Identification of Staphylococcus spp. and detection of

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Phenotypic identification of coagulase-negative staphylococci (CoNS) is difficult and many staphylococcal species carry mecA. This study developed an array ...
Diagnostic Microbiology and Infectious Disease xxx (2016) xxx–xxx

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Research Article

Identification of Staphylococcus spp. and detection of mecA by an oligonucleotide array☆,☆☆ Huan Wen Han a, Hsien Chang Chang a,b,⁎, Tsung Chain Chang c,⁎⁎ a b c

Institute of Biomedical Engineering, College of Engineering, National Cheng Kung University, Tainan, Taiwan Medical Device Innovation Center, National Cheng Kung University, Tainan, Taiwan Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan

a r t i c l e

i n f o

Article history: Received 17 February 2016 Received in revised form 31 May 2016 Accepted 1 June 2016 Available online xxxx Keywords: Staphylococcus Coagulase-negative staphylococci Identification mecA Internal transcribed spacer Array

a b s t r a c t Phenotypic identification of coagulase-negative staphylococci (CoNS) is difficult and many staphylococcal species carry mecA. This study developed an array that was able to detect mecA and identify 30 staphylococcal species by targeting the internal transcribed spacer regions. A total of 129 target reference strains (30 species) and 434 clinical isolates of staphylococci were analyzed. Gene sequencing of 16S rRNA, gap or tuf genes was the reference method for species identification. All reference strains (100%) were correctly identified, while the identification rates of clinical isolates of S. aureus and CoNS were 98.9% and 98%, respectively. The sensitivity and specificity for mecA detection were 99% and 100%, respectively, in S. aureus isolates, and both values were 100% in isolates of CoNS. The assay takes 6 h from a purified culture isolate, and so far it has not been performed directly on patient samples. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Staphylococcus species can cause mild to life-threatening infections (Hirotaki et al., 2011). S. aureus is the most important pathogen in the genus Staphylococcus. Members of the coagulase-negative staphylococci (CoNS) are part of the normal flora of human skin. However, CoNS are being increasingly recognized as an important cause of hospitalacquired infections associated with the use of prosthetic and indwelling devices (Fujita et al., 2005; Mehta et al., 2009; Morfin-Otero et al., 2012; Szabados et al., 2011). Many species of CoNS carry multiple antibioticresistant genes, especially mecA, making treatment of infections caused by these microorganisms challenging (Piette and Verschraegen, 2009; Prere et al., 2006). Methicillin resistance was found in 55% to 75% of isolates of CoNS (Biavasco et al., 2000; Piette and Verschraegen, 2009). In addition to antimicrobial resistance, identification of CoNS to the species level by biochemical tests may be unreliable (Sudagidan et al., 2005).

☆ Conflicts of interest: none. ☆☆ Financial support: This project was supported by grants from the Ministry of Science and Technology (MOST 102-2320-B-026-MY3) and the Multidisciplinary Center of Excellence for Clinical Trial and Research (MOHW105-TDU-B-211-133016), Department of Health and Welfare, Taiwan. These funding organizations had no role in the design or conduct of this research. ⁎ Corresponding author. Tel.: +886 6 2757575x63426. ⁎⁎ Corresponding author. Tel.: +886 6 2353535x5790; fax: +886 6 2363956. E-mail addresses: [email protected] (H.C. Chang), [email protected] (T.C. Chang).

Therefore, an assay that is able to rapidly identify Staphylococcus spp. and to detect mecA has clinical value. Rapid identification of methicillin-resistant S. aureus (MRSA) is important, as MRSA is resistant to virtually all β-lactam antibiotics, and several commercial kits have been developed to achieve this. Reports indicate that mecC (a homologue of mecA) can also cause methicillin resistance in S. aureus (García-Álvarez et al., 2011; Paterson et al., 2014). Therefore, the recent developments in MRSA detection, in addition to mecA, also target mecC to increase sensitivity (Becker et al., 2016; Mendes et al., 2016). However, the inclusion of mecC for MRSA detection is debatable, as the prevalence of mecC in MRSA isolates was only 0.06% (2/3207) in Germany (Schaumburg et al., 2012) and 0.45% (9/2010) in the UK (Paterson et al., 2014), although the prevalence was much higher (1.9% to 2.8%) in Denmark (Petersen et al., 2013). Targeting both the SCCmec–orfX right-extremity junction (MREJ) and mecA/mecC regions to avoid false-positives and false-negatives, respectively, is adopted for the detection of MRSA in newer generations of commercial kits, such as the Xpert MRSA Gen 3 PCR (Becker et al., 2016) and BD MAX StaphSR (Mendes et al., 2016). However, S. aureus that lacks the mecA element (drop-out mutant) can still result in false positives (Blanc et al., 2011), due to the co-presence of methicillinresistant CoNS (Tubbicke et al., 2012). In contrast, strains of MRSA with altered MREJ sequences can produce false-negative reactions (Mendes et al., 2016). The internal transcribed spacer (ITS) region has been successfully used as a sensitive and specific target for molecular identification of bacteria (Sudagidan et al., 2005; Tung et al., 2007). This study aimed

