Simple multiplex PCR assays to detect common pathogens and associated genes encoding for acquired extended spectrum betalactamases (ESBL) or carbapenemases from surgical site specimens in Vietnam
© Trung et al.; licensee BioMed Central. 2015
Received: 8 November 2014
Accepted: 18 March 2015
Published: 12 April 2015
Surgical site infection (SSI) is common in Vietnamese post-operative patients. It contributes to increased morbidity, mortality, hospitalization time and health care expenditure. Bacterial culture is considered the gold standard procedure to identify SSI pathogens and antibiotic resistant properties; however, it can detect microbes that can readily grow and is time-consuming. We propose optimized multiplex PCR assays to diagnose the most relevant microbes and associated genes encoding for acquired extended spectrum betalactamases (ESBL) or carbapenemases from Vietnamese patients with SSI in a hospital setting in Hanoi.
Ninety-one patients (n = 91) were collected in order to identify microbial pathogens and associated genes encoding for acquired extended spectrum betalactamases (ESBL) or carbapenemases by both conventional bacterial culture and in-house multiplex PCR assays.
Result and conclusion
The novel in-house multiplex PCR assays are comparable to the bacterial culture approach in screening for common pathogens causing SSI and for relevant genotypes conferring betalactam/carbapenem resistance for bacteria. This is the first report of Turkey-specific ESBL gene (PER-1) and two Oxacilinase families (Oxa23 and Oxa 58) in Vietnam.
Hospital-acquired bacterial infection is a common problem in post-operative patients and contributes to increased morbidity, mortality, hospitalization time and health care expenditure . Once surgical site infections (SSI) are detected in patients in medical intensive care, antibacterial/antifungal therapy is implemented. However, the efficacy of antimicrobial treatment largely depends on accurate diagnoses of the microbes causing SSI. To date, bacterial culture is considered the gold standard procedure to identify pathogens that causing SSI. Staphylococcus aureus, Escherichia coli, Enterococcus spp., Pseudomonas aeruginosa, Enterobacter spp., Candida albicans, Klebsiella pneumonia, Gram-positive anaerobes and Proteus mirabilis are the most common causative pathogens of SSI . However, the classical blood culture procedure has two intrinsic problems: (i) it detects only culturable microbes, thus reducing the chance to identify infectious microorganisms from patients treated with antibiotics; (ii) it takes 24 to 48 hours to achieve first test results of cultures, which hampers accurate treatment and risks the patient’s life . Although a broad spectrum of antibiotics is administered post-operatively to control SSI, the increasing occurrence of pathogens resistant to a wide spectrum of antibiotics is alarming, such as meticillin-resistant S.aureus (MRSA) or resistance of betalactamases (ESBL)/carbapenemases enterobacteriacea [4,5]. So far, more than one thousand betalactamase genes encoding either ESBL or the carbapenemase phenotype have been recognized  (http://www.lahey.org/studies/webt.asp). The distribution of these clinical phenotypes differs greatly between geographical settings and displays distinct local patterns. If conventional bacterial cultures fail to generate colonies, antibiotic resistance profiles cannot be generated.
Vietnam is a tropical country where the risk of infectious diseases remains high. The incidence of SSI detected by bacterial culture reaches up to 33% . Only a few sporadic reports on SSI-related pathogens and no conclusive data on resistance phenotypes are available from Vietnam. Systematic statistics on the betalactamase genotypes that cause epidemics are lacking. Own studies indicate that Staphylococcus epidermidis, E. coli, Pseudomonas auriginosa, Streptococcus spp., Klebsiella pneumonia, Enterobacter spp., Staphylococcus aureus and Candida spp. are the most culturable SSI-causative pathogens and we suspect resistance patterns in the genes encoding VEB, CTX-M of ESBL group or NDM-1 of carbapenemase. Here, we introduce simple multiplex PCR assays that provide rapid diagnostic applications and supplement classical bacterial cultures. Furthermore, our multiplexed PCR approach enables to estimate the prevalence of ESBL and/or carbapenem resistance phenotypes in Vietnamese patients with deep incisional SSI.
