Establishment of a Novel Target-Based Real-Time Quantitative PCR Method for Acinetobacter baumannii Detection : Diagnostic Molecular Pathology

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00019606-201112000-00008ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2011 Lippincott Williams & Wilkins, Inc.20December 2011 p 242–248Establishment of a Novel Target-Based Real-Time Quantitative PCR Method for Acinetobacter baumannii DetectionOriginal ArticlesZhang, Lei MSc; Ding, Gangqiang MD; Wei, Lanfen BSc; Pan, Xieshang BSc; Mei, Lingling PhD; Zhang, Yanjun MD; Lu, Yiyu PhDCenter for Disease Control and Prevention of Zhejiang Province, Hangzhou, Zhejiang, P.R. ChinaSupported by grants from the Provincial Medical Research Fund of Zhejiang, China (2004B022) and from the Monitor Technology Platform of Infectious Diseases of the State Major Science and Technology Special Projects during the 11th 5-year plan of China (2009ZX10004-210).Reprints: Yanjun Zhang, MD, Center for Disease Control and Prevention of Zhejiang Province, 630 Xin-Cheng Road, Hangzhou, Zhejiang 310051, P.R. China (e-mail: [email protected]).AbstractBiofilm formation is a well-known pathogenic mechanism in infections caused by Acinetobacter baumannii. Recently, a biofilm synthesis-associated gene has been found in A. baumannii ATCC19606. Bioinformatic analysis showed 2 transmembrane structures and an hmsS superfamily domain, which was related to biofilm formation. What is more, high homology sequences of the bfs gene were only present in A. baumannii spp., and the similarities of nucleotide sequences of the bfs gene from A. baumannii strains ATCC17978, ACICU, S1, AB307-0294, and AB0057 compared with the reported sequence of bfs (GenBank accession No.: NZ_GG704572) were all above 95%. The distribution and conservation of the bfs gene from clinically derived A. baumannii strains were verified through conventional polymerase chain reaction (PCR). After this, we established a bfs gene-based real-time quantitative PCR assay to detect A. baumannii. Species specificity and sensitivity assays were designed and validated. By using this method, all the A. baumannii strains separated from clinical samples were identified and showed good accordance with the results from biochemical identification. This study is the first report of developing a bfs gene-based quantitative polymerase chain reaction for rapid, stable, and specific detection of A. baumannii. This method can be applied to clinical laboratory diagnosis, and detection of A. baumannii present on medical instruments.Acinetobacter baumannii is widely distributed in hospital environments and particularly affects critically ill patients in intensive care.1–3 It is now considered to be an important opportunistic nosocomial pathogen. Treatment of A. baumannii infection has become difficult, because many strains are resistant to a wide range of antibiotics.4–6 The emergence and increase in the number of multidrug-resistant strains have been caused by the extensive use of antimicrobial chemotherapy. Antimicrobial susceptibility testing, biological and genomic typing of Acinetobacter isolates have shown the dissemination of drug-resistant strains in various hospitals.7–10 However, A. baumannii can survive severe conditions by forming biofilms that also contribute to the mutidrug resistance. Biofilms are complex matrices that contain proteins, ions, nucleic acids, and polysaccharide polymers.11–15 The ability to form biofilms on medical devices16,17 and to colonize skin and mucosal surfaces of vulnerable hosts is associated with a high prevalence of A. baumannii strains in the hospital in epidemic and endemic situations.18–20 The ability of A. baumannii to adhere to epithelial cells and to form biofilms on glass and plastic surfaces has been studied.21,22 The production of pili and a biofilm-associated protein and the expression of antibiotic resistance are needed for robust biofilm formation on abiotic and biotic surfaces.16 In short, the ability of A. baumannii to adhere to and persist on surfaces as biofilms could be central to its pathogenicity.Identification and quantitative analysis of A. baumannii in environmental samples would provide the opportunity for early intervention. Quick and reliable identification of Acinetobacter spp. remains challenging.23 Cultures from clinical specimens are often considered to be the “gold standard.” However, it is time consuming and has low sensitivity. As a result, modern molecular diagnostic methods need to be developed to facilitate its detection and identification in clinical samples. Quantitative polymerase chain reaction (QPCR) is useful for quick and quantitative analysis of microorganisms in environmental samples.24–26 On the basis of a unique target, QPCR detection could provide a simple and convenient method for A. baumannii identification. This would be easier and more reliable than current methods, such as amplified ribosomal RNA gene restriction analysis, and biochemical identification. As A. baumannii is clinically by far the most significant of the