Continuous evolution and adaptation of a DNA-microarray ... - Alere

Continuous evolution and adaptation of a DNA-microarray-based assay for the rapid identification of carbapenemase and other resistance genes in Gram-negative bacteria Sascha D. Braun1,2, Bushra Jamil4, Muhammad Ali Syed5, Shahid Abbasi6 Daniel Weiß1,2, Stefan Monecke1,2,3, Ines Engelmann1,2, Ralf Ehricht1,2 Technologies GmbH, Jena, Germany, 2InfectoGnostics Research Campus, Jena, Germany, 3Technische Universität Dresden, Medizinische Fakultät „Carl Gustav Carus“, Dresden, Germany, 4 Department of Biosciences, COMSATS Institute of Information Technology, Park Road, Islamabad, Pakistan.5Department of Microbiology, University of Haripur, Haripur, Pakistan, 6Al-Sayed Hospital (Pvt) Ltd. 1-Hill Park, Opp. Ayub Park. Jhelum Road, Rawalpindi, Pakistan.

Method: The functionality of the CarbDetect AS-2 Kit (CDAS2) was verified using a set of 133 genotypically characterized reference strains and clinical isolates from different sources [University Medical Center of Dresden (n=12), University Medical Center of Jena (n=31), German Collection of Microorganisms and Cell Cultures (DSMZ; n=3), University of Fribourg (n=74), Alere in-house collection (n=12) and the German National Reference Laboratory for MultidrugResistance Gram-negative Bacteria at Bochum University (NRL, n=2)]. All reference strains were analyzed and evaluated using the same method as described in Figure 1. An overview of all detectable targets and the concordance between known and detected genotype are written in Table 1. The CDAS2 kit was validated using clinical isolates of Gramnegative bacteria isolated from 7,857 clinical samples. These isolates (n=425) were subjected to susceptibility tests by the standard Kirby-Bauer disc diffusion method and those strains that were resistant to carbapenems were selected for further characterization. For all 82 carbapenem resistant isolates, the carbapenemase, ESBL, narrow spectrum β-lactamase, AmpC, aminoglycoside, macrolide, quinolone and co-trimoxazole genotypes were detected using the new CDAS2 Kit.

DNA-labeling by multiplex singleprimer amplification with biotin-dUTP.

Hybridization and analysis of the microarray signals.

Fully automatic report with information to species, carbapenemase and other AMR genes.

Family-, genus- and speciesspecific genes Carbapenemase genes

isolated genomic DNA

Gene Function

Table 1: Target overview and concordance between detected and known genotype

Introduction: There is an urgent need for diagnostic laboratories to introduce rapid molecular identification of carbapenemase genes in Gramnegative bacteria for infection control and prevention, surveillance and for epidemiological purposes. Misidentification of carbapenem resistance could lead to inappropriate antibiotic treatment and dissemination of resistance. A multitude of carbapenemase genes and their allelic variants are known worldwide. The updated microarray based assay gives us a new option to identify at least 43 globally important carbapenemase genes (including their allelic variants) simultaneously. We described a new DNA-microarray for rapid and accurate identification of carbapenemase genes. We also determined the concordance between the genotype detection and phenotype using clinical samples of a tertiary care hospital in Rawalpindi, Pakistan.

Figure 1: Labeling, hybridization and automated analysis

1Alere

ESBL genes

Other betalactamase genes

AmpC genes

Aminoglycoside resistance

Macrolide resistance Quinolone resistance Sulphonamide resistance

Trimethoprim resistance

Virulence factors Multidrug efflux pump Toxin-antitoxin system a

Target Gene

a

Gene Function

Reference Sequence

Number of Reference Gene Concordance Strains Found (%)

