Bacteriological Assessment of Hospital Air and Selected Surfaces in Three Referral Hospitals in Makurdi Middle Belt Nigeria

Bacteriological Assessment of Hospital Air and Selected Surfaces in ABSTRACT Hospital environment needs more attention because of the influx of people into the environment for medical checkup and other services. This study unveils the incidence of bacteria isolated from air and selected surfaces in three referral hospitals (City hospital, Federal Medical Centre, Wadata and Saint Theresa Hospital) in Makurdi Middle Belt Nigeria. Fifty (50) bacteria were isolated; hospital air (26%), bed rails (16%), door knobs (2%), floor (4%), nurse table top (10%), operation table (4%), sink (18%), stretchers (2%) and toilet seat (12%). In City Hospital, the bacteria isolated include Escherichia coli (6%), Pseudomonas aeruginosa (2%), Staphylococcus aureus and Staphylococcus spp (2%), from Federal Medical Centre, bacteria isolated were Klebsiella spp (10%), Staphylococcus aureus (4%), Staphylococcus spp (8%), Pseudomonas aeruginosa (12%) and E. coli (6%). In STH, bacteria isolated include E. coli (8%), Pseudomonas aeruginosa (4%), Staphylococcus spp (8%) and Staphylococcus aureus (14%). The invitro antibiotics susceptibility pattern shows that Pseudomonas aeruginosa showed highest resistant to the antibiotics while Klebsiella spp was susceptible to majority of the antibiotics but resistant to cloxacillin and erythromycin. The study reveals Klebsiella spp, Escherichia coli, Pseudomonas aeruginosa, S. aureus and other Staphylococcus spp as bacteria commonly associated with hospital environment. This study affirms the presence of resistant bacteria strains and highlighted world-wide problem of hospital borne infections as it concerns the study area and population. This report will create awareness and be a good guide to health care workers, patients and the public about the likeliness of contracting nosocomial infection and how to treat such infection. Major recommendations offered suggests that, healthcare workers should be more careful in carrying out their duty to avoid chance of being infected in the course of their work. Also, the in-vitro antibiotics susceptibility testing on the bacterial pathogens in the study will assist the clinicians in making improvement on the management of nosocomial infections.


