Archives of Clinical Microbiology

Keywords

Airborne bacteria; ESBL; Multidrug resistance; Antibiotic resistance; Hospital theatres

Abbreviations

STEC: Shiga Toxin Producing E. coli; EAE: Effacing Gene; Stxs: Shiga Toxins; Gb3: GlycosphingolipidVTEC: Verotoxigenic E. coli

Introduction

Indoor air quality is a major problem worldwide, both in developed and developing countries. Problems of indoor air quality are recognized as important risk factors for human health since majority spend a substantial fraction of time within buildings [1]. Airborne bacteria in health care facilities is associated with nosocomial infection. Hospital environments provide a reservoir of airborne bacteria, many of which exhibit multidrug-resistant traits. Antibiotic resistance by microorganisms has been a serious challenge in the treatment of various nosocomial pathogens and has jeopardized the current management protocols in practices. Resistance towards antibiotics by microorganisms is not a new phenomenon [2]. Huang et al. [3] reiterated that Pseudomonas aeruginosa represented the most frequently detected and abundant bacterium in air samples; and was the only bacterium exhibiting a positive correlation of the mean counts between air samples and surface samples, which is intrinsically resistant to many antibiotics. Aerobiological survey undertaken in various ward areas of two hospitals indicated that Gram-positive cocci were the most predominant airborne microbial flora followed by Gram-positive rods, Gram – negative rods, and the Diphteroids and Coccobacilli [4]. The evaluation of indoor and outdoor air contamination of hospitals showed that hospitals act as reservoir of multidrugresistant nosocomial pathogens, where labour room was recorded as the most contaminated site, followed by the dressing room and the operation theatre [4].

The increased rate of methicillin-resistant Staphylococcus aureus, followed by vancomycin-resistant enterococci (VRE) or extended-spectrum beta-lactamase – producing microorganism ’ s transmission associated with a prior occupant’s carriage is suggested of routes for transmission of pathogens in intensive care unit (ICU) and other hospital environment [2]. Vulnerable groups of in-patients are especially at high risk of developing antibiotic-resistant infections. Such infections pose serious threat to immunocompromised patients causing increased morbidity, mortality, and medical costs [5]. Because of evolution and emergency of bacterial resistance to antibiotics, and an increase in the number of immunocompromised individuals; HIV infections, chemotherapy, drug therapies and genetic disorders hospitals are now more often facing the problems of antibiotic-resistant nosocomial infections.

Airborne microorganisms are spread from numerous sources including air conditioning systems, respiratory droplets produced by patient's coughing, laughing, sneezing, and ward activities; such as those generated by bed making and mechanical floor cleaning, have been shown to release large number of bacteria into the air [6]. The potential hazards posed by air-borne bacteria depends on the pathogenicity of a specific strain, environmental factor and bacterial gene pool including antibiotic resistance gene. This study was therefore designed to determine the prevalence and antibiotic susceptibility profile of airborne bacteria isolated from surgical and labour theatres in Federal Teaching Hospital Abakaliki (FETHA), Ebonyi State, Nigeria.

Materials and Methods

Sample collection

Forty (40) air samples were collected aseptically from general surgical theatre, (n=26), and labour theatre (n=14) using settle plate method. A blood agar plate was exposed to the air in the hospital theatres; a metre above the floor and a metre from the wall. The plates were then covered after 1 hour of exposure and immediately transported to Department of Applied Microbiology laboratory, Ebonyi State University, for bacteriological analysis.

Ethical clearance

Ethical clearance for this study was approved by the ethical clearance committee of Federal University Teaching Hospital, Abakaliki, Ebonyi State.

Culture and isolation of clinical airborne bacteria

The blood agar plate exposed to the air in the hospital theatres were incubated at 37?C for 18-24 hours before subculturing onto mannitol salt agar, cysteine lactose electrolyte deficient (CLED) agar, nutrient agar and MacConkey agar using standard microbiological technique to obtain discrete colonies. Pure colonies were observed for their colonial morphology, growth on different/selective media, Gram ’ s reaction, coagulase test, catalase test, and motility test. Isolates were further identified by biochemical tests which includes indole test, oxidase test, citrate utilization test, and sugar fermentation test (triple sugar iron agar) [7].