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Please cite this article as: Han HW, et al, Identification of Staphylococcus spp. and detection of mecA by an oligonucleotide array..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.06.003

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H.W. Han et al. / Diagnostic Microbiology and Infectious Disease xxx (2016) xxx–xxx

to develop an oligonucleotide array that has the capability to identify 30 species of staphylococci and to detect mecA, based on hybridization with probes designed from the ITS regions and mecA, respectively.

control (template DNA substituted with pure water) and a positive control (a strain of MRSA) were also performed with each run. 2.3. ITS cloning and sequencing

2. Materials and methods 2.1. Reference strains and clinical isolates A collection of 133 target reference strains belonging to 30 species of Staphylococcus and 434 clinical isolates (10 species) were used in this study (Supplemental Table S1). Clinical isolates of Staphylococcus were obtained from the Department of Pathology, National Cheng Kung University Hospital (Tainan, Taiwan); these isolates were mainly isolated from sterile body fluids, especially blood. Of the clinical isolates, S. aureus was identified by the tube coagulase test. An isolate of CoNS was identified by the VITEK 2 GP identification card (VITEK bioMérieux, Taipei, Taiwan) if it was isolated from both bottles of a pair (aerobic and anaerobic) of blood culture bottles, but an isolate was identified as CoNS if it was only isolated from one of the two bottles. The species names of all reference strains and clinical isolates were further confirmed by sequence analysis of the 16S rRNA gene (Relman, 1993). If the difference between sequence similarities of the best-match and second bestmatch by BLAST search in GenBank was less than 0.1%, the partial gap gene (931 bp) (Yugueros et al., 2001) and/or tuf gene (660 bp) (Bergeron et al., 2011; Carpaij et al., 2011; Li et al, 2012) were sequenced for species determination. Species names determined by gene sequencing were considered the final identifications. In addition, 61 non-target reference strains (38 species) were used for specificity test of the array (Supplemental Table S2). Subspecies-level identification was not considered in this study, as gene sequence analysis cannot identify staphylococci to the subspecies level (Ghebremedhin et al., 2008; Poyart et al., 2001). All strains were subcultured on tryptic soy agar plates for 18–24 h at 35 °C, except for S. saccharolyticus, which was cultured on CDC Anaerobe 5% Sheep Blood Agar (BBL, Becton Dickinson Microbiology Systems, Taipei, Taiwan) and incubated in an anaerobic chamber at 35 °C for 24–48 h. 2.2. Amplification of the ITS, 16S rRNA, gap, tuf, and mecA genes Bacterial DNA was extracted with a DNA extraction kit (Geneaid, Taipei) or extracted by the boiling method (Vaneechoutte et al., 1995). The following primer pairs were used to amplify ITS (2F, TTGTACACACCGCCCGTC; 10R, TTCGCCTTTCCCTCACGGTA) (Chang et al., 2005), 16S rRNA gene (11F, GTTTGATCCTGGCTCAG; 1512R, GGYTACCTTGTTACGACTT, Y = C or T) (Relman, 1993), gap (Gap-1F, ATGGTTTTGGTAGAATTGGTCGTTTA; Gap-2R, GACATTTCGTTATCATAC CAAGCTG) (Yugueros et al., 2001), tuf (Tuf-F, CCAATGCCACAAACTCGT; Tuf-R, CCTGAACCAACAGTACGT) (Li et al., 2012), mecA (mecF, AAAAT CGATGGTAAAGGTTGGC; mecR, AGTTCTGCAGTACCGGATTTGC) (Murakami et al., 1991) and mecA/mecC (mecALGA251 FP, TCACCAGGTT CAACYCAAAA; mecALGA251 RP, CCTGAATCWGCTAATAATATTTC; Y = C or T, W = A or T) (García-Álvarez et al., 2011) by PCR. All genes were amplified under the same thermal conditions. PCR was performed in a total volume of 25 μl containing 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 0.8 mM deoxyribonucleoside triphosphates (0.2 mM each), 0.5 μM of each primer, 1 U of Taq DNA polymerase and 1 to 5 ng of bacterial template DNA. The PCR program consisted of an initial denaturation at 94 °C for 2 min, followed by 35 cycles of denaturation (94 °C, 1 min), annealing (55 °C, 30 s), and extension (72 °C, 45 s), and a final extension step (72 °C, 7 min). For array hybridization, the ITS and mecA were simultaneously amplified by a duplex PCR using the same reaction mixture and thermocycling conditions as described in this section, except that the four primers used to amplify the two genes were included in the PCR reaction mixture, and were labeled with a digoxigenin molecule at the 5′ end of each primer. A negative