Acinetobacter species, the ability to distinguish it rapidly from other members of the genus would be valuable.The purpose of this research was to develop QPCR based on the biofilm synthesis (bfs) gene to identify A. baumannii. This may be useful for monitoring A. baumannii in hospital environments. The extent of environmental contamination by A. baumannii could also be evaluated rapidly by QPCR, and prove highly useful to clinicians and infection control staff.MATERIALS AND METHODSA. baumannii Strains and Culture ConditionsThe A. baumannii ATCC19606 strain and other standard strains listed in Table 1 were maintained by the Center for Disease Control and Prevention of Zhejiang Province, China. Forty clinical isolates of A. baumannii strains were obtained from different inpatients in Hangzhou Third People's Hospital, Zhejiang, China. Sheep blood agar was used for isolation and purification of the A. baumannii strains. All strains were routinely cultured in Luria-Bertani agar or broth at 37°C.17,27JOURNAL/dimp/04.03/00019606-201112000-00008/table1-8/v/2021-02-17T200039Z/r/image-tiffStandard Bacterial Strains Used in This StudyBfs Gene Sequence Analysis and Designation of PrimersThe conservation of the bfs gene and the transmembrane domain in the actual Bfs sequence were analyzed by using Blast software (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and TMHMM Server v2.0 (http://www.cbs.dtu.dk/services/TMHMM/). The Primer software package (Premier, CA) was used for primer designation. All the primers used in this study were synthesized by Invitrogen Co. (Shanghai, China) and their sequences are given in Table 2.JOURNAL/dimp/04.03/00019606-201112000-00008/table2-8/v/2021-02-17T200039Z/r/image-tiffNucleotide Sequences of Primers Used in This StudyConventional PCR Amplification and SequencingGenomic DNA from A. baumannii strain ATCC19606 was extracted by using a Bacterial Genomic DNA Extraction Kit (BioColor, Shanghai, China) and then dissolved in TE buffer. Density and purity of the extracted DNA were detected by ultraviolet spectrophotometry. A High Fidelity PCR Kit (TaKaRa, China) was used to amplify the target gene. The total volume per PCR was 100 μl, which included 20 pmol of each of the primers BSPF and BSPR (Table 2), 2.5 U EX-Taq DNA polymerase, and 100 ng DNA templates. The reaction mixture was initiated by incubation at 94°C for 5 minutes, followed by 30 cycles of amplification at 94°C for 30 seconds, 50°C for 30 seconds and 72°C for 30 seconds, and then incubated at 72°C for 10 minutes. The products were detected in 1.5% ethidium bromide-prestained agarose gel after electrophoresis. To obtain accurate sequence data, the PCR product was purified with a PCR Product Purification Kit (BioColor, Shanghai, China). Purified PCR products were sequenced by Invitrogen. DNA sequences were compared with the National Center for Biotechnology Information database. All PCR amplifications were performed in duplicate.Identification of Clinical Isolates and Distribution of the bfs GeneBiochemical identification of the isolates was performed using the analytical profile index procedure (bioMe´rieux, Marcy, France). After this, species identification was confirmed by amplified fragment length polymorphism.28,29 Genomic DNA from different A. baumannii strains was extracted and used as a template. With optimal amplification conditions, the distribution of the bfs gene in different A. baumannii strains was analyzed.QPCRReal-time QPCR was performed using the SYBR Premix Ex-Taq II Kit (TaKaRa). Each reaction mixture consisted of 200 nM of the primers BfYF and BfYR (Table 2), and 4 μL of template DNA in a final volume of 50 μL. According to the instructions, QPCR reactions were performed with the following program: 95°C for 30 seconds, followed by 40 cycles of 5 seconds at 95°C, and 34 seconds at 60°C. Melt curve analysis (55°C to 95°C in 0.5°C steps for 10-s increments) was accomplished. All reactions were performed in duplicate. Fluorescence signals were measured every cycle at the end of the annealing step and continuously during the melt curve analysis.Specificity of QPCR AssaysTo evaluate whether the species-specific primer pair amplified only the Acinetobacter species of interest, we used DNA extracts from the array of bacterial species listed in Table 1, which were prepared as described above. DNA template from A. baumannii ATCC19606 was used as a positive control. These extracts were analyzed in duplicate reactions on an ABI 7500 sequence detection system by amplifying standards of known concentrations that were processed in the same manner as the unknown samples. A cycles of threshold value of 40 (maximum number of PCR cycles run) indicated that DNA from the test strain was not detected.Sensitivity Determination of QPCRThe numbers of A. baumannii ATCC19606 CFU/milliliter were determined before DNA extraction. As a result, the concentrations of initial target DNA in QPCR amplification mixtures, which were averaged for sample replicates, were converted to the numbers of A. baumannii templates per sample. DNA templates from