Identification of antimicrobial resistance genes in multidrug-resistant clinical Bacteroides fragilis isolates by whole genome shotgun sequencing

Anaerobe xxx (2014) 1e6

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Identification of antimicrobial resistance genes in multidrug-resistant clinical Bacteroides fragilis isolates by whole genome shotgun sequencing
zsef So
ki b, Henrik Hasman c, Mikala Wang d, Thomas Vognbjerg Sydenham a, *, Jo a Ulrik Stenz Justesen , on behalf of ESGAI (ESCMID Study Group on Anaerobic Infections) a

Department of Clinical Microbiology, Odense University Hospital, Odense, Denmark Institute of Clinical Microbiology, Faculty of General Medicine, University of Szeged, Szeged, Hungary The Microbial Genomics and Antimicrobial Resistance Group, National Food Institute, Technical University of Denmark, Lyngby, Denmark d Department of Clinical Microbiology, Aarhus University Hospital, Aarhus, Denmark b c

a r t i c l e i n f o

a b s t r a c t

Article history: Available online xxx

Bacteroides fragilis constitutes the most frequent anaerobic bacterium causing bacteremia in humans. The genetic background for antimicrobial resistance in B. fragilis is diverse with some genes requiring insertion sequence (IS) elements inserted upstream for increased expression. To evaluate whole genome shotgun sequencing as a method for predicting antimicrobial resistance properties, one meropenem resistant and five multidrug-resistant blood culture isolates were sequenced and antimicrobial resistance genes and IS elements identified using ResFinder 2.1 (http://cge.cbs.dtu.dk/services/ResFinder/) and a custom BLAST database. Combinations of cfxA, cepA, cfiA, nimA, nimD, nimE, nimJ, tetQ, ermB, ermF, bexB, linAn2 and mefEn2 genes were identified in the six isolates. blaOXA-347, an open reading frame predicted to be a b-lactamase (Cheng et al., 2012), was identified in one strain. Full length IS elements were identified directly upstream of four genes, but in most cases contigs terminated 100e150 bases upstream of the gene in question. Even though partial IS elements were identified in these short sequences, certain identification could not be ascertained. Full antiobiograms for B. fragilis from genetic data will most likely require complete or nearly complete genomes. Current approaches to this are laborious and/or costly. Emerging technologies such as nanopore based single DNA strand sensing could perhaps provide a solution in the future. © 2014 Published by Elsevier Ltd.

Keywords: Bacteroides fragilis Antimicrobial resistance Whole genome sequencing

1. Introduction Bacteroides fragilis is the most frequently isolated anaerobe bacterium in blood stream infections [1] and rising prevalences of antimicrobial resistance and antibiotic resistance genes have been observed in the past decades, presumably due to increased antibiotic pressure [2,3]. Several genes encoding antimicrobial resistance properties have been identified [4]. cepA encodes a b-lactamase protein that hydrolyzes cephalosporins (except cefoxitin) and penicillins and cfxA is associated with resistance towards cefoxitin. Mechanisms

* Corresponding author. Department of Clinical Microbiology, Odense University Hospital, J.B. Winsløws Vej 21.2, 5000 Odense C, Denmark. Tel.: þ45 65414780; fax: þ45 65414785. E-mail address: [email protected] (T.V. Sydenham).

concerning b-lactam/b-lactamase inhibitor combinations have not been fully elucidated, but appear to be dependant on IS element insertion directly upstream of cepA and concomitant membrane modifications such as porin loss [5]. Carbapenem resistance is caused by expression of a metallo-b-lactamase encoded by the cfiA gene, and metronidazole resistance is associated with the presence of the nitroimidazole resistance genes nimA-H and nimJ [4,6]. While ermB, ermF, and ermG genes that encode a macrolideelincomycinestreptogramin (MLS) mechanism are the main causes of clindamycin resistance, msrSA and mefA-mediated macrolideelincosamid resistance, and lincomycin and erythromycin resistance conferred by a linA type gene (linAn2) have also been demonstrated in Bacteroides spp. [4]. tetQ confers tetracycline resistance in the majority of Bacteroides isolates with tetX and tetX1 being associated with tetracycline resistance and a higher tigecycline MIC [4]. The bexA gene encodes an efflux pump that possibly is

http://dx.doi.org/10.1016/j.anaerobe.2014.10.009 1075-9964/© 2014 Published by Elsevier Ltd.

