Phenotypic and Genotypic Detection of extended-spectrum b-lactamase production by Klebsiella pneumoniae Isolated from Different Clinical samples in Baghdad, iraq

The present study was conducted for the phenotyping of antibiotic resistance patterns among patients infected with Klebsiella pneumoniae, isolated from different clinical sites of the patients admitted to the Medical City Teaching Laboratories in Baghdad, Iraq, and to study the frequencies of the blaCtX-M, blateM, and blaOXA genes in the extended-spectrum b-lactamase (ESBL)-producing isolates. A total of 20 out of 35 (57.14%) K. pneumoniae isolates collected from different clinical samples were identified as ESBL producers using the combination disk test (CDT) against six types of antibiotics, as suggested by the Clinical and Laboratory Standards Institute. All K. pneumoniae isolates were observed for ESBL positivity using the CDT method and screened for blateM, blaCtX-M, and blaOXA genes by PCR using a specific primer. In total, 19/20 (95.0%) ESBL-positive isolates harbored the TEM genes, 18/20 (90.0%) carried CtX-M, while the blaOXA gene, for the first time in Baghdad city, was not reported in any of the isolates. A high occurrence of ESBL-producing K. pneumoniae was observed in our study based on the analysis of the TEM and CTX-M genes. Although molecular methods are more reliable in identifying ESBL production, routine clinical screening for ESBL-producing K. pneumoniae by phenotypic methods, such as CDT tests, must be introduced and encouraged in clinical settings because of its low cost.


Phenotypic methods for ESBL pattern determination
Thirty-five isolates of K. pneumoniae bacteria were previously acquired and identified from different clinical samples from patients admitted to the Medical City Teaching Laboratories, Baghdad, Iraq between November 2020 and January 2021. The isolates were phenotypically investigated for ESBL production using the CDT as a screening test, per the guidelines of the Clinical and Laboratory Standards Institute (CLSI) 8 . Resistance to 6 types of drugs at different concentrations (Bioanalyse, Turkey) ( Table 2) was tested using the disk diffusion method, following the manufacturer's instructions 9 . Genotypic methods for ESBL resistance determination Extraction of K. pneumoniae genome DNA extraction was performed using the steps mentioned in Presto TM Mini gDNA Bacteria Kit (Geneaid, Taiwan). After extraction, the gel electrophoresis method was used to detect the presence of the K. pneumoniae genome. The selected genome of K. pneumoniae was then further investigated for the molecular detection of three types of ESBL genes, blaCTX-M-D1, blaTEM-D1, and bla OXA-D-1.

Polymerase chain reaction (PCR) technique for beta-lactamase encoding gene detection
PCR was performed according to the manufacturer's instructions (Bioneer, South Korea). A PCR assay, which was designed in this study and gave the different sizes of bands, was used to detect CTX-M, TEM, and OXA genes (Table 1) (Sentebiolab, Turkey). The following PCR program was used: one cycle of 3 min, initial denaturation at 94°C, followed by 40 cycles of denaturation at 93°C for 1 min, annealing at 54°C for 1 min, extension at 72°C for 1 min, and a final extension step for 10 min at 72°C. Then, 5 ìL each of the amplified PCR products was loaded onto an agarose gel along with a standard molecular weight DNA ladder (Promega, USA) and subjected to electrophoresis as described by a previous study 10 .

Statistical analysis
Data analysis were performed using MS Excel software (version 2010).

Results ANd disCussiON
Phenotypic analysis for antibiotic resistance determination Determination using CDT In total, 35 K. pneumoniae isolates were tested for ESBL production. The results showed that 20 (57.14%) isolates were ESBL positive, which was indicated by an increase in the score of the inhibition zone diameter e" 5 mm with clavulanic acid than without (Table 2), while 15 (42.85%) isolates were ESBL negative, which was indicated by a decrease in score of inhibition zone < 5 mm with clavulanic acid than without (data not shown).
The results of the present study are in agreement with those of a previous study 11 , in which 58.8% of the isolates produced ESBLs, as determined by CDT. The results of the present study also agree with another previous study 12 , that showed that the resistance rate of ESBL-producing K. pneumoniae isolated from different clinical samples was 88.23%, as determined by the disk diffusion test (DDT), used as a screening test for ESBL detection. It was noted that CDT was easily performed and immediately interpreted. The specificity and sensitivity of this procedure were initially recorded to be 96% and 100%, respectively 5 . The high percentages of ESBL-producing isolates of K. pneumoniae detected in this study may be due to patients taking large amounts of third-generation cephalosporins, sometimes without the guidance of a physician, and the widespread abuse of antibiotic administration by patients as well as physicians.

