Frequency of blaCTX-M and blaTEM Virulence Genes and Antibiotic Resistance Profiles among Klebsiella pneumoniae Isolates in Urinary Tract Infection (UTI) Samples from Hashtgerd, Iran

Background: Klebsiella pneumoniae (K. pneumoniae) is an opportunistic microorganism and one of the most important causes of urinary tract infection. This study aimed to evaluate the frequency of K. pneumoniae producing broad-spectrum beta-lactamase in urinary tract infection and to determine the pattern of drug resistance. Methods: This study was performed on 50 samples of K. pneumoniae isolated from patients with urinary tract infection referred to the Medical Diagnostic Laboratory in Hashtgerd city. The isolates were first evaluated for antibiotic susceptibility by disk diffusion method according to the method proposed by the Clinical and Laboratory Standards Institute (CLSI). Then phenotypic detection of ESBLS was carried out by the DDST method. The frequency of gene blaTEM and blaCTX-M was determined by PCR. Results: The highest resistance was observed to ampicillin (94%) and the highest sensitivity was observed to gentamicin (84%). 22 isolates (44%) were positive for ESBLs production. Of the 50 isolates studied, 34% had blaCTX-M and 28% had blaTEM and 11 (22%) had both genes simultaneously. Also, more than 77% of positive ESBLs isolates had the blaCTX-M gene and approximately 63.64% of positive ESBLs isolates had the blaTEM gene. Conclusions: Given the high prevalence of antibiotic-resistant and ESBL-producing isolates, early identification of these resistant isolates and their follow-up is essential to prevent further outbreaks. It is also important to use appropriate therapeutic strategies and proper and rational administration of antibiotics by physicians.


Introduction
Klebsiella pneumoniae (K. pneumoniae) is an opportunistic pathogen and one of the most important bacteria involved in nosocomial infections (1,2). The mortality rate due to this bacterium is high and causes various diseases such as urinary tract infection, septicemia, pneumonia, and intra-abdominal infections in patients hospitalized in different cities of the hospital (3). The basis of appropriate treatment in urinary tract infections caused by K.
pneumoniae is the selection of a highperformance and not expensive antibiotic but the main problem in the treatment of these infections is the resistance of bacteria to a large number of common antibiotics (4). Antibiotic resistance in these bacteria has caused major problems in the treatment of infected patients (4). Increasing the use of antibiotics, especially third-generation cephalosporin, has led to the spread of extended-spectrum beta-Lactamase (ESBLs) producing bacteria (5). Betalactamases prevent the antimicrobial effect of the drug on the synthesis of bacteria walls by destroying the beta-lactam ring in drugs (6). Cefotaximases (CTX-M-ases) enzymes and beta-lactamase (TEM) are the most widespread beta-lactamases in Escherichia coli (E. coli) and K. pneumoniae that their encoded enzymes confer resistance against treatments. Currently, the TEM enzyme is considered one of the most important resistance mechanisms against betalactam antibiotics among gram-negative bacteria.
Cefotaximases (CTX-M-ases) enzymes are a new family of broad-spectrum plasmid beta-lactamases produced by different genera of the Enterobacteriaceae family, including K. pneumoniae. These enzymes cause resistance to penicillin and a wide spectrum of third generation cephalosporins such as ceftazidime ceftriaxone, cefotaxime, and monobactams such as aztreonam. Extendedspectrum β-lactamase (ESBL) genes are located on large plasmids (more than 100 Kbp), which at the same time carry resistance genes to other antimicrobial agents such as aminoglycosides, chloramphenicol, sulfonamides, and tetracycline (6). Also, the information provided by many articles indicates the key role of these genes in resistance to many antibiotics used in the treatment of urinary tract infection (7). Determination of antibiotic resistance patterns in common pathogenic bacteria is important in treatment of infections (5). Accordingly, this study aimed to isolate K. pneumoniae from patients with urinary tract infection, investigate antibiotic resistance, screening of ESBLproducing isolates by the phenotypic method. In addition, the frequency of genes encoding betalactamase enzymes (TEM and CTX-M) in K. pneumoniae isolates producing these enzymes by Polymerase chain reaction (PCR) were investigated.