Please cite this article in press as: Sydenham TV, et al., Identification of antimicrobial resistance genes in multidrug-resistant clinical Bacteroides fragilis isolates by whole genome shotgun sequencing, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.10.009

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T.V. Sydenham et al. / Anaerobe xxx (2014) 1e6

responsible for fluoroquinolone resistance in B. fragilis and bexB encodes a putative efflux pump also though to mediate fluoroquinolone resistance [4,7]. The genetic background for antimicrobial resistance in B. fragilis is diverse in that insertion of sequence (IS) elements upstream of some resistance genes confer increased expression and antimicrobial resistance [8]. Notable examples of this are the cfiA, cfxA, nim, and erm genes. IS elements carry outwards facing promoters that increase expression when inserted upstream of resistance genes [8]. However, antimicrobial susceptibility cannot always be predicted by the absence of an IS element upstream of a gene. In some resistant strains no IS elements upstream of cfiA or cfxA have been identified suggesting the presence of other mechanisms governing expression than IS element induced promoter activity [9e11]. For other bacterial species, antimicrobial susceptibility prediction by whole genome sequencing and resistance gene identification has been explored with sensitivities and specificities comparable to phenotypical susceptibility testing [12,13]. In the case of B. fragilis, where the presence of IS elements upstream of some key resistance genes is required for a high level antimicrobial resistance, and several different genes are associated with phenotypical resistance, whole genome sequencing and identification of resistance genes with easy-to-use websites or programs could be an attractive alternative to traditional identification with multiple laborious PCR and Sanger sequencing experiments required. We sought to ascertain whether antimicrobial resistance genes relevant to B. fragilis could be identified using whole genome shotgun sequencing (WGS) and easy to use web-based tools. The ability to identify IS elements upstream of resistance genes from WGS data was also explored. Preliminary results for three of the six isolates included in this study were presented at the 24th European Congress of Clinical Microbiology and Infectious Diseases in Barcelona 2014 (Poster P1193). 2. Material and methods

FASTX-toolkit and de novo assembly was performed using the SPAdes 3.0.0 assembler implemented on the Orione Galaxy web server (http://orione.crs4.it/) [15,16]. K values were set to 21, 33, 55, 77, 99, and 127 as suggested in the SPAdes 3.0 manual. Contigs under 200 bp in length were removed using tools on the Orione Galaxy server. During the submission process to the NCBI Whole Genome Shotgun submission portal, contigs tagged as contaminations by the contamination screen performed by NCBI staff were removed. Contigs were trimmed of sequences tagged as adapter sequences, and genomes were annotated using the NCBIs Prokaryote Genome Annotation Pipeline (PGAAP) version 2.0 [17]. Assembly metrics were calculated using the Quast web server (http://quast.bioinf.spbau.ru/) [18] and the crude read depth was calculated by dividing the total number of sequenced nucleotides with the genome length reported by Quast.

2.3. Identification of genes and IS elements The assemblies were submitted to ResFinder 2.1 (http://cge.cbs. dtu.dk/services/ResFinder/) which identifies acquired antimicrobial resistance genes using local nucleotide BLAST [19]. Percent identity (%ID) threshold and minimum length settings were 90% and 60% respectively. Genes not included in the ResFinder database (bexA, bexB, mefE, and linAn2) and known IS elements relevant to Bacteroides identified in the literature [8] and by searching the NCBI nucleotide database were downloaded from NCBI GenBank and incorporated in a custom BLAST database. For included IS elements please see Supplementary Table 1. This database was searched using the draft assemblies as queries using nucleotide BLAST in CLC Main Workbench version 7.0.3 (CLC bio, Aarhus, Denmark). Identified IS elements upstream of resistance genes were noted. In several cases contigs ended approximately 100e150 bases upstream of resistance genes. Several fragments of various IS elements were identified with high scores in these short upstream regions. The IS element fragment with the highest %ID similarity score and the longest hit length was noted as a possible IS element.

