Microbiol Immunol 2014; 58: 77–86 doi: 10.1111/1348-0421.12120
O R I GIN A L A R T I C L E
Rapid microarray‐based DNA genoserotyping of Escherichia coli Lutz Geue1, Stefan Monecke2,3, Ines Engelmann2, Sascha Braun2, Peter Slickers2 and Ralf Ehricht2 1
Friedrich‐Loeffler‐Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, Wusterhausen, 2Alere Technologies GmbH, Jena and 3Technical University of Dresden, Institute for Medical Microbiology, Virology and Hygiene, Dresden, Germany
ABSTRACT In this study, an improvement in the oligonucleotide‐based DNA microarray for the genoserotyping of Escherichia coli is presented. Primer and probes for additional 70 O antigen groups were developed. The microarray was transferred to a new platform, the ArrayStrip format, which allows high through‐put tests in 96‐well formats and fully automated microarray analysis. Thus, starting from a single colony, it is possible to determine within a few hours and a single experiment, 94 of the over 180 known O antigen groups as well as 47 of the 53 different H antigens. The microarray was initially validated with a set of defined reference strains that had previously been serotyped by conventional agglutination in various reference centers. For further validation of the microarray, 180 clinical E. coli isolates of human origin (from urine samples, blood cultures, bronchial secretions, and wound swabs) and 53 E. coli isolates from cattle, pigs, and poultry were used. A high degree of concordance between the results of classical antibody‐based serotyping and DNA‐based genoserotyping was demonstrated during validation of the new 70 O antigen groups as well as for the field strains of human and animal origin. Therefore, this oligonucleotide array is a diagnostic tool that is user‐friendly and more efficient than classical serotyping by agglutination. Furthermore, the tests can be performed in almost every routine lab and are easily expanded and standardized. Key words
Escherichia coli, genoserotyping, oligonucleotide‐based DNA microarray.
Escherichia coli belongs to the family of Enterobacteriaceae. These are gram‐negative, facultative anaerobic, often peritrichous bacteria. Because of their oxygen‐using metabolism, E. coli bacteria play an important role in the symbiotic relationship of a host organism with its intestinal flora. In addition to commensal intestinal E. coli, an increasing number of obligatory pathogenic variants of E. coli have been described. These include both extra‐intestinal pathogenic E. coli strains (uropathogenic variants and those causing neonatal meningitis) and intestinal pathogenic E. coli. The latter are grouped in various pathotypes such as enterotoxigenic E. coli, enteropathogenic E. coli, entero‐invasive E. coli, entero‐
aggregative E. coli and STEC. EHEC is a subgroup of STEC. In humans, infections with EHEC serotypes may result in hemorrhagic or non‐hemorrhagic diarrhea, and can be complicated by the HUS (1). Some STEC strains have a zoonotic potential; ruminants are believed to represent their main reservoir (2–5). Because specific serogroups are associated with certain clinical syndromes, serotyping has a central place in the epidemiology and surveillance of E. coli infections (6). Classical antibody‐based serotyping uses specific antibodies to discriminate variants of O and the H antigens. However, conventional serotyping is largely restricted to specialized laboratories and not suitable for routine
Correspondence Lutz Geue, Friedrich‐Loeffler‐Institute, Federal Research Institute for Animal Health, Institute of Epidemiology, Seestrasse 55, D‐16868 Wusterhausen, Germany. Tel: þ49 33979 80189; fax: þ49 33979 80222; email: lutz.geue@fli.bund.de Received 4 September 2013; revised 22 November 2013; accepted 26 November 2013. List of Abbreviations: EHEC, enterohemorrhagic E coli; HUS, hemolytic‐uremic syndrome; STEC, Shiga toxin‐producing E. coli.
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diagnostics because: (i) a complete and standardized set of all known O and H antigens is very costly; (ii) serotyping requires trained and experienced personnel; (iii) agglutination results are not always unambiguous and cross‐reactions can occur; (iv) a minority of strains is non‐typeable; (v), capsular antigens can mask the O antigens; and (iv) non‐motile strains that do not express the flagellar antigen are observed. Different genetic approaches have been described previously (7–9). Ballmer et al. developed a diagnostic oligonucleotide‐based DNA microarray on the ArrayTube format of Alere Technologies GmbH (Jena, Germany) for detection of 24 of the most epidemiologically relevant O antigens from over 180 known O antigens and for 47 of the 53 different H antigens (10). This oligonucleotide array is a diagnostic tool that is simpler and better than classical serotyping by agglutination. It improves the diagnosis and makes epidemiological studies of E. coli infection easier. Furthermore, Braun et al. later showed that this approach can also be applied to the closely related genus Salmonella and its genoserotyping scheme (11), according to the Kauffman and White classification Scheme (12). Here, we present an improvement in the oligonucleotide‐based DNA microarray for genoserotyping E. coli described by Ballmer et al. (10). We developed and processed primers and probes for additional 70 O antigen groups by biomathematical methods. The microarray was transferred to a completely new platform, the ArrayStrip format of Alere Technologies GmbH, which allows high through‐put tests in 96‐well formats and fully automated microarray analysis by imaging. Moreover, the test protocol was optimized. Subsequently, we verified the performance of the more advanced microarray by testing E. coli reference and field strains of human and animal origin.
MATERIALS AND METHODS Bacterial strains, growth conditions and genomic DNA extraction The microarray was verified with a set of reference strains that had previously been serotyped by conventional agglutination in various reference centers (Table 1). For validation of the microarray, 180 clinical E. coli isolates of human origin were used. These included 56 isolates from urine samples, 50 from blood cultures, 39 from bronchial secretions and 35 from wound swabs; all were obtained in the course of routine diagnostic procedures at the Institute for Medical Microbiology and Hygiene, TU Dresden, Dresden, Saxony, Germany. Furthermore, 51 E. coli isolates of animal origin (42 isolates from cattle, 5 from pigs and 4 from poultry) were included; these 78
Table 1. E. coli reference strains
Serotype
Strain id and origin
Genoserotype
O1:K1:H7 O1:K1:H‐ O1 O2:H1 O2:H25 O3 O4 O4 O4:H5 O6 O6 O6:H49 O7 O7 O8:H9 O8:H51 O8:H20 O9 O9:H19 O11:H33 O11:H52 O13:H11 O13 O15:H4 O17 O18ac:K1:H7 O18:H14 O18ab:H14 O21 O22:H8 O24 O25 O25 O26:H46 O26 O26 O27:H30 O28:H37 O28ac:H‐ O29:H10 O29 O32 O32 O35 O40 O42:H37 O44 O45 O45 O52:H10 O53 O53:H3 O55 O55
157043† 158607‡ 166235§ 123118¶ 162373†† 109775††† 123083¶ 123084¶ 123119¶ 123085¶ 123086¶ 123123¶ 123087¶ 166241§ 123081¶ 123124¶ 123132¶ 123075¶ 123134¶ 123106¶ 123125¶ 157044† 131078‡‡ 123109¶ 168481§§ 157045† 157046† 157047† 166242§ 123098¶ 168492§§ 129044¶¶ 166243§ 123117¶ 123857§ 123888§ 123079¶ 188340†† 157048† 157049† 166248§ 165940‡‡ 168493§§ 168494§§ 168495§§ 123113¶ 168497§§ 131135‡‡ 166245§ 123110¶ 123088¶ 123127¶ 123892§ 109789†††