INTRODUCTION
The microbes operate in different ecosystem because in their world they are exceedingly diverse in their appearance, metabolism, physiology, and genetics. The incredible biodiversity bacteria range from extreme environment in water even unto the atmosphere in air. This variety of species is as well present in a particular environment like hospitals air and some surfaces in hospitals (Prescott, Harley, and Klein, 2008).
Hospital environments are places that need more attention because of the influx of people coming for medical checkup and other services in the environment. Patients and hospital staff get in contact with this environment every day and some of these patients may have little or no knowledge about the bacteriology of the hospital air and various surfaces that are potential source of infections. Coronavirus disease  pandemic has provided a lot of information about air borne infection, there is need to also shed light on bacteria that are capable of causing infection from hospital premises also known as nosocomial infection. These infections often manifested within forty-eight hours after a patient is being admitted or thirty days after being discharge from the hospital (Anusree et al., 2015;WHO, 2002). Therefore, bacteria from air can cause infection on patients with compromised immunity, especially when the patients come close a very close range (that is, neglecting physical distancing) to another individual who is a carrier of bacteria that are potential hospital acquired pathogens. So, exchange of gas due to respiration during conversation or exchange of pleasantry will pave way to inhale potential pathogen breathe out by another person at closed range.
Therefore, there is likeliness of contracting nosocomial infection by patient and, or healthcare staff from the hospital environment which is a major route of nosocomial bacterial pathogens (Tambekar, Gulhane, and Bhokare, 2007). At Kenyatta National Hospital, Kenya, P. aeruginosa, Klebsiella, Citrobacter, S. aureus, S. pneumoniae, Acinetobacter and E. coli were isolated from tracheal aspirate, urine, blood and pus swabs from the intensive care unit (Peter et al., 2017). Some researchers have highlighted a host of nosocomial bacteria which include Streptococcus spp, Acinetobacter spp, Enterococci, Pseudomonas aeruginosa, coagulase-negative Staphylococcus, Staphylococcus aureus, Bacillus cereus, Legionella, Proteus mirablis, Klebsiella pneumonia, E. coli, and Serratia marcescens (Bereket et al., 2012;Drach, 2005;Hassan, Aftab, and Riffat, 2015;Stamm, 2001). Even though, efforts are made in Nigeria through awareness of the general public on airborne infection of COVID -19 which is a viral disease, but there is little awareness on airborne infection caused by bacteria. Hence, the microbiological quality assessment of indoor air is one of the most vital investigations to determine the microbial indoor air pollution. The information on the indoor microbial concentrations of airborne bacteria is necessary both to estimate the health hazard and to create standards for indoor air-quality control (Arzu et al., 2019). Fu and Wang (2016) reported nosocomial infection control in health care settings with respect to protection against emerging infectious diseases; the report revealed that proper control measure should be taken to ensure that nosocomial infections do not occur in designated healthcare settings that accommodate suspected cases suffering from emerging infectious diseases and a comprehensive and detailed evaluation of nosocomial infection control should be conducted in each designated healthcare setting.
Moreover, hospital environment and its air are inseparable from health care worker and patients, bacteriological assessments of the environment such as indoor air quality cannot be underestimated when it comes to human health. The bacteria that live in troposphere, stratosphere and even ionosphere are capable of causing indoor air pollution (Awosika, Olajubu, and Amusa, 2012;Tambekar, Gulhane, and Bhokare, 2007).
Favorable temperature and poor air ventilation of indoors coupled with poorly designed structure gives an ample opportunity for microbial growth. It has been observed that Bacillus, Clostridium and Staphylococci are the most common bacteria isolated from indoor surfaces (Hayleeyesus and Manaye, 2014). Tambekar, Gulhane, and Bhokare (2007) report showed that bacteria can survive in both indoors and outdoors and the maternity and children hospitals had the highest bacterial isolates. It is easier to avoid infection transmitted from formites than that of airborne which is often difficult.
However, lithosphere is conducive for a lot of mixed microbes which may become airborne and exposure to this may cause an epidemic infection of a particular environment because contaminated air is significant in transmission of communicable airborne diseases (Klevens et al., 2007;Tambekar, Gulhane, and Bhokare, 2007). It is obvious that hospital acquired infection is on the increase and the rate of antibiotic resistance of bacterial pathogens associated with these infections is alarming in the public health (Wondemagegn et al., 2012).
Consequently, to avoid the risk of infection by nosocomial bacteria, negative-pressure quarantine ICUs and wards should be built in accordance with the demand in the area. Regulations, standards, procedures, and operational instructions on protection against infections from emerging respiratory, gastrointestinal, body fluid, and insect-borne infectious diseases should be established. Periodical regular trainings for the knowledge of prevention and control emerging or unknown infectious diseases, and emergency exercises regarding nosocomial infection events among medics can also help to strengthen the infection control system (Fu and Wang, 2016).
According to previous investigations, the individual and nations economy do suffer loss due to the menace of this infections thereby causing financial burden, disability of patients, morbidity and eventual death also known as mortality of the hospitalized victims. The victim may be infected via surgical wound, blood stream, respiratory, urinary tract infections (Endalafer, Gebre-Selassie, and Kotisso, 2010;;Wondemagegn et al., 2012). Wondemagegn et al. (2012) isolated 26.2% S. aureus as the leading bacterial isolates followed by E. coli and coagulase negative Staphylococcus spp. which consist of 21.4% each. Wondemagegn et al. (2012) further revealed that almost all the bacteria in the study were resistant to at least two of the antibiotics employed.
In general, bacteriological study of hospital environment has revealed the incidence of some bacteria associated with the environment. This research is aimed at bacteriological assessment of hospital air and selected surfaces in three referral hospitals in Makurdi, middle belt, Nigeria.

The Study Area and Population
The study areas of the research are three referral hospitals which include: City Hospital (CH), Federal Medical Centre Wadata (FMCW) and Saint Theresa Hospital (STH) in Makurdi, Nigeria. One hundred and forty-three (143) samples which include 104 surface swabs and 39 samples from air were collected from the referral hospitals for laboratory analysis within a period of five months.