basC cfa dnaE ecfX efp gad ihfA invA ipaH9.8 khe lacY pld blaBIC blaDIM blaGES blaGIM (consensus) blaGOB (consensus) blaIMI-3 (NmcA) blaIMI-R blaIMP (consensus) blaIMP25 (blaSIM-1) blaIMP35 blaIND (consensus) blaKHM (consensus) blaKPC (consensus) blaNDM (consensus) blaPAM-1 blaSFH-1 blaSMB-1 blaSME (consensus) blaSPM-1 blaTMB-1 blaVIM (consensus) blaVIM-2 blaVIM-7 blaOXA-23-like blaOXA-40-like blaOXA-48-like blaOXA-51-like ISABa1 to blaOXA-51 no ISABa1 to blaOXA-51 blaOXA-54 blaOXA-55 blaOXA-58 blaOXA-134/235/284 blaOXA-143/182/253/255 blaOXA-181/232 blaOXA-214 blaOXA-279 blaOXA-292 blaCME blaCTX-M1/15 blaCTX-M2 blaCTX-M8 blaCTX-M9 blaMOX-CMY9 blaPER-1 blaPER-2 blaVEB (consensus) blaOXA-18 blaOXA-45 blaSHV (consensus) blaTEM (consensus) blaOXA-1 blaOXA-2 (consensus) blaOXA-9 blaOXA-10 (consensus) blaOXA-60 blaMIR blaACC blaACT blaCMY (consensus) blaDHA blaFOX (consensus) blaMOX (consensus) aac(3') (consensus) aac(3')-Ia aac(3')-Ib aac(3')-Ic aac(3')-Ie aac(3')-IVa aac(6') (consensus) aac(6')-31 aac(6')-Ib aac(6')-II aac(6')-IIa aac(6')-IIc aac-aph aadA1 aadA2 aadA4 aadB ant2 aphA armA grm npmA rmtA rmtB rmtC rmtD strA strB mph (consensus)

acinetobactin biosynthesis protein of Acinetobacter baumannii colicin five activity protein of Citrobacter freundii and Citrobacter braakii DNA polymerase III subunit alpha extracytoplasmic function sigma factor of Pseudomonas aeruginosa [LavenirR-JocktaneD-2007] elongation factor P of Acinetobacter baumannii glutamate decarboxylase of Escherichia coli integration host factor subunit alpha invasin A, highly specific for genus Salmonella invasion plasmid antigen klebsolysin of Klebsiella pneumoniae lactose permease; the lacY gene is missing in all Shigella spp. phospholipase D of Acinetobacter baumannii carbapenemase, class A beta-lactamase carbapenemase, class B metallo beta-lactamase carbapenemase, class A beta-lactamase carbapenemase, class B metallo beta-lactamase carbapenemase, class B metallo beta-lactamase carbapenemase, class A beta-lactamase associated with imipenem resistance regulator of blaIMI-3 (NMC-A) carbapenemase, class B metallo beta-lactamase carbapenemase, class B metallo beta-lactamase (synonym: blaSIM) carbapenemase, class B metallo beta-lactamase carbapenemase, class B metallo beta-lactamase of Chryseobacterium carbapenemase, class B metallo beta-lactamase carbepenemase, class A serin beta-lactamase carbapenemase, class B metallo beta-lactamase (New Dehli metallo ß-lactamase) carbapenemase, subclass B3 metallo beta-lactamase (Pseudomonas alcaligenes metallo-ß-lactamase) carbapenemase, class B metallo beta-lactamase carbapenemase, class B metallo beta-lactamase (Serratia marcescens) carbapenemase, class A beta-lactamase (Serratia marcescens) carbapenemase, class B metallo beta-lactamase carbapenemase, class B metallo beta-lactamase carbapenemase, class B metallo beta-lactamase carbapenemase, class B metallo beta-lactamase carbapenemase, class B metallo beta-lactamase carbapenemase, class D beta-lactamase carbapenemase, class D beta-lactamase carbapenemase, class D beta-lactamase carbapenemase, class D beta-lactamase Insertion sequence ABa1 is adjacent to blaOXA-51-like gene. Insertion sequence ABa1 is not adjacent to blaOXA-51-like gene. carbapenemase, class D beta-lactamase carbapenemase, class D beta-lactamase carbapenemase, class D beta-lactamase