O1:H7 O1:H7 O1:H7 O2:H1 O2:H25 O3:H2 O4:H8 O4:H27 O4:H5 O6:H21 O6:H49 O6:H49 O7:H6 O7:H45 O8:H9 O8:H51 O8:H20 O9:H19 O9:H19 O11:H33 O11:H52 O13 þ O129 þ O135:H11 O13 þ O129 þ O135:H12 O15:H4 O17 þ O44 þ O73 þ O77 þ O106:H18 O18:H7 O18:H14 O18:H14 O21:H4 O22:H8 O24:H26 O25:H4 O25:H1 O26:H46 O26:H11 O26:H11 O27:H30 O28 þ O42:H37 O28 þ O42:H7 O29:H10 O29:H12 O32:H37 O32:H19 O35:H10 O40:H4 O28 þ O42:H37 O17 þ O44 þ O73 þ O77 þ O106:H18 O45:H11 O45:H7 O52:H10 O53:H21 O53:H3 O55:H7 O55:H7
continued
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Genoserotyping of E. coli Table 1. Continued
Serotype O58 O58 O63 O66 O70:H42 O71 O73 O75 O75 O77 O77 O77 O78:K80:H‐ O79:H40 O79 O81 O83 O85:H49 O86:H‐ O86:H34 O87 O91 O98 O98 O99 O101:H‐ O103:H25 O104:H12 O104 O105 O105 O106 O106 O107 O107 O109 O111 O111 O112 O112 O112ac:H‐ O113:H4 O113:H6 O113:H4 O113:H21 O114 O114 O114:H32 O115:H10 O117:H16 O118 O119:H8 O121 O121
Table 1. Continued
Strain id and origin ‡‡
131157 168498§§ 188334¶¶ 168499§§ 123116¶ 168500§§ 168501§§ 184296¶¶ 172823¶¶ 166246§ 129654¶¶ 172819¶¶ 157050† 123111¶ 172831¶¶ 168502§§ 166247§ 158608‡ 123093¶ 123107¶ 168503§§ 158611‡ 168504§§ 187726†† 168505V 123067¶ 169705‡‡‡ 123112¶ 142261¶¶ 168506§§ 131094‡‡ 132486¶¶ 168507§§ 131129‡‡ 168508§§ 168509§§ 123094¶ 166249§ 131112‡‡ 166250§ 157051† 123071¶ 123072¶ 123095¶ 123105¶ 123089¶ 123090¶ 123133¶ 158612‡ 123101¶ 94331§§ 123102¶ 166251§ 131122‡‡
Genoserotype O58:H40 O58:H27 O63:H27 O66:H25 O70:H42 O71:H12 O17 þ O44 þ O73 þ O77 þ O106:H31 O75:H5 O75:H7 O17 þ O44 þ O73 þ O77 þ O106:H18 O17 þ O44 þ O73 þ O77 þ O106:H18 O17 þ O44 þ O73 þ O77 þ O106:H18 O78:H4 O79:H40 O79:H40 O81:H27 O83:Hn.t. O85:H49 O86:H21 O86:H34 O87:H12 O91:H14 O98:H8 O98:H25 O99:H33 O101:H9 O103:H25 O104:H12 O104:H4 O105:H8 O105:H4 O17 þ O44 þ O73 þ O77 þ O106:H18 O17 þ O44 þ O73 þ O77 þ O106:H33 O107 þ O117:H27 O107 þ O117:H27 O109:H19 O111:H8 O111:H2 O112:H2 O112:H8 O112ac:Hn.t. O113:H4 O113:H6 O113:H4 O113:H21 O114:H4 O114:H9 O114:H32 O115:H10 O107 þ O117:H16 O118 þ O151:H10 O119:H8 O121:H19 O121:H46
continued
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Serotype O123 O123 O123 O123 O124:H30 O126:H2 O127a:H‐ O127:H6 O128:H2 O128:H‐ O129 O129 O130 O138 O139 O141 O141:K85 O143:H‐ O145 O146:H28 O147 O147 O148:H53 O149 O149 O150 O150 O151 O152:H‐ O152 O157:H12 O157:H38 O157:H18 O157:H43 O157:H7 O159 O164:H‐ O167:H5 O168 O172:H‐ O174:H27 O177 O177 O177
Strain id and origin §
123333 123874§ 131150‡‡ 168510§§ 157052† 157053† 157054† 158604‡ 123069¶ 162374†† 131140‡‡ 168511§§ 168512§§ 168513§§ 168514§§ 187744†† 158610‡ 157055† 109797††† 123077¶ 168515§§ 187748†† 123128¶ 168516§§ 109792††† 131107‡‡ 166252§ 168517§§ 157056† 168518§§ 123073¶ 123080¶ 123082¶ 123091¶ 157057† 168519§§ 157058† 157059† 162371¶¶ 123130¶ 123131¶ 166253§ 97991†† 97996††
Genoserotype O123:H2 O123:H2 O123:H16 O123:H16 O124 þ O164:H30 O126:H2 O127:H6 O127:H6 O128:H2 O128:H2 O13 þ O129 þ O135:H11 O13 þ O129 þ O135:H11 O130:H9 O138:H4 O139:H1 O141:H4 O141:H4 O143:Hn.t. O145:H34 O146:H28 O147:H19 O147:H14 O148:H53 O149:H10 O149:H10 O150:H8 O150:H21 O118 þ O151:H10 O152:Hn.t. O152:Hn.t. O157:H12 O157:H38 O157:H18 O157:H43 O157:H7 O159:H20 O124 þ O164:H7 O167:H5 O168:H4 O172:H25 O174:H27 O177:H25 O177:H25 O177:H25
†
Leibniz Institute DSMZ‐German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany. ‡F Gunzer, Institute for Medical Microbiology and Hygiene, Dresden, Germany. §E. Bingen, Service de Microbiology, Paris, France. ¶H. Hächler, Institute for Food Safety and Food Hygiene, Zürich, Switzerland. ††L. Geue, Friedrich Loeffler Institute, Wusterhausen, Germany. ‡‡S. Blum, Kimron Veterinary Institute, Bet Dagan, Israel. §§S. Chappell, Animal Health and Veterinary Laboratories Agency, Weybridge, UK. ¶¶S. Monecke, Institute for Medical Microbiology and Hygiene, Dresden, Germany. †††L. Beutin, Federal Institute for Risk Assessment, Berlin, Germany. ‡‡‡T. Lindbäck, Norwegian School of Veterinary Science, Oslo, Norway.
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originated from clinical diagnostics or epidemiological studies. The strains were cultivated on tryptone yeast agar (VWR International GmbH, Darmstadt, Germany). A full 1 mL loop (diameter 1 mm) of clonal colony material of each strain was picked from solid medium, resuspended in 200 mL lysis reagents (lysis enhancer A2 dissolved in 200 mL lysis buffer A1, Alere Technologies GmbH) and incubated for 30–60 min at 37°C and 550 rpm in a thermomixing device (Eppendorf GmbH, Hamburg, Germany). RNA‐free unfragmented genomic DNA was extracted with a Qiagen DNeasy Blood & Tissue kit (Qiagen GmbH, Heiden, Germany) according to the manufacturer's instructions. The DNA concentration was determined spectrophotometrically at 260 nm and finally analyzed for fragmentation by electrophoresis in a 1% non‐denaturing agarose gel. Multiplex linear DNA amplification and labeling for hybridization to prepared ArrayStrips For multiplex linear DNA amplification, a set of 252 primers was used. One hundred and three of these primers have been described elsewhere (10). A list of the additional new 149 primers is shown in Table S1. These primers are non‐overlapping, but located as close as possible, on the complementary strand, upstream of the position of the oligonucleotide probe. For labeling and biotinylation of the genomic DNA, a site‐specific labeling approach was used (13). A primer elongation reaction was performed using a primer mixture, the HybPLUSKit (Alere) and 0.5–1.5 mg unfragmented RNA‐free genomic DNA of the E. coli isolates according to the manufacturer's instructions. The reaction was started with denaturation (5 min, 96°C), followed by 50 cycles of 60 s at 96°C, 20 s at 50°C, and 40 s at 72°C. The sample was then cooled down to 4°C. Hybridization of the DNA‐based serotyping E. coli ArrayStrips The ArrayStrips, which were spotted with the probes for serotyping (see Table S1), were produced by Alere Technologies GmbH. Details on the layout of the ArrayStrips are provided in the Results Section. For hybridization, a HybPLUSKit (Alere) was used with the following adapted protocol. Each ArrayStrip was initially washed with 200 mL double‐distilled water and then with 150 mL of buffer C1 using a thermomixing device (BioShake IQ; Qinstruments GmbH, Jena, Germany; each 5 min, 50°C, 550 rpm). The hybridization sample consisted of 10 mL labeled probe and 90 mL buffer C1. It was transferred into the ArrayStrip and incubated 80
(60 min, 50°C, 550 rpm). The sample was then removed from the tube and the array washed twice (10 min, 45°C, 550 rpm), with buffer C2. Next, 100 mL conjugate solution (1 mL C3 HRP conjugate plus 99 mL C4 conjugation buffer) was added for 10 min at 30°C and 550 rpm followed by a washing step with 200 mL buffer C5 for 5 min at 30°C and 550 rpm. The ArrayStrip was then stained with buffer D1 (100 mL, 10 min, no agitation). After removal of the liquids, it was photographed using an ArrayMate instrument (Alere) and automatically analyzed. Mean signal intensity (mean) and local background were measured for each probe position and values calculated by the following formula: value ¼ 1 mean/local background. Breakpoints for the interpretation of signals were defined based on a series of experiments with known, characterized reference strains (DSM10728, DSM1058, DSM10720, DSM10784, DSM11753, DSM9025, DSM9026, DSM5212, DSM9027, DSM9031, DSM8701, DSM8702, DSM9028, DSM9030, DSM17076, DSM9034, DSM9033‐ Leibniz Institut DSMZ‐Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). Resulting values below 0.1 were considered negative and above 0.3 as positive. Values between 0.1 and 0.3 were regarded as inconclusive.
