**2. Materials and methods**

## **2.1 Study site**

The study took place at the MCS-NSIF (Medico-Social Centre of National Social Insurance Fund of Maroua) in Maroua, which is located between 10°35'North latitude and 14°19<sup>0</sup> East longitude [7]. The MCS-NSIF is one of the hospitals in the distric of Maroua I first. It is adjacent to the road that connects the *'Djarma'* crossroads to the third and northernmost entrance of the SODECOTON company. This hospital receives among its patients, those who clinical examinations require the realisation of the antibiogram.

The requirement of an antibiogram by the clinician was the criteria retained for the choice of the samples to be analysed. In compliance with this requirement, a sample of 318 biological samples was taken from patients received at the MCS-NSIF laboratory in Maroua. This sample consisted of 123 urine samples, 71 vaginal samples, 70 stool samples, 27 blood samples, 13 osteitis pus samples, 9 urethral samples and 5 wound pus samples. The material collection were done between the month of january and febuary in 2018.

#### **2.2 Isolation, purification and selection of resistant carbapenem strains**

## *2.2.1 Isolation of enterobacterial strains*

The plating technique carried out near a flame maintained by a Bunsen burner was used to isolate the strains of interest [8]. Biological material that remained attached to the sterile loop handle was streaked onto MacConkey agar in a Petri dish. For the microorganisms to be isolated from the blood, a pre-culture in bovine heart-brain infusion incubated at 37°C for 18 hours in an oven preceded the implementation of the technique.

*Phenotypic Characterisation of Carbapenemases Produced by Enterobacteria Isolated from… DOI: http://dx.doi.org/10.5772/intechopen.102969*

#### *2.2.2 Purification of enterobacterial strains*

The quadrant method was used to purify the strains of interest [8]. One of the colonies from among those having the same appearance during the isolation phase was used for this purpose. The first quadrant was formed by inoculation in tight streaks using a sterile loop. The loop used at this stage of the operation is flamed to red and cooled by touching an unused area of the agar. The Petri dish is rotated at an angle of 90° and the loop is passed once through the first quadrant to form the second quadrant. The same procedure is used to form the other two quadrants.

#### *2.2.3 Selection of strains of interest*

The standardised method for determining the susceptibility of bacteria to antibiotics using ertapenem 10 μg, imipenem 10 μg and meropenem 10 μg was used to select the strains of interest [9]. First, a suspension containing 106 CFU/mL of bacteria for each of the purified strains was prepared. Swabbing for each of the prepared suspensions was performed on Müller-Hinton (MH) contained in a petri dish. The carbapenem discs were placed at a distance of 3 cm from each other in each of the seeded Petri dishes. All prepared Petri dishes were incubated at 37°C for 18 hours in an oven. It should be noted that the disc quality test was validated on E. coli ATCC 29522 reference strains classified as susceptible. The determination of resistant, intermediate or susceptible traits was based on the comparison between the inhibition diameters obtained and those of the EUCAST reference [9].

#### **2.3 Determination of the enzymatic character of carbapenem resistance**

The Carba NP test which is a biochemical colorimetric test was used to demonstrate the enzymatic activity of carbapenem resistance in the strains of interest [10]. A 100 L volume of Tris–HCl B-PER II (Bacterial Protein Extraction Reagent) lysis buffer, 20 mM, pH 7.5 and one colony of bacteria were introduced into each of two 1.5 mL Eppendorf tubes prepared for each strain to be tested. The resulting mixture was homogenised using a 1000 L micropipette. Subsequently, 100 L of solution (A) containing 0.54% (W/V) phenol red and 0.2 mM zinc sulphate was introduced into control tube 1. The same volume of solution (A), this time containing concentrated carbapenem 6 mg/mL, was introduced into test tube 2. Both tubes were incubated at 37°C in the incubator for 2 hours. The appearance of a yellow coloration was interpreted as positive and therefore the presence of carbapenemase, whereas the red coloration was interpreted as negative and therefore the absence of carbapenemase.

#### **2.4 Determination of carbapenemase classes produced by the strains of interest**

The classes of carbapenemases produced by the Enterobacteriaceae were determined using phenotypic inhibition and synergy tests [9]. After swabbing on MH, the antibiotics were arranged with a distance of 3 cm between them. These were imipenem (IMP), ertapenem (ETP), meropenem (MRP), amoxicillin (AMX), cefotaxime (CTX), ceftazidime (CAZ), EDTA, clavulanic acid (CMA), cloxacillin (CXC), cefepime (CFP), piperacillin-tazobactam (PIT) and aztreonam (AZT). The elements used in the algorithm to identify the types of carbapenemases produced by the strains of interest were arranged as follows (**Figure 1**):

#### **Figure 1.**

*Layout of the elements of the carbapenemase class identification algorithm.*


#### **Table 1.**

*Resistance phenotypes resulting from the expression of carbapenemases reported in Enterobacteriaceae without or with extended-spectrum lactamases.*

The classes of carbapenemases produced by the isolated Enterobacteriaceae were determined from the algorithm (**Table 1**).

#### **2.5 Determination of minimum inhibitory concentrations of carbapenems**

The E-test, an agar diffusion technique, was used to determine the minimum inhibitory concentrations (MICs) [12]. The commercially available strip was adapted with easily accessible blotting paper. This blotting paper, cut to the size of 10 cm x 1 cm, was divided into 10 zones of equal size (widthwise) by lines obtained with a pencil. This paper was sterilised in an autoclave at 125°C for 15 minutes. To determine the MICs of the ETP, 640 g of antibiotic was introduced into a sterile 10 mL volumetric flask. This mass was dissolved in 5 mL of sterile distilled water measured with a pipette. After complete dissolution, the volume was made up to the mark to obtain a concentrated solution C1 64 mg/L. Solution C1/2 was obtained by removing 1 mL of solution C1 and adding it to a tube containing the same volume of sterile distilled water. Concentrated solution C1/2n was obtained by pipetting 1 mL of the prepared concentrated solution C1/n into 1 mL of distilled water. Once the 10 dilutions had

*Phenotypic Characterisation of Carbapenemases Produced by Enterobacteria Isolated from… DOI: http://dx.doi.org/10.5772/intechopen.102969*

been obtained, 25 L was taken from each tube using a 50 L micropipette and arranged along with the blotting paper in the corresponding zones respecting the gradient (C1, C1/2, C1/4, C1/8, C1/16, C1/32, C1/64, C1/128, C1/256, C1/512). The same procedure was adopted for the determination of MICs for MRP and IMP.

#### **2.6 Identification of carbapenemase-producing strains**

The identification of carbapenemase-producing strains of Enterobacteriaceae has followed a three-stage procedure [13].
