0-10) 1.3% 0 0.0% 3 (0-10) 0.6% NR 191 100 (0-400) 35% 83 (0-140) 29% 5 (0-20) 1.7% 3 (0-10) 1% 95 (0-380) 33.3% SOR: Surgical operation room; ICU: Intensive care Unit; NR: Neonatal Room; CFU: Colony Forming Unit; CNS: Coagulase Nagative Staphylococci 25 Table 3.3: Colony Forming Unit of air collected from various hospital wards by active and passive air sampling according to the type of medium. Room passive air sampling CFU/m 2 /h Active air sampling CFU/m 3 Blood Agar Mannitol Salt Agar Tryptic Soy Agar Macconkey agar Tryptic Soy Agar SOR1 5893 1886 2043 0 570 SOR2 6599 4713 6441 0 250 SOR3 7962 4924 6129 52 108 SOR4 5892 3142 3771 0 100 SOR5 8721 8446 7935 0 947 SOR6 4675 2200 3496 0 160 ICU 4793 3981 4425 52 444 NR 4085 1925 3398 39 200 SOR: Surgical operation room; ICU: Intensive care Unit; NR: Neonatal Room; CFU: Colony Forming Unit. 26 Table 3. 4: Antibiotic resistance of bacterial isolates recovered from air from various hospital wards by active and passive air sampling Antibiotics S. aureus n =20 CoNS and Micrococcus spp n =17 Bacillus spp. n =10 E. coli n =2 Klebsiella spp. n =1 Corynobacteria spp. n =1 Ciprofloxacin 35% 17.6% 0% 50% 0% 0% Norfloxacin 35% 23.5% NT NT NT NT Trimethoprim/ Sulfamethoxazole 65% 47% 0% 0% 0% 100% Tetracycline 10% 5.9% 10% 0% 0% 0% Cefotaxime 75% 52.9% 60% 0% 0% 100% Oxacillin 70% 94.1% NT NT NT NT Ceftriaxone NT NT 60% 100% 0% 0% Aztreonam NT NT 100% 0% 0% 100% Nalidixic acid NT NT 70% 0% 0% 100% ND: Not tested Table 3.5: T test to differentiate between active and passive sampling methods. Sig. df t n s.d mean *0.000 62 12.85 32 6017.46 14071.9 Passive 32 399.85 367.9 active *significant at 0.05 level 27 Figure1. DNA fingerprints generated by ERIC PCR analysis of 10 bacterial isolates (S. aureus and CNS and/or Micrococcus spp.) recovered from clinical and air samples on 1.5% agarose gel. L: 100 bp ladder; lanes 1 and 9 referring to CNS and/or Micrococcus spp. isolated from air samples; lanes 2-8 and 10 referring to S. aureus isolated from air samples and lanes 10-14 referring to S. aureus isolated from clinical samples. Figure 2. DNA fingerprints generated by ERIC PCR analysis of 10 bacterial isolates (E. coli and Klebsiella spp.) recovered from clinical and air samples on 1.5% agarose gel. L: 100 bp ladder; lanes 1 and 2 referring to E. coli isolated from air; lanes 3-7 referring to E. coli isolated from clinical samples; lane 8 referring to Klebsiella spp. isolated from air sample and lanes 9 and 10 referring to Klebsiella spp. isolated from clinical samples. 28 Chapter Four Discussion Air can play a vital role as a reservoir for both pathogenic and non pathogenic living microorganisms. Microbial contamination of air hospitals is considered as a source of hospital-associated infections (Borghesi et al., 2008). These infections may pose high financial load on governments and healthcare systems and may cause considerable morbidity and mortality rate among patients admitted for post-operative surgery, patients in intensive care units with multi-drug resistant strains like methicillin- resistant S. aureus (MRSA) show difficult control of these organism (Napoli et al., 2012). Microbiological contamination of air in the surgical operation rooms is considered to be one of the most important high risk factor for infections of surgical site in clean surgery (Pasquarella et al., 2004). Therefore, recognition, monitoring and control of air microbial contamination of hospital wards is very necessary especially for airborne pathogens postulated to cause hospital-associated infections (Perdelli et al., 2006), and can be done routinely by microbiological sampling and particle counting (Javed et al., 2008). It could be hypothesized that decreasing the load of these airborne pathogens in the hospital parts could help lower the risk of infection (Guriz et al., 2008). There are many factors that play a role to keep the lowest possible airborne microorganism levels in hospital parts. Airborne pathogen levels in hospital parts affected by weather conditions, season, the outdoor microbial load and the number of occupants and visitors, surgical team and their activities, indoor ventilation system 29 design, cleaning procedures and relative humidity (Sonmez et al., 2011; Park et al., 2013). Although airborne microorganisms encountered in hospital wards are apparently non pathogenic to healthy people, but they can cause adverse health problems in immune-compromised people. Approximately, it is up to 10% of the hospital acquired infections in both healthy and immunecompromised patients are determined by airborne bacteria (Gioffre et al., 2007). There are 2 most common techniques used for air sampling, the active method carried out by using air sampler and the passive method performed by using settle plate technique. However, there is no accepted opinion about which method is more suitable (Sonmez et al., 2011). It was reported that the level of contamination in the air is directly proportional with the number of colonies isolated by passive method (Górny and Dutkiewicz, 2002). The advantages of passive method over