2.1. Isolates and their antibiotic resistance determination One isolate resistant to meropenem and five multidrugresistant B. fragilis blood culture isolates were included in the study; O17 (DCMOUH0017B), O18 (DCMOUH0018B), S01 (DCMSKEJBY0001B), O42 (DCMOUH0042B), O67 (DCMOUH0067B), and O85 (DCMOUH0085B). A case report has previously been published for isolate O18 [14] and a case report concerning isolate S01 is being prepared (Ank et al., in preparation). Species identification was confirmed using Matrix Assisted Laser Desorption and Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) on the Biotyper platform (Bruker Daltonics, Bremen, Germany). Antimicrobial susceptibility testing was performed
rieux, Craponne, France) on using the Etest gradient method (BioMe Brucella blood agar supplemented with 5 mg/L hemin and 1 mg/L vitamin K1 (Becton, Dickinson GmbH, BD Diagnostics, Heidelberg, Germany) according to the manufacturer's instructions and interpreted according to EUCAST clinical breakpoints version 4.0 (www. eucast.org/clinical_breakpoints/). 2.2. Whole genome shotgun sequencing and de novo assembly Genomic DNA was purified using the MasterPure DNA Purification kit (Epicentre Biotechnologies, Madison, WI, USA) following the manufactures instructions and libraries were prepared using the Nextera XT kit (Illumina, Essex, United Kingdom). 250 bp (basepair) paired-end sequencing was performed using the Illumina MiSeq platform. Read quality metrics was evaluated using the

3. Results 3.1. Whole genome shotgun sequencing Six isolates were sequenced using the MiSeq platform. Contigs per assembly ranged from 186 to 367. Draft genome lengths varied from 5,117,284 to 5,509,612 bp with crude read depths of 39e171 and contig N50 values ranging from 101,349 to 184,070 bp. DDBJ/ EMBL/GenBank accession numbers, assembly versions used in this paper, and SRA accession numbers can be found in Supplementary Table 2. Please see Supplementary Table 3 for WGS quality and assembly metrics specified for each isolate.

3.2. Antimicrobial susceptibility and identification of resistance genes Main results from antimicrobial susceptibility testing and identification of known resistance genes and IS elements are presented in Table 1. Five strains were resistant towards meropenem with four of these isolates also displayed resistance to imipinem. Four strains were resistant towards metronidazole, two were resistant towards piperacillin/tazobactam, and four showed resistance towards clindamycin. Combinations of cfxA, cepA, cfiA, nimA, nimD, nimE, nimJ, tetQ, ermB, ermF, blaOXA-347, bexB, mefEn2, and linAn2 were identified in the six strains at >98 %ID.

Please cite this article in press as: Sydenham TV, et al., Identification of antimicrobial resistance genes in multidrug-resistant clinical Bacteroides fragilis isolates by whole genome shotgun sequencing, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.10.009

T.V. Sydenham et al. / Anaerobe xxx (2014) 1e6

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Table 1 Antimicrobial susceptibility testing and resistance gene and IS element identification for the six Bacteroides fragilis strains. Antibiotic susceptibility testinga Strain

Antibiotic

O17

MEM IPM MTZ

Etest MIC (mg/L) >32 >32 >32

Resistance gene and IS element identificationb Result

Gene

R R R

cfiA

Upstream IS element *IS614B

nimJ *IS614B tetQ bexB

CLI PTZ O18

MEM IPM MTZ

0.094 >256 >32 16 16

R R R

cfiA

6

R

ermF

*ISBf12 nimD

IS4351 LinAn2 tetQ bexB mefEn2 >256

R

MEM IPM MTZ

>32 16 64

R R R

nimE

CLI

>32

R

ermF

PTZ S01

cfiA *IS1187 *ISBf6 *IS1187 tetQ tetQ bexB

O42

PTZ

6

S

MEM IPM

0.094 0.25

S S

MTZ

8

R

blaOXA-347 cepA nimA

>256

R

ermF

ISBf13 CLI

*IS613B LinAn2 tetQ bexB mefEn2

O67

PTZ

0.38

S

MEM IPM MTZ CLI

8 0.5 0.19 0.38

R S S S

PTZ

2

S

O85

cfiA *IS1187 LinAn2 tetQ bexB mefEn2

cfxA IS614B MEM IPM MTZ CLI

PTZ

HSP/query length (%)