RESULTS Target gene selection and construction of array The oligonucleotide array‐based detection of 24 of the over 180 known O antigens (O antigens 4, 6–9, 15, 26, 52, 53, 55, 79, 86, 91, 101, 103, 104, 111, 113, 114, 121, 128, 145, 157 and 172) by use of the target sequences of wzx (an O antigen flipase) or wzy (an O antigen polymerase) was developed by Ballmer et al. (10). In the present study, the same strategies were applied to search in public sequence databases (GenBank, EMBL) for additional wzy and wzx sequences and other previously described O‐specific sequences, such as wzm, wzt, rfbU, rfbE, isla29, sil‐inv, sil‐1, sil‐2, wbdA, wbdH, wbdM, wbdU, gtrB and gtrX. Ballmer et al. evaluated probes for 47 of the 53 known H antigens by using the fliC locus‐specific sequences (10). All these probes were re‐used for the expanded microarray described herein. New 23 to 31 bp oligonucleotide probes were designed as described previously (10). The new probe sequences were designated using a code consisting of the gene name and the serotype or strain number (Table S1; Fig. S1). Seven wzx or wzy gene probes covered multiple O groups as follows: wzx_O13 þ O129 þ O135/wzy_O13 þ O129 þO135 (O13, O129, O135), wzx_O17 þ O44 þ O73 þO77 þ O106/ © 2013 The Societies and Wiley Publishing Asia Pty Ltd
Genoserotyping of E. coli
wzy_O17 þ O44 þ O73 þO77 þ O106 (O17, O44, O73, O77, O106), wzx_O28 þ O42/wzy_O28 þ O42 (O28 and O42), wzx_O107 þO117/wzy_O107 þ O117 (O107 and O117), wzx_O118 þ O151/wzy_O118 þ O151 (O118 and O151), wzx_O124 þ O164/wzy_O124 þ O164 (O124 and O164) and wzx_O152 þ O173/wzy_O152 þ O173 (O152 and O173). Additionally, an individual‐specific spot for O152 is present. The array comprises a total of 259 different probes, each probe being spotted in duplicate. A staining control (Biotin‐Marker), family‐specific controls for Enterobacteriaceae (hp_rrs_611, hp_rrs_612, hp_dnaE_613 and prob_gapA_611) and species‐specific controls for E. coli (hp_dnaE_612 and gad_10) complete the array. DNA‐free spotting buffer was used as a negative control (Fig. S1). Initial validation of the diagnostic microarray for the new O antigen groups The performance for the new O antigen groups of the microarray was tested for each spot with a series of reference strains (Table 1). The system detected the available sequences for O antigens with a high degree of reliability (Table S1). The wzx or wzy gene probes had a specificity and sensitivity of 100%. All 70 new O antigen groups were correctly identified with each corresponding reference strain. Unspecific reactions were rare and limited to a small number of spots exclusively representing O antigen‐specific genes other than wzx or wzy. Most cross‐reactions were found for the target sequences isla29‐ O145_11, sil‐inv‐O145_11, rfbU‐O157_11, sil‐inv‐O157_11, sil1‐O157_11 and sil2‐O157_11. False‐positive signals on spots of these target sequences were found in 24.6% of the reference strains. Correct identification of 47 H antigen groups has already been demonstrated by Ballmer et al. (10). In the present study, all serotyped H antigen groups of the reference strains were identified correctly. Further validation of the diagnostic microarray using E. coli field strains For further validation of the microarray, 180 clinical E. coli isolates of human origin (56 from urine, 50 from blood cultures, 39 from bronchial secretions and 35 from wound swabs) were tested. All isolates were characterized as E. coli by VITEK‐II (bioMérieux, Nuertingen, Germany/Marcy l'Etoile, France), a commercially available system for routine identification of clinically relevant bacteria based on metabolic profiles and for susceptibility tests using a miniaturized agar dilution approach. However, classical antibody‐based serotyping was not performed on these isolates. All isolates were identified as E. coli; and 132 of the 180 isolates (73.3%) were assigned unambiguously to 26 O antigen groups (O1, O2, O4, O6–O9, O11, O15, O18, © 2013 The Societies and Wiley Publishing Asia Pty Ltd
O21, O22, O24, O25, O58, O63, O75, O78, O79, O83, O86, O91, O101, O130, O143 and O150) using the wzx or wzy gene probes. Most isolates were grouped as O25 (24 isolates), O8 (15 isolates), O2 (13 isolates), O6 (13 isolates), O9 (11 isolates), and O101 (8 isolates). E. coli isolates of the other O groups were detected much less frequently (Table 2). For 10 isolates positive signals were observed with DNA gene probes for several serogenotypes (wzx_O107 þ O117/wzy_O107 þ O11, one isolate; wzx_O17 þ O44þ O73þ O77þ O106/wzy_O17 þ O44 þ O73 þ O77 þ O106, nine isolates). This occurred because the sequences of the wzx/wzy genes of these O groups are so similar that exact separation is not possible. Additional positive reactions, besides the wzx and wzy gene probes, were found in only 6 of the 180 isolates (3.3%). Here, positive signals were obtained for the wzx_O9/wzy_O9 and wz_O101_11 gene probes. It should be noted that this wz gene probe is not an allele of wzx/wzy (Table 2). No signals were detected for 32 isolates (17.8%). Six probes (isla29‐O145_11, sil‐inv‐ O145_11, rfbU‐O157_11, sil‐inv‐O157_11, sil1‐O157_11 and sil2‐O157_11) showed strong cross‐reactions with about 65% of all strains. For 176 of the 180 E. coli field strains (97.8%), the H antigen groups were unambiguously identified. The isolates were assigned to 25 H antigen groups (H1, H2, H4–H10, H12, H16, H18–H21, H25, H27, H28, H30, H34, H40, H41, H42, H45 and H51). Most isolates were grouped as H4 (39 isolates), H9 (21 isolates), H1 (20 isolates), H7 (16 isolates), H18 (14 isolates) and H6 (13 isolates). E. coli isolates of the other H antigen groups were detected much less frequently (Table 2). Cross‐reactions were not observed. For just four isolates no H antigen groups could be identified because no signals were obtained with the fliC probes. All results for the strains of human origin are shown in Table 2. Additionally, 51 E. coli isolates of animal origin were tested. Complete classical antibody‐based O:H serotyping results were known for 37 of the 42 cattle isolates. For the E. coli strains isolated from pigs and poultry, only the O antigen groups had been determined by antibody‐based serotyping. A very good concordance was found between the results of classical antibody‐based serotyping and the DNA‐based genoserotyping by oligonucleotide microarray. Different results were only obtained for the O antigen groups of three E. coli strains. One O136:H49 isolate showed a positive signal with the DNA probes wzx_O6_11/wzy_O6_11 (Table 3) whereas fliC for H49 was correctly identified. A second discrepancy, a clearly positive signal with the K‐12‐specific DNA gene probes (wzx_O150‐K12/wzy_O150‐K12), was detected for an E. coli isolate that had originally been reported as O69:H (Table 3). Furthermore, no signal for any wzx/wzy target sequence was detected for an O98:H‐, although DNA 81
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Table 2. Results of genoserotyping E. coli isolates of human origin
O antigen group (wzx/wzy) 1 2 4 6 7 8 9 9,101 101 11 15 18 21 22 24 25 58 63 75 78 79 83 86 91 130 143 O017 þ O044 þ O073 þ O077 þ O106 O107 þ O117 O150_K‐12 — Total H antigen group (fliC) 1 2 4 5 6 7 8 9 10 12 16 18 19 20 21 25 27 28
Number
Isolates from urine
Isolates from bronchial secretions
7 13 3 13 1 15 11 6 9 1 5 6 1 1 4 24 1 1 6 1 1 1 2 1 1 1 9 1 2 32 180
2 5 2 3 0 2 6 2 3 0 1 1 1 0 1 8 0 1 3 0 1 0 0 1 0 0 4 0 1 8 56
1 2 0 3 0 8 0 0 2 0 0 2 0 1 1 9 1 0 0 0 0 0 1 0 0 0 1 0 0 7 39
4 1 0 4 1 2 3 3 4 0 3 1 0 0 2 2 0 0 2 1 0 0 1 0 1 1 2 1 0 11 50
0 5 1 3 0 3 2 1 0 1 1 2 0 0 0 5 0 0 1 0 0 1 0 0 0 0 2 0 1 6 35
20 1 39 6 13 16 3 21 7 3 2 14 6 1 3 9 1 2
7 0 11 2 5 5 0 5 2 1 1 5 2 0 0 4 1 1
2 0 13 0 2 3 1 6 2 0 0 2 1 0 2 3 0 0
6 1 9 3 1 4 2 9 1 1 1 6 1 1 0 1 0 0
5 0 6 1 5 4 0 1 2 1 0 1 2 0 1 1 0 1
Isolates from blood cultures
Isolates from wound swabs
continued
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Genoserotyping of E. coli Table 2. Continued
30 34 40 41 42 45 51 — Total
Number
Isolates from urine
Isolates from bronchial secretions
3 1 1 1 1 1 1 4 180
1 0 1 0 1 0 0 1 56
0 0 0 0 0 0 0 2 39
Isolates from blood cultures
Isolates from wound swabs
1 1 0 0 0 0 1 0 50
1 0 0 1 0 1 0 1 35
Table 3. Results of genoserotyping compared to the classical antibody‐based serotyping for E. coli isolates of animal origin Number 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Host
O antigen group (wzx, wzy)
H antigen group (fliC)
Classical antibody‐based serotyping
Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle
26 28 þ 42 — 128 6 98 172 172 — — 157 35 177 26 107 þ 117 40 — — — 40 — — 40 — — — — — — 8 8 103 174 O118 þ O151 150 174
11 37 9 2 49 25 25 25 4 2 7 2 25 11 27 2 25 25 25 2 25 25 2 25 8 25 5 25 2 21 21 2 21 16 2 21
O26:H11 O28:H37 O136:H‐ O128:H2 O136:H49 O98:H‐ O172:H‐ O172:H‐ O98:H‐ O84:H‐ O157:H7 O35:H2 Orough:H25 O26:H11 O107:H27 Not tested O165:H25 O165:H25 O165:H25 Not tested O165:H25 O165:H25 Not tested O165:H25 Not tested O156:H25 Not tested O156:H25 Orough:H‐ Ont:H‐ Ont:H‐ O103:H2 O174:H‐ Ont:H16 O150:H‐ O174:H‐
continued
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L. Geue et al. Table 3. Continued Number 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
Host
O antigen group (wzx, wzy)
H antigen group (fliC)
Classical antibody‐based serotyping
Cattle Cattle Cattle Cattle Cattle Cattle Pig Pig Pig Pig Pig Poultry Poultry Poultry Poultry
K‐12 174 174 103 177 174 149 141 149 149 147 1 1 2 2
2 21 21 2 25 21 10 4 10 10 14 15 15 6 9
O69:H‐ O174:H‐ O174:H‐ O103:H2 O177:H‐ O174:H‐ O149 O141 O149 O149 O147 O1 O1 O2 O2
probes for O98 (wzx_O98/wzy_O98) are present on the microarray (Table 3). The microarray revealed the O groups of five strains that had not been identified by classical antibody‐based serotyping (rough forms or untypeable). Because the target sequences for these O antigen groups are not yet known and therefore not covered by the microarray, correct positive signals for wzx/wzy DNA probes were lacking on the microarray for six O165:H25, two O156:H25, one O136:H‐ and one O84:H‐ strain. Three of the five strains without classical serotyping data were characterized as O40:H2, whereas only the H groups (H5 and H8) were identified in the remaining two isolates. Full accordance of the O antigen identifications by classical antibody‐based serotyping and by microarray was found for all strains isolated from pigs and poultry. There was also full accordance for H antigen group identifications obtained with the two different methods. Importantly, for 17 strains that had been reported to be non‐motile, clear results for a fliC gene, which apparently was not expressed, were found (Table 3). Unspecific reactions were rare, being limited to a small number of spots in seven strains (13.7%) exclusively representing O antigen‐specific genes other than wzx or wzy. All results are listed in Table 3. The datasets of mean values, background values and calculated signals of detected spots are shown in Tables S2, S3, and S4.