As almost all species of staphylococci possess multiple ITS fragments with different lengths and sequences, the ITS amplicons of 21 staphylococcal species without ITS sequences in public databases were cloned by using the pOSI-T PCR cloning kit (Genemark, Taipei, Taiwan) according to the manufacturer's instructions. The ITS fragments of positive clones were re-amplified and sequenced. The ITS sequences thus determined were submitted to GenBank (Table 1). 2.4. Probe design A total of 34 oligonucleotide probes were designed to identify 30 species of staphylococci and to detect mecA (Table 1). The speciesspecific probes were based on the ITS sequences available in GenBank or determined in this study. The designed probes were checked for internal repeats, secondary structure, self-binding, and GC content by using the software Vector NTI (Invitrogen Corporation, Carlsbad, CA). Several additional bases of thymine were added to the 3′ or 5′ ends of some probes to increase the hybridization signals (Brown and Anthony, 2000) (Table 1). In addition, a positive control probe (code PC) that is bacteria-specific was designed from the 3′ end of bacterial 16S rRNA genes. All probes were synthesized by GeneMark Inc (Taipei, Taiwan). An irrelevant probe (code M, dig-TCCTCCGCTTATTGATATGC) labeled with a digoxigenin molecule at its 5′ end was used as a position marker on the array. 2.5. Array fabrication The oligonucleotide probes were diluted 1:1 (final concentration 10 μM) with a tracking dye solution containing 0.15% (wt/vol) bromophenol blue. The probes in a round-bottom 96-well microtiter plate were spotted onto a positively charged nylon membrane (Roche, Mannheim, Germany) using a solid pin (400 μm in diameter) by an automatic arrayer (model SR-A300; EZlife Technology Co., Taipei, Taiwan), as described previously (Hsiao et al., 2005). The layout of the probes on the array (7 × 7 mm) is shown in Fig. 1. 2.6. Hybridization procedures The reagents and procedures used for pre-hybridization, hybridization (50 °C for 90 min), and color development with enzymeconjugated anti-digoxigenin antibodies were described previously (Hsiao et al., 2005). The hybridized spots could be read by the naked eye. A strain was identified to the species level when both the positive control probe and the species-specific probe (or probes) were hybridized. However, due to intra-species ITS sequence variation, S. aureus was identified if at least two of the three probes (SA5N, SA6g, and SA9a) were hybridized, while S. xylosus were identified if any one of the two probes (SXY1b and SXY2d) was hybridized (Table 1). 2.7. Methicillin drug susceptibility testing The disc (cefoxitin) diffusion method was used to determine the methicillin susceptibility of S. aureus and CoNS according to the CLSI guidelines (CLSI, 2016), and the results were considered the gold standard in this study. Discrepant results between the array (detection of mecA) and drug susceptibility testing were analyzed for the presence of mecA (Murakami et al., 1991) and mecA/mecC (García-Álvarez et al., 2011) by PCR followed by sequencing of the amplicons.