Ethical Clearance
The consent of the administrative authorities of Federal Medical Centre Wadata, Saint Theresa Hospital and City Hospital were obtained before commencement of the research in the hospitals.

Collection of Samples
Air samples were taken by use of plate exposure methods while the surface swabs of the hospitals environments were collected aseptically by the use of sterile swabs sticks by trained personnel and researchers in the morning before commencement of work and transported immediately to the Microbiological Laboratory of Federal University of Agriculture, Makurdi. The parts of the hospital environment where patients, hospital staff and even visitors do have contact with almost every time were used as sample site due to likelihood of being infected. Thus, the analyses of samples collected are as follows: (i) 40 samples were taken from City Hospital (C): Thirty (30) samples from selected surfaces and 10 from hospital air. The samples collected from the surfaces include nurse table tops (4), bed rails (3), door knobs (4), from stretchers (3), sinks (4), operation table (4), hospital floor (4), and toilet seats (4), while 10 samples were collected from the hospital air. (ii) 47 samples were collected from Saint Theresa Hospital (S): 32 surface swabs which include nurse table tops (4), bed rails (4), door knobs (4), stretchers (4), sinks (4), operation table (4), hospital floor (4), toilet seats (4) and 15 samples were collected from the air of maternity ward (labour room), female medical ward, male medical ward and pediatrics ward. (iii) A total of 56 were collected from Federal Medical Centre Wadata (F): 42 samples were collected from the surfaces and 14 samples from the hospital air. The samples collected from the surface swabs include nurse table tops (6), bed rails (6), door knobs (6), stretchers (6), sinks (6), hospital floor (6) and toilet seats (6) and air samples (14) were obtained from Federal Medical Centre.

Procedure for Sterilization
All glass wares were sterilized as described by Cheesbrough (2006).

Sampling Design and Techniques
Air samples were taken by exposure of prepared media in plates (set plates) into air for 20 minutes while surface swabs were collected using sterile swab sticks. The selected surfaces analyzed include hospital floors, door knobs, nurse table tops, bedrails, stretchers, operation tables, toilet seats and sinks for the purposes of isolating microorganisms from these sources and transported with sealed sterile polythene bag to the laboratory for analysis within thirty minutes. These samples were inoculated into nutrient agar plates and blood agar under aseptic condition as described by Cheesbrough (2006).

Cultural method
The swabs collected from the selected surfaces by the researchers and other trained medical laboratory scientist were streaked on the bacteriological media with the use sterile swab sticks to make an inoculum on the plates. These swabs samples were taken before cleaning of the hospitals while, the bacteria from air samples were isolated by plate exposure method before incubation and later streaked with the use of a sterile wire loop to make axenic culture (Prescott, Harley, and Klein, 2008).

Isolation and characterization of isolates
The organisms cultured from the air and the swabs were directly inoculated on nutrient agar by the use of sterile inoculating loop and later sub-cultured on blood agar, chocolate and MacConkey agar near bunsen burner. The inoculated media were incubated at 35 o C for 24 hours and then examined for bacterial growth. The organisms isolated were identified by cultural characteristics, microscopy (Gram stain) and biochemical tests which include catalase, coagulase test, indole test, citrate test, oxidase test and carbohydrates fermentation (Prescott, Harley, and Klein, 2008).

In-vitro Determination of Antibiotic Susceptibility
Antibiotic susceptibility of the bacterial isolates was performed by disk diffusion method according to Clinical Laboratory Standards Institute guidelines. The bacteria were sub-cultured to prepare the inoculum with the sterile inoculating loop. Then, one isolated colony was transferred into 10ml of sterile distilled water prepared in the test tube and mixed properly. The liquid now serve as source of inoculum containing 10 6 CFU/ml of bacterial suspension. A sterile swab stick was properly dipped into mixed inoculum and excess fluid was removed before rotating the swab on the surface of the prepared Mueller-Hinton agar plates. This spread was done four different planes by rotating the planes 180 o each time. This was to ensure that every part of the plate was inoculated. Forceps was flammed until red hot and cooled and then used to pick the antibiotics disc and it was also used to place the discs on the inoculated plates. The discs were pressed down on the agar so as to be in complete contact with agar. The plates were incubated at 37°C for 18 hours. The antibiotics multidisc employed contained Ofloxacin (OFL) 5μg, Ceftriazone (CTR) 30μg, Gentamicin (GEN) 10μg, Cefuroxime (CRX) 10μg, Cloxacillin (CXC) 10μg, Augmentin (AUG) 30μg, Ceftazidime (CAZ) 30μg, and Erythromycin (ERY) 30μg. Zones of inhibition were used to determine the level of susceptibility of the isolates to these test antibiotics (CLSI, 2012).

