- Email: [email protected]
Rapid identification of ST131 Escherichia coli by a novel multiplex real-time allelic discrimination assay
Journal of Microbiological Methods 140 (2017) 12–14
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
Rapid identification of ST131 Escherichia coli by a novel multiplex real-time allelic discrimination assay
MARK
Patrice Françoisa,b,⁎, Eve-Julie Bonettia, Carolina Fankhauserc, Damien Bauda, Abdessalam Cherkaouib, Jacques Schrenzela,b, Stephan Harbarthc a b c
Genomic Research Laboratory, University of Geneva Hospitals and Faculty of Medicine, Geneva, Switzerland Bacteriology Laboratory, University of Geneva Hospitals and Faculty of Medicine, Geneva, Switzerland Infection Control Programme, University of Geneva Hospitals and Faculty of Medicine, Geneva, Switzerland
A R T I C L E I N F O
A B S T R A C T
Keywords: Escherichia coli Genotyping Sequence type 131 Allelic discrimination Real-time PCR Molecular assay
Escherichia coli sequence type 131 is increasingly described in severe hospital infections. We developed a rapid real-time allelic discrimination assay for the rapid identification of E. coli ST131 isolates. This rapid assay represents an affordable alternative to sequence-based strategies before completing characterization of potentially highly virulent isolates of E. coli.
Escherichia coli colonize our lower intestinal tract but also represent a leading cause of infection. A wide variety of infections is caused by E. coli including secretory or hemorrhagic colitis, urinary tract infection, and bacteremia (Jaureguy et al., 2008). Clinical outcomes are related to host factors and to virulence gene determinants present in the infecting strain. Different genotype schemes have been proposed in the past to characterize pathogen and commensal isolates (Clermont et al., 2000) or to obtain clinically relevant genotypes (Jaureguy et al., 2008). Recently, several studies have shown an important increase in specific bacterial clones associated with severe infections (Karfunkel et al., 2013; Kim et al., 2016; Peirano et al., 2012; Pitout and DeVinney, 2017). For example, E. coli from sequence type 131 (ST131) and harboring extended-spectrum beta-lactamase (ESBL) genes showed increasing prevalence and particular capacity to spread in the community and hospitals. Nowadays, diagnostic tests and related clinical decisions are generally concentrated on strains responsible for infection, since many countries have abandoned special infection control measures and screening policies for carriers of ESBL-producing E.coli (Tschudin-Sutter et al., 2017). However, the increasing prevalence of highly virulent ESBL-producing E.coli in the healthcare environment reemphasizes the need for inexpensive ESBL E.coli screening assays, to be implemented at a large scale to assess the colonization status of high-risk patients. To date, the identification of strains with high virulence potential (ST131 H30-Rx) is a multistep molecular process. Following identification of E. coli, the determination of the sequence type and the
⁎
presence of ESBL enzymes are mandatory steps before detection of specific additional markers or phenotype. Only sequence-based methods are used to diagnose E. coli strains belonging to the ST131 lineages before identifying subtypes. Sequence-based methods are generally costly and require Sanger or next-generation sequencers, devices not frequently available in routine laboratories but only in large university centers. The aim of this study was to develop a novel procedure for the rapid and possibly real-time identification of ST131 E. coli strains. Five single nucleotide polymorphisms constituting a unique signature of ST131 were targeted and allelic discrimination oligonucleotides were selected and validated against reference strains. The reliability of the assay was evaluated on a collection of 89 sequenced strains responsible for bloodstream infections, from highly diverse genetic backgrounds. Strain collection- A collection of 89 isolates was obtained from our institution representing all bloodstream infections due to ESBL-E. coli identified in 2015. Strains were maintained on LB agar. The identification of all isolates was confirmed using matrix-assisted desorption ionization–time of flight mass spectrometry (MALDI-TOF MS; Maldi Biotyper 2.0, Bruker Daltonics, Bremen, Germany) (Cherkaoui et al., 2015) according to the manufacturer's instructions. The MALDI-TOF MS score values ranged between 2.4 and 2.5. DNA extraction and sequence analysis- Genomic DNA was extracted from one colony suspended in 200 μl Tris-EDTA buffer (10 mM Tris, 1 mM EDTA). A total of 100 mg of glass beads (diameter, 100 μm;
Corresponding author at: Genomic Research Laboratory, Geneva University Hospitals, Switzerland. E-mail address: [email protected] (P. François).
http://dx.doi.org/10.1016/j.mimet.2017.06.018 Received 11 May 2017; Received in revised form 23 June 2017; Accepted 24 June 2017 Available online 26 June 2017 0167-7012/ © 2017 Elsevier B.V. All rights reserved.