AY571146.1 U09771.1 U00096.3 DQ996558.1 CP001172.1 AE014075.1 U00096.3 CP000026.1 AF047365.1 AF293352.1 U00096.2 CP000521.1 GQ260093.1 KC004136.2 AY219651.1 consensus consensus AY780889.1 Z21956.1 consensus EU686387.1 JQ432564.1 Consensus consensus consensus consensus AB858498.1 AF197943.1 AB636283.1 consensus AY341249.1 FR771847.1 Consensus AF191564.1 AJ536835.1 AJ132105.1 AF509241.1 AY236073.2 CP000863.1 CP001921.1 CU459141.1 AY500137.1 AY343493.1 AY665723.1

25 7 33 15 25 11 56 5 2 25 34 24 2 1 5 2 2 1 1 12 3 1 5 0 8 14 0 0 0 1 1 1 24 1 0 9 5 11 24 4 17 1 1 6

25 7 33 15 25 11 56 5 2 25 34 24 2 1 5 2 2 1 1 12 3 1 5 0 8 14 0 0 0 1 1 1 24 1 0 9 5 11 24 4 17 1 1 6

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 n.d. 100 100 n.d. n.d. n.d. 100 100 100 100 100 n.d. 100 100 100 100 100 100 100 100 100

carbapenemase blaOXA-134-like, class D beta-lactamase

AYHO01000005.1

2

2

100

carbapenemase blaOXA-40-like, class D beta-lactamase carbapenemase blaOXA-48-like, class D beta-lactamase carbapenemase, class D beta-lactamase

GQ861437.1 CP000469.1 JN861783.1

5 3 1

5 3 1

100 100 100

carbapenemase, class D beta-lactamase

APOK01000044.1

0

0

n.d.

carbapenemase, class D beta-lactamase extended spectrum beta-lactamase, class A extended spectrum beta-lactamase, class A extended spectrum beta-lactamase, class A extended spectrum beta-lactamase, class A extended spectrum beta-lactamase, class A extended-spectrum beta-lactamase precursor, class C extended-spectrum beta-lactamase, class A beta-lactamase PER-1 extended-spectrum beta-lactamase, class A beta-lactamase PER-2 extended-spectrum beta-lactamase, class A extended spectrum beta-lactamase, class D extended spectrum beta-lactamase, class D class A beta-lactamase - blaSHV genes, including extended-spectrum beta-lactamases class A beta-lactamase - blaTEM genes, including extended-spectrum beta-lactamases narrow spectrum beta-lactamase, class D consensus probe for extended and narrow spectrum class D beta-lactamases narrow spectrum beta-lactamase, class D consensus probe for extended and narrow spectrum class D beta-lactamases narrow spectrum beta-lactamase, class D AmpC beta-lactamase AmpC beta-lactamase AmpC beta-lactamase AmpC beta-lactamase AmpC beta-lactamase AmpC beta-lactamase AmpC beta-lactamase

APRH01000012.1 AF033200.1 X92506.1 AF286192.1 AY750914.2 FQ482074.1 AF381617.1 Z21957.1 X93314.1 consensus EU503121.1 AJ519683.1 consensus consensus AY458016.1 consensus M55547.1 consensus AF525303.2 M37839.2 EF554600.1 U58495.2 consensus EF406115.1 consensus consensus

0 2 15 2 2 4 0 4 2 6 2 1 13 54 16 7 17 14 1 1 1 3 15 1 0 0

0 2 15 2 2 4 0 4 2 6 2 1 13 54 16 7 17 14 1 1 1 3 15 1 0 0

n.d. 100 100 100 100 100 n.d. 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 n.d. n.d.