DISCUSSION We here present an improved version of the oligonucleotide‐based DNA microarray for E. coli genoserotyping described by Ballmer et al. (10). We have expanded the microarray to cover 94 O antigen groups (O antigens 1– 4, 6–9, 11, 13, 15, 17, 18, 21, 22, 24–29, 32, 35, 40, 42, 44, 45, 52, 53, 55, 56, 58, 63, 66, 70, 71, 73, 75, 77–79, 81, 83, 85–87, 91, 98, 101, 103–107, 111–115, 112ac, 117– 84
119, 121, 123, 124, 126–130, 135, 138, 139, 141, 143, 145– 152, 157, 159, 164, 167, 168, 172–174 and 177). Ballmer et al. evaluated probes of 47 of the 53 known H antigens (H antigens 1–12, 14–16, 18–21, 23–34, 37–43, 45, 46, 48, 49, 51–54, and 56) (10). We used all these probes in the improved microarray. In addition, we transferred the microarray to the ArrayStrip format of Alere Technologies GmbH, which allows high through‐put tests in 96‐well formats and fully automated microarray analysis during imaging. Initial validation against agglutination procedures as the “gold standard,” showed a sensitivity and specificity of 100% using the wzx or wzy gene probes for the new O antigen groups in addition to the panel of probes. All new 70 O antigen groups were correctly identified by each corresponding reference strain. Also, we correctly identified all H antigen groups of serotyped reference strains. Furthermore, validation of the microarray with field strains of human and animal origin yielded excellent results. We unambiguously identified more than 70% of the O antigen groups and nearly 98% of the H antigen groups of the E. coli human isolates. For the remaining strains, we could not include DNA probes in the microarray because the target sequences are unknown. We found no cross‐reactions either using the wzx/wzy DNA probes or for H antigen group determinations by fliC gene probes. Moreover, we found a high degree of concordance between the results of classical antibody‐based serotyping and DNA‐based genoserotyping by oligonucleotide microarray for the E. coli strains of animal origin. We obtained different results regarding the O antigen groups for only three strains. It is possible that the classical antibody‐ based serotyping was incorrect for these strains. It is also possible that the cultures were polyclonal, that is contaminated with a second strain, and that one © 2013 The Societies and Wiley Publishing Asia Pty Ltd
Genoserotyping of E. coli
clone was tested serologically whereas another one was subjected to genotyping. Genes selected for detection of the O antigen, that is, wzx and wzy, are the most promising target sequences for an oligonucleotide DNA microarray‐based typing approach, the results of which correlated strongly with those of the classical agglutination method. The agreement of the results of the genotyping assay for wzx, wzy, and fliC with those of serotyping is so high that the microarray technology may replace serotyping as the “gold standard” once relevant sequence data for all serotypes are available. The alternative genes proved to be less specific. We detected most cross‐reactions for the target sequences isla29‐ O145_11, sil‐inv‐O145_11, rfbU‐O157_11, sil‐inv‐O157_11, sil1‐O157_11 and sil2‐O157_11. We found false‐positive signals for these target sequences in a quarter of the reference strains. Among clinical isolates of human origin, we detected up to 65% cross‐hybridization signals with unrelated serotypes. This indicates that the genes of the regions selected for probe design are less suitable than the highly discriminatory wzx and wzy genes. In summary, genoserotyping is very important from a scientific and practical view. We and other authors have previously shown this for E. coli (10) and other bacterial species (5, 11, 14–16). In this study, we detected an excellent match between the results of classical antibody‐ based serotyping and DNA‐based genoserotyping both in validation of the new 70 O antigen groups as well as for the field strains of human and animal origin. The advantages of geroserotyping are obvious, namely: (i) unambiguous results for more than half of all known O antigen groups and nearly all H antigen groups within a few hours, starting from a single colony; (ii) parallel detection of O and H antigens of a single E. coli isolate in a single experiment; and (iii) simultaneous typing of a considerable number of E. coli isolates by use of the ArrayStrip format. Thus, the O and H oligonucleotide microarray is an easy and fast technique and provides a helpful and powerful tool for characterization of pathogenic and non‐pathogenic E. coli.
ACKNOWLEDGMENTS We thank F. Gunzer (Institute for Medical Microbiology and Hygiene), E. Bingen (Service de Microbiology, Paris, France), H. Hächler (Institute for Food Safety and Food Hygiene, Zürich, Switzerland), S. Blum (Kimron Veterinary Institute, Bet Dagan, Israel), S. Chappell (Animal Health and Veterinary Laboratories Agency, Weybridge, UK), L. Beutin (Federal Institute for Risk Assessment, Berlin, Germany), and T. Lindbäck (Norwegian School of Veterinary Science, Oslo, Norway) for kindly providing reference strains. © 2013 The Societies and Wiley Publishing Asia Pty Ltd
DISCLOSURE S. Monecke, I. Engelmann, P. Slickers, S. Braun, and R. Ehricht are employees of Alere Technologies, the company that manufactures the microarrays also used in this study. This had no influence on study design, data collection, and analysis, and did not alter the authors' adherence to all Microbiology and Immunology's policies on sharing data and materials. The other authors do not declare any conflict of interest.
REFERENCES 1. Johnson K.E., Thorpe C.M., Sears C.L. (2006) The emerging clinical importance of non‐O157 Shiga toxin‐producing Escherichia coli. Clin Infect Dis 43(12): 1587–95. 2. Blanco J., Blanco M., Blanco J.E., Mora A., Gonzalez E.A., Bernardez M.I., Alonso M.P., Coira A., Rodriguez A., Rey J., Alonso J.M., Usera M.A. (2003) Verotoxin‐producing Escherichia coli in Spain: prevalence, serotypes, and virulence genes of O157: H7 and non‐O157 VTEC in ruminants, raw beef products, and humans. Exp Biol Med (Maywood) 228(4): 345–51. 3. Geue L., Segura‐Alvarez M., Conraths F.J., Kuczius T., Bockemuhl J., Karch H., Gallien P. (2002) A long‐term study on the prevalence of Shiga toxin‐producing Escherichia coli (STEC) on four German cattle farms. Epidemiol Infect 129(1): 173– 85. 4. Schilling A.K., Hotzel H., Methner U., Sprague L.D., Schmoock G., El‐Adawy H., Ehricht R., Wohr A.C., Erhard M., Geue L. (2012) Zoonotic agents in small ruminants kept on city farms in southern Germany. Appl Environ Microbiol 78(11): 3785– 93. 5. Zweifel C., Blanco J.E., Blanco M., Blanco J., Stephan R. (2004) Serotypes and virulence genes of ovine non‐O157 Shiga toxin‐ producing Escherichia coli in Switzerland. Int J Food Microbiol 95(1): 19–27. 6. Kaper J.B., Nataro J.P., Mobley H.L. (2004) Pathogenic Escherichia coli. Nat Rev Microbiol 2(2): 123–40. 7. Coimbra R.S., Grimont F., Lenormand P., Burguiere P., Beutin L., Grimont P.A. (2000) Identification of Escherichia coli O‐ serogroups by restriction of the amplified O antigen gene cluster (rfb‐RFLP). Res Microbiol 151(8): 639–54. 8. Machado J., Grimont F., Grimont P.A. (2000) Identification of Escherichia coli flagellar types by restriction of the amplified fliC gene. Res Microbiol 151(7): 535–46. 9. Prager R., Strutz U., Fruth A., Tschäpe H. (2003) Subtyping of pathogenic Escherichia coli strains using flagellar (H)‐antigens: serotyping versus fliC polymorphisms. Int J Med Microbiol 292(7/ 8): 477–86. 10. Ballmer K., Korczak B.M., Kuhnert P., Slickers P., Ehricht R., Hachler H. (2007) Fast DNA serotyping of Escherichia coli by use of an oligonucleotide microarray. J Clin Microbiol 45(2): 370–9. 11. Braun S.D., Ziegler A., Methner U., Slickers P., Keiling S., Monecke S., Ehricht R. (2012) Fast DNA serotyping and antimicrobial resistance gene determination of Salmonella enterica with an oligonucleotide microarray‐based assay. PLoS ONE 7(10): e46489. 12. Grimont P.A., Weill F.X. (2007). Antigenic Formulae of the Salmonella serovars. Paris: Institute Pasteur: WHO Collaborating Centre for Reference and Research on Salmonella.