Journal of Microbiological Methods 140 (2017) 12–14
P. François et al.
concordance. Note that among these 56 isolates 2 were susceptible to fluoroquinolone, a prevalence which is in accordance with a previous report (Johnson et al., 2009). Fluoroquinolone susceptible and resistant ST131 isolates were identified by our assay with similar efficacy. Whole genome sequencing allowed obtaining in silico sequence types for the totality of strains of our collection which shows appreciable diversity. In addition to the ST131 isolates the collection is composed of; ST88 (n = 10), ST38 (n = 5), ST69 (n = 3), ST95 (n = 2), ST117 (n = 2), ST2013 (n = 1), ST2279 (n = 1), ST361 (n = 1), ST410 (n = 1), ST457 (n = 1), ST48 (n = 1), ST517 (n = 1), ST648 (n = 1), ST6834 (n = 1), ST10 (n = 1), ST73 (n = 1), ST998 (n = 1). Note that the ST2279, a single locus variant of ST131 was detected as ST131 by using our assay. Overall, the time required to obtain ST information from isolation of the strain from the agar plate was approximately 2 h. In terms of cost, one batch of oligonucleotides allowing 500 PCR determinations to be performed at a unitary cost of < 8 US$/strain which is cheaper than current methods requiring complete MLST determination by using Sanger sequencing of several amplicons. This assay offers the possibility to identify in real-time the prevalence of E. coli belonging to ST131 and to develop adapted infection control strategies. Additional determinations are mandatory to identify the most virulent isolates, such as detection of padB (Clermont et al., 2009) but it simplifies considerably the detection of ST131 compared to MLST or other sequenced-based method. Note that we developed a unique protocol for the 5 allelic discrimination assays which is amenable to high-throughput determinations. In summary, this report describes a rapid, specific, and reliable allelic discrimination assay allowing the detection of ST131 E. coli strains, without the use of costly and time consuming Sanger sequencing. The moderate turnaround time and affordable reagent cost appear compatible with the utilization of this assay in routine laboratories, allowing rapid molecular detection and surveillance of a clinically relevant pathogen.
Table 1 Context sequence of targeted mutations used to identify ST131 E. coli isolatesa. Assay ID (Bustin et al., 2011)
Name
Targeted SNP
5′ Dye
AHLJ2JQ
mdh_1
AHMS0PY
mdh_2
AHN1YV6
gyrB-1
AHPAW2E
gyrB-2
AHQJU8M
gyrB-3
GCCGGCATCGT GCCGGTATCGT ATTGGCGGTCA ATTGGTGGTCA CGCGACAAGCGC CGCGATAAGCGC TACTTCTCCAC TACTTTTCCAC TCCACTGAAAA TCCACCGAAAA
FAMb VICc FAM VIC FAM VIC FAM VIC FAM VIC
a Conditions for the amplification on the CFX96 (BioeRad) were the following: time 1 (t1), 10 min at 95 °C; t2, 15 s at 95 °C; and t3, 60 s at 60 °C (t2 and t3 were repeated 40 times). The volume of the PCR mixtures (ABsolute, Thermo Scientific, Carlsbad, CA, USA) was 25 μl and contained all primers and probes at 600 nM and 133 nM, respectively. b FAM; 6-carboxyfluorescein c VIC; proprietary molecule to Life Technologies, chemical structure is not publicly available.