Hit strand

Contig

Contig length (bp)

Hit start (nt)

Hit end (nt)

Hit length (bp)

99.6 100.0 99.4 94.4 99.3 91.2

99.2 7.9 100.0 7.8 99.3 100.0

M P M P P M

Contig177 Contig177 Contig16 Contig16 Contig184 Contig194

12,656 12,656 1653 1653 63,401 180,804

11,774 12,530 1017 1529 1889 964

12,517 12,656 1514 1653 3849 2328

744 127 498 126 1961 1365

100.0 100.0 99.2 99.4 99.8 99.8 100.0 99.8 91.1 99.8

100.0 7.9 100.0 99.8 72.0 100.0 100.0 100.0 100.0 100.0

M P P P M M P M M P

Contig167 Contig167 Contig251 Contig251 Contig168 Contig168 Contig39 Contig246 Contig162 Contig39

101,303 101,303 7349 7349 106,254 106,254 77,145 125,751 34,537 77,145

100,384 101,178 6749 5429 88,121 88,698 15,558 8958 967 16,095

101,133 101,303 7243 6740 88,697 89,879 16,070 10,883 2331 17,300

750 126 495 1314 577 1181 513 1926 1365 1206

99.2 94.8 100.0 100.0 99.5 100.0 90.5 99.8 91.1

100.0 13.9 100.0 40.6 100.0 9.9 100.0 100.0 100.0

M M M M M M M P P

Contig109 Contig109 Contig125 Contig125 Contig104 Contig104 Contig104 Contig115 Contig111

31,468 31,468 8458 8458 22,876 22,876 22,876 10,163 74,184

30,547 31,297 7612 8073 21,944 22,751 19,732 75,973 63,700

31.296 31,468 8046 8458 22,744 22,866 21,657 77,898 65,064

750 163 435 386 801 116 1926 1926 1365

100.0 100.0 98.3 100.0 99.5 100.0 100.0 100.0 99.1 99.8

100.0 100.0 100.4 100.0 100.0 8.0 100.0 100.0 100.0 100.0

P P P M P P P P M P

Contig74 Contig208 Contig1 Contig1 Contig74 Contig74 Contig104 Contig212 Contig213 Contig104

2087 53,110 8433 8433 2087 2087 107,515 222,964 422,215 107,515

1131 36,310 3373 2121 128 1 58,444 44,695 219,072 58,981

1955 37,212 3905 3339 928 127 58,956 46,668 220,436 60,186

825 903 533 1219 801 127 513 1974 1365 1206

100.0 95.5

100.0 9.5

P P

Contig301 Contig301

135,251 135,251

66,868 66,728

67,617 66,839

750 112

100.0 100.0 90.9 99.8

100.0 100.0 100.0 100.0

M P P M

Contig292 Contig112 Contig23 Contig292

39,473 175,335 83,642 39,473

1682 116,174 30,107 452

2194 118,099 31,471 1657

513 1926 1365 1206

99.9 99.0 100.0 93.6

100.0 93.2 100.0 9.1

M P P M

Contig55 Contig55 Contig55 Contig55

193,255 193,255 193,255 193,255

186,708 187,686 76,886 76,673

187,673 189,181 77,635 76,858

966 1497 750 186

99.7 90.5 99.8 90.9

98.8 100.0 100.0 100.0

P M M M

Contig54 Contig171 Contig60 Contig51

249,202 42,614 172,757 455,838

206,597 13,157 14,304 370,536

207,325 15,082 16,229 371,900

729 1926 1926 1365

S R

IS1169 CLI

%ID

32 1 0.25 >256

R S S R

2

S

cfiA *IS1188 ermB tetQ tetQ bexB

a

Breakpoints (S/R) defined by EUCAST: Meropenem (MEM) 2/>8, imipinem (IPM) 2/>8, metronidazole (MTZ) 4/>4, clindamycin (CLI) 4/>4, and piperacillin/tazobactam (PTZ) 8/>16. Other abbreviations: MIC ¼ minimal inhibitory concentration; R ¼ resistant; S ¼ susceptible. b Identified genes are displayed next to relevant the antibiotic. IS elements identified upstream of a gene follow the respective gene in the table. Partial IS elements are marked with an asterisk (*). Hit strand indicates the strand orientation on which the identified gene/IS element is located by M (Minus) or P (Plus). Contig indicates the assembled contig on which the gene/IS element is located. Hit start and hit end indicate the first and last nucleotide of the BLAST hit corresponding to the Plus strand of the contig. Other abbreviations: IS ¼ insertion sequence; %ID ¼ percent identity; HSP ¼ high scoring segment pairs; bp ¼ base pairs; nt ¼ nucleotide position.