Please cite this article as: Han HW, et al, Identification of Staphylococcus spp. and detection of mecA by an oligonucleotide array..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.06.003

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Table 1 Oligonucleotide probes used to identify 30 species of Staphylococcus and mecA. Microorganism or gene

Probe codea

Probe sequence (5′ to 3′)b

Length (nucleotide)

GC%

Tm c (°C)

Location

Accession no.d

mecA S. arlettae S. aureusf

mecA SAR4h SA5N SA6g SA9a SAU3a SCA1e SCE3d SCR4e SCH3fg SCE2d SCT3g SCR4e SDE3d SE3b SEQ2 SFE3 SGA2b SHA5a SHO3 SHY6a SIN4 SLE4a SLG2gN SNE2 SCE2d SPA2dg SA5N SPS2 SSA1d SSC1c SSR3c SA6g SSI4c SWA4a SA5N SXY1b SXY2d PC

TGATGGTATGCAACAAGTCG GTCGAAAATTTATACACTtttttttttt e CGTTTCCTGTAGGATGGAAACATAGATTAAtttttttttt CGTTATTCCGCATCTTCTGAAGAAGAttttt CGAAGCCGTATGTTAACGTTTGACtttttt AGAAATGTATCGCTTAGACGAAGCATCttttt GTCACGTTATCCCTTTCATCTTGGAAGAAG CCAACATGAGTGATGTCATGTttttttt CCGTACTGTACAGTTGGACCAGAGttttt AATTCGAAAGCCGAATGTAA-AATTCGAAAGCCGAATGTAAttttt CGGTTGTCGAGTTGAAAGCAAATAGATt GTAACTCCATGCAGAGTGTCC-GTAACTCCATGCAGAGTGTCCtttttttttt CCGTACTGTACAGTTGGACCAGAGttttt GCGCAGTGAAGTTTACTTTCTGtttttt TTGAATAACAATTCAAAATATGGTGGAttttttttttt TGTCAATGTCGTTCCTCTTCATCTTCttttt TGGTGATGATAAGTGTGAAAGCtttttt TCAATGTACAATCTTTGAACCCTCTAATGAGC CAAACAATTACATCAAAATAGTATATCtttttttttt GAATATATTAAACGAATCATCTtttttttttt tttttttttt GCAACTGAGTGATTTGTGCCGCGATG ATTTTGGGAAAAGTGGATTTCGtttttttttt CTGGTAAAGGTTAGTTAAACATGGTTGTATCTTCG GTTTGTCAGGCGAGGCGTTGAAGCATTtttttttttt TATCGCTGCGTAAATACATAGCGtttttttttt CGGTTGTCGAGTTGAAAGCAAATAGATt ATACAACATTCAAACATTTATTGTAC- ATACAACATTCAAACATTTATTGTACtttttttt CGTTTCCTGTAGGATGGAAACATAGATTAAtttttttttt TTCGGTAGACGACATGATGtttttttt GGAGTATTCATTGCATAATTTTGttttttttt CATGTTAATTCCGTCTATCACCTGTTGGTG TCTTAAACGTTGTGTATAAGGAAATATAGtttttttttt CGTTATTCCGCATCTTCTGAAGAAGAttttt CGACGGCAAACGATTACTCACAATAtttttttttt CGCTTGACTGGAAATAGAACTTGTACATTG CGTTTCCTGTAGGATGGAAACATAGATTAAtttttttttt GAAGATGACAGAGGAATAACATTGACtttttttttt CATTGCATTGTTTTGTACATTGAAAtttttttttt TGGGGTGAAGTCGTAACAAGGTAGCCGTAtttttttttt

20 18 30 26 24 27 30 21 24 40 27 42 24 22 27 26 22 32 27 22 26 22 35 27 23 27 52 30 19 23 30 29 26 25 30 30 26 25 29

45.0 27.8 36.7 42.3 45.8 40.7 43.3 42.9 54.2 35.0 40.7 52.4 54.2 45.5 25.9 42.3 40.9 37.5 22.2 22.7 53.8 36.4 37.1 51.9 43.5 40.7 23.1 36.7 47.4 30.4 43.3 27.6 42.3 44.0 40.0 36.7 38.5 28.0 51.7

49.2 32.9 59.2 59 56.8 57.5 62.2 47.5 54.5 72.4 59.8 75.4 54.5 51.4 55.1 57.5 49.4 61.8 46.5 40.1 67.0 54.0 62.3 67.4 54.3 59.8 67.3 59.2 46.0 48.2 62.4 51.1 59 59.1 60.5 59.2 52.2 53.8 65.8

mecA ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS ITS 16S rRNA gene

AB033763 KU555844 U11783 U11783 U39769 KU555845 KU555847 KU555848 KU555852 U39770 KU555850 KU555854 KU555852 KU555856 U39771 KU555858 KU555860 KU555863 KU588175 KU555864 U90016 KU588177 KU555866 KU555868 KU588178 KU555850 KU555872 U11783 KU555873 KU555875 KU555878 KU555880 U11783 U39418 KU555884 U11783 U39773 U39773 EU13667