active method were reported as cheap, easy to perform, available everywhere, ability to detect and measure harmful part of airborne contamination, many samples can be taken at the same time from different locations, significant outcomes concerning critical surfaces, comparable and usually reliable outcomes, the airflow is not disturbed, and microorganism growth under the natural conditions. The disadvantages of this method can be listed as unknown volume of the sampled air, long sampling time, insufficient for fungal spore evaluation, requirement of large particles, and it is not always accepted by the official guidelines (Pasquarella et al., 2000; Sonmez et al., 2011). Several studies have been done to compare between the values of microbial 30 loads obtained by both active and passive methods, in some cases there was no correlation (Petti et al., 2003), while in others there was a significant correlation (Perdelli et al., 2000). Due to air sampling protocols are not consolidated, it is not easy to compare results from different studies (Pasquarella et al., 2008). Both active and passive methods can be used for monitoring and controlling of airborne contamination for a routine surveillance programs in hospitals and medical centers (Napoli et al., 2012). In particular, passive sampling is better to use than active sampling if the air sampling performed to monitor the risk of microbial wound contamination during surgery. On the contrary, the active method should be preferred if the sampling is achieved to assess and evaluate the load of all inhalable viable microorganisms (Napoli et al., 2012). The load and type of airborne microorganisms in hospitals or their parts can be used to determine the degree of cleanliness. Results from this study showed that indoor hospital air has a low or middle microbial load based on the recommendation published in AHEM (Acta Hygienica, Epidemiologica et Microbiologica) No. 1/2002, State Health Institute, Prague (Vackova et al., 2011). Results showed that 3 of rooms SOR1and SOR5 (during an operation) and ICU had a middle bacterial contamination and total bacterial level ranged 533 CFU/ m 3 -1085 CFU/ m 3 , while other rooms were of low bacterial contamination and the total bacterial level ranged 116 CFU/ m 3 - 250 CFU/ m 3 . At the same time detection of fungi and yeast in these rooms had a low fungal contamination, based onthe recommendation published in AHEM. The total fungal level ranged from 0 CFU/ m 3 95 CFU/ m 3 . It is 31 recommended that for conventional operation rooms the microbiological concentration should not be greater than 35 CFU/m 3 in an empty room or 180 CFU/m 3 during an operation. It is also mentioned that for ultra-clean operation rooms the microbiological concentration should be less than 1.0 CFU/m 3 in the middle of an empty room and less than 10 CFU/m 3 during an operation and should not be greater than 20 CFU/m 3 at the periphery. Results showed that the microorganisms load from one ward to another in the same hospital can vary (Abdolahi et al., 2009). However, the airborne bacterial load in a modern ventilated operation room should not exceed 30 cfu/ m 3 (Javed et al., 2008). A number of studies have been done in operation rooms to determine relation between total airborne bacterial load in operation rooms and risk of infection. It has been observed that loads in the range of 700-1800/m 3 were significantly associated to risk of infection and the risk was slight when the loads were less than 180/m 3 (Parker, 1978). Results of this study could be used to learn the lesson that such microorganisms levels in different hospital wards could be inappropriate; a suitable actions should be taken in order to lower the contamination level and to protect the susceptible people who generally use hospital wards. Indoor air of hospitals has a wide range of infectious microorganisms (Ekhaise et al., 2008; Vackova et al., 2011; Sonmez et al., 2011; Awosika et al., 2012; Hoseinzadeh et al., 2013; Gebremariam et al., 2015). Results of this report were consistent with previous reports (Kaur and Hans, 2007; Javed et al., 2008; Qudiesat et al. 2009; Vackova et al., 2011; Huseinzadeh et al., 2013; Gebremariam et al., 2015), which showed that the majority of 32 bacterial findings in the indoor air were Gram-positive bacteria, Gram- negative bacteria were sporadic as well as incidence of microscopic fungi and yeast. This may be explained that patients, medical staff, seeing of the sick and all the activities in hospital environments as well as surgical operation rooms, intensive care units and neonatal rooms is the main source of the most bacterial organisms (Beegs, 2003; Chow and Yang, 2005; Vackova et al., 2011; Park et al., 2013). Coagulase-negative staphylococci are opportunistic pathogens which could cause infection in immunocompromised patients. Gram-positive bacteria can survive for long time in the form of aerosol than Gram-negative bacteria. This may be due to differences in cell wall structure. Detection of some kinds of microscopic fungi and yeasts in the indoor air where immunosuppressive