Schieritz and Hauenstein, Switzerland) was added to the suspension, and bacteria were lysed by vortexing at maximum power for 45 s. The liquid phase was cleared from beads and bacterial debris by centrifugation and diluted 50-fold, and a 5-μl aliquot was used for real-time multiplex PCR assays. Sequencing and genotyping- Whole-genome sequencing and assembly of the 89 E. coli strains was performed using an Illumina HiSeq2500 device as previously described (Von Dach et al., 2016). Sequencing experiments were not performed for the purpose of the current study but for internal reasons. Genome assembly data was obtained as described (Hernandez et al., 2014) and used to perform in silico MLST determinations for comparison purpose with the newly developed allelic discrimination PCR assay. Sequence analysis, primer and probe selection- Based on 2 previous studies, we developed allelic discrimination assay to characterize a unique combination of point mutations (Johnson et al., 2009; Kim et al., 2016) in the sequences of mdh and gyrB (identification numbers AMC96129 and AMC96591, respectively). Five different mutations were probed in the assay using the oligonucleotides depicted in Table 1 to identify allelic form at position C288T and C525T for mdh and C621T, C729T and T735C, for gyrB. None of the mutation assessed in the gyrB gene conferred resistance to fluoroquinolone. The design of type-specific oligonucleotides was performed in collaboration with ThermoFisher Scientific (San Francisco, CA, USA) in variable regions using the software PrimerExpress (PE Biosystems, Foster City, CA). Based on these observations, minor groove binder (MGB) probes coupled to dark quenchers were designed to ensure optimal specificity (Kutyavin et al., 2000) between the different alleles of the 2 genes. The context and characteristics of these 5 positions is summarized Table 1. Briefly, to assess 1 strain, our assay requires 5 different mixtures in order to establish the allele for the 5 positions to assess. Each sample contains 2 primers and 2 oligonucleotide probes coupled to different fluorescent dyes. Each pair of probes has the same sequence except the variable position (see Table 1). Each analysis was performed in triplicate; the nucleic acids (50 pg of purified DNA per test) from the reference strains (ST131 and non-ST131) were simultaneously assayed in each run. The amplification runs were performed in a CFX96 (Bio-Rad, Hercules, USA) in a total volume of 25 μl (see details on Table 1). Ratio of fluorescent values recorded during the PCR protocol was calculated and analyzed using default parameters (Bio-Rad) using CFX manager software (Bio-Rad). All reactions were analyzable and alleles were unambiguously identifiable for all genes (Supplementary Fig. 1). The results were compared to the results of MLST deduced from whole genome sequencing analysis for the 89 isolates. A total number of 56 ST131 isolates was obtained with the two methods, with perfect
Conflicts of interest No conflict of interest. Acknowledgments The evaluation of this diagnostic test was made possible by a financial contribution from the Office of the Medical Director, Geneva University Hospitals (CGR405111 and CGR440330), Geneva. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mimet.2017.06.018. References Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J., Wittwer, C.T., 2011. Primer sequence disclosure: a clarification of the MIQE guidelines. Clin. Chem. 57, 919–921. Cherkaoui, A., Diene, S.M., Emonet, S., Renzi, G., Francois, P., Schrenzel, J., 2015. Ampicillin-resistant Haemophilus influenzae isolates in Geneva: serotype, antimicrobial susceptibility, and beta-lactam resistance mechanisms. Eur. J. Clin. Microbiol. Infect. Dis. 34, 1937–1945. Clermont, O., Bonacorsi, S., Bingen, E., 2000. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl. Environ. Microbiol. 66, 4555–4558. Clermont, O., Dhanji, H., Upton, M., Gibreel, T., Fox, A., Boyd, D., Mulvey, M.R., Nordmann, P., Ruppe, E., Sarthou, J.L., Frank, T., Vimont, S., Arlet, G., Branger, C., Woodford, N., Denamur, E., 2009. Rapid detection of the O25b-ST131 clone of Escherichia coli encompassing the CTX-M-15-producing strains. J. Antimicrob. Chemother. 64, 274–277. Hernandez, D., Tewhey, R., Veyrieras, J.B., Farinelli, L., Osteras, M., Francois, P., Schrenzel, J., 2014. De novo finished 2.8 Mbp Staphylococcus aureus genome assembly from 100 bp short and long range paired-end reads. Bioinformatics 30, 40–49. Jaureguy, F., Landraud, L., Passet, V., Diancourt, L., Frapy, E., Guigon, G., Carbonnelle,