Please cite this article in press as: Sydenham TV, et al., Identification of antimicrobial resistance genes in multidrug-resistant clinical Bacteroides fragilis isolates by whole genome shotgun sequencing, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.10.009

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cfiA genes were identified in all five meropenem resistant isolates. In two meropenem resistant isolates, O67 and O85, cfiA was identified and the uninterrupted upstream region did not contain a full length IS element. In the other three meropenem resistant isolates, possible IS elements were identified in the short sequences upstream of the cfiA genes. nim genes were identified in the four metronidazole resistant isolates and not in the two susceptible isolates. In strains O18 and O42 full length IS elements were identified upstream of the nim gene, in strain S01 a partial ISBf6 that was cut short after 386 bp due to contig termination was identified upstream of nimE. In strain O17 the contig with nimJ terminated 140 bp upstream of the gene, with a partial IS614B identified in the short upstream sequence. Of the six isolates, four were resistant to clindamycin, with erm genes identified in all four isolates. A full length IS4351 was found upstream of the truncated ermF gene in isolate O18. In isolates S01 and O42 partial IS elements were identified in the short upstream sequences, and in isolate O85 no IS element was identified in the uninterrupted upstream region of ermB. linAn2 was identified in the clindamycin resistant isolates O18 and O42, but also in the susceptible isolate O67. No IS elements was identified upstream of the linAn2 in the resistant isolates. The two piperacillin/tazobactam resistant isolates were also highly carbapenem-resistant and all harbored cfiA-genes and not cepA.

(GenBank: KFX74121.1) spanning almost the entire region dividing the two partial ermF sequences. O18 is resistant to clindamycin at a lower level (MIC ¼ 6 mg/L) than isolates S01, O42, and O85 that contain full length erm-genes, so it is possible that the partial ermF gene is expressed and the resulting protein product confers clindamycin resistance albeit at a lower level than a full length protein. Along with a certain ID of tetQ in strains S01 and O85, hits with 90.5 %ID and 100% high scoring segment pair (HSP)/query length to tetQ were found. The submission of the tetQ-like gene nucleotide sequences to NCBI BLAST-n resulted in two hits with 99 %ID; a gene titled elongation factor Tu GTP binding domain protein in Tannerella forsythia ATCC 43037, complete genome (GenBank: CP003191.1 location 1,331,023e 1,332,960) and a gene titled small GTP-binding protein domain in Bacteroides xylanisolvens XB1A draft genome (GenBank: FP929033.1 location 4,261,675e4,4263,600). According to the accessions, both genomes were annotated using prediction methods. Searching the NCBI BLAST nr protein sequence database using blastx resulted in 100% ID hits to GenBank: WP_008645586.1, a non-redundant multispecies tetQ protein sequence, as the highest hit, making it apparent that strains S01 and O85 harbor two copies of tetQ. bexB was identified in strain O42 with 99.1 %ID and hits were noted with 90.9e91.2 %ID to bexB in the other five genome sequences. Aligning the five bexB-like sequences using the alignment feature in the CLC Main Workbench, resulted in a perfect gapless alignment with almost 100% conservation at all nucleotide positions (not shown). The five sequences shared 97.1e99.9 %ID, and the bexB-like genes of O18, S01, O67 and O85 showed very high similarity with 99.5e99.9 %ID. The two highest %ID scores when submitting the sequence of the possible bexB-like gene of strain O17 to NCBI nucleotide BLAST were 91.3 %ID with B. fragilis 638R genome (GenBank: FQ31204.1 location 2,664,849e2,663,485) and 91.2 %ID to the complete bexB gene in B. fragilis strain 86-5443-2-2 (GenBank: AY375536.1 location 5963e4599), the latter being the bexB sequence used in the custom BLAST database used in this study. Searching the NCBI BLAST nr protein sequence database using blastx resulted in 100 %ID hits to GenBank: WP_005794219.1 multidrug transporter MatE [Bacteroides] for the bexB-like genes in all five strains. This protein shares 100% homology to GenBank: AAQ92937.1, the protein product of the bexB sequence used in the custom BLAST database in this study. A gene annotated as blaOXA-347 was identified by ResFinder in strain O42. The blaOXA-347 sequence referenced by ResFinder is GenBank: JN086160.1 (location 1583e2407). The predicted b-lactamase gene in JN086160.1 was identified using amoxicillin screening of a fosmid metagenomic library generated from gut microbiota cloned into Escherichia coli, and the closest match to the predicted protein was with 53% identity to a class D b-lactamase from Riemerella anatipestifer [20]. This is to our knowledge the first published identification of a possible blaOXA gene in Bacteroides spp.