3-N-aminoglycoside acetyltransferase; associated with resistance to astromicin; gentamicin; sisomicin 3-N-aminoglycoside acetyltransferase; associated with resistance to astromicin; gentamicin; sisomicin 3-N-aminoglycoside acetyltransferase; associated with resistance to astromicin; gentamicin; sisomicin 3-N-aminoglycoside acetyltransferase; associated with resistance to astromicin; gentamicin; sisomicin 3-N-aminoglycoside acetyltransferase; associated with resistance to astromicin; gentamicin; sisomicin 3-N-aminoglycoside acetyltransferase; associated with resistance to apramycin; gentamicin; aminoglycoside 6'-N-acetyltransferase, associated with resistance to amikacin; dibekacin aminoglycoside 6'-N-acetyltransferase; associated with resistance to streptomycin, spectinomycin aminoglycoside 6'-N-acetyltransferase; associated with resistance to streptomycin, spectinomycin aminoglycoside 6'-N-acetyltransferase; associated with resistance to streptomycin, spectinomycin aminoglycoside 6'-N-acetyltransferase; associated with resistance to streptomycin, spectinomycin aminoglycoside 6'-N-acetyltransferase; associated with resistance to streptomycin, spectinomycin 6'-aminoglycoside-N-acetyltransferase/2''-aminoglycoside phosphotransferase aminoglycoside adenyltransferase; associated with resistance to streptomycin, spectinomycin aminoglycoside adenyltransferase; associated with resistance to streptomycin, spectinomycin aminoglycoside adenyltransferase; associated with resistance to streptomycin, spectinomycin 2''-aminoglycoside nucleotidyltransferase aminoglycoside (2'') adenylyltransferase; associated with resistance to gentamicin; kanamycin; aminoglycoside 3'-phosphotransferase; kanamycin resistance protein 16S rRNA methylase, associated with aminoglycoside resistance 16S rRNA methylase, associated with gentamicin resistance 16S rRNA methylase, associated with aminoglycoside resistance 16S rRNA methylase, associated with aminoglycoside resistance 16S rRNA methylase, associated with aminoglycoside resistance 16S rRNA methylase, associated with aminoglycoside resistance 16S rRNA methylase, associated with aminoglycoside resistance aminoglycoside-3''-phosphotransferase (locus A); associated with resistance to streptomycin aminoglycoside-6''-phosphotransferase; associated with resistance to streptomycin

consensus U90945.1 KJ679408.1 AJ511268.1 AY458224.1 EU784152.1 consensus AJ640197.1 M21682.1 EF127959.1 EU912537.1 EU855788.1 AE017171.1 EU704128.1 EU704128.1 Z50802.3 L06418.4 L06418.4 AY260546.3 AB117519.1 M55521.1 AB261016.1 AB083212.2 DQ345788.1 AB194779.2 DQ914960.2 EF090911.1 EF090911.1

13 9 1 2 1 2 69 1 67 0 0 2 0 47 13 7 23 22 22 6 1 0 1 1 1 1 15 45

13 9 1 2 1 2 69 1 67 0 0 2 0 47 13 7 23 22 22 6 1 0 1 1 1 1 15 45

100 100 100 100 100 100 100 100 100 n.d. n.d. 100 n.d. 100 100 100 100 100 100 100 100 n.d. 100 100 100 100 100 100

macrolide 2'-phosphotransferase

consensus

23

23

100

mrx (consensus) qepA qnrA1 qnrB qnrC qnrD qnrS sul1 sul2 sul3 dfrA1 dfrA12 dfrA13 dfrA14 dfrA15 dfrA17 dfrA19 dfrA5 dfrA7 intI1 intI2 intI3 tnpISEcp1

