A total of 41 Enterobacteriaceae strains from patients with SSI (19 E. coli, 8 Enterobacter sp., 9 Citrobacter sp., and 5 Serratia sp.) were studied. Isolates were received from Hôpital de Référence Saint Joseph (Kinshasa). The clinical samples were collected for diagnostic purposes by the bacteriology laboratories of this hospital, and were from hospitalized patients.
Isolated strains were identified using microbiological conventional methods including Gram staining, oxidase tests, indole and urease production, citrate utilization, hydrogen sulphide, gas production and fermentation of sugars, phenylalanine deaminase, lysine decarboxylase (L.D.C.), ornithine decarboxylase (O.D.C.), arginine dihydrolase (A.D.H.) tests, and methyl red reaction. In our laboratory, strains were picked on MacConkey agar (Liofilchen, Roseto degli Abruzzi, Italy), and were confirmed as Enterobacteriaceae species using the same tests. All cultures were maintained on trypticase soy agar (Liofilchen, Roseto degli Abruzzi, Italy).
Antibiotic susceptibility tests
Antibiograms of each isolated Enterobacteriaceae strains using the diffusion method on Mueller Hinton Agar were realized with the following antibiotic disks (Liofilchen, Roseto degli Abruzzi, Italy): ampicillin (30 µg), amoxicillin-clavulanic acid (30 µg), amikacin (30 µg), cefotaxime (30 µg), imipenem (10 µg), norfloxacin (5 µg), ciprofloxacin (5 µg), kanamycin (30 µg). After incubation of plates at 37°C for 24 hours, diameters of zone of inhibition were measured. Evaluation of the results was done according to the criteria of Clinical Laboratory Standards Institute (CLSI) [19]. E. coli ATCC 25922 were used for quality control. The results of antibiotic susceptibility tests from hospital were confirmed in our laboratory using the same antibiotic disks.
Biofilm formation assay
In present study, we screened all isolates for their ability to form biofilm by Crystal Violet Staining Method (CVSM) as previously described [20], with modifications. A suspension equivalent to the McFarland 0.5 turbidity standard was prepared in trypticase soya broth (Liofilchen, Roseto degli Abruzzi, Italy) for each strain. Accuracy of bacterial counts in the suspension was confirmed by serial dilution in log steps. Polystyrene sterile strips were inoculated with 200 μL of each calibrated bacterial suspension and incubated for 24 hours at 35°C in a humid atmosphere. A control well was inoculated with sterile medium. Each strain was evaluated in triplicate. Medium was removed from the wells which were washed 3 times with 200 μL sterile distilled water. The strips were air- dried for 45 min and the adherent cells were stained with 200 μL of 0.1% Crystal violet solution. After 45 min, the dye was eliminated and the wells were washed 5 times with 300 μL of sterile distilled water to remove excess stain. The dye incorporated by the cells forming a biofilm was dissolved with 200 μL of 33% (v/v) glacial acetic acid and the absorbance of the well was obtained by means of enzyme-linked immunosorbent assay (ELISA) reader, at the wavelength of 540 nm. The results were expressed as variation of Optical Density (OD)540 nm (OD540 nm sample - OD540 nm control). These OD values were considered as an index of bacteria adhering to surface and forming biofilms. For interpretation of biofilm production, the average of the three wells was calculated, and the criterion proposed by Ramos-Vivas et al. [21] was adopted: OD ≤ 0.05, non-biofilm producer; OD > 0.05–0.1 weak biofilm producer; OD > 0.1–0.3 moderate biofilm producer; and OD > 0.3 strong biofilm producer.
Detection of OXA-48 producers
OXA- 48-producing Enterobacteriaceae were detected on ChromaticTM OXA-48 chromogenic medium (Liofilchem, Roseto degli Abbruzzi, Italy). After incubation at 37°C/24 - 48 hours, the color and the morphology of the colonies were observed and the results interpreted according to the supplier’s instructions as follow: red colony (E. coli-producing OXA-48), blue-violet colony (Klebsiella sp. producing OXA-48), blue-green (Enterobacter sp. producing OXA-48), blue colony with red halo (Citrobacter sp. producing OXA-48). E. coli ATCC 25922 was used for quality control.
Statistical analysis
GraphPad software package was used for statistical analysis. Chi-square test was applied. P-value < 0.05 was considered statistically significant.