Statistical Analysis
Statistical Package for the Social Sciences (SPSS) software version 23 was used for data analysis through the use of descriptive statistics which include frequency tables, percentage, pie chart and bar chart as described by Pallant (2007).

RESULTS AND DISCUSSION
The bacteriological assessment of the three referral hospitals is presented below. Figure 1 depicts the distribution of bacteriological samples while Figure 2 represents the occurrence of the bacterial isolates in relation to air sample and surface swab. Figure 3 reveals the incidence of bacteria isolated from the air sample and selected surfaces of the referral hospitals. One hundred and forty-three (143) bacteriological samples were collected for analysis and fifty bacterial isolates were obtained which consist of 13 (26%) and 37 (74%) bacterial isolates from air sample and surface swabs respectively (Figure 2). The selected surfaces comprise bed rails, doorknobs, floor, nurse table top, operation tables, sinks, stretchers and toilet seats. The bacteriological assessment of the indoor air in different wards and the selected surfaces is shown in Figure 3. Thus, bacteria isolated from hospital air include 13 (26%), bed rails 8 (16%), door knobs 1 (2%), floor 2 (4%), nurse table top 5 (10%), operation table 2 (4%), sink 9 (18%), stretchers 1 (2%) and toilet seat 6 (12%). The cultural, morphological and biochemical characteristics of the bacterial isolates are shown in Table 1 as described in Bergey's Manual 9th edition (Bergey, 2000).
The Gram-negative bacteria are Klebsiella spp (10%), Pseudomonas aeruginosa (18%) and Escherichia coli (20%). This is also in agreement with findings by Hassan, Aftab and Riffat, (2015) that, 3-7% of nosocomial bacterial infections are related to Klebsiella pneumonia. Klebsiella is a Gram-negative, nonmotile, encapsulated, rod-shaped bacillus present within the nasopharynx and gastrointestinal tract of humans and nonhuman primates. E. coli is one of the common Gramnegative bacteria often associated with wound sepsis and endotoxin-induced shock (Hassan, Aftab and Riffat, 2015). It often causes urinary tract and wound infections in patient with compromised immunity. Drach (2005) revealed Klebsiella spp and E. coli among others as the microorganisms related to urinary catheterization. Bereket et al. (2012) reported that Pseudomonas aeruginosa can transiently colonize the respiratory and gastrointestinal tracts of hospitalized individuals. Furthermore, giving the preponderance of these organisms in the present study, there is a high possibility of patients contracting such infections. According to Brooks et al. (2010), Klebsiella can cause respiratory tract infection, urinary tract infections as well as wounds infections in a poor ventilated environment.
The percentage of occurrence of Staphylococcus aureus were (34%) and other Staphylococcuss spp were (18%). Out of 26 (Twenty-six) Gram positive cocci isolated, Staphylococcus aureus were 17 (65.4%) while Staphylococcus spp were 9 (34.6%). This report agrees with the findings by Hassan, Aftab and Riffat (2015) that Staphylococcus spp had the least occurrence among the Gram-positive bacteria associated with hospital acquired infections. Staphylococcus aureus being among normal microbiota of human may be responsible for higher number of incidence (65.4%).
Therefore, the nine sources in this study which include air and other eight selected surfaces shown in Figure 3 and Table  3 are multi-reservoirs being responsible for hospital contamination. This was also the same with the findings of Wilks, Michels and Keevil (2005) and Michels (2006) that inanimate objects can allow survival of microorganisms for a particular period of time. However, this work is in contrast to a report by Derese et al. (2016) where the main isolates in a similar finding executed in selected wards of a referral hospital in Ethiopia were coagulase negative Staphylococci (CoNs) 44%, followed by S. aureus, 37.4%, and Klebsiella spp at 11.6%.
Meanwhile, E. coli isolated from Federal Medical Centre was sensitive to considerable number of the antibiotics with sensitivity to ofloxacin and augmentin streaking ( Table 5). Whereas this was in contrast to the strain of E. coli isolates from Saint Theresa Hospital (Table 6). This corroborates with the reports of Gelaw et al. (2013) and Dessie et al. (2016) that stated E. coli among gram-negative isolates found resistant to antimicrobial tested.
The highest incidence of Staphylococcus aureus in the work give an ample opportunity of looking into antibiogram of the isolates. It was observed that Staphylococcus aureus isolated from City Hospital were highly sensitive to ofloxacin (inhibitors of nucleic acid synthesis) and bacteriological assessment of Staphylococcus aureus isolated from Saint Theresa Hospital reveals that the bacteria is sensitive to cloxacillin (cell wall inhibitor antimicrobial agent). Staphylococcus aureus isolated from Federal Medical Centre Wadata was also sensitive to ofloxacin (a member of fluoroquinolones) which is capable of inhibiting nucleic acid synthesis. This is in agreement with the report of Pollack (2010), that the effectiveness of antimicrobial drugs in chemotherapy is based on the susceptibility of the target microbes.
In general, predisposing factors that make patients susceptible to nosocomial infections had been reported by some researchers, the length of hospital stay remains the most important factors of nosocomial infections (Kleven et al., 2007;Lahsaeizadeh et al., 2009). Assessment of environmental quality of hospital wards should be based on comprehensive analysis with multiple indicators, because there may be imbalances in the microbial diversity in the hospital wards, therefore, monitoring of the environmental quality of hospitals is important in the prevention of nosocomial infections (Hart et al., 2015;Ling and Hui, 2019;Loupa, et al., 2016). This research is in line with the study on nosocomial bacterial infections and their antimicrobial susceptibility patterns among patients in Ugandan intensive care units (Peter et al., 2017). It is also consistent with the study on evaluation of the complex indoor environment of hospital wards through the composition of bacteria and microbial ecology of the hospital wards (Ling and Hui, 2019).