Antibiotics susceptibility test

Antibiotic susceptibility test on isolated airborne bacteria was determined using the Kirby-Bauer disc diffusion method according to the recommendations of the Clinical and Laboratory Standard Institute [8]. A standardized inoculum equivalent to 0.5 McFarland standard of the isolate was inoculated aseptically on the surface of prepared Mueller- Hinton agar plates. The inoculated plates were allowed for prediffusion of the antibiotics for 10-15 minutes. The following standard antibiotic discs were tested against the isolates; ampicillin (10 μg), cefotaxime (30 μg), nofloxacin (5 μg), erythromycin (5 μg), tetracycline (30 μg), trimethoprim/ sulfamethoxazole (75 μg), ceftazidime (30 μg), cefoxitin (30 μg), clindamycin (10 μg), muporocin (10 μg), tobramycin (10 μg), ticarcillin (30 μg), doripenem (10 μg), vancomycin (10 μg), chloramphenicol (15 μg), penicillin (10 μg) (Oxoid, UK). Sterilized forceps were used to place the antibiotic discs evenly on the inoculated Mueller-Hinton agar so that the disc should be about 15 mm from the edge of the plate and not closer than 25 mm from disc to disc. After 30 minutes, the plates were inverted and incubated for 24 hours. A ruler was used to measure the diameter of each zone of inhibition in mm on the underside of the plate. The inhibitory zone diameter was interpreted as susceptible or resistant according to the criteria of CLSI [8].

Determination of ESBL-producing Gramnegative bacteria

Screening the Gram-negative bacterial isolates for ESBL production was done by observing their sensitivities to 2nd and 3rd generation cephalosporins; such as aztreonam (30 μg), cefepime (30 μg), cefotaxime (30 μg), cefoxitin (30 μg), ceftazidime (30 μg) and ceftriaxone (30 μg). These antibiotics were aseptically placed at a distance of 30 mm apart on Mueller-Hinton agar (Oxoid, UK) plate that was previously inoculated with standardized inoculum of the test bacterium using a sterile swab stick in order to get a confluent growth. The plates could stand for about 30 minutes for pre-diffusion of the antibiotics and after which was incubated for 18-24 hours at 37?C. After the incubation time, the zones of inhibition were measured in millimeter using a metre rule and results were interpreted according to Clinical and Laboratory Standard Institute (CLSI) chart. ESBL production was suspected if any of the test bacteria showed reduced susceptibility or resistance to any of the antibiotics used for the screening studies according to the CLSI guidelines [8,9].

Phenotypic confirmation of ESBL from isolated Gram-negative bacteria using double disk synergy test (DDST)

All the Gram-negative bacterial isolates that showed reduced susceptibility to any of the 2nd and 3rd generation cephalosporins were phenotypically confirmed for ESBL production using the double disc synergy test [9]. Sterile swab sticks were dipped into bacterial suspension (s) standardized to 0.5 McFarland turbidity standards and was inoculated on MH agar plates. Antibiotic disc of amoxicillin/clavulanic acid (20/10 g) was placed at the center of the MH agar plate and antibiotic discs containing cefotaxime (30g) and ceftazidime (30g) each was placed at a distance of 15 mm (center to enter) from the central disc, amoxicillin/clavulanic acid (20/10g) and the plates was incubated at 37?C for 18-24 hours. ESBL production was suspected phenotypically when the zones of inhibition of the cephalosporins (cefotaxime 30g and ceftazidime 30g) increased in the presence of amoxicillin/ clavulanic acid disk (20/10g). A 5mm increase in the inhibition zone diameter for either of the cephalosporins (cefotaxime and ceftazidime) tested in combination with amoxycillinclavulanic acid versus its zone when tested alone confirmed ESBL production phenotypically [9].

