WANG Yi-qian, LIU Kai, LI Pei-jing, XIONG Yanwen, YE Chang-yun

(State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable DiseaseControl and Prevention, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases,Chinese Center for Disease Control and Prevention, Beijing 102206, China)

Genetic diversity ofEnterobactercloacaestrains isolated from hospital in China and TaqMan probe real-time polymerase chain reaction for rapid detection of it

WANG Yi-qian, LIU Kai, LI Pei-jing, XIONG Yanwen, YE Chang-yun

(StateKeyLaboratoryofInfectiousDiseasePreventionandControl,NationalInstituteforCommunicableDisease
ControlandPrevention,CollaborativeInnovationCenterforDiagnosisandTreatmentofInfectiousDiseases,
ChineseCenterforDiseaseControlandPrevention,Beijing102206,China)

Enterobactercloacaeare clinically important as nosocomial pathogens. In order to investigate the genetic diversity of the clinicalE.cloacae, 237 isolates obtained from routine diagnostic laboratory were examined with analysis of heat shock protein 60 gene (hsp60) sequence. Based on the neighbor-joining tree of thehsp60 gene sequence, ten genetic clusters ofE.cloacaecould be isolated from the clinical samples. Three genetic clusters (III, VI and VIII) represent almost 71% of the isolates; cluster I accounts for 11%; cluster VII, X and XII were absent. The remaining six clusters are minority in our study, which totally accounted for 18% of all strains. Based on out membrane protein X (ompX) gene sequence analysis of 237 strains, two sets of primers and probes were designed which were specific for ten clusters and cluster I respectively. The limit of detections of the assay were 3.6×101copies/μL for ten clusters and 2.1×101copies/μL for cluster I strains within 40 cycles. This method was also successfully applied to detect ten clusters and cluster I strains from swab samples, the limit detection for swab samples with inoculated bacteria were 104CFU/mL. In the study, we analyzed the genetic clusters ofE.cloacaeisolated from hospital setting, and developed a novel real-time polymerase chain reaction method for rapid detection of ten clusters and cluster I.

heat shock protein 60; TaqMan real-time polymerase chain reaction; out membrane protein X

Background

In recent years,Enterobactercloacae, usually causes respiratory infection and urinary infection, has emerged as an important pathogen in hospital[1-5]. Based on heat shock protein (hsp60) genotype,E.cloacaecould be divided into 12 genetic clusters (I-XII) and an unstable sequence cluster (XIII)[6]. Over the last few years, six new species (E.asburiae,E.kobei,E.ludwigii,E.hormaechei,E.cloacae,E.nimipressuralis) have been reassigned to the clusters (I, II, III, V, VI, VII, VIII, X and XI respectively) ofE.cloacae[3]. Up to date, thirteen clusters were generally identified asE.cloacaeby phenotypic characteristics, and there was rare report about the genetic structure of the nosocomialE.cloacaestrains in China. The objectives of this study were to describe the genetic diversity ofE.cloacae, and develop a novel method for fast identification using TaqMan probe real-time polymerase chain reaction (qPCR) assays.

Based on sequencing technologies and population genetic studies, real-time polymerase chain reaction (PCR) method and the application of DNA and RNA probes have been developed as important supplement to conventional biochemical identification of pathogens[7-9]. Several studies have reported the use of real-time PCR inE.cloacaedetection. Using the ITS sequence as target region,Enterobacter(cloacae/aerogenes) strains could be identified, but always were interpreted asKlebsiella(pneumoniae/oxytoca) due to sequence homologies[9]. A novel multiplex PCR test, which allows identification of 20 bacterial and fungal species directly from blood, only can identifyEnterobacterlevel (E.cloacaeandE.aerogenes)[10]. TargetingdnaAandnstl, a duplex real-time PCR was developed, which can definedE.cloacaesubsp.cloacae(cluster XI) from other genetic clusters of theE.cloacae[11]. Usinghsp60 gene as target,E.hormaechei(cluster VI, VII and VIII) andE.cloacaeIII could be specifically identified in nosocomial samples[12]. However, there was no method for rapid and accurate detection all clusters ofE.cloacae. Outer membrane protein (OmpX) is a virulence factor ofE.cloacae, and could be involved in invasion[13]. Here, we design and validate a rapid identification method targeting onompXgene ofE.cloacaefor ten genetic clusters and cluster I respectively.

