Received date: October 16, 2016; Accepted date: November 07, 2016; Published date: November 25, 2016
Citation: Satir SB, Elkhalifa AI, Ali MA, El Hussein ARM, Elkhidir IM, et al. (2016) Detection of Carbepenem Resistance Genes among Selected Gram Negative Bacteria Isolated from Patients in –Khartoum State, Sudan. Clin Microbiol 5:266. doi: 10.4172/2327-5073.1000266
Copyright: © 2016 Satir SB, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Background: Carbapenem-resistant Gram-negative rods (CR-GNR) are gaining increasing importance in healthcare settings, especially in high-dependency units and among critically ill patients. These bacteria are frequently resistant to all antibiotics except colistin, some aminoglycosides and variably tigecycline, posing a serious challenge for treatment. CR-GNR cause infections associated with significant morbidity and mortality. Data on the prevalence of carbapenem resistant genes in Sudan is limited. This study, aimed to determine the prevalence of (CR-GNR) isolated from clinical specimens in khartoum, Sudan during January 2015 to August 2015. Methods: A total of 83 Carbapenem resistant clinical isolates (Klebsiella pneumoniae n=21 Escherichia coli n=7, Pseudomonas aeruginosa n=15, citrobacter n=2, proteus n=1 and Acinetobacter baumannii n= 37 were screened for the presence of carbapenemases (blaTEM, blaVIM, blaIMP, blaSHV, blaCTX and blaKPC genes) by using Multiplex PCR. Results: Out of 83 isolates 68 were Tem gene positive while, 50 isolates were Vim gene positive, Imp gene was present in 42 isolates, Kpc gene in 41 isolates, Ctx gene present in 40 isolates and Shv gene in 15 isolates. TEM gene was the predominant gene among the positive (antibiotic resistant) species. Conclusion: Detection of the genes related with carbapenemase production indicated widespread prevalence and multiplicity of these genes in carbapenem resistant clinical isolates. The results also showed that the multiplex PCR as reliable, fast method for the detection of these genes.
Tem; Vim; Kpc; Imp; Shv; Ctx; Carbapenemases; Multiplex PCR
Multidrug resistance in Gram-negative rod (GNR) is an increasing problem and might lead to dangerous limitations of treatment options. Carbapenems are considered as the most potent agents used for chemotherapy of infectious diseases caused by (GNR) due to their high affinity for PBP 2, stability to most β-lactamases including extendedspectrum β-lactamases, and excellent permeation across bacterial outer membranes . Carbapenemases are versatile β-lactamases that have the ability to hydrolyse penicillin, cephalosporin, and monobactams. Carbapenems exhibit a broader spectrum of antibacterial activity towards Gram-positive and Gram-negative bacteria than other beta-lactams . Resistance to carbapenems, which is caused mainly by carbapenemase production or by porin loss, combined with the expression of beta-lactamases like an extended spectrum beta-lactamase (ESBL) or AmpC . The most prevalent carbapenemases in (GNR) are KPC, VIM, IMP, TEM, and CTX . So far, SHV are rarely found in other members of Gram-negative bacteria . These resistant genes are located on transferable plasmids and can be freely transferred between bacteria, from one region to another and from one country to the other and then spread all over the world . Carbapenemase-producing (GNR) have been associated with increasing mortality and with serious hospital outbreaks that present major therapeutic and infection control challenges . CR-GNR has been associated with the use of medical devices such as: intravenous catheters, ventilators, urinary catheters, and through wounds caused by injury or surgery . Detection of Carbapenems resistance genes producing bacteria may be difficult based on routine antibiotic susceptibility testing. Therefore it is essential to implement efficient infection control actions to limit the spread of these pathogens . Tests based on molecular techniques are considered the standard tests for the identification of carbapenemase genes . Considering the fact that information on the subject is limited in our country, utilization of a suitable method for determination of the carbapenemase production is important in the microbiology laboratory . Therefore, the aim of the present study was to test the feasibility of nucleic acid extraction and a multiplex PCR amplification assay for identification of blaTEM, blaSHV blaIMP, blaKPC, blaVIM and blaCTX genes in a series of clinical isolates of (CR-GNR). Such data serve an important role in understanding the spread of carbapenem-resistant gram-negative pathogens.
A total number of 83 CR-GNR isolates including (Psedumonas aeruginosa, Acintobacter bumannii, Klebsiella pneumonia, Echerichia coli, Citobacter frendii and Proteus mirabilis) and 12 negative controls isolated from three private hospitals, during January till August 2015, Khartoum, Sudan were investigated using multiplex PCR. The isolates were identified using microbiological and biochemical methods.
Antibiotic resistance investigation
Measurement of antibiotic resistance was conducted by disk diffusion method for the following antibiotics: Ceftazidime (CAZ), Cefteriaxon (CRO), Cefotaxime (CTX), Cephalexin (CN30), Cefixime (CFM), Penicillin (P), Cefuroxime (CXM), Meropenem (MRP) and Imipenem (IMP) according to the standards of Clinical and Laboratory Standards Institute (CLSI).