Screening for methicillin-resistant Staphylococcus aureus (MRSA)

Oxacillin screening agar test: A standardized inoculum equivalent to 0.5 McFarland standard of the isolate was inoculated aseptically on Mueller-Hinton agar containing 4% sodium chloride and 1μg oxacillin ml-1 (Hi media) and incubated at 35?C for 18-24 hours. Any isolate showing growth on the plate containing oxacillin was resistant to methicillin [10].

Determination of multiple antibiotic resistance index (MARI)

The multiple antibiotic resistance index was calculated as the ratio of the number of antibiotics to which the isolates were resistant/the total number of antibiotics against which the isolates were tested.

Results

A total of 138 airborne bacterial isolates were obtained from 40 blood agar samples exposed to air in the general surgical and labour theatres (Table 1). Eighty seven (87) bacterial isolates were obtained from the general surgery theatre while 51 isolates were obtained from labour theatre (Table 1). Out of the 138 airborne bacterial isolates obtained, S. aureus [40 (29%)] was the most predominant species. This was followed by coagulase-negative staphylococci (CONS) [26 (18.8%)]. Micrococcus spp. had prevalence frequency of 16 (11.6%) each; Klebsiella spp. had prevalence frequency of 8 (5.8%); P. aeruginosa had prevalence frequency of 9 (6.5%) while Streptococcus spp. and Acinetobacter had the least prevalence frequency of 7 (5.1%) each (Table 1). Antibiotic susceptibility results showed that S. aureus isolates were completely resistant (100%) to trimethoprim/sulfamethoxazole. Resistance (50-85.7%) was also recorded against clindamycin, erythromycin, mupirocin, and vancomycin in the two theatres. Isolates were completely susceptible (100%) to ticarcillin and tobramycin in the two theatres (Table 2).

Bacterial isolates	General surgery theatre	Labour theatre	Total n (%)
S. aureus	26	14	40 (29%)
CONS	16	10	26 (18.8%)
Streptococcus spp.	4	3	7 (5.1%)
Micrococcus spp.	10	6	16 (11.6%)
Bacillus spp.	15	10	25 (18.1%)
Acinetobacter spp.	4	3	7 (5.1%)
Klebsiella spp.	5	3	8 (5.8%)
P. aeruginosa	7	2	9 (6.5%)
Total	87	51	138

Key: CONS=Coagulase negative staphylococci

Table 1: Frequency Distribution of the Bacteria Isolated from air samples in theatres.

Wards	Pattern	Antibiotics
		DA	E	MUP	NOR	OX	P	SXT	TIC	TOB	VAN
General Theatre	S	62.5	37.5	75	100	62.5	68.8	56.3	100	100	75
	R	37.5	62.5	25	0	37.5	31.3	43.8	0	0	25
Labour Theatre	S	80	80	70	100	30	60	50	100	100	80
	R	20	20	30	0	70	40	50	0	0	20

Table 2: Antibiotic Susceptibility Pattern of Staphylococcus aureus isolates.

There was no statistically significant difference in the antibiotic resistance frequencies of S. aureus isolates from the two theatres (P=0.641 at p<0.05). Antibiotic susceptibility results showed that CONS isolates were resistant (50-62.5%) to trimethoprim/sulfamethoxazole and erythromycin (62.5%). In contrast, CONS isolates were completely susceptible (100%) to ticarcillin and tobramycin in the two theatres (Table 3). Susceptibility (62.5-80%) was also recorded with clindamycin, muporocin, nofloxacin, penicillin, and vancomycin (Table 3). There was no statistically significant difference in the antibiotic resistance frequencies of CONS isolates from the two theatres (P=0.35 at p<0.05).

Wards	Pattern	Antibiotics
		DA	E	MUP	NOR	OX	P	SXT	TIC	TOB	VAN
General Theatre	S	62.5	37.5	75	100	62.5	68.8	56.3	100	100	75
	R	37.5	62.5	25	0	37.5	31.3	43.8	0	0	25
Labour Theatre	S	80	80	70	100	30	60	50	100	100	80
	R	20	20	30	0	70	40	50	0	0	20

Table 3: Antibiotic Susceptibility Pattern of Coagulase-negative Staphylococci (CONS) isolated from General Surgical and Labour Theatre in FETHA.

