The frequency of Klebsiella pneumonia encoding genes for CTX-M, TEM-1 and SHV-1 extended-spectrum beta lactamases enzymes isolated from urinary tract infection

Background The extended- spectrum β-lactamase producing bacteria are widely spread worldwide. The productions of these enzymes cause bacterial resistance to a wide range of antibiotics. The aim of this study was to investigated the frequency of K. pneumonia encoding genes for CTX-M, TEM-1 and SHV-1 extended-spectrum beta lactamases enzymes isolated from urinary tract infection. Methods This study is cross-sectional study. All K. pneumonia isolates from urine samples, which had grown on media culture more than 105 were delivered to the medical microbiology laboratory. K. pneumonia susceptibility of 198 samples were confirmed by disk diffusion. The gene frequency of genes was determined using PCR, and analyzed using SPSS version 21 software. Finding Most of the K. pneumonia isolated from urine producing β-lactamase were resistant to cotrimoxazole (53.2%) followed by cefotaxime (50%), ceftazidime, ceftriaxone (40.3%), nalidixic acid (17.8%), amikacin and imipenem (1.6%) and meropenem (0%) respectively. Out of the 198 confirmed isolates of K. pneumonia, 62 cases (31.3%) have the gene phenotype of broad spectrum β-lactamase enzymes and highest frequency of gene phenotype was related to the SHV-1 gene (85.5%). Then in the terms of abundance from highest to lowest CTXM-3 (56.5%), CTXM-1 (27.4%), TEM-1 (16.1%) and CTXM-2 (8.1%), were respectively. Conclusion This study showed that K. pneumonia isolated from urine producing β-lactamase were resistance to a wide range of antibiotics. Due to the increasing resistance of most antibiotics, control and supervision in the use of antibiotics and identification of broad spectrum β-lactamase enzymes by phenotypic methods appears to be essential.

These enzymes are derived from SHV-1. They are the first enzymes of SHV class which are found in K. pneumonia and are responsible for 20% of ampicillin resistance in the present samples [8,9].
There exists a structural likeness between SHV-1 and TEM-1 in general, and a 68% amino acid similarity. More than 50 SHV varieties have come to be known based on the replacement of amino acid combinations.

β-Lactamases CTX-M type (class A)
CTX is representative of the high hydrolysis power of β-lactamase against cefotaxime. More than 40 CTX-M enzymes are known so far, which can be classified in five groups. These enzymes are capable of cephalothin hydrolysis better than penicillin and cefaloxine, and more than Ceftazidime. CTXM type is predominantly found in three geographic areas; South America, The Far East, and Europe [10,11].
The aim of present study was to indicate the frequency of Klebsiella isolates encoding SHV-1, TEM-1, CTXM genes and the broad spectrum β-lactamase enzymes in urinary tract infection (UTI) patients in Yasuj City, Kohgiluyeh and Boyer-Ahmad province, Iran, during 2014-2015.

Methods
This study sought to determine the prevalence of K. pneumonia isolates encoding SHV-1, TEM-1, CTXM genes and the broad spectrum β-lactamase enzymes in UTI patients in Yasuj City during 2014-2015. In this sense, statistical, cross sectional, and descriptive analysis methods were employed.

Ethics approval and consent to participate
Ethics approval (MEC: 93.05.06.07) was obtained prior to the start of this study.

Sampling and culture methods
This study was carried out during October 2014 to August 2015. Urinary samples of patients confirmed with UTI from three laboratories around the city were sent to microbiology laboratory of Yasuj University of Medical Science. Then, each sample was cultured on EMB media. Further diagnostic tests followed as described below:

Phenotyping test for ESBL production
Confirmatory tests for ESBL production based on CLSI standards were carried out on the bacteria. First, bacteria were inoculated on MHA media culture containing ceftazidime and cefotaxime disks. Both plates were inoculated alike and kept on incubator for 18-24 h. The zone of inhibition diameter (ZOID) was measured around each disk and ZOIDs more than 5 mm were considered positive [12].

Polymerase chain reaction (PCR)
Bacterial genome extraction by boiling method: One colony from fresh bacteria was solved in 300 ml distilled water and homogenized with vortex and kept on thermo block in 100° C for 10 min, and then centrifuged in 12,000 rpm for 10 min and the supernatant containing genome was used for PCR [13].
Samples were put into gel wells which had already been prepared. Then the electrodes were connected (red wire was the sign of positive electric charge which was placed in the bottom and black wire was the sign of negative electric charge sign which was placed next to gel holes). After the connection of electrodes, the instrument was adjusted to 100 voltage for 1 h with 700 mA flow (current strength), and then turned on.