tenfold serial dilutions of A. baumannii were analyzed by QPCR, and the results were used to generate a standard curve.QPCR Detection of Clinical IsolatesGenomic DNA extracted from all the A. baumannii strains separated from clinical samples was used as templates. With the primers (BfYF and BfYR), the QPCR developed in this study was applied to identify A. baumannii. The amplification procedure was the same as described above.RESULTSBioinformatic AnalysisAfter acquiring the bfs gene from Genebank, the sequence of the bfs gene from A. baumannii ATCC19606 was subjected to homology analysis through blast in the National Center for Biotechnology Information. We found that the bfs gene sequence was only conserved in A. baumannii spp. The similarities of nucleotide sequences of the bfs gene from A. baumannii strains ATCC17978, ACICU, S1, AB307-0294, AB0057, and AYE compared with the reported sequence of bfs in A. baumannii strain ATCC19606 (GenBank accession No.: NZ_GG704572) were 97% to 98% (Fig. 1). The conservation of the bfs gene in A. baumannii spp. not only suggested that it may play an important role, but also indicated that it may be a useful marker for A. baumannii identification. Conserved domain searching and transmembrane structure prediction were also accomplished (Fig. 2). Two transmembrane structures were predicted in Bfs. Besides, an hmsS superfamily domain related to biofilm formation was found in the product of the bfs gene (Fig. 3). As a result, we suggested that Bfs is an outer membrane protein associated with biofilm formation by A. baumanni.JOURNAL/dimp/04.03/00019606-201112000-00008/figure1-8/v/2021-02-17T200039Z/r/image-jpegHomology comparison of bfs gene sequences from different Acinetobacter baumannii strains. A, bfs gene sequence from A. baumannii ATCC19606. B to G, bfs gene sequences from A. baumannii ATCC17978, ACICU, S1, AB307-0294, AB0057, and AYE.JOURNAL/dimp/04.03/00019606-201112000-00008/figure2-8/v/2021-02-17T200039Z/r/image-jpegDiagrams of predicted transmembrane structures of Acinetobacter baumannii Bfs protein. Two significant transmembrane structures were found between amino acids 35 to 57 and 77 to 99.JOURNAL/dimp/04.03/00019606-201112000-00008/figure3-8/v/2021-02-17T200039Z/r/image-jpegDiagrams of predicted conserved domain structure present in Bfs protein; an HmsS domain structure was found in Bfs.PCR Amplification and Distribution of the bfs GeneAfter PCR amplification using genomic DNA from A. baumannii ATCC19606 as template, the product was visualized on a 1.5% agarose gel (Fig. 4). According to the size of the bfs gene described above, the size of the amplification product was correct. The PCR product was purified before sequencing, and it was found that the sequence of the target product was the same as the reported bfs gene. This confirmed that, with the primers BfS and BfR, the bfs gene from A. baumannii ATCC19606 was amplified. Furthermore, the amplification conditions were optimized. Hence, this procedure can be applied to identify the distribution of the bfs gene in A. baumannii from clinical samples. Using the genomic DNA from clinical isolates as templates for amplification, the PCR product sizes were determined from agarose gels. The amplification results were the same and consistent with the size expected in each case (Table 2). This further confirmed that the bfs gene was present in every clinical A. baumannii strain.JOURNAL/dimp/04.03/00019606-201112000-00008/figure4-8/v/2021-02-17T200039Z/r/image-jpegPolymerase chain reaction (PCR) amplification results. M indicates marker; 1, negative control; 2, PCR amplification product of the entire bfs gene from Acinetobacter baumannii ATCC 19606 using primers BSPF and BSPR; 3, PCR amplification of a fragment of the bfs gene from A. baumannii ATCC 19606 using primers BfYF and BfYR.Optimization of QPCR ConditionsBefore the primers designed according to the sequences of the bfs gene were further used in QPCR, a conventional PCR was performed. The results (Fig. 4) suggested the specificity of the primers. Sequencing of the amplified target product was also accomplished, and showed good agreement with the reported corresponding sequence of the bfs gene. On the basis of conventional PCR, the optimal annealing temperature for each assay was determined. The highest temperature that generated significant specific product, as visualized on an agarose gel, was selected as the annealing temperature for establishing QPCR. The melting curve from each product was examined. As shown previously, the primers (BfYF/BfYR) were used and a significant primer dimer melting peak was found (data not shown). However, with the concentration of the primers reduced, clean melting curves were obtained.Specificity and Sensitivity of QPCR AssayGenebank was explored to find out whether the primer sequences paired with sequences other than bfs from A. baumanni, but after searching through BLAST, no close hits from genes other than bfs were found. Then, the specificity of the targeting of primers BfYF and BfYR to the biofilm synthetase gene bfs was also