Antibiotic susceptibility results showed that Streptococcus spp. isolates were resistant (60-80%) to mupriocin, norfloxacin, and penicillin but susceptible (60-100%) to clindamycin, trimethoprim/sulfamethoxazole, ticarcillin, erythromycin, tobramycin, and vancomycin (Table 4). There was no statistically significant difference in the antibiotic resistance frequencies of Streptococcus spp. isolates from the two theatres (P=0.41 at p<0.05). Antibiotic susceptibility results showed that micrococcus spp. isolates were resistant (50-100%) to clindamycin, trimethoprim/sulfamethoxazole, ticarcillin, norfloxacin, and penicillin but susceptible (66.7-100%) to mupriocin, erythromycin, tobramycin, and vancomycin (Table 5). There was no statistically significant difference in the antibiotic resistance frequencies of micrococcus spp. isolates from the two theatres (P=0.89 at p<0.05). Antibiotic susceptibility results showed that Bacillus spp. isolates were completely resistant (100%) to clindamycin, mupirocin, and penicillin but susceptible (50-90%) to erythromycin, trimethoprim/sulfamethoxazole, ticarcillin, norfloxacin, tobramycin, and vancomycin (Table 6).

Wards	Pattern	Antibiotics
		DA (%)	E (%)	MUP (%)	NOR (%)	OX (%)	P (%)	SXT (%)	TIC (%)	TOB (%)	VAN (%)
General Theatre	S	100	80	40	40	80	20	100	100	60	80
	R	0	20	60	60	20	80	0	0	40	20
Labour Theatre	S	100	100	66.7	33.3	100	100	100	100	100	0
	R	0	0	33.3	66.7	0	0	0	0	0	100

Table 4: Antibiotic susceptibility pattern of Streptococcus spp. isolates.

Wards	Pattern	Antibiotics
		DA (%)	E (%)	MUP (%)	NOR (%)	OX (%)	P (%)	SXT (%)	TIC (%)	TOB (%)	VAN (%)
General Theatre	S	100	70	70	30	50	30	0	0	100	100
	R	0	30	30	70	50	70	100	100	0	0
Labour Theatre	S	50	66.7	83.3	33.3	33.3	0	0	0	100	100
	R	50	33.3	16.7	66.7	66.7	100	100	100	0	0

Table 5: Antibiotic Susceptibility Pattern of Micrococcus species isolated from General Surgical and Labour Theatre in FETHA.

Wards	Pattern	Antibiotics
		DA (%)	E (%)	MUP (%)	NOR (%)	OX (%)	P (%)	SXT (%)	TIC (%)	TOB (%)	VAN (%)
General Theatre	S	0	80	0	86.7	0	0	53.3	66.7	66.7	66.7
	R	100	20	100	13.3	100	100	46.7	33.3	33.3	33.3
Labour Theatre	S	0	80	0	90	0	0	50	80	60	70
	R	100	20	100	10	100	100	50	20	40	30

Table 6: Antibiotic Susceptibility Pattern of Bacillus species isolates.

There was no statistically significant difference in the antibiotic resistance frequencies of Micrococcus spp. isolates from the two theatres (P=0.06 at p<0.05). Antibiotic susceptibility results showed that Acinetobacter spp. isolates were resistant (50% -100%) to ampicillin, chloramphenicol, mupirocin, norfloxacin, trimethoprim/sulfamethoxazole, tetracycline, ticarcillin, and tobramycin but susceptible to doripenem (66.7%) and ceftazidime (75%) (Table 7). There was no statistically significant difference in the antibiotic resistance frequencies of Acinetobacter spp. isolates from the two theatres (P=0.38 at p<0.05). Antibiotic susceptibility results showed that Klebsiella spp. isolates were resistant (66.7-100%) to ampicillin, ceftazidime, mupirocin, norfloxacin, trimethoprim/sulfamethoxazole, and tetracycline but susceptible (60-100%) to chloramphenicol, doripenem, ticarcillin, and tobramycin (Table 8). There was no statistically significant difference in the antibiotic resistance frequencies of Klebsiella spp. isolates from the two theatres (P=0.09 at p<0.05).