S. S. S. S. S. S. S.

auricularis capitis caprae carnosus chromogenes cohnii condimentih

S. S. S. S. S. S. S. S. S. S. S. S.

delphini epidermidis equorum felis gallinarum haemolyticus hominis hyicus intermedius lentus lugdunensis nepalensish

S. pasteurih S. S. S. S. S. S. S.

pseudintermedius saprophyticus schleiferi sciuri simiae simulans warnerih

S. xylosusi Positive controlj a b c d e f g h i j

Oligonucleotide probes are positioned on the array as depicted in Fig. 1. The underlined nucleotide is a mismatch base that was intentionally incorporated into the probe to avoid cross hybridization with other staphylococci. Tm, melting temperature. The accession no. with a prefix of KU is the ITS sequence determined in this study and submitted to GenBank. Several bases of thymine were added to the 3′ or 5′ end of the probe to increase the hybridization signal. S. aureus was identified by the hybridization of at least two of the three probes (SA5N, SA6g, and SA9a). A repeat of the same sequence was designed for the probe to increase the hybridization signal. The species was identified by simultaneous hybridization of the two probes. S. xylosus was identified if any one of the two probes was hybridized. The positive control probe was specific to all bacteria.

2.8. Statistical analysis Statistical calculations were done using a two-sided χ 2 test. A comparison of the results of mecA detection by the array and drug susceptibility testing was made in clinical isolates of staphylococci. A P value of b0.05 was considered statistically significant. 3. Results 3.1. Identification of reference strains by the array A collection of 133 target strains (30 species) of Staphylococcus (Supplemental Table S1) and 61 non-target strains (38 species) (Supplemental Table S2) were used for sensitivity and specificity testing, respectively, by the array. After confirmation of the species names of the target reference strains by sequence analyses of 16S rRNA, gap and/or tuf genes, S. cohnii BCRC 15224, S. intermedius BCRC 15235,

S. intermedius BCRC 15236, and S. xylosus BCRC 15251 were found to be misidentifications of Macrococcus caseolyticus, S. pseudointermedius, S. pseudointermedius, and S. gallinarum, respectively (Han et al., 2015). After excluding the four strains with wrong species names, the sensitivity of the array for identification of reference strains was 100% (129/ 129). The hybridization patterns of the 30 target staphylococci are shown in Fig. 2. S. pasteuri, S. simiae, and S. warneri are closely related species of S. aureus (Pantucek et al., 2005; Suzuki et al., 2012; Takahashi et al., 1999). S. aureus was identified by hybridization with at least two of the three probes (SA5N, SA6g, and SA9a), while S. pasteuri, in addition to its specific probe (SPA2d), also hybridized with one (SA5N) of the three probes used for identification of S. aureus. S. simiae is a sister species derived from S. aureus (Pantucek et al., 2005; Suzuki et al., 2012), and it only hybridized with one (SA6g) of the three probes, but S. warneri, in addition to its specific probe (SWA4a), also hybridized with one (SA5N) of the three probes for S. aureus (Table 1 and Fig. 2).

Please cite this article as: Han HW, et al, Identification of Staphylococcus spp. and detection of mecA by an oligonucleotide array..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.06.003

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1

2

3

4

5

6

7

A

mecA

SA5N SAR4h

M

SGA2b SHA5a SHO3

B

SA9a

SA6g SAU3a

M

SHY6a

C

PC

SCT3

SNE2

M

D

M

M

M

SIN4

SLG2gN SPA2d M

M

SLE4a SPS2 M

E

SCA1e SCR4e SCE2d

M

SSA1d SSC1c SSR3c

F

SCE3d SCH3f SDE3d

M

SSI4c SWA4a

M

SXY2d SXY1b

G

SE3b

SEQ2

SFE3

NC

Fig. 1. Layout of oligonucleotide probes on the array (7 × 7 mm). The probe “PC” (C1) is bacteria-specific and was designed from the 16S rRNA genes of bacteria. The probe “NC” (G7) is a negative control (tracking dye only). The probe “M” is a position marker that is an irrelevant probe (dig-TCCTCCGCTTATTGATATGC) labeled with a digoxigenin molecule at its 5′ end. The corresponding sequences of all probes are listed in Table 1.