tested by conventional PCR. DNA from A. baumannii, Escherischia coli, Staphyllococcus aureus, Candida albicans, and Pseudomonas aeruginosa was used as templates. The target product was only amplified from the DNA of A. baumannii. As a result, the primers were specific for the bfs gene of A. baumanni. On the basis of the amplification results of conventional PCR, the specificity of the primers used in this study were further confirmed by QPCR. In QPCR, only the A. baumannii ATCC19606 strain gave cycles of threshold values over the detection limit, and therefore the results of QPCR also showed the specificity of the primers for A. baumannii strains. The QPCR products were also visualized on an agarose gel to confirm that they were the correct size (Table 2) and were sequenced to confirm their identity. As a conclusion, primers BSPF and BSPR were specific to the bfs gene from A. baumannii strains. To estimate the sensitivity of QPCR, series dilutions of genomic DNA templates from A. baumannii ATCC19606 were used for standard curve analysis by QPCR, each in triplicate. The detection range of the QPCR method was at least from 101 to 106 cfu/mL (Fig. 5). Furthermore, slight variations were seen in the amplification efficiency among different templates, which suggested that the QPCR was stable in detection of the bfs gene. Data were then subjected to a log-linear analysis to generate a standard curve for calculation of unknowns (Fig. 5). The standard curves regularly showed high R2 values (>0.98).JOURNAL/dimp/04.03/00019606-201112000-00008/figure5-8/v/2021-02-17T200039Z/r/image-jpegStandard curve for the bfs gene-based quantitative polymerase chain reaction (QPCR). Acinetobacter baumannii ATCC 19606, in serial dilutions from 1 to 7 log10 copies, was amplified by QPCR. R2=0.9987.Identification of Bacterial Strains Isolated From Clinical SamplesClinical samples were obtained from the skin of patients and the surface of medical instruments. Forty strains isolated from these samples were first discriminated by biochemical methods and were further identified by the QPCR established in this study. DNA from A. baumannii ATCC19606 was used as a positive control and QPCR results showed that 36 of the strains gave a positive amplification. The other 4 strains were again identified by biochemical methods and showed negative results (data not shown). This suggested that QPCR not only takes less time at lower cost but also provides a precise approach to strain identification.DISCUSSIONA. baumannii, which has a particular ability to develop antimicrobial resistance and cause nosocomial outbreaks of infection, is a significant worldwide nosocomial pathogen.30 As therapeutic options are limited for multidrug-resistant Acinetobacter infection, the development or discovery of new therapies, well-controlled clinical trials of existing antimicrobial regimens and combinations, and greater emphasis on the prevention of health care-associated transmission of multidrug-resistant Acinetobacter infections are essential.4 As a result, a quick and simple detection method has to be established for routine monitoring of A. baumannii present in the medical environment. Recently, based on antimicrobial resistance genes or integrase genes, some PCR methods have been established for pathogenic A. baumannii identification. It was found that integrons are useful markers for epidemic strains of A. baumannii and that integron typing provides valuable information for epidemiological studies. The combination of these markers detected in the multiplex provides powerful information on A. baumannii identification and allows, to some extent, prediction of the likely genotype.31,32 However, the large proportion of A. baumannii that have no antimicrobial resistance genes or integrase genes also have epidemic potential. Hence, real-time QPCR is generally accepted as an accurate and cost-effective method for organism identification. We set out to design QPCR assays to identify and quantify A. baumannii present in the medical environment.Biofilm formation is related to the antimicrobial resistance of A. baumannii and is a well-known pathogenic mechanism during infections associated with medical devices, such as vascular catheters, cerebrospinal fluid shunts, or Foley catheters.33 Furthermore, biofilm on abiotic surfaces facilitates the survival of some microorganisms when faced with severe environmental conditions. It has been shown that the pgaABCD locus of A. baumannii, which encodes poly-β-1-6-N-acetylglucosamine, is critical for biofilm formation. Moreover, the OmpA protein of A. baumannii ATCC19606 also plays an important role in biofilm formation on abiotic surfaces. However, as far as we know, the mechanism of biofilm formation in A. baumannii has not yet been clarified.After scanning the genome sequence of A. baumannii strain ATCC 19606, a biofilm synthesis gene (bfs) was found. Bioinformatic analysis of the bfs gene showed that it was only conserved in A. baumannii strains, and the similarity of bfs genes from different strains was high. This strongly indicated