Wards	Pattern	Antibiotics
		AMP (%)	C (%)	CAZ (%)	DOX (%)	MUP (%)	NOX (%)	SXT (%)	TE (%)	TIC (%)	TOB (%)
General Theatre	S	0	25	75	75	0	50	0	25	25	0
	R	100	75	25	25	100	50	100	75	75	100
Labour Theatre	S	0	0	66.7	66.7	0	33.3	0	33.3	0	66.7
	R	100	100	33.3	33.3	100	66.7	100	66.7	100	33.3

Table 7: Antibiotic Susceptibility Pattern of Acinetobacter spp. Isolates.

Wards	Pattern	Antibiotics
		AMP (%)	C (%)	CAZ (%)	DOX (%)	MUP (%)	NOR (%)	SXT (%)	TE (%)	TIC (%)	TOB (%)
General Theatre	S	0	100	0	60	0	0	0	20	60	60
	R	100	0	100	40	100	100	100	80	20	40
Labour Theatre	S	0	100	0	100	0	0	0	33.3	66.7	66.7
	R	100	0	100	0	100	100	100	66.7	33.3	33.3

Table 8: Antibiotic Susceptibility pattern of Klebsiella spp. Isolates.

Antibiotic susceptibility results showed that Pseudomonas aeruginosa isolates were resistant (80%-100%) to ampicillin, ceftazidime, mupirocin, trimethoprim/sulfamethoxazole, and tetracycline but susceptible (57.1-100%) to chloramphenicol, doripenem, norfloxacin, ticarcillin, and tobramycin (Table 9). There was no statistically significant difference in the antibiotic resistance frequencies of Pseudomonas aeruginosa isolates from the two theatres (P=0.49 at p<0.05).

Wards	Pattern	Antibiotics
		AMP (%)	C (%)	CAZ (%)	DOX (%)	MUP (%)	NOR (%)	SXT (%)	TE (%)	TIC (%)	TOB (%)
General Theatre	S	0	57.1	20	85.7	14.3	85.7	0	0	100	100
	R	100	42.9	80	14.3	85.7	14.3	100	100	0	0
Labour Theatre	S	0	100	20	66.7	0	100	0	0	100	100
	R	100	0	80	33.3	100	0	100	100	0	0

Table 9: Antibiotic Susceptibility Pattern of Pseudomonas aeruginosa isolates.

A total 17 Gram-negative bacterial isolates (2 Acinetobacter spp., 7 Klebsiella spp., and 8 P. aeruginosa) were ESBL-positive while 30 out of the S. aureus isolates were methicillin-resistant S. aureus (MRSA) in the two theatres (Table 10).

Wards	MRSA Positive isolates	ESBL positive isolates
		Acinetobacter spp	Klebsiella spp	Pseudomonas spp
General Theatre	18	0	4	6
Labour Theatre	12	2	3	2
Total	30 (75%)	2	7	8

Table 10: Frequency Distribution of Methicillin-resistant S. aureus (MRSA) and ESBL-positive Gram-negative bacteria in General surgery and Labour theatres.

Discussion

The control and effective prevention of antibiotic resistance nosocomial infections requires a better identification of airborne bacteria that are potentially harmful to patients. This information is critical to implementing more appropriate control measures against the spread of airborne hospital- acquired infections. In this study, analysis of air samples carried out by plate gravitational settling (passive) method in general surgical and labour theatres at Federal Teaching Hospital Abakaliki (FETHA), has shown the preponderance of airborne bacteria. Gram-positive bacteria such as Staphylococcus aureus (29%), Coagulase-negative staphylococci (18.8%), Streptococus spp. (5.1%), Micrococcus spp. (11.6%), and Bacillus spp. (18.1%) were isolated from the general surgery and labour theatres while Gram-negative bacteria such as P. aeruginosa (6.5%), Klebsiella spp. (5.8%), and Acinetobacter spp. (5.1%) were also isolated. Our study revealed that Gram-positive bacteria were more prevalent than Gram-negative bacteria among the bacterial isolates obtained from the air samples of the two theatres. This agrees with the findings of Ekhaise et al. [5] who also reported that