member of macrolide inactivation gene cluster mphA-mrx-mphR QepA - fluoroquinolone/quinolone efflux pump quinolone or fluoroquinolone resistance protein quinolone or fluoroquinolone resistance protein quinolone or fluoroquinolone resistance protein quinolone or fluoroquinolone resistance protein quinolone or fluoroquinolone resistance protein dihydropteroate synthetase type 1 dihydropteroate synthetase type 2 dihydropteroate synthetase type 3 dihydrofolate reductase type 1 dihydrofolate reductase type 12 dihydrofolate reductase type 13 (synonym A21) dihydrofolate reductase type 14 dihydrofolate reductase type 15 dihydrofolate reductase type 17 dihydrofolate reductase type 19 dihydrofolate reductase type 5 dihydrofolate reductase type 7 class 1 integron integrase class 2 integron integrase class 3 integron integrase transposase for the transposon ISEcp1

consensus AM886293.1 AY931018.1 AB281054.1 EU917444.1 FJ228229.1 AM234722.1 AJ698325.1 DQ464881.1 AJ459418.2 AJ884723.1 AB154407.1 Z50802.3 AJ313522.1 Z83311.1 AF169041.1 AJ310778.1 AB188269.1 AB161450.1 AY260546.3 AY183453.1 EF469602.1 AB543698.1

11 1 5 19 1 1 19 81 28 1 21 9 2 7 3 7 4 2 5 61 4 0 29

11 1 5 19 1 1 19 81 28 1 21 9 2 7 3 7 4 2 5 61 4 0 29

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 n.d. 100

oqxA

OqxA - membran fusion protein, component of RND-type multidrug efflux pump,

EU370913.1

25

25

100

oqxB higA higB splA splT

OqxB - integral membrane protein, component of RND-type multidrug efflux pump higA is the antitoxin of the translation-dependent mRNA interferase toxin higB Ectopic expression of higB causes inhibition of cell growth which is alleviated by co-expression of higA splA is the antitoxin of the translation-dependent mRNA interferase toxin splT Ectopic expression of splT causes inhibition of cell growth which is alleviated by co-expression of splA

EU370913.1 U43847.1 U43847.1 EU294228.1 EU294228.1

text highlighted in red are targets of the previous CarbDetect AS-1 Version

22 22 0 0 0 0 21 21 21 21 Overall concordance

100 n.d. n.d. 100 100 100

Results and Discussion: The functionality of CDAS2 Kit was verified using 133 different reference strains with known resistance genotypes. A comparison between all characterized reference strains and the detected genotype is listed in the Table 1. The concordance was 100% between detected genotype and the known genotype from the reference strains. Of 425 isolates collected in a clinical setting, 82 (19%) were preliminary identified as carbapenem resistant using disk diffusion assays. According to the confirmatory tests performed with the VITEK-2 system, 76 (93 %) were resistant to fourth generation (cefepime), 80 (96%) against third generation cephalosporins and 72 (87%) were

resistant

to

meropenem

and

imipenem.

Carbapenemase genes were detected in 61 of the 72 carbapenem-resistant isolates using the CDAS2 Kit (Fig.2), where the most prevalent carbapenemase gene was blaNDM (n=31) followed by blaOXA-181/232 (n=15). In 12

isolates, multiple carbapenemase genes were detected. The following combinations were found, blaNDM/blaOXA-48-like (n=9), blaNDM/blaGES (n=2) and blaVIM/blaGES (n=1). The concordance between genotype and phenotype for carbapenem resistant isolates was 95.2%. There was a high concordance between genotype and phenotype. The microarray proved a suitable tool for the confirmation of carbapenem resistance and for identification of the responsible genes. It can be used to obtain epidemiological data on the distribution and prevalence of resistance markers. Such data are of high relevance for decisions on initial therapeutic regimens as well as for the design of new point-of-care tests for identification of, e.g., carriers of carbapenemase-producing organisms.

Figure 2: Detected β-lactam genes in clinical isolates

Carbapenemases

ESBL

Other & AmpC