patients are treated can be a serious risk factor for the incidence of infectious complication (Soubani et al., 2004; Vandewoude et al., 2004). In addition, attention to fungal spores presence in hospital air is very important, allergic reactions have been recorded following inhalation of these spores (Omoigberale et al., 2014). Humidity and temperature significantly affected fungi loads in air of these rooms (Park et al., 2013). Staphylococcus aureus is known to be easily harbored in many sites included the throat, nasopharynx, boils, skin, nails and cuts. This microorganism can contribute to the normal microbial flora in the hospital environment causing several infections such as respiratory tract infections, bed sore, post-operative infections and food poisoning under favorable conditions. 33 Results of this study was in agreement with previous report (Qudiesat et al. 2009), which showed that, results from 2 hospitals (a private and a public) in Jordan, S. aureus, Micrococcus and CoNS were among the most common bacteria identified, whereas fungal species were isolated in both private and a public hospitals. Recently, in cross sectional research from 30 wards in five educational hospitals, it was reported that the highest bacterial population was CoNS, Bacillus spp., Micrococcus spp. and S. aureus, respectively (Hoseinzadeh et al., (2013). Staphylococcu aureus, S. epidermidis, Micrococus sp and Bacillus sp. were the most frequently occurring airborne bacterial isolates in the two hospitals in Benin City, Nigeria (Ekhaise and Ogboghodo, 2011). Singh et al., 2013 recorded high air contamination in general surgery ward of CoNS 100%, S. aureus 66.66% and Bacillus spp. 50%, whereas Gynecology ward contain the high contamination of CoNS spp (100%) and S. aureus (50%) with least concentration (33.3%) of Bacillus spp. These results were consistent with results of this research, which showed that Gram-positive bacteria were the predominant contaminants in these hospital wards. In other study, CoNS, Micrococcus spp., Bacillus spp., and diphtheroid bacillus were the most frequently isolated microorganisms from autopsy room, respectively, and Gram-positive. Acinetobacter spp., Proteus mirabilis, and E. coli were the most frequently isolated microorganisms.For the Gram-negative groups (Sonmez et al., 2011). Bacillus species commonly exist in the air, in the soil, in dusty environments and also existing as normal intestinal flora in human and animals. Microorganisms associated to this genus are spore 34 forming bacteria that can survive for long periods in the environment. Bacillus species other than B. anthracis, could not cause medical problems except for those suffering from immune deficiency. Diphteroid bacillus as well Corynebacterium spp. may exist in the soil, in the air, in the skin and in mucous membranes. However, these species, except Corynebacterium diphtheriae, are considered as non pathogenic microorganisms for the patients except for those suffering from immune deficiency (Sonmez et al., 2011). In the present study, isolated Gram- negative bacteria; E. coli and Klebsiella spp by passive air sampling showed in very low load count. These results were in agreement with recent report (Park et al., 2013), which showed very low level of Gram-negative bacteria (17 CFU/ m 3 ) in hospital lobbies. This may be explained that these bacteria are susceptible for dryness and they are not expected to exist in the air (Sonmez et al., 2011). Klebsiella spp and Escherichia coli are associated with urinary tract infections among catheterized patients. Antibacteria resistance results in increased illness, death cases and health care costs. Hospitalized patients requiring intensive care and extended treatments are at increased risk hazardous exposure to bacterial air contamination. This risk of health setting infection is increased rapidly by the increasing prevalence of antibiotic-resistant microorganisms and multi- drug resistant (MDR) pathogens such as MRSA (Huang et al., 2013). This study paid particular attention to the presence of drug-resistant species in MRSA and CoNS. In general staphylococcal isolates in this study showed high level of resistance, particularly to Oxacillin. This may indicate that 35 CoNS could be a natural reservoir for disseminating antibiotic resistance genes including methicillin resistant genes into community (Pamuk et al., 2010). ERIC PCR profile revealed that clinical bacterial strains S. aureus, E. coli and Klebsiella spp and airborne strains collected simultaneously were not clonally related. This might be due to that the short period of the study and not covered all parts of the hospital. A number of studies have indicated that biological indoor air pollutants pose potential hazards to patients, medical staffs, and visitors in hospitals (Javed et al., 2008; Ortiz et al., 2009; Wan et al., 2011; Napoli et al., 2012; Wirtanen et al., 2012; Huang et al., 2013; Omoigberale et al., 2014). Strategies can be adapted to decrease spreading of microbial contaminants. 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