Gram-positive bacteria were more prevalent than Gram- negative bacteria, with Staphylococcus being the most predominant Gram-positive bacteria in air samples collected from hospital environment. Pseudomonas aeruginosa was the predominant Gram-negative bacteria isolated. Reports published earlier suggested that high prevalence of Gram- positive bacteria in air samples could be attributed to their ability to survive environmental stress as they possess pigments and photo-reactivation mechanisms that provide protection from sunlight. The high peptidoglycan content in their cell walls have also been known to provide protection from drying and heat stress, thereby spreading across considerable distance which in turn results in infections [11]. Gram-negative bacteria have been reported to be less prevalent in air samples due to their susceptibility to environmental stress because of low peptidoglycan contents in their cell wall [12]. The antibiotic susceptibility pattern of airborne bacterial isolate revealed that vancomycin, erythromycin, ticarcillin, and tobramycin were generally the most active antibiotics against Gram-positive bacteria isolated from the general surgery and labour theatres while doripenem, chloramphenicol, ticarcillin, and tobramycin were the active antibiotics against Gram-negative bacteria. Interestingly, ticarcillin and tobramycin were continually active against all the Gram-positive and Gram-negative bacteria isolated from the two theatres. Virtually all the bacterial isolates in this study were multidrug-resistant as they were resistant to at least two different classes of antibiotics. Our study agrees with the report indicated that airborne bacteria are a risk factor for surgical site infections. Our study also revealed that 17 Gram-negative bacteria (2 Acinetobacter spp., 7 Klebsiella spp., and 8 P. aeruginosa) were ESBL-positive while 30 (75%) out of the S. aureus isolates were methicillin-resistant S. aureus (MRSA) in the two theatres. The observed trend in prevalence/incidence of these pathogen in hospital infections further highlights them as important public health challenge with increasing economic and human impact. This is in line with  the  study  of  Stetzenbach  [13],  who  reported  that the increasing prevalence of antibiotic resistance among various pathogens such as methicillin-resistant Staphyloccocus aureus (MRSA) and extended spectrum beta-lactamases (ESBLs) by Gram-negative bacteria such as Klebsiella pneumoniae and multidrug resistant Acinetobacter species has made the incidence of hospital infections critical. Their existence in air may be attributed to the improper handling and disposal of hospital wastes and the climatic conditions prevailing in hospital theatres [13,14].

Conclusion

Our study has shown that airborne Gram-positive (Staphylococcus aureus, Coagulase-negative Staphylococci, Bacillus spp., Micrococcus spp., and Streptococcus spp.) and Gram-negative bacteria (Pseudomonas aeruginosa, Klebsiella spp., and Acinetobacter spp.) were present in general surgical and labour theatres of Federal Teaching Hospital Abakaliki (FETHA). Our study also showed that Gram-positive bacteria, especially S. aureus were more predominant among the airborne bacterial isolates. Gram-negative bacterial isolates in this study were also positive for extended-spectrum beta- lactamase (ESBL) production. Virtually all isolates in this study were multidrug-resistant. Interestingly, vancomycin, erythromycin, ticarcillin, and tobramycin were generally the most active antibiotics against Gram-positive bacteria isolated from the general surgery and labour theatres while doripenem, chloramphenicol, ticarcillin, and tobramycin were the most active against Gram-negative bacteria. The findings of this study imply that the high bacterial load of indoor air judges it as a risk factor for surgical site infections. The presence of airborne bacterial isolates in surgical and theatre wards threatens patients’ health status as it puts them at risk of post-operative infections. There is need for hospitals to encourage periodic microbial load review of surgical and labour theatres, and their antibiotic sensitivity pattern. Therefore, to ensure good indoor air quality for patients and healthcare workers, it is imperative for hospitals to properly disinfect/sterilize hospital environment and encourage periodic microbial load review of surgical and labour theatres. This will greatly help in the prevention of nosocomial infections.

Conflict of Interest

None to declare.

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