Samples collecting and detecting bacteria
This cross-sectional study was performed on 50 K. pneumoniae isolated from patients with urinary tract infection referred to a medical diagnostic laboratory in Hashtgerd, Iran, in 2019-2020. Specific biochemical tests used to differentiate species based on fermentation characteristics of sugars by bacteria (in TSI media), indole production, MR and VP test results, movement in SIM, urease, oxidase test, and lysine decarboxylase test.

Identification of ESBLs producing bacteria by combined disk method
To identify bacteria producing ESBLs, the screening test was performed according to CLSI (2019) guidelines using ceftazidime and cefotaxime antibiotic disks (Mast Group Ltd UK) (8). To do this, the inhibition zone of each antibiotic was measured for all bacteria. The combination disk method was used in the confirmatory test. For this purpose, ceftazidime-clavulanic acid (30/10 μg) and cefuroxime-clavulanic acid (30/10 μg) were used by the Disk-diffusion method. E. coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as negative and positive control, respectively.

Polymerase chain reaction (PCR)
Total DNA of bacteria was extracted by boiling method and then PCR was performed for the detection of beta-lactamase, blaTEM, and blaCTX-M genes (9). The reaction mixture consists of 12.5 μl of 2x PCR Master Mix purchased from Amplicon Company (Tris-HCl 0.5 M, MgCl2 2 mM, dNTPs 1.6 mM, Taq 0.04 Units/μl, 0.5 μl of Forward primer and 0.5 μl of each Reverse Primer (0.2 μM) were used for each gene plus 2 μl of DNA (20 ng), and 9.5 μl of sterilized distilled water.
Each PCR program consisted of 30 cycles under the following conditions: Initial denaturation at 94 °C for 10 minutes, 30 thermal cycles including denaturation at 94 °C for 60 seconds, annealing (60 °C for blaTEM and 61°C for blaCTX-M), extension at 72 °C for 60 seconds, and a final extension at 72 °C for 10 minutes. Then, PCR products were evaluated by electrophoresis on agarose gel 1% and after staining with SAFE Stain DNA (CinnaGen co. Iran). Deionized distilled water was used as a negative control and the DNA of K. pneumoniae ATCC 700603 strain was used as a positive control.

Frequency of blaCTX-M and blaTEM genes using PCR
All 50 isolates were subjected to PCR and the results showed that 34% of isolates were blaCTX-M positive and 28% contained blaTEM (Fig. 2). The analysis showed that out of 22 isolates that were phenotypically producing beta-lactamase enzymes, 11 isolates had both blaCTX-M and blaTEM genes, simultaneously (Fig. 3). All isolates with blaCTX-M and blaTEM genes were phenotypically ESBL positive and these genes were not observed in any of the ESBL negative isolates. All 17 and 14 isolates that had blaCTX-M and blaTEM genes, respectively, were also ESBL positive, and out of 22 ESBL positive isolates, 20 isolates (90.9%) had at least one of the two genes, 11 isolates, both genes (50%) and only two ESBL positive isolates (9.1%) lacked both studied genes.

