Study area
The study was conducted at Wolaita Sodo University Teaching and Referral Hospital (WSUTRH), Sodo, located South Central Ethiopia. It is serving people in catchment’s area of 2 million people. The hospital has 320 beds for inpatient service which are on medical, pediatrics, surgical, intensive care unit, gynecology and obstetrics wards.
Study design and period
A hospital based cross sectional study was conducted to determine the prevalence and pattern of antibiotic resistance, extended spectrum and metallo beta-lactamase producing P. aeruginosa and A. baumannii isolates from restricted settings of indoor air hospital environment. The study was undertaken from December 1, 2015 to April 30, 2016 in WSUTRH.
Sampling techniques
The Air samples were collected during the first week of the months, twice a week during Monday’s and Friday’s. All microbiological procedures were conducted in Wolaita Sodo University microbiology laboratory which is an accredited laboratory with bio-safety cabinet two and vitek 2 microbiology apparatus. The laboratory built independently 5 km far from the clinical departments where air samples were conducted.
Active air sampling
Active air sampler, Anderson six state cascade impactor, which sucks 28.3 l of air per minute, was used and the Petridish was placed in the impactor for 5 minutes [13]. After that the Petridish was shipped to Wolaita Sodo university microbiology laboratory. Petri dishes were labeled with sample number, hospital ward, date and time (hour, minute and second) of sample collection.
Three agar plates were placed at various distances in each of the selected wards with five meter apart. Self-contamination was prevented by wearing sterile surgical gloves, mouth masks, and protective gown.
Processing of specimens and preliminary identification
Following collection, colonies on tryptic soya agar were inoculated into MacConkey agar, and blood agar plates. The inoculated plates were incubated at 35 °C for 24–48 h. Then the growth was inspected to identify the bacteria.
P. aeruginosa isolates were presumptively identified by gram staining, colony morphology, pigment formation, mucoid, haemolysis on blood agar, positive oxidase test, grape-like odour, growth at 42 °C on nutrient agar, and positive motility [14].
Genus Acinetobacter was identified by Gram staining, cell and colony morphology, positive catalase test, negative oxidase test and absence of motility. Suspected A. baumanii isolates were confirmed by API-20 NE kit (biomerieux, France) system.
Antibiotic susceptibility testing
The drug susceptibility testing of the isolates was done by Kirby-Bauer disc diffusion method [15] following Clinical Laboratory Standards Institute (CLSI) guide lines. The grades of susceptibility pattern were recognized as sensitive, intermediate and resistant by comparison of the zone of inhibition as indicated by CLSI, 2014 [16]. Intermediate isolates were taken as sensitive for the purpose of this study. The antibiotic discs were obtained from Oxoid, England, with the following concentrations: amikacin (30 μg), cefotaxime (30 μg), cefepime (30 μg), azetronam (30 μg) amoxicillin-clavulanic acid (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg), ciprofloxacin (10 μg), meropenem (10 μg), gentamicin (10 μg), imipenem (10 μg), trimethoprim-sulphamethoxazole (25/1.25 μg). Antibiotics were selected based on local availability, their effectiveness, guideline provided by CLSI and from literatures.
Phenotypic detection of extended spectrum beta-lactamase producing bacteria
Extended spectrum beta-lactamase (ESBL) production was detected by double disc synergy test (DDST) [17]. Accordingly, 3–5 selected colonies were taken from a pure culture and transferred to a tube containing 5 ml sterile nutrient broth and mixed gently until a homogenous suspension was formed. The suspension was incubated for 4–6 h at 37 °C until the turbidity was matched with the 0.5 McFarland standards. A sterile cotton swab was then used to distribute the bacteria evenly over the entire surface of Mueller Hinton agar (Oxoid, England).
Amoxicillin-clavulanic acid disc was placed in the center of the plate whereas ceftriaxone, ceftazidime and cefotaxime (30 μg each) discs were placed at a distance of 20 mm (center to center) from the amoxicillin-clavulanic acid disk. The plates were then incubated at 37 °C for 24 h and results were read. Enhancement of zone of inhibition of the cephalosporin disc towards clavulanic acid containing disc was inferred as synergy and the strain considered as ESBL producer.
Phenotypic detection of metalo-beta lactamase producing bacteria
Imipenem-resistant isolates were screened for producing MBL. The double disk method was used to detect this enzyme. Colonies from overnight cultures on blood agar plates were suspended in Mueller-Hinton broth and the turbidity standardized to equal that of a bacterial concentration of 1:100 suspensions of the 0.5 McFarland standards. Then the suspension was streaked onto Mueller-Hinton agar plates (Hi Media, Mumbai, India). A disc of Imipenem alone (10 μg) and Imipenem (10 μg) in combination with EDTA (750 μg/disc) was placed at the distance of 20 mm (centre to centre). After overnight incubation at 35 °C, a ≥ 7 mm increase in the inhibition zone of diameter around Imipenem-EDTA discs, as compared to imipenem discs alone, interpreted as indicative of MBL production [18].
Operational definitions
MDR was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories.
Pan resistance-resistance for all antibiotics tested.
High MDR: resistance rate of the isolates for more than 60% of the antibiotics.
Quality controls
Standard operating procedures were prepared and followed from sample collection to reporting. Culture medias were prepared based on the manufacturers’ instruction then the sterility was checked by incubating 5% of the batch at 35-37 °C for overnight and observing bacterial growth. Those Media which showed growth were discarded. Anderson air sampler was handled by environmental microbiologist and as per the manufacturer’s instruction.
Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as control strains.
Data analysis
Statistical analysis was performed by using SPSS version 20 software program and descriptive statistics were used.