Investigation of carbapenem resistance genes by PCR
Carbapenem resistant isolates were used to investigate bla-VIM, bla-IMP, blaTEM, blaSHV, blaKPC and blaCTX genes by PCR. For DNA extraction the boiling method was applied . Firstly, three to five colonies were picked from fresh culture medium and then a suspension was prepared using 200 ml of distilled water boiled at 100?C for 30 minutes. The suspension was then centrifuged at 12000 rpm for 30 minutes, and the supernatant containing DNA was transferred to new Eppendorf tubes for PCR in order to amplify the genes, (bla-VIM, bla-IMP, blaKPC, blaTEM and bla-CTX ). The primers used to amplify the genes are shown in the Table 1.
To perform PCR, 2 μl from the primers, 5 μl of the extracted DNA, 13 μl of distilled water was added to the PCR Master Mix (Maxime PCR Permix Kit, Korea) with a final volume of 20 μl. Thermal cycling (Aeris Machine Pelter technology Thermo Assisst) for 30 cycles was done at 94°C for 1 min, 60°C for 1 min and 72°C for one and half min. The final extension step was performed for 5 min at 72°C. The PCR products were applied and electrophoresed in 2% agarose gel along with ladder DNA and then stained using ethidium bromide. The result was observed by transillumiator system (Biometer an analytical Jena company) (Figure 1 and Figure 2).
The results of the Multiplex PCR for the six target genes are shown in Table 2.
Table 2: Results of Genotypic test (Multiplex PCR).
Total of 99 clinical isolates which successfully grew, 83 were identified to be resistant to Meropenem, while 12 isolates were susceptible to Meropenem.
Out of 83 CR-GNB 37 (44.5%) were A. bumanii as the most predominant species followed by K. pneumonia 22 (26.5%), P. aeruginosa 14 (16.8%), E. coli 7 (8.4%), C. frendii 2 (2.4%) and P. mirabilis 1 (1.2%). The prevalence of the resistance genes in the 83 resistant isolates were as follows TEM (81.9%) was the most predominant gene, VIM (56.6%), IMP (50.6%), KPC (45.7%), CTX (48.1%) and SHV (18.1%). A. bumannii, K. pneumoniae, P. aeruginosa, E. coli isolates harbored all six resistance genes (TEM, KPC, CTX, IMP, VIM, SHV), C. freundii contained five of the genes but lacked (SHV ) gene, While P. mirabilis on the other hand contained only one gene (CTX ).
Different antibiotics including beta lactamases, aminoglycosides and quinolones are applied to treat the infections caused by Acintobacter baumannii, Klebsiella pneumonia, Pseudomona aeruginosa, E. coli, Proteus mirabilis and Citrobacter freundii. However, it is becoming a great challenge to treat infections caused by these bacteria due to its resistance against drugs and the rapid changes in the pattern of resistance. Additionally, the resistance of the bacteria against antibiotics fluctuates, especially regarding Imipenem, Carbapenems and Beta lactam antibiotics are being used increasingly to treat infections due to CR-GNR since they are resistant against most of the beta lactamases and have great membrane permeability . Carbapenems and Beta lactam antibiotics are used considerably to treat infections due to multidrug-resistant Gram Negative bacteria. Resistance of Carbapenem agents is due to carbapenemase and presence of other resistance mechanisms, such as ESBLs, and porin mutations . In the current study, these genes were mostly detected in a high percentage of antibiotic resistant isolates of A. bumannii , K. pneumoniae, P. aeruginosa, E. coli, C. freundii and P. mirabilis. findings where TEM is reported as the commonest MBL to be found (81.9%), followed by VIM (56.6%), IMP (50.6%), KPC (45.7%), CTX (48.1%), while SHV (18.1%) had the lowest prevalence in the tested resistant isolates. Of 83 bacterial isolates detected of having carbapenemase genes, 70 had multiple genes coding for carbapenem resistance especially in A. baumanni and K. pneumoniae. The presence of multiple resistance genes in one strain provides selection advantage of these strains; such phenomenon has not been commonly detected in a large number of studies probably due to the limited number of genes studied since most of the studies research on one or two genes. Since various reports indicate the increased resistance of these bacteria against antibiotics (especially imipenem), proper use of these antibiotics and time-consuming identification of isolates generating MBL should be considered. This can lead to successful treatment and prevents propagation of resistant genes, which can be seriously harmful for societies . Testing of carbapnem resistant genes among MDR-GNB isolates should routinely be used to determine these species in clinical laboratories. In addition, increased effort at discovery of more effective antimicrobial compounds with new effecting mechanisms should be noted. Finally, as the most of MDRGNB infection are hospital acquired every effect should be exerted to prevent spread of these bacteria in our health facilities.
Identification of carbapenem resistance and their resistance pattern of genes are necessary for the surveillance of their transmission in hospitals to overcome the problems associated with G-ve carbapenem resistance. The multiplex PCR described here is a reliable and rapid method for detection of the most prevalent carbapenemase genes.
The authors declare that they have no competing interests.
SBS carried out the molecular genetic studies and drafted the manuscript. AIE conceived of the study and contributed to the conception and design of the study. AME, IME and MAA revised the manuscript. KAE contributed to the conception and design of the studyand acquisition of funding. All authors read and approved the final manuscript.
The current study was supported by Central laboratory.
Authors wish to thank the manager of the center and his colleague for useful discussions and critical reading of the manuscript. We thank also Microbiology department, RCIH for their efforts specially Prof. Samia A.Gomaa.