The right medication can decrease the fee that takes a lot of money when the rapid screening techniques for the detection of extended-spectrum -lactamase (ESBL) producing by bacteria. In the current study, we used the genotypic methods for detection the prevalence of four genes (blaTEM, blaSHV, blaCTX-M, and blaOXA) in Gram-negative bacteria in hemodialysis patients that may be causing increases to spend to treat of infection by bacteria that can produce ESBLs enzymes that lead to in many cases patient dead.
A total of 27 clinical isolates of Gram-negative bacteria that collected from hemodialysis (blood samples, were screened for the prevalence of ESBL genes by used four specific primers to amplification blaTEM, blaSHV, blaCTX-M, and blaOXA genes. Results showed that 24 (88.9 %) out of 27 Gram-negative bacteria were ESBL positive. The highest rate of blaOXA gene was 17 (70.8%) while blaTEM, blaSHV were 13 (54.2%) and black X-M was the lowest (8.3%) out of 24 ESBL positive isolates.
Keywords: ESBLs, blaTEM, blaSHV, blaCTX-M, blaOXA, Haemodialysis.
Extended-spectrum -lactamases (ESBLs) are classically defined as ?-lactamases enzymes with the ability to hydrolyze extended-spectrum cephalosporins (ESCs), such as ceftriaxone (CRO), ceftazidime (CAZ), cefotaxime (CTX), and the aztreonam (ATM) (Livermore and Brown, 2001; Rupp and Paul, 2003; Lal et al.
,2007; Peirano and Pitout, 2010). ESBLs are found in the Gram-negative bacteria and are plasmid-mediated enzymes, In addition, they have been derived from mutations that occurred to the original -lactamases (Pfaller and Segreti, 2006; Kiiru et al., 2012). ESBLs can be blocked in vitro by -lactamase inhibitors such as clavulanic acid and usually retain sensitivity to the cephamycins (i.e. cefotetan and cefoxitin) or carbapenems (i.e. ertapenem, meropenem, and. imipenem) (Nathisuwan et al.,2001; Tham, 2012), that produced by the Gram-negative bacteria and it have been identified among members of the family Enterobacteriaceae and Pseudomonadaceae in different sites but more frequently in Escherichia coli and Klebsiella pneumonia (Rupp and Paul, 2003; Lal et al.,2007; Peirano and Pitout, 2010). A major source of morbidity and mortality among hemodialysis (HD) patients the Infectious complications of the vascular access area. Abundant reports implicate vascular access in up to 48-73% of all HD bacteriemia patients (Nassar and Ayus, 2001; Allon, 2004). Controversy remains regarding the optimal treatment, and few clinical reports comparing the treatment efficacy of antibiotic in HD patients (Pitout and Laupland, 2008). Because not found real studies in Iraq that tackle the causes that lead of increased of morbidity and mortality among hemodialysis (HD) patients in Basrah province hospitals, in the south of Iraq this study suggests the screening techniques for detection of four genes (blaTEM, blaSHV, blaCTX-M and blaOXA) producing by Gram-negative bacteria in haemodialysis patients that may cause increases spend to treated of infection by bacteria that can produce ESBLs enzymes that lead to in many cases patient dead.
A total of 27 Gram-negative bacteria isolated from 147 blood samples from 147 hemodialysis patients in the industrialized kidney unit in the Basrah General Hospital and AL-Sader teaching Hospital in Basrah province of Iraq during the period of October 2017 to January 2018, the patients aged between (9 -78) years old. 1cc of blood was collected from laboratory and nursing staff and before the patient was given anticoagulation substance.
The collected blood was inoculated directly into 10 ml of the brain, heart infusion broth in a sterile condition, after that, the blood was transferred to the bacteriological laboratory at the biology department of science college in Basrah University, and incubation in 37 °C for 7 days with making a subculture each 24 h, on the MacConky agar and subculture repeated for 7 days before discarding the sample (Cheesbrough, 2000). The identification of bacterial isolates was done by using a Vitek2 system test.
Plasmid DNA was extracted from (27) bacterial samples according to (Pure YieldTM Plasmid Miniprep System, Promega, USA).
Five microliters of the template DNA were added to each PCR mixture. The PCR mixture consisted of 25 µl of a mixture of 12.5 microliters Promiga Mastermix, one microliter for each forward and reverse and a final volume completed free nuclease water. The program that used for blaTEM, blaSHV and blaCTX-M PCR amplification consisted of an initial heating step at 95°C for 2 minutes, followed by 30 cycles of 45 s denaturation step at 95°C, a 45s annealing step at 62°C, and a 1min extension at 72°C; and a final 5min extension step at 72°C. While, the program that used for blaOXA PCR amplification consisted of an initial heating step at 96°C for 5 minutes, followed by 35 cycles of 1min denaturation step at 96°C, a 1min annealing step at 60°C, and a 2min extension at 72°C; and a final 10min extension step at 72°C. PCR products were resolved by electrophoresis and visualized with ethidium bromide on 1.5% agarose gels.