Materials and methodsBacterial strains and DNA extraction

Genomic DNA was extracted following the instruction of the High Pure PCR Template Preparation kit (Roche Applied Science, Mannheim, Germany). Purity and concentration of the genomic DNA were analyzed with the NanoDropTM1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, Delaware).

Hsp60-genotype

Amplification ofhsp60 gene was performed with TaKaRa ExTaq (TAKARA Code DRR001A China). The primers were:hsp60-F 5′-GGTAGAAGAAGGCGTGGTTGC-3′,hsp60-R 5′-ATGCATTCGGTGGTGATCATCAG-3′)[6]. Sequences ofhsp60 were obtained from 237 isolates, and genotype of each isolate was assigned by the sequence analysis of 272 bp fragment of thehsp60 gene[6,14-16].

OmpX gene sequence analysis

A pair of primers was designed for amplifying the full length ofompXof reference strain ATCC 13047 and 237 isolates ofE.cloacae:ECompX-LF (5′-ATGAAAAAAATTGCATGTCTTTCAG-3′) and ECompX-LR (5′-TTAGAAGCGGTAACCTACGCC-3′). The PCR products were sequenced and aligned with the homologous sequences in the GenBank. Multiple alignments were generated with DNAMAN program (Lynnon Biosoft, USA).

Real-time PCR detection of E. cloacae using TaqMan probe

Primers and probes were designed based on multiple sequence alignments ofompXof 237 isolates and the homologous sequences deposited in GenBank. 5-hexachloro-fluorescein (HEX) and 6-carboxy-fluorescein (FAM) were used as a reporter moiety in probes respectively. The sequences of primers and probes were following:ECompX-F (5′-CRAGCATCTACGGTGTTGTAGGTG-3′),ECompX-R (5′-CCRTAGTCGCTGTTGCTTGC-3′),ECompX-HEX (5′-HEX-CAGCAGACYGAAAAC-MGB-3′) for ten genetic clusters ofE.cloacae, andECaompX-FAM (5′-FAM-ACTGAAAACCAGAGCGAAA-MGB-3′) just only for genetic cluster I. TaqMan real-time PCR reactions (20 μL) were performed: 10 μL Premix ExTaq (2×) buffer (TaKaRa Code DRR039A, China), 1 μL template DNA, 0.2 μL each of the 10 μM primers, 0.4 μL 2 μM probe and 8.2 μL sterile water. One well was blank control, which contained all reagents except template DNA. The real-time PCR were run on Rotor-Gene Q System (Qiagen Co., Germany) using the following program: denaturation at 95 ℃ for 30 s, 40 cycles consisted of denaturation at 95 ℃ for 5 s, 60 ℃ for 20 s for annealing and extension.

Specificity evaluation

Reference ATCC 13047 and 237E.cloacaeisolates clustered byhsp60 genotypes were subjected to real-time PCR. In addition, 30 reference strains of other genus species were used for specificity test.

Sensitivity and efficiency evaluation

To evaluate the sensitivity of real-time PCR, standard positive control was constructed. Using reference strain ATCC 13047 and one strain of cluster I (CN11EC0003) as templates, the conventional PCR products (usingECompX-LF andECompX-LR as primers, size at 516 bp) were cloned into pMD 18-T Vector (2 682 bp) respectively (TaKaRa Code D101A, China). Plasmids harboringompXgene were isolated from transformed cells and measured using a NanoDrop 2 000 spectrophotometer. Serial dilution were constructed (100to 108copies/μL) using an Easy Dilution kit (TaKaRa Code 9160, China) and used for evaluating the efficiency and linearity of the real-time PCR assay. All reactions were performed in triplicate.

To determine the sensitivity and efficiency of the real-time PCR, swab samples with ten-fold serial dilution of reference strain ATCC 13047 and CN11EC0003 were constructed by counting the colony forming units (101-108CFU/mL). The 100 μL of each dilution was spread for counting the bacteria concentrations. Using QIAamp DNA Blood Mini Kit (QIAGEN Code No. 51104), the genomic DNA of 400 μL of each dilution was extracted and eluted by 150 μL elution buffer for real-time PCR. The study performed each dilution in triplicate.