Discussion
The resistance frequency to ampicillin antibiotic we found here was similar to the study conducted by Abdolahi et al. They reported 88% resistance to ampicillin antibiotic (10). In Mansouri et al study, the highest amoxicillin resistance (100%) was detected (11). The susceptibility to gentamicin in this study was 84%, which is consistent with the results by Mohajeri (19). Also, the results of the present study differed from a study conducted by Shams et al in 2015, in which high resistance to ciprofloxacin was reported among K. pneumoniae isolates (20). The results of resistance to ceftazidime and cefotaxime antibiotics in this study with the results of Sedaghatpishe et al who stated resistance to these two antibiotics 54% and 36% respectively and with Asgari et al study in which 24% of K. pneumoniae isolates to two antibiotics ceftazidime and cefotaxime were identified to be slightly different (21,22). However, there are several reasons for the variability of drug susceptibility outcomes of a bacterium in different studies, including study time, differences in geographical logic and origin of samples, the number of isolates studied, as well as the type of disks studied, and the sensitivity of the method studied (23). Another aim of this study was to evaluate the presence of beta-lactamase genes in K. pneumoniae isolates obtained from human urinary tract infections. In the present study, for initial screening, the isolates were first evaluated for resistance to ceftazidime and cefotaxime antibiotics, and then the DDST phenotypic test was performed for confirmatory testing to identify positive ESBLS isolates. Based on the results; 22 isolates (44%) were positive for ESBLs. In the study of Mansury et al, it was shown that 26.3% of the 144 K. pneumoniae isolated from different samples are ESBLs, which is almost different from our study (24). However, the results of this study are consistent with the results of Nematzadeh et al, who stated that 40.8% of K. pneumoniae isolates studied by ESBLs are positive (25). Also, the rate of ESBLs-producing isolates in the studies of consistent with the results of the present study and some of the results are more and less than the present study. The reason for the difference in results can be more related to the number of samples studied and the time and differences in geographical logic and the origin of the samples.
In this study, the PCR method was used to identify blaTEM and blaCTX-M genes. The obtained results showed that; Of the 50 isolates studied, 34% had the blaCTX-M gene and 28% had the blaTEM gene and 11 isolates (22%) had both genes at the same time. Of the 22 isolates in which ESBLs were positive, 20 isolates (90.9%) had at least one of the two genes studied and 11 isolates (50%) had both genes; Only 2 isolates of positive ESBLs (9.1%) did not have both genes studied and also more than 77% of isolates of positive ESBLs had the blaCTX-M gene and approximately 63.64% of the isolates had ESBLs were positive for blaTEM gene. Statistical analysis did not show a significant relationship between these isolates in terms of resistance to the studied antibiotics and the presence of the studied genes. But studies have shown that ESBL was positive in all bacteria containing these genes. However, the two ESBL-positive bacteria did not have any of the two genes studied, which could indicate the role of other genes in resistance to beta-lactam antibiotics. The information provided by many articles indicates the key role of these genes in resistance to many of the antibiotics used to treat urinary tract infections (22)(23)(24)(25)(26)(27)(28). The frequency of the mentioned genes in different studies in Iran is very different and these differences can be due to the clonal release of organisms that produce these enzymes in different regions or hospitals or even wards (21). Comparison of the results of the present study with the study of other researchers has similarities and differences, including the study of Shahcheraghi et al in 2007 in Tehran showed that 32% of K. pneumoniae isolates are resistant to cefotaxime and 31% to ceftazidime and the prevalence of blaSHV and blaTEM to the equations were 69.6% and 32.1%, respectively, which is almost close to the results of this study (29). In the study of Mansouri et al, the prevalence of blaCTX-M and blaTEM beta-lactamase genes was 19% and 16%, respectively, which was lower than the present study (11). Also, the study of Asgari et al showed that 20 isolates out of 25 isolates of positive ESBLs (80%) contained the blaTEM gene and 22 isolates (88%) contained blaSHV gene and 68% contained both genes (22). In the study of Nasehi et al, blaTEM was observed in 26% of Klebsiella isolates (26). In the study of Nematzadeh et al, in 2011 in Tehran hospitals, it was shown that out of 250 isolates of K. pneumoniae isolates, 102 isolates were ESBLs, which is completely consistent with the results of this study and with molecular analysis of the blaCTX-M gene (71.5%). In particular, CTXM-1 and CTXM-3 had 35.61% and 21.9% frequency, respectively. According to their results, the blaCTX-M gene is the most abundant among isolated Klebsiella isolates; which was similar to the results of the present study (25).
The above results show that the indiscriminate use of antibiotics has led to bacterial resistance to antibiotics and the increasing prevalence of ESBL-producing bacteria. ESBL-producing K. pneumoniae prevalent in society, it seems that screening for infections caused by ESBL production will lead to a more effective antibiotic treatment. The results also showed that genes blaCTX-M and blaTEM in the prevalence of ESBLs bacteria have a key role and a blaCTX-M gene more quickly than other beta-lactamaseproducing genes, is increasing; therefore, it is suggested that by changing the strategy of antibiotic use, suitable conditions be provided in different wards of the hospital and, as far as possible, the transmission of these resistances and the spread of related genes be prevented.