13
Journal of Microbiological Methods 140 (2017) 12–14
P. François et al.
Hedgpeth, J., 2000. 3′-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res. 28, 655–661. Peirano, G., van der Bij, A.K., Gregson, D.B., Pitout, J.D., 2012. Molecular epidemiology over an 11-year period (2000 to 2010) of extended-spectrum beta-lactamase-producing Escherichia coli causing bacteremia in a centralized Canadian region. J. Clin. Microbiol. 50, 294–299. Pitout, J.D., DeVinney, R., 2017. Escherichia coli ST131: a Multidrug-resistant Clone Primed for Global Domination. 6 F1000Res. Tschudin-Sutter, S., Lucet, J.C., Mutters, N.T., Tacconelli, E., Zahar, J.R., Harbarth, S., 2017. Contact precautions for preventing nosocomial transmission of ESBL-producing Escherichia coli - a point/counterpoint review. Clin. Infect. Dis (cix258). (doi: 10.1093/cid/cix258). Von Dach, E., Diene, S.M., Fankhauser, C., Schrenzel, J., Harbarth, S., Francois, P., 2016. Comparative genomics of community-associated methicillin-resistant Staphylococcus aureus shows the emergence of clone ST8-USA300 in Geneva, Switzerland. J. Infect. Dis. 213, 1370–1379.
E., Lortholary, O., Clermont, O., Denamur, E., Picard, B., Nassif, X., Brisse, S., 2008. Phylogenetic and genomic diversity of human bacteremic Escherichia coli strains. BMC Genomics 9, 560. Johnson, J.R., Menard, M., Johnston, B., Kuskowski, M.A., Nichol, K., Zhanel, G.G., 2009. Epidemic clonal groups of Escherichia coli as a cause of antimicrobial-resistant urinary tract infections in Canada, 2002 to 2004. Antimicrob. Agents Chemother. 53, 2733–2739. Karfunkel, D., Carmeli, Y., Chmelnitsky, I., Kotlovsky, T., Navon-Venezia, S., 2013. The emergence and dissemination of CTX-M-producing Escherichia coli sequence type 131 causing community-onset bacteremia in Israel. Eur. J. Clin. Microbiol. Infect. Dis. 32, 513–521. Kim, S.Y., Park, Y.J., Johnson, J.R., Yu, J.K., Kim, Y.K., Kim, Y.S., 2016. Prevalence and characteristics of Escherichia coli sequence type 131 and its H30 and H30Rx subclones: a multicenter study from Korea. Diagn. Microbiol. Infect. Dis. 84, 97–101. Kutyavin, I.V., Afonina, I.A., Mills, A., Gorn, V.V., Lukhtanov, E.A., Belousov, E.S., Singer, M.J., Walburger, D.K., Lokhov, S.G., Gall, A.A., Dempcy, R., Reed, M.W., Meyer, R.B.,
14
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
Rapid identification of ST131 Escherichia coli by a novel multiplex real-time allelic discrimination assay
MARK
Patrice Françoisa,b,⁎, Eve-Julie Bonettia, Carolina Fankhauserc, Damien Bauda, Abdessalam Cherkaouib, Jacques Schrenzela,b, Stephan Harbarthc a b c
Genomic Research Laboratory, University of Geneva Hospitals and Faculty of Medicine, Geneva, Switzerland Bacteriology Laboratory, University of Geneva Hospitals and Faculty of Medicine, Geneva, Switzerland Infection Control Programme, University of Geneva Hospitals and Faculty of Medicine, Geneva, Switzerland
A R T I C L E I N F O
A B S T R A C T
Keywords: Escherichia coli Genotyping Sequence type 131 Allelic discrimination Real-time PCR Molecular assay
Escherichia coli sequence type 131 is increasingly described in severe hospital infections. We developed a rapid real-time allelic discrimination assay for the rapid identification of E. coli ST131 isolates. This rapid assay represents an affordable alternative to sequence-based strategies before completing characterization of potentially highly virulent isolates of E. coli.