ResultsHsp60 genotype analysis of E. cloacae isolates in clinical samples

By the sequence analysis of 272 bp of thehsp60 gene, 237E.cloacaeisolates were assigned to its respective genotype. Ten of thirteen genetic clusters were isolated from clinical samples, and all frequencies were summarized (Table 1). Three clusters were prominent in our collection, together representing 71% (cluster III, 60 strains; cluster VI, 42 strains; and cluster VIII, 67 strains). Tweety-six isolates of cluster I accounts for 11%; and the remaining six clusters were minority in our study, which totally accounted for 18% of all strains. Cluster VII, X and XII were absent in this study.

Tab.1 Genotypes and real-time PCR results of theE.cloacaeand cluster I strains in clinical samples

ClustersNo.ofisolatesECompX-HEXECaompX-FAMI26(11%)++II8(3%)+-III60(25%)+-IV10(4%)+-V8(3%)+-VI42(17%)+-VII0nnVIII67(28%)+-IX6(3%)+-X0nnXI3(2%)+-XII0nnXIII7(3%)+-Total23721126

Note: n, indicated no data.

OmpX gene analysis of E. cloacae isolates in clinical samples

The full length ofompXgene for 237 isolates ofE.cloacaewas sequenced. Multiple sequences alignment showed a conserved region of ten clusters ofE.cloacae, which was located in 295-388 bp (Figure 1). Compared withCronobactersakazakii(GenBank accession no. CP012253),Citrobacterkoseri(GenBank accession no. LK931336) andSalmonellaenteric(GenBank accession no. CP012151),E.cloacaeshowed low similarities of nucleotide acid sequences with other bacteria, and ten clusters had a conserved sequence (CAGCAGACC/TGAAAAC) (Figure 1). It's suggested that the conserved region could be used to design the primers and probe of TaqMan PCR. Cluster I strains had its unique sequence in the region (CAGAGCGAA), we also designed a specific probe for cluster I.

Identical nucleotide acid was shared with black. The number of strains is marked in brackets.Fig.1 Multi-alignments of ompX gene sequence of 237 isolates of E. cloacae with Cronobacter sakazakii, Citrobacter koseri and Salmonella enteric

Specificity of primers and probes

Specificity of the real-time PCR assay was determined using reference strain ATCC 13047 and 237 clinicalE.cloacaeisolates. DNA extractions of all isolates were subjected to the real-time PCR. Reference strain ATCC 13047 and 237 clinical isolates of 10 clusters were positive forECompX-HEX probe (Table 1). Twenty-six strains of cluster I were positive for ECaompX-FAM probe. Thirty non-E.cloacaestrains were also tested for the specificity of the primers and probes, and there was no measurable fluorescent signal detected from all strains within 40 cycles (Table 2). Accordingly, the specificity of real-time PCR assay performance was 100%, the two pairs of primer and probe could be used to detect ten clusters and cluster I strains specifically.

Sensitivity and efficiency of the real-time PCR

Sensitivity and efficiency was determined by measuring DNA dilution series (100-108copies/μL) with the real-time PCR assays in triplicate. The amplification curve for the real-time PCR showed that the results displayed a linear log correlative strand curve. The detection limit was 3.6×101copies/μL for ATCC 13047 (ten clusters ofE.cloacae) in 34 cycles (Figure 2A) and 2.1×101copies/μL for CN11EC0003 (cluster I ofE.cloacae) in 37 cycles (Figure 2B). The amplification curve was good, and the Ct value was distributed equally. The PCR assays displayed a linear log correlative standard curve with a slope of 3.073 05 and R2values of 0.998 92, resulting in a PCR efficiency of 1.115 48 for ten clusters of E. cloacae (Figure 2C); and a slope of 3.216 28 and R2values of 0.999 04, resulting in a PCR efficiency of 1.046 06 for cluster I strains (Figure 2D). These results indicated that the TaqMan probe real-time PCR assay for ten clusters and cluster I strains was established successfully.