In addition, S. carnosus and S. condimenti are genetically related species (Misawa et al., 2015; Probst et al., 1998), and they were differentiated by two probes (SCR4e and SCT3). S. carnosus only hybridized with one (SCR4e) of the two probes, but S. condimenti hybridized with both. Similarly, S. cohnii and S. neplanesis are genetically related species (Novakova et al., 2006; Spergser et al., 2003), and were also differentiated by two probes (SCE2d and SNE2). S. cohnii only hybridized with one (SCE2d) of the two probes, while S. neplanesis hybridized with both (Table 1 and Fig. 2).

3.2. Identification of clinical isolates by array Initially, a collection of 434 clinical isolates (10 species) of staphylococci were analyzed by array (Table S1). Two isolates of S. aureus and one isolate of S. epidermidis were not identified (only the positive control probe hybridized). Four isolates of S. epidermidis were misidentified as S. capitis. Discrepant analysis of the seven clinical isolates is shown in Supplemental Table S4. Other clinical isolates of CoNS were correctly identified by the array. The correct identification rate of the S. aureus isolates was 98.9% (186/188), with the remaining 1.1% (two isolates) being not identified (Table 2). The average identification rate of the 10 species of CoNS was 98% (241/246), ranging from 95% to 100% in different species (Table 2). The rates of no identification and misidentification of CoNS were 0.4% (1/246) and 1.6% (4/246), respectively.

Fig. 2. The hybridization patterns of 30 species of Staphylococcus and detection of mecA in each of the 30 species. The arrays are alphabetically arranged according to the species names. The corresponding probes hybridized on the arrays are indicated in Fig. 1, and the corresponding sequences of the hybridized probes are shown in Table 1. The last 3 arrays show the hybridization patterns of mecA-positive reference strains.

Please cite this article as: Han HW, et al, Identification of Staphylococcus spp. and detection of mecA by an oligonucleotide array..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.06.003

H.W. Han et al. / Diagnostic Microbiology and Infectious Disease xxx (2016) xxx–xxx Table 2 Results of identification of clinical isolates of Staphylococcus by the array. Species

S. aureus (n = 188) S. capitis (n = 45) S. caprae (n = 12) S. cohnii (n = 8) S. epidermidis (n = 100) S. haemolyticus (n = 42) S. hominis (n = 19) S. lugdunensis (n = 16) S. saprophyticus (n = 1) S. warneri (n = 3) All isolates of CoNS (n = 246)

No. of isolate (%) with identification results of: Species

No identification

Misidentification

186 (98.9) 45 (100) 12 (100) 8 (100) 95 (95) 42 (100) 19 (100) 16 (100) 1 (100) 3 (100) 241 (98)

2 (1.1) 0 (0) 0 (0) 0 (0) 1 (1) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (0.4)

0 (0) 0 (0) 0 (0) 0 (0) 4 (4) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 4 (1.6)

5

The distribution of methicillin resistance in different species of clinical isolates is shown in Supplemental Table S5. The mecA gene was found in 51.1% of S. aureus isolates. Some species of CoNS had high carrying rates of mecA, such as S. haemolyticus (95.2%, 40/42), S. capitis (88.9%, 40/45), and S. epidermidis (75.8%, 72/95), but none of the three isolates of S. warneri carried mecA. 3.5. Limits of detection of the array The limits of detection for S. aureus BCRC 14957, S. epidermidis BCRC 10785, S. saprophyticus BCRC 14839, and mecA of S. aureus BCRC 14976 by the array were 25 pg, 250 pg, 25 pg, and 25 pg per assay, respectively. 4. Discussion

3.3. Specificity of the array A total of 61 non-target strains (38 species), including eight species (23 strains) of Staphylococcus and other bacteria, were used for the specificity test (Supplemental Table S2). No cross-hybridization was found in any of these strains by the array, resulting in a specificity of 100%.