Photography from gel
The gel was placed inside the device and then the device was turned on with the correct setting (Table 1).

Data analysis method
For data analysis, SPSS software version 21 was applied. Both descriptive statistics such as frequency, mean, standard deviation, graph, and statistical inferential tests such as Chi square, fishers exact test, independent T test, and one-way variance analytic test were used. It is worth noting that in this study, before inferential statistical tests with Kolmogorov-Smirnov test, variable study from normal distribution was reviewed and approved.

Finding
Out of 986 samples suspicious to K. pneumonia UTI, 198 (20.1%) were confirmed based on Klebsiella diagnostic test. The results are summarized in Table 2.
Based on Table 2 out of 198 samples, 138 were female patients and 60 were males. The minimum and maximum ages were 1 month to 68 years respectively with the standard deviation of 16.33 ± 17.92.
Frequency distribution of K. pneumonia isolates according to broad spectrum β-lactamase phenotype and sex are revealed in Table 4.
All K. pneumonia isolates were susceptible to meropenem and most antibiotic resistance in both genders were observed with Co-trimoxazole. No statistically significant difference was seen between antibiotic resistance of isolates by gender or age. Also, presence of β-lactamase genes such as TEM-1, SHV-1,CYXM-2, and CTXM-3 confirmed with PCR. No statistically significantly differ was observed among individuals of various ages. As Table 6 showed the most commonly ESBL producing gene was belonged to SHV-1(85.5%), followed by highest

Discussion
Klebsiella pneumonia is a common cause of nosocomial infections such as UTI. The importance of UTI is that if not diagnosed or diagnosed too late it will lead to renal failure. β-Lactam drugs are one of the most effective drugs in UTI treatment. The ESBL bacteria, with inactivation of a wide range of β-lactam drugs especially cephalosporin and monobactam, cause treatment failure and increase healthcare costs. Therefore, the study of gene resistance to β-lactamase is important. It seems that the emergence and spread of these bacteria are due to prolonged hospitalization, increased consumption of β-lactam antibiotics (especially ceftazidime), use of catheters, and experimental treatments against antibiotic-resistant [16].
The aim of this study was to determine the prevalence of K. pneumonia producing ESBL and molecular diagnosis of B lactamase genes such as β-lactamase CTX-M, β-lactamase TEM, and β-lactamase SHV).
In present study out of 198 samples, 62 (31.3%) were ESBL productive (27 separated from males and 35 from females). Published result from different scientific researches related to ESBL production in Iran is very diverse ranging from 9.8% to 75.7% depending on infection control system and therapeutic regimens. Another study from Bazaz et al. reported the prevalence of ESBLs in K. pneumonia and E. coli bacteria as high as 59.2%. In another research on 218 bacterial strains, 70 (32%) strains of Klebsiella and 49 (70%) strains of K. pneumonia contained ESBL [13]. Reports on ESBL production rate in K. pneumonia isolates from different countries also show significant variations. In a study in Japan and USA the prevalence rate was reported 40 and 44% respectively. However, in Southeast Asia, the prevalence rates of ESBL reported varied from 20% to more than 60%. ESBL production in India, Taiwan, Thailand, Pakistan, and Korea were reported 44, 8-29, 64, 36-56 and 22% respectively [14,15].
The prevalence rate of ESBL production was reported by Ajas et al. in Lahore Pakistan 71% [16].
Due to emergence of bacterial resistance and ESBL production In a region compared to other regions is different. Also, the treatments of this infection with cephalosporins and other broad-spectrum antimicrobials have led to the emergence of multi-drug resistance. In this study, bacterial resistance was shown to be 30.4, 50, and 61.1% respectively in the case of cephalosporin-ceftriaxone and imipenem.
Ishim in Japan determined that the imipenem as an effective antibiotic for K. pneumonia treatment [18].
PCR results in our research on β-lactamase Drug resistance to antibiotics in different regions of Iran and worldwide is differs due to genetic variations in causative strains and the use of antibiotics, access to broad spectrum and new antibiotics, and difference of climate conditions.

Conclusion
Due to insufficient information about ESBL gene plasmid abundance and genetic pattern in Iran, the diagnosis of K. pneumonia strains containing β-lactamase enzyme for better treatment and prevention of the spread of these genes to other bacteria is essential by genotyping and phenotyping methods. However, this study showed that this bacterium is one the most health problem in south west of Iran and more cure should be done for prevention of infection and resistance spread to other area.