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13. Monecke S., Ehricht R. (2005) Rapid genotyping of methicillin‐ resistant Staphylococcus aureus (MRSA) isolates using miniaturised oligonucleotide arrays. Clin Microbiol Infect 11(10): 825–33. 14. Ruettger A., Feige J., Slickers P., Schubert E., Morre S.A., Pannekoek Y., Herrmann B., de Vries H.J., Ehricht R., Sachse K. (2011) Genotyping of Chlamydia trachomatis strains from culture and clinical samples using an ompA‐based DNA microarray assay. Mol Cell Probes 25(1): 19–27. 15. Sachse K., Laroucau K., Hotzel H., Schubert E., Ehricht R., Slickers P. (2008) Genotyping of Chlamydophila psittaci using a new DNA microarray assay based on sequence analysis of ompA genes. BMC Microbiol 8: 63. 16. Sachse K., Laroucau K., Vorimore F., Magnino S., Feige J., Müller W., Kube S., Hotzel H., Schubert E., Slickers P., Ehricht R. (2009) DNA microarray‐based genotyping of Chlamydophila psittaci strains from culture and clinical samples. Vet Microbiol 135(1/2): 22–30.
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Supporting Information
Additional supporting information may be found in the online version of this article at the publisher's web site. Fig. S1. Layout of the oligonucleotide microarray. Table S1 Reference strains, chip layout and validation of results Table S2. Data concerning mean values of the detected spots (mean_sero.xlsx) Table S3. Data concerning background values of detected spots (background_sero.xlsx) Table S4. Data concerning calculated signals of detected spots (signal_data_sero.xlsx)
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O R I GIN A L A R T I C L E
Rapid microarray‐based DNA genoserotyping of Escherichia coli Lutz Geue1, Stefan Monecke2,3, Ines Engelmann2, Sascha Braun2, Peter Slickers2 and Ralf Ehricht2 1
Friedrich‐Loeffler‐Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, Wusterhausen, 2Alere Technologies GmbH, Jena and 3Technical University of Dresden, Institute for Medical Microbiology, Virology and Hygiene, Dresden, Germany
ABSTRACT In this study, an improvement in the oligonucleotide‐based DNA microarray for the genoserotyping of Escherichia coli is presented. Primer and probes for additional 70 O antigen groups were developed. The microarray was transferred to a new platform, the ArrayStrip format, which allows high through‐put tests in 96‐well formats and fully automated microarray analysis. Thus, starting from a single colony, it is possible to determine within a few hours and a single experiment, 94 of the over 180 known O antigen groups as well as 47 of the 53 different H antigens. The microarray was initially validated with a set of defined reference strains that had previously been serotyped by conventional agglutination in various reference centers. For further validation of the microarray, 180 clinical E. coli isolates of human origin (from urine samples, blood cultures, bronchial secretions, and wound swabs) and 53 E. coli isolates from cattle, pigs, and poultry were used. A high degree of concordance between the results of classical antibody‐based serotyping and DNA‐based genoserotyping was demonstrated during validation of the new 70 O antigen groups as well as for the field strains of human and animal origin. Therefore, this oligonucleotide array is a diagnostic tool that is user‐friendly and more efficient than classical serotyping by agglutination. Furthermore, the tests can be performed in almost every routine lab and are easily expanded and standardized. Key words
Escherichia coli, genoserotyping, oligonucleotide‐based DNA microarray.
Escherichia coli belongs to the family of Enterobacteriaceae. These are gram‐negative, facultative anaerobic, often peritrichous bacteria. Because of their oxygen‐using metabolism, E. coli bacteria play an important role in the symbiotic relationship of a host organism with its intestinal flora. In addition to commensal intestinal E. coli, an increasing number of obligatory pathogenic variants of E. coli have been described. These include both extra‐intestinal pathogenic E. coli strains (uropathogenic variants and those causing neonatal meningitis) and intestinal pathogenic E. coli. The latter are grouped in various pathotypes such as enterotoxigenic E. coli, enteropathogenic E. coli, entero‐invasive E. coli, entero‐
aggregative E. coli and STEC. EHEC is a subgroup of STEC. In humans, infections with EHEC serotypes may result in hemorrhagic or non‐hemorrhagic diarrhea, and can be complicated by the HUS (1). Some STEC strains have a zoonotic potential; ruminants are believed to represent their main reservoir (2–5). Because specific serogroups are associated with certain clinical syndromes, serotyping has a central place in the epidemiology and surveillance of E. coli infections (6). Classical antibody‐based serotyping uses specific antibodies to discriminate variants of O and the H antigens. However, conventional serotyping is largely restricted to specialized laboratories and not suitable for routine
Correspondence Lutz Geue, Friedrich‐Loeffler‐Institute, Federal Research Institute for Animal Health, Institute of Epidemiology, Seestrasse 55, D‐16868 Wusterhausen, Germany. Tel: þ49 33979 80189; fax: þ49 33979 80222; email: lutz.geue@fli.bund.de Received 4 September 2013; revised 22 November 2013; accepted 26 November 2013. List of Abbreviations: EHEC, enterohemorrhagic E coli; HUS, hemolytic‐uremic syndrome; STEC, Shiga toxin‐producing E. coli.
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diagnostics because: (i) a complete and standardized set of all known O and H antigens is very costly; (ii) serotyping requires trained and experienced personnel; (iii) agglutination results are not always unambiguous and cross‐reactions can occur; (iv) a minority of strains is non‐typeable; (v), capsular antigens can mask the O antigens; and (iv) non‐motile strains that do not express the flagellar antigen are observed. Different genetic approaches have been described previously (7–9). Ballmer et al. developed a diagnostic oligonucleotide‐based DNA microarray on the ArrayTube format of Alere Technologies GmbH (Jena, Germany) for detection of 24 of the most epidemiologically relevant O antigens from over 180 known O antigens and for 47 of the 53 different H antigens (10). This oligonucleotide array is a diagnostic tool that is simpler and better than classical serotyping by agglutination. It improves the diagnosis and makes epidemiological studies of E. coli infection easier. Furthermore, Braun et al. later showed that this approach can also be applied to the closely related genus Salmonella and its genoserotyping scheme (11), according to the Kauffman and White classification Scheme (12). Here, we present an improvement in the oligonucleotide‐based DNA microarray for genoserotyping E. coli described by Ballmer et al. (10). We developed and processed primers and probes for additional 70 O antigen groups by biomathematical methods. The microarray was transferred to a completely new platform, the ArrayStrip format of Alere Technologies GmbH, which allows high through‐put tests in 96‐well formats and fully automated microarray analysis by imaging. Moreover, the test protocol was optimized. Subsequently, we verified the performance of the more advanced microarray by testing E. coli reference and field strains of human and animal origin.