3.4. Detection of mecA by the array After excluding the seven clinical isolates with no identification or misidentification (Supplemental Table S4), 427 clinical isolates (186 isolates of S. aureus and 241 isolates of CoNS) were used for mecA detection by the array. Using conventional drug susceptibility testing (the disc diffusion method) as the gold standard, the performance of the array in detecting mecA is shown in Table 3. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the array for S. aureus isolates were 99%, 100%, 100%, and 98.9%, respectively, while all the respective values for CoNS were 100% (Table 3). One isolate (S. aureus 868) was found to be mecA negative by the array, but it was found to be methicillin-resistant by drug susceptibility testing, and was mecA positive by PCR. Sequence analysis of the mecA fragment amplified by PCR revealed that the isolate had a single nucleotide change in the region of the probe used to detect mecA (Table 2), resulting in a false-negative reaction by the array. No significant difference (two-sided χ 2 test) was found between the array and drug susceptibility testing for detection of methicillin resistance in isolates of S. aureus and CoNS.

The use of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) can greatly reduce the time needed for bacterial identification, but the cost of the instrument is high (Bergeron et al., 2011; Clerc et al., 2014; Matsuda et al., 2012; Richter et al., 2012; Spanu et al., 2011). The GeneXpert MRSA (Cepheid, Sunnyvale, CA) is a fully automated system able to identify S. aureus and to detect mecA simultaneously (Clerc et al., 2014). A coupling of MALDI-TOF MS for rapid identification of S. aureus in positive blood cultures, followed by the use of GeneXpert MRSA for mecA detection, has thus been proposed to accelerate the detection of MRSA bacteremia (Clerc et al., 2014). Although 30 species of Staphylococcus of references strains were analyzed in this study, only 10 species of CoNS from clinical isolates were evaluated by the array, as only a limited number of species of CoNS are commonly encountered in clinical settings (Richter et al., 2012; Rychert et al., 2013). The 16S rRNA gene sequence similarity is relatively high among staphylococcal species, ranging from 90 to 99% (Becker et al., 2004; Ghebremedhin et al., 2008; Huletsky et al., 2004). Compared with the 16S rRNA gene, gene sequences of gap and tuf were found to be more discriminative for identification of staphylococci (Bergeron et al., 2011; Carpaij et al., 2011; Yugueros et al., 2001). The ITS region is also a good target for identifying a variety of bacteria (Sudagidan et al., 2005; Tung et al., 2007). However, the ITS sequences of CoNS in public databases are very limited. This scarceness of staphylococcal ITS sequences is because almost every staphylococcal species contains multiple ITS fragments that have different lengths and sequences, and thus direct sequencing of ITS after PCR is not possible (Couto et al., 2001). Four isolates of S. epidermidis (5273N, 5696A, 8619N, and 3011-2) were identified as S. capitis in this study (Supplemental Table S4). S. capitis, S. caprae, S. epidermidis, and S. saccharolyticus are closely related species, as demonstrated in the phylogenic trees constructed from 16S rRNA gene sequences (Takahashi et al., 1999). In addition, two

Table 3 Comparison of the array and drug susceptibility testing for detection of methicillin resistance in clinical isolates of Staphylococcus. Detection of mecA by the arraya

S. aureus (n = 186) Positive Negative CoNS (n = 241) Positive Negative Total (n = 427) Positive Negative a b c

No. of isolates determined by drug susceptibility testing

Performance of the array, % (95% CI)

Agreement, %

Resistant

Susceptible

Sensitivity

Specificity

PPVb

NPVc

95 1

0 90

99.0 (94.3–100)

100 (96.0–100)

100 (96.2–100)

98.9 (94.0–100)

99.5

179 0

0 62

100 (98.0–100)

100 (94.2–100)

100 (98.0–100)

100 (94.2–100)

100

274 1

0 152

99.6 (98.0–100)

100 (97.6–100)

100 (98.7–100)

99.4 (96.4–100)

99.8

Clinical isolates not identified or misidentified by the array were excluded for detection of mecA by the array. PPV = positive predictive value. NPV = negative predictive value.

