One hundred and forty-two bacterial isolates were isolated from 534 environmental specimens obtained in the wards of NAUTH, Nnewi, a major referral centre serving individuals from most parts of South-East, Nigeria. The bacteria were collected from January to June 2018. The specimens included swabs collected from; patients beds, bedside tables, bedside cupboards, trolleys, sphygmomanometers, water taps, antiseptics, disinfectants, hand wash solutions, hand sanitisers, forceps, wheelchairs, kidney dishes, door handles, drip stands, drug mortars, methylated spirits, suction tubes, nurses desks, doctors desks and pulse oximeters.
Duplicate swabs were collected by rolling moistened sterile swab sticks over the sites mentioned above for about 5 seconds. These swabs were sent to the laboratory immediately after collection and cultured on chocolate and Mac Conkey agar (Oxoid, UK) and incubated at 35–37°C for 24 hours [10, 12]. The isolates were Gram-stained, and the Gram‑negative rods were subjected to confirmatory identification of K. pneumoniae using the MicrobactTM Gram-negative bacteria identification kit (Oxoid, UK) .
Antimicrobial Susceptibility testing
The Modified Kirby-Bauer antimicrobial susceptibility testing technique was performed on all isolates confirmed as K. pneumoniae [13, 14]. A lawn of each bacterial inoculum equivalent to 1.5 X 108 CFU/ml, was made on the surface of a Mueller-Hinton agar (Oxoid, UK) plate using a sterile swab stick and left to dry for 3-5 minutes. Antibiotics were then placed on the lawn, and the plates incubated aerobically at 35-37oC for 16-18 hours. The zones of growth inhibition around each antibiotic disc were measured and reported based on the guidelines of the CLSI .
Screening for suspected carbapenemase production
This involved placing 10μg carbapenem discs viz meropenem and ertapenem (Oxoid, UK) on the surface of Mueller Hinton agar (Oxoid, UK) plates inoculated with each isolate. Following incubation for 16-18 hours at 35-370C, zones of growth inhibition around each antibiotic were read off.
K. pneumoniae isolates that showed a zone of inhibition ≤ 22mm in diameter for meropenem or ≤ 21mm for ertapenem were considered as suspected carbapenemase producers and were subjected to phenotypic confirmation by the modified Hodges test (MHT) [11, 14].
Phenotypic confirmation of carbapenemase production (MHT)
In this method, a suspension of E. coli ATCC 25922 equivalent to 0.5 McFarland turbidity standard was prepared. The E. coli suspension was then diluted 1:10 by adding 0.5 ml of the E. coli suspension to 4.5 ml of saline. A lawn of the 1:10 dilution of E. coli ATCC 25922 was evenly streaked onto Mueller Hinton agar plates using sterile cotton swabs and then allowed to dry for 3-5 minutes. One disc of meropenem (10µg), was placed on the centre surface of the MHA plate. In a straight line, using a sterilised wire loop, the test organisms were streaked from the edge of each Meropenem disc to the edge of the plate. The plates were incubated at 37oC for 24 hours. After incubation, they were examined for a cloverleaf type indentation at the intersection of the test organism and E. coli ATCC 25922 within the zone of inhibition of the meropenem disc as described by the CLSI. K. pneumoniae ATCC 1705 and K. pneumoniae ATCC 1706 were used as positive and negative controls .
Molecular Detection of blaKPC
Bacteria DNA from the K. pneumoniae isolates was extracted using a previously described boiling method for DNA extraction with slight modifications [15, 16]. The extracted DNA was quantified and tested for purity using the NanoDrop® ND-1000 spectrophotometer (Additional file 1: Table S1). The blaKPC gene was detected using a conventional PCR reaction that was based on the protocols and primer sequences previously published by Shanmugam et al., with slight modifications . (Additional file 2: Table S2).
The PCR conditions for blaKPC detection were as follows: initial denaturation at 94ºC for 3 minutes, followed by 30 cycles of denaturation at 94ºC for 1 minute, annealing at 60ºC for 1 minute, extension at 72ºC for 1 minute, then final extension at 72º C for 5 minutes. The products were then resolved at 130V for 25 minutes on 1.5% agarose gel stained with 0.5μg/ml ethidium bromide solution (Nippon Genetics, Europe GmbH) in an electrophoresis tank containing one mMol Tris-Borate EDTA (TBE) buffer. The gels were observed under UV gel Transilluminator (UV DOC, England) at 280nm, and the band pattern observed.
Statistical analysis was done using STATA version 13 (Stata Corp LP, Texas, USA). Prevalence was determined using frequency distribution tables.