MATERIALS AND METHODS Bacterial strains, growth conditions and genomic DNA extraction The microarray was verified with a set of reference strains that had previously been serotyped by conventional agglutination in various reference centers (Table 1). For validation of the microarray, 180 clinical E. coli isolates of human origin were used. These included 56 isolates from urine samples, 50 from blood cultures, 39 from bronchial secretions and 35 from wound swabs; all were obtained in the course of routine diagnostic procedures at the Institute for Medical Microbiology and Hygiene, TU Dresden, Dresden, Saxony, Germany. Furthermore, 51 E. coli isolates of animal origin (42 isolates from cattle, 5 from pigs and 4 from poultry) were included; these 78
Table 1. E. coli reference strains
Serotype
Strain id and origin
Genoserotype
O1:K1:H7 O1:K1:H‐ O1 O2:H1 O2:H25 O3 O4 O4 O4:H5 O6 O6 O6:H49 O7 O7 O8:H9 O8:H51 O8:H20 O9 O9:H19 O11:H33 O11:H52 O13:H11 O13 O15:H4 O17 O18ac:K1:H7 O18:H14 O18ab:H14 O21 O22:H8 O24 O25 O25 O26:H46 O26 O26 O27:H30 O28:H37 O28ac:H‐ O29:H10 O29 O32 O32 O35 O40 O42:H37 O44 O45 O45 O52:H10 O53 O53:H3 O55 O55
157043† 158607‡ 166235§ 123118¶ 162373†† 109775††† 123083¶ 123084¶ 123119¶ 123085¶ 123086¶ 123123¶ 123087¶ 166241§ 123081¶ 123124¶ 123132¶ 123075¶ 123134¶ 123106¶ 123125¶ 157044† 131078‡‡ 123109¶ 168481§§ 157045† 157046† 157047† 166242§ 123098¶ 168492§§ 129044¶¶ 166243§ 123117¶ 123857§ 123888§ 123079¶ 188340†† 157048† 157049† 166248§ 165940‡‡ 168493§§ 168494§§ 168495§§ 123113¶ 168497§§ 131135‡‡ 166245§ 123110¶ 123088¶ 123127¶ 123892§ 109789†††
O1:H7 O1:H7 O1:H7 O2:H1 O2:H25 O3:H2 O4:H8 O4:H27 O4:H5 O6:H21 O6:H49 O6:H49 O7:H6 O7:H45 O8:H9 O8:H51 O8:H20 O9:H19 O9:H19 O11:H33 O11:H52 O13 þ O129 þ O135:H11 O13 þ O129 þ O135:H12 O15:H4 O17 þ O44 þ O73 þ O77 þ O106:H18 O18:H7 O18:H14 O18:H14 O21:H4 O22:H8 O24:H26 O25:H4 O25:H1 O26:H46 O26:H11 O26:H11 O27:H30 O28 þ O42:H37 O28 þ O42:H7 O29:H10 O29:H12 O32:H37 O32:H19 O35:H10 O40:H4 O28 þ O42:H37 O17 þ O44 þ O73 þ O77 þ O106:H18 O45:H11 O45:H7 O52:H10 O53:H21 O53:H3 O55:H7 O55:H7
continued
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Genoserotyping of E. coli Table 1. Continued
Serotype O58 O58 O63 O66 O70:H42 O71 O73 O75 O75 O77 O77 O77 O78:K80:H‐ O79:H40 O79 O81 O83 O85:H49 O86:H‐ O86:H34 O87 O91 O98 O98 O99 O101:H‐ O103:H25 O104:H12 O104 O105 O105 O106 O106 O107 O107 O109 O111 O111 O112 O112 O112ac:H‐ O113:H4 O113:H6 O113:H4 O113:H21 O114 O114 O114:H32 O115:H10 O117:H16 O118 O119:H8 O121 O121
Table 1. Continued
Strain id and origin ‡‡
131157 168498§§ 188334¶¶ 168499§§ 123116¶ 168500§§ 168501§§ 184296¶¶ 172823¶¶ 166246§ 129654¶¶ 172819¶¶ 157050† 123111¶ 172831¶¶ 168502§§ 166247§ 158608‡ 123093¶ 123107¶ 168503§§ 158611‡ 168504§§ 187726†† 168505V 123067¶ 169705‡‡‡ 123112¶ 142261¶¶ 168506§§ 131094‡‡ 132486¶¶ 168507§§ 131129‡‡ 168508§§ 168509§§ 123094¶ 166249§ 131112‡‡ 166250§ 157051† 123071¶ 123072¶ 123095¶ 123105¶ 123089¶ 123090¶ 123133¶ 158612‡ 123101¶ 94331§§ 123102¶ 166251§ 131122‡‡
Genoserotype O58:H40 O58:H27 O63:H27 O66:H25 O70:H42 O71:H12 O17 þ O44 þ O73 þ O77 þ O106:H31 O75:H5 O75:H7 O17 þ O44 þ O73 þ O77 þ O106:H18 O17 þ O44 þ O73 þ O77 þ O106:H18 O17 þ O44 þ O73 þ O77 þ O106:H18 O78:H4 O79:H40 O79:H40 O81:H27 O83:Hn.t. O85:H49 O86:H21 O86:H34 O87:H12 O91:H14 O98:H8 O98:H25 O99:H33 O101:H9 O103:H25 O104:H12 O104:H4 O105:H8 O105:H4 O17 þ O44 þ O73 þ O77 þ O106:H18 O17 þ O44 þ O73 þ O77 þ O106:H33 O107 þ O117:H27 O107 þ O117:H27 O109:H19 O111:H8 O111:H2 O112:H2 O112:H8 O112ac:Hn.t. O113:H4 O113:H6 O113:H4 O113:H21 O114:H4 O114:H9 O114:H32 O115:H10 O107 þ O117:H16 O118 þ O151:H10 O119:H8 O121:H19 O121:H46
continued
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Serotype O123 O123 O123 O123 O124:H30 O126:H2 O127a:H‐ O127:H6 O128:H2 O128:H‐ O129 O129 O130 O138 O139 O141 O141:K85 O143:H‐ O145 O146:H28 O147 O147 O148:H53 O149 O149 O150 O150 O151 O152:H‐ O152 O157:H12 O157:H38 O157:H18 O157:H43 O157:H7 O159 O164:H‐ O167:H5 O168 O172:H‐ O174:H27 O177 O177 O177
Strain id and origin §
123333 123874§ 131150‡‡ 168510§§ 157052† 157053† 157054† 158604‡ 123069¶ 162374†† 131140‡‡ 168511§§ 168512§§ 168513§§ 168514§§ 187744†† 158610‡ 157055† 109797††† 123077¶ 168515§§ 187748†† 123128¶ 168516§§ 109792††† 131107‡‡ 166252§ 168517§§ 157056† 168518§§ 123073¶ 123080¶ 123082¶ 123091¶ 157057† 168519§§ 157058† 157059† 162371¶¶ 123130¶ 123131¶ 166253§ 97991†† 97996††
Genoserotype O123:H2 O123:H2 O123:H16 O123:H16 O124 þ O164:H30 O126:H2 O127:H6 O127:H6 O128:H2 O128:H2 O13 þ O129 þ O135:H11 O13 þ O129 þ O135:H11 O130:H9 O138:H4 O139:H1 O141:H4 O141:H4 O143:Hn.t. O145:H34 O146:H28 O147:H19 O147:H14 O148:H53 O149:H10 O149:H10 O150:H8 O150:H21 O118 þ O151:H10 O152:Hn.t. O152:Hn.t. O157:H12 O157:H38 O157:H18 O157:H43 O157:H7 O159:H20 O124 þ O164:H7 O167:H5 O168:H4 O172:H25 O174:H27 O177:H25 O177:H25 O177:H25
†
Leibniz Institute DSMZ‐German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany. ‡F Gunzer, Institute for Medical Microbiology and Hygiene, Dresden, Germany. §E. Bingen, Service de Microbiology, Paris, France. ¶H. Hächler, Institute for Food Safety and Food Hygiene, Zürich, Switzerland. ††L. Geue, Friedrich Loeffler Institute, Wusterhausen, Germany. ‡‡S. Blum, Kimron Veterinary Institute, Bet Dagan, Israel. §§S. Chappell, Animal Health and Veterinary Laboratories Agency, Weybridge, UK. ¶¶S. Monecke, Institute for Medical Microbiology and Hygiene, Dresden, Germany. †††L. Beutin, Federal Institute for Risk Assessment, Berlin, Germany. ‡‡‡T. Lindbäck, Norwegian School of Veterinary Science, Oslo, Norway.
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originated from clinical diagnostics or epidemiological studies. The strains were cultivated on tryptone yeast agar (VWR International GmbH, Darmstadt, Germany). A full 1 mL loop (diameter 1 mm) of clonal colony material of each strain was picked from solid medium, resuspended in 200 mL lysis reagents (lysis enhancer A2 dissolved in 200 mL lysis buffer A1, Alere Technologies GmbH) and incubated for 30–60 min at 37°C and 550 rpm in a thermomixing device (Eppendorf GmbH, Hamburg, Germany). RNA‐free unfragmented genomic DNA was extracted with a Qiagen DNeasy Blood & Tissue kit (Qiagen GmbH, Heiden, Germany) according to the manufacturer's instructions. The DNA concentration was determined spectrophotometrically at 260 nm and finally analyzed for fragmentation by electrophoresis in a 1% non‐denaturing agarose gel. Multiplex linear DNA amplification and labeling for hybridization to prepared ArrayStrips For multiplex linear DNA amplification, a set of 252 primers was used. One hundred and three of these primers have been described elsewhere (10). A list of the additional new 149 primers is shown in Table S1. These primers are non‐overlapping, but located as close as possible, on the complementary strand, upstream of the position of the oligonucleotide probe. For labeling and biotinylation of the genomic DNA, a site‐specific labeling approach was used (13). A primer elongation reaction was performed using a primer mixture, the HybPLUSKit (Alere) and 0.5–1.5 mg unfragmented RNA‐free genomic DNA of the E. coli isolates according to the manufacturer's instructions. The reaction was started with denaturation (5 min, 96°C), followed by 50 cycles of 60 s at 96°C, 20 s at 50°C, and 40 s at 72°C. The sample was then cooled down to 4°C. Hybridization of the DNA‐based serotyping E. coli ArrayStrips The ArrayStrips, which were spotted with the probes for serotyping (see Table S1), were produced by Alere Technologies GmbH. Details on the layout of the ArrayStrips are provided in the Results Section. For hybridization, a HybPLUSKit (Alere) was used with the following adapted protocol. Each ArrayStrip was initially washed with 200 mL double‐distilled water and then with 150 mL of buffer C1 using a thermomixing device (BioShake IQ; Qinstruments GmbH, Jena, Germany; each 5 min, 50°C, 550 rpm). The hybridization sample consisted of 10 mL labeled probe and 90 mL buffer C1. It was transferred into the ArrayStrip and incubated 80
(60 min, 50°C, 550 rpm). The sample was then removed from the tube and the array washed twice (10 min, 45°C, 550 rpm), with buffer C2. Next, 100 mL conjugate solution (1 mL C3 HRP conjugate plus 99 mL C4 conjugation buffer) was added for 10 min at 30°C and 550 rpm followed by a washing step with 200 mL buffer C5 for 5 min at 30°C and 550 rpm. The ArrayStrip was then stained with buffer D1 (100 mL, 10 min, no agitation). After removal of the liquids, it was photographed using an ArrayMate instrument (Alere) and automatically analyzed. Mean signal intensity (mean) and local background were measured for each probe position and values calculated by the following formula: value ¼ 1 mean/local background. Breakpoints for the interpretation of signals were defined based on a series of experiments with known, characterized reference strains (DSM10728, DSM1058, DSM10720, DSM10784, DSM11753, DSM9025, DSM9026, DSM5212, DSM9027, DSM9031, DSM8701, DSM8702, DSM9028, DSM9030, DSM17076, DSM9034, DSM9033‐ Leibniz Institut DSMZ‐Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). Resulting values below 0.1 were considered negative and above 0.3 as positive. Values between 0.1 and 0.3 were regarded as inconclusive.