Molecular detection of the ESBL genes
The results of the ESBL phenotyping of the investigated isolates were highly correlated with the genotyping test results. Based on the  results of the CDT test, only 20 (57.14%) out of the 35 K. pneumoniae isolates were ESBL-producing. All three K. pneumoniae genes associated with ESBL production, namely the blaTEM, blaCTX-M, and blaOXA genes, were screened using PCR. Genotypically, 95% (19/20) of the ESBL-positive isolates produced the TEM ESBLs, whereas 90% (18/20) produced CTX-M ESBLs. In contrast, no isolate showed the production of OXA ESBLs ( Table 2 and Figure 1-6). According to the type of the sample, the results of this study showed that genotypically, the highest number of resistance patterns were observed among the K. pneumoniae isolated from urine samples. Locally, the findings of this study were consistent with those of a study in Najaf City 13 that used PCR methods and reported that 60.1% of the isolated K. pneumoniae carried CTX-M genes. In another study in Erbil City, the ESBL-producing K. pneumoniae showed a lower resistance rate than those obtained in this study, with 64.7% harboring blaTEM ESBL genes while 41.1% harboring the blaCTX-M genes 14 .
Another study performed in Najaf City focused on studying ESBL genotypic patterns among patients with urinary tract infections without kidney disease and those with chronic kidney disease. The study revealed that the percentages of the blaTEMand blaCTX-M-harboring ESBL-positive K. pneumoniae isolates were 83.3%, 88.2%, 61.1%, and 6.4% 15 . Genotypically, the percentage found in the current study was higher than that found in Turkey in a previous study 12 that demonstrated that the TEM-type ESBLs were found in 73.33% of K. pneumoniae isolates from different clinical samples. In a study conducted in India, 115 (38%) of 304 K. pneumoniae isolates harbored blaTEM 16 .  With respect to the ESBL blaOXA gene, our results showed that no isolate harbored the OXA genes. The production of beta-lactamases, which inactivate and hydrolyze b-lactam antibiotics, has become the most important resistance mechanism of different bacterial species, particularly among the Enterobacteriaceae family 17 . In this study, ESBL-positive isolates were detected using CDT and PCR. Twenty K. pneumoniae isolates were positive in the CDT test and 19/20 and 18/20 K. pneumoniae isolates were positive for the TEM and CTX genes, respectively, as determined by PCR. The observation of negative ESBLs in the two isolates using PCR confirmed the accuracy of the DDT. SHV and TEM b-lactamases are particularly present in K. pneumoniae and E. coli but may be found in other members of the Enterobacteriaceae family and in non-enteric microorganisms, such as Acinetobacter species 18 . Rapid laboratory identification of these strains is necessary because of their resistance to available antibiotics, the genes responsible for which can be passed to other strains. Molecular typing can identify the type of ESBLs that exist in each isolate. Molecular identification and detection of beta-lactamases are important for the accurate epidemiological detection of antimicrobial resistance.

CONClusiON
In this study, the isolated K. pneumoniae exhibited high resistance to most antibiotics, especially the beta-lactam group, and were considered multidrug-resistant bacteria. Furthermore, K. pneumoniae produced the TEM and CTX-M ESBLs at high rates of occurrence, reaching 95 % and 90%, respectively. This highlights the need to adopt strict protocols related to antibiotic administration in hospitals to control the emergence of extensive antibiotic resistance in different species of bacteria. Because of the significance of ESBL-producing K. pneumoniae and the difficulties involved in the therapy of infections caused by these bacteria, clinical laboratories should adopt a simple test based on the suggestions of the CLSI for the rapid identification and confirmation of ESBL production in Enterobacteriaceae.