Escherichia coli colonize our lower intestinal tract but also represent a leading cause of infection. A wide variety of infections is caused by E. coli including secretory or hemorrhagic colitis, urinary tract infection, and bacteremia (Jaureguy et al., 2008). Clinical outcomes are related to host factors and to virulence gene determinants present in the infecting strain. Different genotype schemes have been proposed in the past to characterize pathogen and commensal isolates (Clermont et al., 2000) or to obtain clinically relevant genotypes (Jaureguy et al., 2008). Recently, several studies have shown an important increase in specific bacterial clones associated with severe infections (Karfunkel et al., 2013; Kim et al., 2016; Peirano et al., 2012; Pitout and DeVinney, 2017). For example, E. coli from sequence type 131 (ST131) and harboring extended-spectrum beta-lactamase (ESBL) genes showed increasing prevalence and particular capacity to spread in the community and hospitals. Nowadays, diagnostic tests and related clinical decisions are generally concentrated on strains responsible for infection, since many countries have abandoned special infection control measures and screening policies for carriers of ESBL-producing E.coli (Tschudin-Sutter et al., 2017). However, the increasing prevalence of highly virulent ESBL-producing E.coli in the healthcare environment reemphasizes the need for inexpensive ESBL E.coli screening assays, to be implemented at a large scale to assess the colonization status of high-risk patients. To date, the identification of strains with high virulence potential (ST131 H30-Rx) is a multistep molecular process. Following identification of E. coli, the determination of the sequence type and the
⁎
presence of ESBL enzymes are mandatory steps before detection of specific additional markers or phenotype. Only sequence-based methods are used to diagnose E. coli strains belonging to the ST131 lineages before identifying subtypes. Sequence-based methods are generally costly and require Sanger or next-generation sequencers, devices not frequently available in routine laboratories but only in large university centers. The aim of this study was to develop a novel procedure for the rapid and possibly real-time identification of ST131 E. coli strains. Five single nucleotide polymorphisms constituting a unique signature of ST131 were targeted and allelic discrimination oligonucleotides were selected and validated against reference strains. The reliability of the assay was evaluated on a collection of 89 sequenced strains responsible for bloodstream infections, from highly diverse genetic backgrounds. Strain collection- A collection of 89 isolates was obtained from our institution representing all bloodstream infections due to ESBL-E. coli identified in 2015. Strains were maintained on LB agar. The identification of all isolates was confirmed using matrix-assisted desorption ionization–time of flight mass spectrometry (MALDI-TOF MS; Maldi Biotyper 2.0, Bruker Daltonics, Bremen, Germany) (Cherkaoui et al., 2015) according to the manufacturer's instructions. The MALDI-TOF MS score values ranged between 2.4 and 2.5. DNA extraction and sequence analysis- Genomic DNA was extracted from one colony suspended in 200 μl Tris-EDTA buffer (10 mM Tris, 1 mM EDTA). A total of 100 mg of glass beads (diameter, 100 μm;
Corresponding author at: Genomic Research Laboratory, Geneva University Hospitals, Switzerland. E-mail address: [email protected] (P. François).
http://dx.doi.org/10.1016/j.mimet.2017.06.018 Received 11 May 2017; Received in revised form 23 June 2017; Accepted 24 June 2017 Available online 26 June 2017 0167-7012/ © 2017 Elsevier B.V. All rights reserved.
Journal of Microbiological Methods 140 (2017) 12–14
P. François et al.
concordance. Note that among these 56 isolates 2 were susceptible to fluoroquinolone, a prevalence which is in accordance with a previous report (Johnson et al., 2009). Fluoroquinolone susceptible and resistant ST131 isolates were identified by our assay with similar efficacy. Whole genome sequencing allowed obtaining in silico sequence types for the totality of strains of our collection which shows appreciable diversity. In addition to the ST131 isolates the collection is composed of; ST88 (n = 10), ST38 (n = 5), ST69 (n = 3), ST95 (n = 2), ST117 (n = 2), ST2013 (n = 1), ST2279 (n = 1), ST361 (n = 1), ST410 (n = 1), ST457 (n = 1), ST48 (n = 1), ST517 (n = 1), ST648 (n = 1), ST6834 (n = 1), ST10 (n = 1), ST73 (n = 1), ST998 (n = 1). Note that the ST2279, a single locus variant of ST131 was detected as ST131 by using our assay. Overall, the time required to obtain ST information from isolation of the strain from the agar plate was approximately 2 h. In terms of cost, one batch of oligonucleotides allowing 500 PCR determinations to be performed at a unitary cost of < 8 US$/strain which is cheaper than current methods requiring complete MLST determination by using Sanger sequencing of several amplicons. This assay offers the possibility to identify in real-time the prevalence of E. coli belonging to ST131 and to develop adapted infection control strategies. Additional determinations are mandatory to identify the most virulent isolates, such as detection of padB (Clermont et al., 2009) but it simplifies considerably the detection of ST131 compared to MLST or other sequenced-based method. Note that we developed a unique protocol for the 5 allelic discrimination assays which is amenable to high-throughput determinations. In summary, this report describes a rapid, specific, and reliable allelic discrimination assay allowing the detection of ST131 E. coli strains, without the use of costly and time consuming Sanger sequencing. The moderate turnaround time and affordable reagent cost appear compatible with the utilization of this assay in routine laboratories, allowing rapid molecular detection and surveillance of a clinically relevant pathogen.