that this gene could be a good marker for A. baumannii identification. Conserved domain and transmembrane structures have been found in Bfs. An Hmms domain associated with biofilm synthesis was present in Bfs. Furthermore, a significant transmembrane structure was found, which suggested that Bfs is an outer membrane protein and could act through interacting with the extracellular environment. The adherence ability of Bfs to abiotic surfaces or extracellular matrix needs further study. In conclusion, bioinformatics analysis indicated that Bfs is an important factor for biofilm formation by A. baumannii, and provides a clue for studying the mechanism of biofilm formation.Another aim of our study was to establish QPCR for A. baumannii identification, and to investigate whether this method could be used in clinical samples. On the basis of the informatic analysis, a conventional PCR was first used to confirm these results. Then, on the basis of the bfs gene, another pair of primers was designed and used for QPCR. The standard curve showed that the amplification efficiency of templates with different concentrations of DNA was almost the same. Specificity and sensitivity investigation of the QPCR assay showed that only templates from A. baumannii showed a positive result, and the maximum sensitivity was 10 cfu/mL. Therefore, this QPCR was stable and reliable in A. baumannii detection. Finally, the QPCR established in this study was further used to identify bacterial strains isolated from clinical samples. Interestingly, 4 strains among 40 showed negative results after QPCR detection, which was not in accordance with the results given by biochemical identification. However, the other 4 strains were reidentified by the biochemical method and showed negative results. Thus, compared with the shortcomings of the biochemical method, the QPCR was more stable and precise in A. baumannii identification. Although further investigations of the efficacy and cost effectiveness of various infection control strategies to prevent transmission of Acinetobacter infection are needed, application of this QPCR to the evaluation of medical equipment and environment disinfection could be useful in the prevention of health care-associated transmission of Acinetobacter infection.In conclusion, the bfs gene from A. baumannii ATCC19606, which plays an important role in biofilm synthesis, provided the basis for QPCR. This is a rapid, cost-effective procedure that can be easily used in routine clinical microbiology laboratories for the identification and detection of the bfs gene in A. baumannii isolates from patients and environments. This is important for the immediate introduction of specific infection control measures in the hospital setting to limit the nosocomial spread of A. baumannii.REFERENCES1. Bergogne-Berezin E, Towner KJ. 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Nosocomial infections caused by Acinetobacter baumannii : a major threat worldwide Infect Control Hosp Epidemiol.. 2006;27:645–646[Context Link]31. Turton JF, Kaufmann ME, Glover J, et al. Detection and typing of integrons in epidemic strains of Acinetobacter baumannii found in the United Kingdom J Clin Microbiol.. 2005;43:3074–3082[Context Link]32. Jane FT, Neil W, Judith G, et al. Identification of Acinetobacter baumannii by detection of the blaOXA-51-like carbapenemase gene intrinsic to this species J Clin Microbiol.. 2006;44:2974–2976[Context Link]33. Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms Lancet.. 2001;358:135–138[Context Link]Acinetobacter baumannii; bfs gene; bioinformatic analysis; QPCRStandard Bacterial Strains Used in This StudyNucleotide Sequences of Primers Used in This StudyHomology comparison of bfs gene sequences from different Acinetobacter baumannii strains. A, bfs gene sequence from A. baumannii ATCC19606. B to G, bfs gene sequences from A. baumannii ATCC17978, ACICU, S1, AB307-0294, AB0057, and AYE.Diagrams of predicted transmembrane structures of Acinetobacter baumannii Bfs protein. Two significant transmembrane structures were found between amino acids 35 to 57 and 77 to 99.Diagrams of predicted conserved domain structure present in Bfs protein; an HmsS domain structure was found in Bfs.Polymerase chain reaction (PCR) amplification results. M indicates marker; 1, negative control; 2, PCR amplification product of the entire bfs gene from Acinetobacter baumannii ATCC 19606 using primers BSPF and BSPR; 3, PCR amplification of a fragment of the bfs gene from A. baumannii ATCC 19606 using primers BfYF and BfYR.Standard curve for the bfs gene-based quantitative polymerase chain reaction (QPCR). Acinetobacter baumannii ATCC 19606, in serial dilutions from 1 to 7 log10 copies, was amplified by QPCR. R2=0.9987.Establishment of a Novel Target-Based Real-Time Quantitative PCR Method for <em xmlns:mrws="http://webservices.ovid.com/mrws/1.0">Acinetobacter baumannii</em> DetectionZhang Lei MSc; Ding, Gangqiang MD; Wei, Lanfen BSc; Pan, Xieshang BSc; Mei, Lingling PhD; Zhang, Yanjun MD; Lu, Yiyu PhDOriginal ArticlesOriginal Articles420p 242-248