RESULTS Target gene selection and construction of array The oligonucleotide array‐based detection of 24 of the over 180 known O antigens (O antigens 4, 6–9, 15, 26, 52, 53, 55, 79, 86, 91, 101, 103, 104, 111, 113, 114, 121, 128, 145, 157 and 172) by use of the target sequences of wzx (an O antigen flipase) or wzy (an O antigen polymerase) was developed by Ballmer et al. (10). In the present study, the same strategies were applied to search in public sequence databases (GenBank, EMBL) for additional wzy and wzx sequences and other previously described O‐specific sequences, such as wzm, wzt, rfbU, rfbE, isla29, sil‐inv, sil‐1, sil‐2, wbdA, wbdH, wbdM, wbdU, gtrB and gtrX. Ballmer et al. evaluated probes for 47 of the 53 known H antigens by using the fliC locus‐specific sequences (10). All these probes were re‐used for the expanded microarray described herein. New 23 to 31 bp oligonucleotide probes were designed as described previously (10). The new probe sequences were designated using a code consisting of the gene name and the serotype or strain number (Table S1; Fig. S1). Seven wzx or wzy gene probes covered multiple O groups as follows: wzx_O13 þ O129 þ O135/wzy_O13 þ O129 þO135 (O13, O129, O135), wzx_O17 þ O44 þ O73 þO77 þ O106/ © 2013 The Societies and Wiley Publishing Asia Pty Ltd
Genoserotyping of E. coli
wzy_O17 þ O44 þ O73 þO77 þ O106 (O17, O44, O73, O77, O106), wzx_O28 þ O42/wzy_O28 þ O42 (O28 and O42), wzx_O107 þO117/wzy_O107 þ O117 (O107 and O117), wzx_O118 þ O151/wzy_O118 þ O151 (O118 and O151), wzx_O124 þ O164/wzy_O124 þ O164 (O124 and O164) and wzx_O152 þ O173/wzy_O152 þ O173 (O152 and O173). Additionally, an individual‐specific spot for O152 is present. The array comprises a total of 259 different probes, each probe being spotted in duplicate. A staining control (Biotin‐Marker), family‐specific controls for Enterobacteriaceae (hp_rrs_611, hp_rrs_612, hp_dnaE_613 and prob_gapA_611) and species‐specific controls for E. coli (hp_dnaE_612 and gad_10) complete the array. DNA‐free spotting buffer was used as a negative control (Fig. S1). Initial validation of the diagnostic microarray for the new O antigen groups The performance for the new O antigen groups of the microarray was tested for each spot with a series of reference strains (Table 1). The system detected the available sequences for O antigens with a high degree of reliability (Table S1). The wzx or wzy gene probes had a specificity and sensitivity of 100%. All 70 new O antigen groups were correctly identified with each corresponding reference strain. Unspecific reactions were rare and limited to a small number of spots exclusively representing O antigen‐specific genes other than wzx or wzy. Most cross‐reactions were found for the target sequences isla29‐ O145_11, sil‐inv‐O145_11, rfbU‐O157_11, sil‐inv‐O157_11, sil1‐O157_11 and sil2‐O157_11. False‐positive signals on spots of these target sequences were found in 24.6% of the reference strains. Correct identification of 47 H antigen groups has already been demonstrated by Ballmer et al. (10). In the present study, all serotyped H antigen groups of the reference strains were identified correctly. Further validation of the diagnostic microarray using E. coli field strains For further validation of the microarray, 180 clinical E. coli isolates of human origin (56 from urine, 50 from blood cultures, 39 from bronchial secretions and 35 from wound swabs) were tested. All isolates were characterized as E. coli by VITEK‐II (bioMérieux, Nuertingen, Germany/Marcy l'Etoile, France), a commercially available system for routine identification of clinically relevant bacteria based on metabolic profiles and for susceptibility tests using a miniaturized agar dilution approach. However, classical antibody‐based serotyping was not performed on these isolates. All isolates were identified as E. coli; and 132 of the 180 isolates (73.3%) were assigned unambiguously to 26 O antigen groups (O1, O2, O4, O6–O9, O11, O15, O18, © 2013 The Societies and Wiley Publishing Asia Pty Ltd
O21, O22, O24, O25, O58, O63, O75, O78, O79, O83, O86, O91, O101, O130, O143 and O150) using the wzx or wzy gene probes. Most isolates were grouped as O25 (24 isolates), O8 (15 isolates), O2 (13 isolates), O6 (13 isolates), O9 (11 isolates), and O101 (8 isolates). E. coli isolates of the other O groups were detected much less frequently (Table 2). For 10 isolates positive signals were observed with DNA gene probes for several serogenotypes (wzx_O107 þ O117/wzy_O107 þ O11, one isolate; wzx_O17 þ O44þ O73þ O77þ O106/wzy_O17 þ O44 þ O73 þ O77 þ O106, nine isolates). This occurred because the sequences of the wzx/wzy genes of these O groups are so similar that exact separation is not possible. Additional positive reactions, besides the wzx and wzy gene probes, were found in only 6 of the 180 isolates (3.3%). Here, positive signals were obtained for the wzx_O9/wzy_O9 and wz_O101_11 gene probes. It should be noted that this wz gene probe is not an allele of wzx/wzy (Table 2). No signals were detected for 32 isolates (17.8%). Six probes (isla29‐O145_11, sil‐inv‐ O145_11, rfbU‐O157_11, sil‐inv‐O157_11, sil1‐O157_11 and sil2‐O157_11) showed strong cross‐reactions with about 65% of all strains. For 176 of the 180 E. coli field strains (97.8%), the H antigen groups were unambiguously identified. The isolates were assigned to 25 H antigen groups (H1, H2, H4–H10, H12, H16, H18–H21, H25, H27, H28, H30, H34, H40, H41, H42, H45 and H51). Most isolates were grouped as H4 (39 isolates), H9 (21 isolates), H1 (20 isolates), H7 (16 isolates), H18 (14 isolates) and H6 (13 isolates). E. coli isolates of the other H antigen groups were detected much less frequently (Table 2). Cross‐reactions were not observed. For just four isolates no H antigen groups could be identified because no signals were obtained with the fliC probes. All results for the strains of human origin are shown in Table 2. Additionally, 51 E. coli isolates of animal origin were tested. Complete classical antibody‐based O:H serotyping results were known for 37 of the 42 cattle isolates. For the E. coli strains isolated from pigs and poultry, only the O antigen groups had been determined by antibody‐based serotyping. A very good concordance was found between the results of classical antibody‐based serotyping and the DNA‐based genoserotyping by oligonucleotide microarray. Different results were only obtained for the O antigen groups of three E. coli strains. One O136:H49 isolate showed a positive signal with the DNA probes wzx_O6_11/wzy_O6_11 (Table 3) whereas fliC for H49 was correctly identified. A second discrepancy, a clearly positive signal with the K‐12‐specific DNA gene probes (wzx_O150‐K12/wzy_O150‐K12), was detected for an E. coli isolate that had originally been reported as O69:H (Table 3). Furthermore, no signal for any wzx/wzy target sequence was detected for an O98:H‐, although DNA 81
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Table 2. Results of genoserotyping E. coli isolates of human origin
O antigen group (wzx/wzy) 1 2 4 6 7 8 9 9,101 101 11 15 18 21 22 24 25 58 63 75 78 79 83 86 91 130 143 O017 þ O044 þ O073 þ O077 þ O106 O107 þ O117 O150_K‐12 — Total H antigen group (fliC) 1 2 4 5 6 7 8 9 10 12 16 18 19 20 21 25 27 28
Number
Isolates from urine
Isolates from bronchial secretions
7 13 3 13 1 15 11 6 9 1 5 6 1 1 4 24 1 1 6 1 1 1 2 1 1 1 9 1 2 32 180
2 5 2 3 0 2 6 2 3 0 1 1 1 0 1 8 0 1 3 0 1 0 0 1 0 0 4 0 1 8 56
1 2 0 3 0 8 0 0 2 0 0 2 0 1 1 9 1 0 0 0 0 0 1 0 0 0 1 0 0 7 39
4 1 0 4 1 2 3 3 4 0 3 1 0 0 2 2 0 0 2 1 0 0 1 0 1 1 2 1 0 11 50
0 5 1 3 0 3 2 1 0 1 1 2 0 0 0 5 0 0 1 0 0 1 0 0 0 0 2 0 1 6 35
20 1 39 6 13 16 3 21 7 3 2 14 6 1 3 9 1 2
7 0 11 2 5 5 0 5 2 1 1 5 2 0 0 4 1 1
2 0 13 0 2 3 1 6 2 0 0 2 1 0 2 3 0 0
6 1 9 3 1 4 2 9 1 1 1 6 1 1 0 1 0 0
5 0 6 1 5 4 0 1 2 1 0 1 2 0 1 1 0 1
Isolates from blood cultures
Isolates from wound swabs
continued
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Genoserotyping of E. coli Table 2. Continued
30 34 40 41 42 45 51 — Total
Number
Isolates from urine
Isolates from bronchial secretions
3 1 1 1 1 1 1 4 180
1 0 1 0 1 0 0 1 56
0 0 0 0 0 0 0 2 39
Isolates from blood cultures
Isolates from wound swabs
1 1 0 0 0 0 1 0 50
1 0 0 1 0 1 0 1 35
Table 3. Results of genoserotyping compared to the classical antibody‐based serotyping for E. coli isolates of animal origin Number 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Host
O antigen group (wzx, wzy)
H antigen group (fliC)
Classical antibody‐based serotyping
Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle
26 28 þ 42 — 128 6 98 172 172 — — 157 35 177 26 107 þ 117 40 — — — 40 — — 40 — — — — — — 8 8 103 174 O118 þ O151 150 174
11 37 9 2 49 25 25 25 4 2 7 2 25 11 27 2 25 25 25 2 25 25 2 25 8 25 5 25 2 21 21 2 21 16 2 21
O26:H11 O28:H37 O136:H‐ O128:H2 O136:H49 O98:H‐ O172:H‐ O172:H‐ O98:H‐ O84:H‐ O157:H7 O35:H2 Orough:H25 O26:H11 O107:H27 Not tested O165:H25 O165:H25 O165:H25 Not tested O165:H25 O165:H25 Not tested O165:H25 Not tested O156:H25 Not tested O156:H25 Orough:H‐ Ont:H‐ Ont:H‐ O103:H2 O174:H‐ Ont:H16 O150:H‐ O174:H‐
continued
© 2013 The Societies and Wiley Publishing Asia Pty Ltd
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L. Geue et al. Table 3. Continued Number 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
Host
O antigen group (wzx, wzy)
H antigen group (fliC)
Classical antibody‐based serotyping
Cattle Cattle Cattle Cattle Cattle Cattle Pig Pig Pig Pig Pig Poultry Poultry Poultry Poultry
K‐12 174 174 103 177 174 149 141 149 149 147 1 1 2 2
2 21 21 2 25 21 10 4 10 10 14 15 15 6 9
O69:H‐ O174:H‐ O174:H‐ O103:H2 O177:H‐ O174:H‐ O149 O141 O149 O149 O147 O1 O1 O2 O2
probes for O98 (wzx_O98/wzy_O98) are present on the microarray (Table 3). The microarray revealed the O groups of five strains that had not been identified by classical antibody‐based serotyping (rough forms or untypeable). Because the target sequences for these O antigen groups are not yet known and therefore not covered by the microarray, correct positive signals for wzx/wzy DNA probes were lacking on the microarray for six O165:H25, two O156:H25, one O136:H‐ and one O84:H‐ strain. Three of the five strains without classical serotyping data were characterized as O40:H2, whereas only the H groups (H5 and H8) were identified in the remaining two isolates. Full accordance of the O antigen identifications by classical antibody‐based serotyping and by microarray was found for all strains isolated from pigs and poultry. There was also full accordance for H antigen group identifications obtained with the two different methods. Importantly, for 17 strains that had been reported to be non‐motile, clear results for a fliC gene, which apparently was not expressed, were found (Table 3). Unspecific reactions were rare, being limited to a small number of spots in seven strains (13.7%) exclusively representing O antigen‐specific genes other than wzx or wzy. All results are listed in Table 3. The datasets of mean values, background values and calculated signals of detected spots are shown in Tables S2, S3, and S4.