Table 1 Context sequence of targeted mutations used to identify ST131 E. coli isolatesa. Assay ID (Bustin et al., 2011)
Name
Targeted SNP
5′ Dye
AHLJ2JQ
mdh_1
AHMS0PY
mdh_2
AHN1YV6
gyrB-1
AHPAW2E
gyrB-2
AHQJU8M
gyrB-3
GCCGGCATCGT GCCGGTATCGT ATTGGCGGTCA ATTGGTGGTCA CGCGACAAGCGC CGCGATAAGCGC TACTTCTCCAC TACTTTTCCAC TCCACTGAAAA TCCACCGAAAA
FAMb VICc FAM VIC FAM VIC FAM VIC FAM VIC
a Conditions for the amplification on the CFX96 (BioeRad) were the following: time 1 (t1), 10 min at 95 °C; t2, 15 s at 95 °C; and t3, 60 s at 60 °C (t2 and t3 were repeated 40 times). The volume of the PCR mixtures (ABsolute, Thermo Scientific, Carlsbad, CA, USA) was 25 μl and contained all primers and probes at 600 nM and 133 nM, respectively. b FAM; 6-carboxyfluorescein c VIC; proprietary molecule to Life Technologies, chemical structure is not publicly available.
Schieritz and Hauenstein, Switzerland) was added to the suspension, and bacteria were lysed by vortexing at maximum power for 45 s. The liquid phase was cleared from beads and bacterial debris by centrifugation and diluted 50-fold, and a 5-μl aliquot was used for real-time multiplex PCR assays. Sequencing and genotyping- Whole-genome sequencing and assembly of the 89 E. coli strains was performed using an Illumina HiSeq2500 device as previously described (Von Dach et al., 2016). Sequencing experiments were not performed for the purpose of the current study but for internal reasons. Genome assembly data was obtained as described (Hernandez et al., 2014) and used to perform in silico MLST determinations for comparison purpose with the newly developed allelic discrimination PCR assay. Sequence analysis, primer and probe selection- Based on 2 previous studies, we developed allelic discrimination assay to characterize a unique combination of point mutations (Johnson et al., 2009; Kim et al., 2016) in the sequences of mdh and gyrB (identification numbers AMC96129 and AMC96591, respectively). Five different mutations were probed in the assay using the oligonucleotides depicted in Table 1 to identify allelic form at position C288T and C525T for mdh and C621T, C729T and T735C, for gyrB. None of the mutation assessed in the gyrB gene conferred resistance to fluoroquinolone. The design of type-specific oligonucleotides was performed in collaboration with ThermoFisher Scientific (San Francisco, CA, USA) in variable regions using the software PrimerExpress (PE Biosystems, Foster City, CA). Based on these observations, minor groove binder (MGB) probes coupled to dark quenchers were designed to ensure optimal specificity (Kutyavin et al., 2000) between the different alleles of the 2 genes. The context and characteristics of these 5 positions is summarized Table 1. Briefly, to assess 1 strain, our assay requires 5 different mixtures in order to establish the allele for the 5 positions to assess. Each sample contains 2 primers and 2 oligonucleotide probes coupled to different fluorescent dyes. Each pair of probes has the same sequence except the variable position (see Table 1). Each analysis was performed in triplicate; the nucleic acids (50 pg of purified DNA per test) from the reference strains (ST131 and non-ST131) were simultaneously assayed in each run. The amplification runs were performed in a CFX96 (Bio-Rad, Hercules, USA) in a total volume of 25 μl (see details on Table 1). Ratio of fluorescent values recorded during the PCR protocol was calculated and analyzed using default parameters (Bio-Rad) using CFX manager software (Bio-Rad). All reactions were analyzable and alleles were unambiguously identifiable for all genes (Supplementary Fig. 1). The results were compared to the results of MLST deduced from whole genome sequencing analysis for the 89 isolates. A total number of 56 ST131 isolates was obtained with the two methods, with perfect