Please cite this article as: Han HW, et al, Identification of Staphylococcus spp. and detection of mecA by an oligonucleotide array..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.06.003

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H.W. Han et al. / Diagnostic Microbiology and Infectious Disease xxx (2016) xxx–xxx

isolates of S. aureus and one isolate of S. epidermidis were not identified by the array (Table 2). The reason for the misidentification and no identification of these isolates is probably the high inter-species similarity of ITS sequences in some species, and ITS polymorphisms in strains of a single species were found in staphylococci (Couto et al., 2001). Detection of mecA in clinical isolates by the array had high sensitivity, specificity, PPV, NPV, and agreement between the array and drug susceptibility testing (Table 3). For mecA detection, the array produced one false negative (S. aureus 868) (Table 3) which had a single nucleotide change in the region of mecA probe, as determined by gene sequencing. In conclusion, the array used in this study is able to identify 30 species of staphylococci and to detect mecA, something that has not yet been achieved by other approaches. This method might thus have practical value in clinical microbiology laboratories. However, the feasibility of applying the array for direct identification of Gram-positive cocci in clinical specimens needs further evaluation. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.diagmicrobio.2016.06.003. Acknowledgements This project was supported by grants from the Ministry of Science and Technology (MOST 102-2320-B-026-MY3) and the Multidisciplinary Center of Excellence for Clinical Trial and Research (MOHW105TDU-B-211-133016), Department of Health and Welfare, Taiwan. The funding organizations had no role in the design or conduct of this research. We thank Ay-Huey Huang at the Department of Pathology, National Cheng Kung University Hospital, Tainan, Taiwan, for her collection of clinical isolates. References Becker K, Harmsen D, Mellmann A, Meier C, Schumann P, Peters G, et al. Development and evaluation of a quality-controlled ribosomal sequence database for 16S ribosomal DNA-based identification of Staphylococcus species. J Clin Microbiol 2004;42: 4988–95. Becker K, Denis O, Roisin S, Mellmann A, Idelevich EA, Knaack D, et al. Detection of mecAand mecC-positive methicillin-resistant Staphylococcus aureus (MRSA) isolates by the new Xpert MRSA Gen 3 PCR. J Clin Microbiol 2016;54:180–4. Bergeron M, Dauwalder O, Gouy M, Freydiere AM, Bes M, Meugnier H, et al. Species identification of staphylococci by amplification and sequencing of the tuf gene compared to the gap gene and by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Eur J Clin Microbiol Infect Dis 2011;30:343–54. Biavasco F, Vignaroli C, Varaldo PE. Glycopeptide resistance in coagulase-negative staphylococci. Eur J Clin Microbiol Infect Dis 2000;19:403–17. Blanc DS, Basset P, Nahimana-Tessemo I, Jaton K, Greub G, Zanetti G. High proportion of wrongly identified methicillin-resistant Staphylococcus aureus carriers by use of a rapid commercial PCR assay due to presence of staphylococcal cassette chromosome element lacking the mecA gene. J Clin Microbiol 2011;49:722–4. Brown TJ, Anthony RM. The addition of low numbers of 3′ thymine bases can be used to improve the hybridization signal of oligonucleotides for use within arrays on nylon supports. J Microbiol Methods 2000;42:203–7. Carpaij N, Willems RJ, Bonten MJ, Fluit AC. Comparison of the identification of coagulasenegative staphylococci by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and tuf sequencing. Eur J Clin Microbiol Infect Dis 2011;30: 1169–72. Chang HC, Wei YF, Dijkshoorn L, Vaneechoutte M, Tang CT, Chang TC. Identification of Acinetobacter isolates of the A. calcoaceticus-A. baumannii complex by sequence analysis of the 16S-23S rRNA gene spacer region. J Clin Microbiol 2005;43:1632–9. Clerc O, Prod'hom G, Senn L, Jaton K, Zanetti G, Calandra T, et al. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and PCR-based rapid diagnosis of Staphylococcus aureus bacteraemia. Clin Microbiol Infect 2014;20: 355–60. Clinical and Laboratory Standards Institute. M100S-26. Performance standards for antimicrobial disk susceptibility tests; approved standard12th ed. ; 2016 [Wayne: PA]. Couto I, Pereira S, Miragaia M, Sanches IS, de Lencastre H. Identification of clinical staphylococcal isolates from humans by internal transcribed spacer PCR. J Clin Microbiol 2001;39:3099–103. Fujita S, Senda Y, Iwagami T, Hashimoto T. Rapid identification of staphylococcal strains from positive-testing blood culture bottles by internal transcribed spacer PCR followed by microchip gel electrophoresis. J Clin Microbiol 2005;43:1149–57. García-Álvarez L, Holden MTG, Lindsay H, Webb CR, Brown DFJ, Curran MD, et al. Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study. Lancet Infect Dis 2011;11:595–603.

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