A 150 μL of genomic DNA were extracted from 400 μL swab sample of each dilution, and 1 μL were amplified with the real-time PCR assays in triplicate. The result showed a linear log correlative standard curve, and the limit detection for swab samples with inoculated bacteria were 104CFU/mL (Figure 3). Plate counting for 100 μL of the 103CFU/mL were 93±14 CFU forE.cloacaeATCC13047 and 71±9 CFU for cluster I strains CN11EC0003 (n=3).

Tab.2 Information of the strains used in this study

SpeciesSourceNo.ofstrainsECompX-HEXECaompX-FAME.cloacaeATCC130471+-E.cloacaeclusterICN11EC00031-+SalmonellaentericATCC107181--CitrobacterkoseriATCCBAA-8951--CronobactersakazakiiClinical1--EnterobacteraerogenesATCC130481--SerratiamarcescensClinical1--EscherichiacoliEDL9335--ListeriamonocytogenesATCC540031--ShigellaflexneriCMCC515721--ShigellasonneiClinical1--ShigelladysenteriaeCMCC513291--ShigellaboydiiCMCC511051--StreptococcussuisClinical1--CitrobacterFreundiiClinical1--EnterococcusFaeciumATCCBAA-4721--EnterococcusfaecalisATCC512991--AeromonashydrophilaClinical1--BacilluscereusClinical1--YersiniaenterocoliticaATCC237151--YersiniapseudotuberculosisClinical1--legionellapneumophilaClinical1--KlebsiellaoxytocaClinical1--KlebsiellapneumoniaeClinical1--neisseriameningitidisClinical1--StreptococcuspneumoniaeClinical1--AcinetobacterbaumaniiClinical1--StaphylococcusaureusATCC65381--VibriocholeraeClinical1--vibrioparahaemolyticusATCC178021--VibriovulnificusClinical1--EberthellatyphosaClinical1--

Serial dilutions of plasmids with PCR products insert were used in individual real-time PCR assays to generate the standard curves.

The R2values, slopes and efficiencies were shown for each reaction.

(A) The amplifications of 10-fold serial dilution from 3.6×100to 3.6×108copies/μL forE.cloacaeATCC 13047;(B) The amplifications of 10-fold serial dilution from 2.1×100to 2.1×108copies/μL for cluster I strain CN11EC0003;(C) Standard curves forE.cloacaeATCC 13047 of TaqMan real-time PCR;

(D) Standard curves for cluster I strain CN11EC0003 of TaqMan real-time PCR;

1-9: Plasmid DNA templates of 108-100copies /μL.

Fig.2 Real-time PCR forE.cloacaeand cluster I strains using a dilution series of plasmid pMD-18T harboringompXgene

Standard curve showing linearity of the assay for known concentration.

(A, C) Fluorescence detection curves forE.cloacaeATCC 13047;

(B, D) Fluorescence detection curves for cluster I strain CN11EC0003;

1-6: Genomic DNA templates of 108-103CFU/ml.

Fig.3 Sensitivity of TaqMan probe real-time PCR detection forE.cloacaeATCC 13047 and cluster I strain CN11EC0003 from swab samples

Discussion

Recent years, the genetic heterogeneity and the clinical importance ofE.cloacaewere well studied. However, little is known about the genetic diversity of clinicalE.cloacaestrains in China. In this work, we investigated the distribution of the genetic clusters of nosocomial strains in a Tertiary A hospital. All strains evaluated in this study (n=237) could be assigned to ten genetic clusters, and the prevalence of the genetic clusters are concordant with those published previously[4,6,15,17]. Cluster III, cluster VI and cluster VIII account for most of the clinical isolates; some genetic clusters were absent in our study. On the basis ofhsp60 gene analysis, the results show the prevalence of the genetic clusters in clinical samples, and the genetic clustering might serve as a framework for the epidemiological study of theE.cloacaespecies.