The results of the Vitek2 test show that Acinetobacter spp. Recorded the highest rates that represented 8 27(29.6%). Pseudomonas spp. Was the second most frequent bacterial isolates with 727(25.9%) isolates, Whereas Klebsiella pneumoniae and Burkholderia cepacia group represented 4 (14.8%) for each other, while Sphingomonas paucimobilis and Ralstonia mannitoilyfica represented 3 (11.1%) and 1 (3.7%) respectively out of 27 bacterial isolates.
The amplification genes that responsible for production of ESBLs enzymes showed that represent (blaTEM, blaSHV, blaCTX-M and blaOXA) genes in 24 (88.9%) out of (27) Gram-negative bacterial isolates, highest incidence in Pseudomonas luteola which have 3 genes (blaTEM, bla SHV, and blaOXA) and two isolates of Klebsiella pneumoniae (18a) and (18b) which have (blaTEM, blaSHV, blaCTX-M and blaOXA) and (blaTEM, blaSHV, and blaCTX-M) genes respectively, followed by one isolate of Pseudomonas aeruginosa (isolate No.132) and Sphingomonas paucimobilis (isolate No. 76) that have (3) genes (blaTEM, blaSHV, and blaOXA) for each other, whereas other isolates variance to have 1 or 2 ESBLs genes (blaTEM, blaSHV, and/or blaOXA), as shown in (table 1).
Whereas the percentage of enzyme-encoded genes in isolates, among the 4 genes, blaOXA recorded the highest percentage in 17 (70.8%), while blaTEM and blaSHV were 13 (54.2%), while blaCTX-M recorded the lowest percentage in 2 (8.3%) out of 24 isolates.
Recent research showed that the emergence of bacteria has ability to produce ESBL enzymes is more common in patients with a past history of exposure to antibiotics, hospitalization, urogenital surgical procedure, and catheterization (Azap and Arslan, 2010).
The study of enzyme detection at the gene level is one of the few studies carried out of Iraq, especially in the cases of hospital-acquired infections and the detection of four genes belonging to the ESBL enzyme groups, blaTEM, blaSHV and blaCTX-M which inhabited by clavulanic acid, and blaOXA which low or not inhabited by clavulanic acid.
Although the phenotypic method is useful and certified to detect the presence of the ESBL enzyme, it is not possible through which any of the genes that belong to the ESBL enzymes are already present.
This study was conducted to investigate the production of the ESBL enzyme by the genes and using PCR technique to detect the spread of these enzymes. Numerous research or reference laboratories use genotypic techniques amplification of ESBL gene by PCR for the identity of the particular gene responsible for the production of the ESBL enzymes, that have the extra capacity to detect low-level resistance (i.e., Maybe neglected by using phenotypic methods).
Moreover, molecular assays additionally have the ability to be accomplished without delay on clinical samples without culturing the bacteria, with the next reduction of time which needed for detection. In addition to the fact that the genetic method to detect the production of ESBL enzyme is more efficient, accurate and give results more reliable than the phenotypic methods, and avoid the problems that may occur when use phenotype methods such as technical failure and problems of storage. In this study, all Gram-negative bacteria obtained from the blood culture were genetically tested after extraction of plasmid and investigation of ESBL genes (blaTEM, blaSHV, blaCTX-M and blaOXA) presence in plasmid by using PCR technique, and the percentage of ESBL was 88.9 % (24 out of 27).
There are many studies around the world that included study the prevalence of ESBL enzymes, in a study in 2010 by Bali and Co-workers detected the ESBL in Gram-negative bacteria deals with different samples (Urine, Blood, Sputum, Wound, Catheter, CSF and Abscess) by using genotypic methods was 71.3%. Another study by Osman et al.,2017 also in the same line with our result that recorded (73.9%) 28. In Iran, 2009, blaTEM, blaSHV and blaCTX-M was, 18%, 7.5% and24. 5%, respectively (Nasehi et al., 2010). In India both blaTEM and blaSHV were, 67.3%, 20%, respectively (Lal et al., 2007). While this study shows the 54.2% for blaTEM, blaSHV equally and 8.3% for blaCTX-M. A study by Al-Diywania Iraq recorded the prevalence of blaSHV and blaCTX-M in K. Pneumonia in patients undergoing urinary infections was 92%(3538), 78.9% (3038) respectively (Saleh et al., 2018). The prevalence of ESBL -Producing bacteria suggest over use and irrational use of third generation cephalosporins and fluoroquinolones, provide the world brand new resistance bacteria which can produce many different antibiotic resistance enzymes (Soraas et al., 2013).
Using the genetic method and gene amplification can avoid the technical errors that the phenotype requires. It is also a reliable and accurate method for detecting the spread of the enzyme even if the ESBL activity is low.
High prevalence of blaOXA followed blaTEM and blaSHV and low incident of blaCTX-M which, revealed by molecular analysis, among ESBL-Producing Gram-negative bacteria isolate collected from the blood of hemodialysis patients.
Acknowledgment: The authors kindly thank the Research council of Basrah University of life science and the industrial kidney unit in the Basrah General Hospital and AL-Sader teaching Hospital in Basrah province of Iraq to support and overcome difficulties to achieve this study.
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