Material and methods
Eligible study participants were patients hospitalized at Tran Hung Dao Hospital, Hanoi, Vietnam, between February 2012 and December 2012 with at least one of the following conditions: (1) purulent incisional drainage from deep layers of soft tissue within 30 days of the operation or within 1 year of the operation if a prosthesis was implanted; (2) local signs and symptoms of pain or tenderness, swelling, and erythema, with the incision opened by the surgeon or confirmed by the attending surgeon or physician. Patients provided informed consent and the study was approved by the ethics committee of Tran Hung Dao Hospital. Exudates from deep incisional surgical infection sites (aspirate beneath the incision area) were collected using sterile syringes and, in parallel, subjected to bacterial culture or stored at -80°C for molecular diagnostics. Ninety-one patients (n = 91) were enrolled in the study.
For optimization of multiplex PCR assays, well-characterized, both biochemically and molecularly, colonies of Candida albicans, Acinetobacter baumanni, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Staphylococcus epidermidis and Escherichia coli were isolated from clinical isolates at the Department of Clinical Microbiology, Tran Hung Dao Hospital, Hanoi, Vietnam.
For the establishment of bacterial cultures we followed the Manual of Clinical Microbiology . Briefly, 500 μl aliquots of the purulent incisional drainage from individual biopsies was vortexed well in 800 μl sterilized phosphate buffer saline (PBS). The resulting suspension was streaked onto 4 solid media: sheep blood agar, MacConkey agar, chocolate agar and buffered charcoal-yeast extract agar (BCYE) under aerobic conditions at 37°C for 7 days. Colonial growth was confirmed by medical microbiologists at the Department of Clinical Microbiology, Tran Hung Dao Hospital, Hanoi, Vietnam.
Design of genus specific primers for multiplex PCR assays to detect microorganisms
Design of family specific primers for multiplex PCR assays to screen for relevant ESBL and carbapenemase encoding genes
Primer sequence used for screening of genes coding ESBL or carbapenemase
SHPT@ESBL-1(SHV, TEM, CTX-M)
Final concentration (mM)
SHPT@ESBL-2(VEB, GES, PER)
CTGAGCA ACC TGC GCA ATR ATA GCT T
SHPT@Carba-1(NDM, SPM, VIM)
SHPT@Carba-2(IMP, AIM, KPC/BIC, DIM)
SHPT@Carba-3(Oxa23 like group, Oxa48 like group, Oxa58 like group)
DNA extraction and multiplex PCR
Target genes and primer sequences used for screeningof infectious microorganisms
Forward/Reverse Primer (5′ to 3′)
Final concentration (mM)
Multiplex PCR assays enhance positive rate of SSI
Positive rates of Vietnamese SSI specimens detected by multiplex PCR assays and bacterial cultures
Total patient samples
Genotypic prevalence of betalactamases detected from SSI specimens
Hospital acquired bacterial infections after surgical interventions and antibiotic resistances are a major public health threat. Here, we report two optimized multiplex PCR assays that can detect genetics materials of eight most common microbial pathogens, namely Candida albicans, Acinetobacter baumanni, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Staphylococcus epidermidis and Escherichia coli. The assays have detection limits ranging from 10 to 50 CFU/ml human blood (unpublished data). The bacterial identification capability of our multiplex PCR is comparable to that of the conventional blood culture approach, especially in some patient biopsies. PCR was more sensitive than blood cultures.
Genotypic prevalence of betalactamases detected from SSI specimens
Bacterial strain bearing the betalactamse genes
Detected betalactamse genes by multiplex PCR assays
E. coli, K. pneumonia, A. baumanii
E. coli, K. pneumonia, A. baumanii, P. aurigninosa
E. coli, P. aurigninosa
P. aurigninosa, S. aureus, Ecoli
Although PCR based assays are much sensitive and can detect microbes causing SSI, conventional blood culture procedures should not be ignored for obvious reasons. In addition, PCR based assays alone will not allow to get a comprehensive profile SSI related pathogens or SSI associated antibiotic resistance profiles. Therefore, both methodologies should be considered which shall provide vital information on appropriate administration of drugs during SSI.
In summary, we developed simple optimized multiplex PCR assays that clearly enhance the positive diagnostic rate of deep incisonal SSI compared to the conventional bacterial culture approach. Our multiplex PCR assays also quickly detected important associated genotypes that confer betalactam resistance for bacteria. Also this is the first report of Turkey-specific ESBL gene (PER-1) and two Oxacllinase families (Oxa23 and Oxa 58) in Vietnam.
We would like to acknowledge the funding from KC-10.43/11-15 for this study.
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