DISCUSSION We here present an improved version of the oligonucleotide‐based DNA microarray for E. coli genoserotyping described by Ballmer et al. (10). We have expanded the microarray to cover 94 O antigen groups (O antigens 1– 4, 6–9, 11, 13, 15, 17, 18, 21, 22, 24–29, 32, 35, 40, 42, 44, 45, 52, 53, 55, 56, 58, 63, 66, 70, 71, 73, 75, 77–79, 81, 83, 85–87, 91, 98, 101, 103–107, 111–115, 112ac, 117– 84
119, 121, 123, 124, 126–130, 135, 138, 139, 141, 143, 145– 152, 157, 159, 164, 167, 168, 172–174 and 177). Ballmer et al. evaluated probes of 47 of the 53 known H antigens (H antigens 1–12, 14–16, 18–21, 23–34, 37–43, 45, 46, 48, 49, 51–54, and 56) (10). We used all these probes in the improved microarray. In addition, we transferred the microarray to the ArrayStrip format of Alere Technologies GmbH, which allows high through‐put tests in 96‐well formats and fully automated microarray analysis during imaging. Initial validation against agglutination procedures as the “gold standard,” showed a sensitivity and specificity of 100% using the wzx or wzy gene probes for the new O antigen groups in addition to the panel of probes. All new 70 O antigen groups were correctly identified by each corresponding reference strain. Also, we correctly identified all H antigen groups of serotyped reference strains. Furthermore, validation of the microarray with field strains of human and animal origin yielded excellent results. We unambiguously identified more than 70% of the O antigen groups and nearly 98% of the H antigen groups of the E. coli human isolates. For the remaining strains, we could not include DNA probes in the microarray because the target sequences are unknown. We found no cross‐reactions either using the wzx/wzy DNA probes or for H antigen group determinations by fliC gene probes. Moreover, we found a high degree of concordance between the results of classical antibody‐based serotyping and DNA‐based genoserotyping by oligonucleotide microarray for the E. coli strains of animal origin. We obtained different results regarding the O antigen groups for only three strains. It is possible that the classical antibody‐ based serotyping was incorrect for these strains. It is also possible that the cultures were polyclonal, that is contaminated with a second strain, and that one © 2013 The Societies and Wiley Publishing Asia Pty Ltd
Genoserotyping of E. coli
clone was tested serologically whereas another one was subjected to genotyping. Genes selected for detection of the O antigen, that is, wzx and wzy, are the most promising target sequences for an oligonucleotide DNA microarray‐based typing approach, the results of which correlated strongly with those of the classical agglutination method. The agreement of the results of the genotyping assay for wzx, wzy, and fliC with those of serotyping is so high that the microarray technology may replace serotyping as the “gold standard” once relevant sequence data for all serotypes are available. The alternative genes proved to be less specific. We detected most cross‐reactions for the target sequences isla29‐ O145_11, sil‐inv‐O145_11, rfbU‐O157_11, sil‐inv‐O157_11, sil1‐O157_11 and sil2‐O157_11. We found false‐positive signals for these target sequences in a quarter of the reference strains. Among clinical isolates of human origin, we detected up to 65% cross‐hybridization signals with unrelated serotypes. This indicates that the genes of the regions selected for probe design are less suitable than the highly discriminatory wzx and wzy genes. In summary, genoserotyping is very important from a scientific and practical view. We and other authors have previously shown this for E. coli (10) and other bacterial species (5, 11, 14–16). In this study, we detected an excellent match between the results of classical antibody‐ based serotyping and DNA‐based genoserotyping both in validation of the new 70 O antigen groups as well as for the field strains of human and animal origin. The advantages of geroserotyping are obvious, namely: (i) unambiguous results for more than half of all known O antigen groups and nearly all H antigen groups within a few hours, starting from a single colony; (ii) parallel detection of O and H antigens of a single E. coli isolate in a single experiment; and (iii) simultaneous typing of a considerable number of E. coli isolates by use of the ArrayStrip format. Thus, the O and H oligonucleotide microarray is an easy and fast technique and provides a helpful and powerful tool for characterization of pathogenic and non‐pathogenic E. coli.
ACKNOWLEDGMENTS We thank F. Gunzer (Institute for Medical Microbiology and Hygiene), E. Bingen (Service de Microbiology, Paris, France), H. Hächler (Institute for Food Safety and Food Hygiene, Zürich, Switzerland), S. Blum (Kimron Veterinary Institute, Bet Dagan, Israel), S. Chappell (Animal Health and Veterinary Laboratories Agency, Weybridge, UK), L. Beutin (Federal Institute for Risk Assessment, Berlin, Germany), and T. Lindbäck (Norwegian School of Veterinary Science, Oslo, Norway) for kindly providing reference strains. © 2013 The Societies and Wiley Publishing Asia Pty Ltd
DISCLOSURE S. Monecke, I. Engelmann, P. Slickers, S. Braun, and R. Ehricht are employees of Alere Technologies, the company that manufactures the microarrays also used in this study. This had no influence on study design, data collection, and analysis, and did not alter the authors' adherence to all Microbiology and Immunology's policies on sharing data and materials. The other authors do not declare any conflict of interest.
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13. Monecke S., Ehricht R. (2005) Rapid genotyping of methicillin‐ resistant Staphylococcus aureus (MRSA) isolates using miniaturised oligonucleotide arrays. Clin Microbiol Infect 11(10): 825–33. 14. Ruettger A., Feige J., Slickers P., Schubert E., Morre S.A., Pannekoek Y., Herrmann B., de Vries H.J., Ehricht R., Sachse K. (2011) Genotyping of Chlamydia trachomatis strains from culture and clinical samples using an ompA‐based DNA microarray assay. Mol Cell Probes 25(1): 19–27. 15. Sachse K., Laroucau K., Hotzel H., Schubert E., Ehricht R., Slickers P. (2008) Genotyping of Chlamydophila psittaci using a new DNA microarray assay based on sequence analysis of ompA genes. BMC Microbiol 8: 63. 16. Sachse K., Laroucau K., Vorimore F., Magnino S., Feige J., Müller W., Kube S., Hotzel H., Schubert E., Slickers P., Ehricht R. (2009) DNA microarray‐based genotyping of Chlamydophila psittaci strains from culture and clinical samples. Vet Microbiol 135(1/2): 22–30.
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Supporting Information
Additional supporting information may be found in the online version of this article at the publisher's web site. Fig. S1. Layout of the oligonucleotide microarray. Table S1 Reference strains, chip layout and validation of results Table S2. Data concerning mean values of the detected spots (mean_sero.xlsx) Table S3. Data concerning background values of detected spots (background_sero.xlsx) Table S4. Data concerning calculated signals of detected spots (signal_data_sero.xlsx)
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