A descriptive study was carried out. Microorganisms were preserved in the Collection of Microorganisms of Universidad Católica de Manizales (CM-CUM).

The data was collected from a prior study conducted in patients undergoing mechanical ventilation at the ICU of a hospital in Manizales, Colombia), between 2013 and 2014. The sample consisted of 18 isolates of K. pneumoniae and 7 isolates of E. coli obtained from the supraglottic region of said patients. In addition, 5 isolates of K. pneumoniae obtained from blood cultures, external secretion and peritoneal fluid of patients admitted to the same ICU, but not included in the first study, were also considered for molecular analysis.

The preserved strains were initially grown on nutrient agar and were subsequently inoculated on a selective medium (MacConkey agar). These preserved strains were confirmed in terms of gender and species through the automated system VITEK^® 2 compact (BioMérieux) at the UCM laboratory.

After confirming the identification, antimicrobial susceptibility was determined by means of the disk diffusion method on selective medium (Mueller Hinton Agar), in accordance with the recommendations of the Clinical and Laboratory Standards Institute (CLSI), ¹³ including the following antibiotics for detecting possible ESBL-producing strains: aztreonam, cefotaxime, cefotetan, ceftazidime and ceftriaxone.

For preparing the inoculum, 4 to 5 colonies of similar morphology were selected from each culture and were homogenized in 5mL of saline solution, while the resulting turbidity in the solution was adjusted by using sterile saline solution until a density equivalent to 0.5 on the McFarland standard was achieved. Within 15 minutes after adjusting the inoculum, the spread plate technique on a Mueller Hinton agar was performed by using a sterile swab, and then, antibiotic disks were placed on the surface of the inoculated agar through sterile forceps. The disks were distanced at a 24mm average length. Afterwards, plates were incubated at 37°C for 16 to 18 hours. Finally, diameters of inhibition zones were measured according to the CLSI criteria. ¹³ To confirm ESBL-producing strains, double-disk diffusion method was used in accordance with the the CLSI standard and the following breakpoints: cefpodoxime (≤22mm), aztreonam (≤27mm), ceftazidime (≤22mm), cefotaxime (≤27mm) or ceftriaxone (≤25mm) ¹³.

ESBL production by the strains studied was confirmed through growth measurement against cefotaxime and ceftazidime alone and in combination with clavulanic acid. A ≥5mm difference between the diameter of the disk where ceftazidime + clavulanic acid were used and the disk where only ceftazidime was used, or between the disk were cefotaxime + clavulanic acid were applied and the one where only cefotaxime was used, was considered as ESBL production.

Bacterial DNA was extracted by using the UltraClean^® Blood DNA Isolation Kit (NON-SPIN), which was provided by MOBIO laboratories, Inc. Subsequently, the DNA obtained was stored in Eppendorf tubes at -20°C until its amplification by polymerase chain reaction (PCR) was done.

BOX-PCR typing was performed with the AR1-5'-CTACGGCAAGGCGACGCTGACG-3' primer under the following conditions: 100ng of DNA were amplified in a mini thermal cycler (Bio Rad^®) and were arranged in a reaction volume of 25μL, (14,15) which contained 0.2mM of DNTPs, 2mM of MgCl₂, 1.5μM of primer, 0.10mg/mL of BSA, 10% of DMSO and 1U/μL of Taq DNA polymerase (BIOLINE^®). For the BOX-PCR amplification, first the DNA was denatured for 5 minutes at 95°C and then underwent 30 denaturation cycles (92°C, 30 sec), an association cycle (60°C, 1 min), and one extension cycle (65°C, 8 min), which was followed by a final extension cycle at 65°C for 8 min. Amplified products were separated by electrophoresis on 2% agarose gel with 1.0 X TBE buffer, for 3.5 h at 4.6 V/cm. Banding patterns were recorded in photos ^14,15.

Finally, visual inspection of electrophoretic profiles was done and matrices of presence and absence of bands for the K. pneumoniae and E. coli isolates were constructed by using Microsoft Office Excel software.

The analysis of the matrices was done by using NTSYSpc 2.2 version software. A dendrogram with the unweighted pair group method with arithmetic averages algorithm and Jaccard similarity coefficient was created.

The similarity coefficient (S) was obtained based on the following equation: S=2*N_ab/a+b, where N_ab is the total number of similar bands between 2 isolates and a+b is the total sum of the number of bands of isolates a and b.

The microorganisms used in this study were collected in a prior research that was approved by the Research Ethics Committee of the Universidad Católica de Manizales on April 18, 2012, this approval was updated in September 2018 by this committee, it should be noted that due to the university policies the approvals issued by said ethics committee are not numbered. In addition, participants' informed consent was duly obtained in said research.

Out of the 18 K. pneumoniae strains analyzed, sensitivity to aztreonam, cefotaxime, ceftriaxone, cefotetan and ceftazidime was observed in 11 (61 %), while the 7 remaining strains (3 9%) were resistant to these antibiotics. Intermediate sensitivity was not observed in any of the isolations (Figure 1).

Figure 1 Source: Own elaboration

Then, 12 potentially ESBL-producing strains were selected and they were assigned the following GIBI codes: 104, 106, 162, 157, 335, 344, 330, 338, 340, 332, 331, and 343. Through the disk diffusion technique, an ESBL test was done for each of the strains, the results are as follows: 11 negative isolates and 1 ESBL-producing strain (Table 1).

Table 1

CTX: Cefotaxime; CAZ: Ceftazidime; CA: Clavulanic acid.

Out of 7 E. coli strains analyzed, 6 (86%) showed sensitivity to aztreonam, cefotaxime, ceftriaxone, cefotetan, and ceftazidime. Intermediate sensitivity was not observed in any of the isolations (Figure 2).

Figure 2 Source: Own elaboration

Later, 4 potentially ESBL-producing strains were selected and were given the following GIBI codes: 160, 169, 171, and 334. Results after performing the ESBL production confirmatory test were as follows: negative for 3 isolates, and positive for 1 strain (Table 2).

Table 2

CTX: Cefotaxime; CAZ: Ceftazidime; CA: Clavulanic acid.

Regarding the results obtained through the BOX-PCR fingerprinting technique, out of23 K. pneumoniae isolates, 2 groups with a divergence greater than 50% were observed. In the first group, constituted by 12 isolates, 3 subgroups were identified: subgroup 1, with isolates 62 and 344; subgroup 2, with isolates 72 and 331, and subgroup 3, where isolates 73, 104, 102, 92, 332, 335, 330, and 112 were found. It is noteworthy that isolate 112 had a divergence greater than 50% in comparison with the other isolates of this subgroup. In addition, a 100% similarity between isolates 73 and 104 was observed, as well as between isolates 92, 332, and 335, while similarity among other isolates was less than 90% (Figure 3).

Figure 3 Source: Own elaboration

On the other hand, in the second group, made up by 11 isolates, 3 subgroups were found: isolates 79 and 338 in subgroup 1, isolate 103 in subgroup 2, and strains 164, 168, 106, 157, 163, 162, 340, and 343 in subgroup 3. A 100% similarity between isolates 157 and 163 was observed, while similarity among other isolates in the remaining subgroups was less than 90% (Figure 3).

According to the results obtained after performing the BOX-PCR test, a divergence greater than 50% in the 7 isolates was found. A 92% similarity between isolates 159 and 160 was observed, while for isolates 161 and 334 a 91% similarity was reported (Figure 4).

Figure 4 Source:Own elaboration