Conflicts of interest No conflict of interest. Acknowledgments The evaluation of this diagnostic test was made possible by a financial contribution from the Office of the Medical Director, Geneva University Hospitals (CGR405111 and CGR440330), Geneva. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mimet.2017.06.018. References Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J., Wittwer, C.T., 2011. Primer sequence disclosure: a clarification of the MIQE guidelines. Clin. Chem. 57, 919–921. Cherkaoui, A., Diene, S.M., Emonet, S., Renzi, G., Francois, P., Schrenzel, J., 2015. Ampicillin-resistant Haemophilus influenzae isolates in Geneva: serotype, antimicrobial susceptibility, and beta-lactam resistance mechanisms. Eur. J. Clin. Microbiol. Infect. Dis. 34, 1937–1945. Clermont, O., Bonacorsi, S., Bingen, E., 2000. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl. Environ. Microbiol. 66, 4555–4558. Clermont, O., Dhanji, H., Upton, M., Gibreel, T., Fox, A., Boyd, D., Mulvey, M.R., Nordmann, P., Ruppe, E., Sarthou, J.L., Frank, T., Vimont, S., Arlet, G., Branger, C., Woodford, N., Denamur, E., 2009. Rapid detection of the O25b-ST131 clone of Escherichia coli encompassing the CTX-M-15-producing strains. J. Antimicrob. Chemother. 64, 274–277. Hernandez, D., Tewhey, R., Veyrieras, J.B., Farinelli, L., Osteras, M., Francois, P., Schrenzel, J., 2014. De novo finished 2.8 Mbp Staphylococcus aureus genome assembly from 100 bp short and long range paired-end reads. Bioinformatics 30, 40–49. Jaureguy, F., Landraud, L., Passet, V., Diancourt, L., Frapy, E., Guigon, G., Carbonnelle,
13
Journal of Microbiological Methods 140 (2017) 12–14
P. François et al.
Hedgpeth, J., 2000. 3′-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res. 28, 655–661. Peirano, G., van der Bij, A.K., Gregson, D.B., Pitout, J.D., 2012. Molecular epidemiology over an 11-year period (2000 to 2010) of extended-spectrum beta-lactamase-producing Escherichia coli causing bacteremia in a centralized Canadian region. J. Clin. Microbiol. 50, 294–299. Pitout, J.D., DeVinney, R., 2017. Escherichia coli ST131: a Multidrug-resistant Clone Primed for Global Domination. 6 F1000Res. Tschudin-Sutter, S., Lucet, J.C., Mutters, N.T., Tacconelli, E., Zahar, J.R., Harbarth, S., 2017. Contact precautions for preventing nosocomial transmission of ESBL-producing Escherichia coli - a point/counterpoint review. Clin. Infect. Dis (cix258). (doi: 10.1093/cid/cix258). Von Dach, E., Diene, S.M., Fankhauser, C., Schrenzel, J., Harbarth, S., Francois, P., 2016. Comparative genomics of community-associated methicillin-resistant Staphylococcus aureus shows the emergence of clone ST8-USA300 in Geneva, Switzerland. J. Infect. Dis. 213, 1370–1379.
E., Lortholary, O., Clermont, O., Denamur, E., Picard, B., Nassif, X., Brisse, S., 2008. Phylogenetic and genomic diversity of human bacteremic Escherichia coli strains. BMC Genomics 9, 560. Johnson, J.R., Menard, M., Johnston, B., Kuskowski, M.A., Nichol, K., Zhanel, G.G., 2009. Epidemic clonal groups of Escherichia coli as a cause of antimicrobial-resistant urinary tract infections in Canada, 2002 to 2004. Antimicrob. Agents Chemother. 53, 2733–2739. Karfunkel, D., Carmeli, Y., Chmelnitsky, I., Kotlovsky, T., Navon-Venezia, S., 2013. The emergence and dissemination of CTX-M-producing Escherichia coli sequence type 131 causing community-onset bacteremia in Israel. Eur. J. Clin. Microbiol. Infect. Dis. 32, 513–521. Kim, S.Y., Park, Y.J., Johnson, J.R., Yu, J.K., Kim, Y.K., Kim, Y.S., 2016. Prevalence and characteristics of Escherichia coli sequence type 131 and its H30 and H30Rx subclones: a multicenter study from Korea. Diagn. Microbiol. Infect. Dis. 84, 97–101. Kutyavin, I.V., Afonina, I.A., Mills, A., Gorn, V.V., Lukhtanov, E.A., Belousov, E.S., Singer, M.J., Walburger, D.K., Lokhov, S.G., Gall, A.A., Dempcy, R., Reed, M.W., Meyer, R.B.,
14