CONCLUSION
This investigation has unveiled the hide out of pathogenic bacteria in hospital premises from hospital floor even to the air. Incidence of Gram-negative bacteria is less than incidence of Gram bacteria which mean, patients are prone to contract bacterial infection associated with Gram positive bacteria than Gram negative. This study reveals Klebsiella spp as the predominant bacteria in hospital air. It further reveals E. coli, Pseudomonas aeruginosa, S. aureus and other Staphylococcus spp as bacteria commonly associated with hospital environment and these bacteria are capable of causing nosocomial infections on patients with compromised immunity. Antibiotics susceptibility pattern of the bacterial isolates reveals the presence of highly resistant species of P. aeruginosa and Klebsiella in the hospitals which has high public health implication. This report will create awareness and be a good guide to health care workers, patients and the public about the likeliness of contracting nosocomial infection and how to treat such infection.

RECOMMENDATIONS
Based on the findings and conclusion reached above, the following recommendations are hereby made: 1. Healthcare workers should be more careful in carrying out their duty to avoid been infected in the course of work.
2. The in-vitro antibiotics susceptibility testing on the bacterial pathogens in this study shows that erythromycin, Ceftazidime, Cefuroxime and Cloxacillin may not be effective in the treatment of the hospital acquired infections caused by bacterial pathogens. Therefore, information on resistance patterns of isolates encountered in this study will assist the clinicians in making improvement in management of nosocomial infections.
3. Health care workers, patients and any other individual who visit hospitals should keep proper hygiene through hand wash and the use of hand sanitizer.

4.
Use of antiseptics to clean inside hospital environments surfaces more than once in a day and use of recommended air fresheners in hospital wards will ameliorate the high loads of the pathogens and reduces the chances of infection by immuno-compromised individuals.

FUTURE WORK
There should be sound epidemiology and health information system, properly documented for surveillance in hospitals to be able to monitor, control in order to tackle the rate of hospital acquired infections.