We also developed a real-time PCR assay for rapid detection ofE.cloacaeby targeting onOmpXgene, and evaluated its application byE.cloacaereference strain ATCC 13047, cluster I strain CN11EC0003, 237 clinical isolates and 30 other genus strains. The result shows that the primers and probes are specific for ten genetic clusters of ten clusters and cluster I strains respectively. For the swab sample analysis, the diagnostic sensitivity of TaqMan probe real-time PCR is 104CFU/mL, which is higher than the actual concentration of most pathogens in clinical specimen. Thus, enrichment culture is necessary for the swab samples before the real-time PCR detection. Meanwhile, it reduces the detection time to 2 hours comparing with conventional methods. Therefore, TaqMan probe real-time PCR is a cost-effective and highly sensitive method for detecting ofE.cloacaefrom clinical specimens. This is the first report on the application of TaqMan probe real-time PCR in ten clusters and cluster I strain detection, this method could help clinical laboratory to establish the diagnosis ofE.cloacae, and prompt the interventions for infection control.

Acknowledgments

We acknowledge the financial supports of the grants (Mega Project of Research on the Prevention and Control of HIV/AIDS, Viral Hepatitis Infectious Diseases 2011ZX10004-001, 2013ZX10004-101 to Ye Chang-yun) from the Ministry of Science and Technology, China.

Ethical considerations

All samples were acquired with the written informed consent from donors. This study was reviewed and approved by the ethics committee of the National Institute for Communicable Disease Control and Prevention, China CDC, according to the medical research regulations of the Ministry of Health, China (Approval No. ICDC-2014003).

Authors' contributions

Wang Yi-qian, Xiong Yan-wen and Ye Chang-yun designed the project and wrote the paper. Wang Yi-qian, Wang Yan and Zhao Ai-lan carried out the experimental work. Liu Kai and Li Pei-jing isolatedE.cloacaestrains from samples. All authors read and approved the final manuscript.

Conflicts of interests

The authors declare that they have no competing interests.

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Received:2016-03-03；Editor: LIN Dan

train ATCC 13047 and 237 isolates ofE.cloacaeobtained from routine diagnostic laboratory were used in this study. All isolates were subjected tohsp60 gene andompXgene amplification.

醫(yī)院臨床標本中陰溝腸桿菌遺傳多樣性分析及應用TaqMan熒光定量PCR方法快速檢測陰溝腸桿菌的研究

王怡倩，劉 凱，李培京，熊衍文，葉長蕓

目的 調(diào)查醫(yī)院臨床標本中陰溝腸桿菌基因群的構(gòu)成比例，并建立TaqMan 熒光定量PCR方法對陰溝腸桿菌進行特異、靈敏、快速檢測。方法 通過hsp60基因分型分析臨床標本中基因群的構(gòu)成，并以外膜蛋白基因(ompX)為靶基因設計引物及FAM探針，建立TaqMan 熒光定量PCR方法對陰溝腸桿菌基因群檢測，并評價該方法的特異性、靈敏性和穩(wěn)定性。結(jié)果 237株臨床標本共歸為10個基因群。其中群III，VI和VIII菌株數(shù)量最多，占總菌株數(shù)的71%；群I占11%；其他6個群總共占18%。無基因群VII，X和XII。TaqMan 熒光定量PCR方法能對陰溝腸桿菌不同基因群進行特異檢測：對十個基因群和群I的質(zhì)粒標準品的檢測下限分別為36 拷貝/μL和21 拷貝/μL，對糞便模擬標本的檢測下限為104個菌落形成單位/μL；TaqMan 熒光定量PCR方法對質(zhì)粒標準品和糞便模擬標本檢測的擴增曲線良好；結(jié)論 醫(yī)院臨床標本中可以檢測到十個基因群，具有遺傳多樣性的特征。本研究建立的TaqMan 熒光PCR方法特異性好、靈敏度高，能夠用于陰溝腸桿菌的快速檢測。

熱休克蛋白60；TaqMan 熒光定量PCR；外膜蛋白基因X

R378.2

A

1002-2694(2017)01-0001-08

葉長蕓，Email: yechangyun@icdc.cn

中國疾病預防控制中心傳染病預防控制所，傳染病預防控制國家重點實驗室，感染性疾病診治協(xié)同創(chuàng)新中心，北京 102206

10.3969/j.issn.1002-2694.2017.01.001

Ye Chang-yun, Email address: yechangyun@icdc.cn

國家重大傳染病防治科技重大專項(No.2011ZX10004-001，No.2013ZX10004-101)