3.5. Novel resistance gene alleles

4. Discussion

In strain O18 ermF was identified with 99.8 %ID and 72% hit length (557/801 nucleotides) on the complement strand of contig168. On closer examination another partial 238 bp ermF was discovered 1309 bp downstream on the complement strand. Joining the two partial ermF hits and submitting the 815 bp result to BLAST-n resulted in a 98.2 %ID hit to ermF in several accessions also including ermF in GenBank: M17124. BLAST alignment showed a gap of 15 bases in an otherwise perfectly aligned sequence (not shown). This led us to the conclusion that an insertion event could have occurred disrupting the ermF gene into two parts. Indeed PGAAP annotation had resulted in a predicted transposase gene

The significant antimicrobial resistance genes relevant to B. fragilis could be identified using WGS and the ResFinder website. bexA, bexB, linA and mefE are not included in the ResFinder 2.1 database, but could be added in future versions. In the six isolates included in this study, four to eight resistance genes were identified in each isolate. cfiA and nim genes were identified in all isolates with meropenem and metronidazole resistance respectively. erm genes were identified in the four clindamycin resistant isolates. linAn2 was identified in one clindamycin resistant isolate, but was also found in a clindamycin susceptible isolate. Research data on the degree of linA contribution to clindamycin resistance in

3.3. Identification of IS elements Full length IS elements were identified in the upstream regions of four genes; IS1169 and IS4251 upstream of nimD and the truncated ermF-gene in isolate O18, ISBf13 upstream of nimA in isolate O42, and IS614B upstream of cfxA in isolate O85. ISBf6 was identified with 100.0 %ID and an HSP/Query length of 40.6%. The partial length hit was due to termination of the contig 386 bp into the IS element. Upstream of cepA in isolate O42, cfiA in O67 and O85, and ermB in isolate O85, the contigs continued thousands of bases upstream of the resistance genes and no full length IS elements were identified. Concerning the genes where partial or whole IS elements were identified upstream of the gene, 62% (8/13) of contigs terminated just approximately 100e200 bp upstream of the identified antimicrobial resistance gene. Partial BLAST hits for IS elements were recorded upstream of several resistance genes with upstream regions terminated by contig ends. A partial hit for IS1187 with a hit length of 112 bases was noted upstream of cfiA in isolate O67 and a partial hit of 186 bases of IS1188 was found directly upstream of O85 where the contigs do not terminate just upstream of the cfiA gene. Several of the IS elements and partial IS elements were identified on the non-sense/opposite strand of the gene, but still “upstream” of the gene. 3.4. Correlation between resistance profile and identified genes and IS elements

Please cite this article in press as: Sydenham TV, et al., Identification of antimicrobial resistance genes in multidrug-resistant clinical Bacteroides fragilis isolates by whole genome shotgun sequencing, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.10.009

T.V. Sydenham et al. / Anaerobe xxx (2014) 1e6

Bacteroides is scarce. Our results do indicate that the presence of linA alone does not confer clindamycin resistance. In several cases IS elements or partial IS elements were identified on the opposite strand of the resistance gene. Previous studies have identified IS elements in the upstream regions of the resistance gens, and shown that the presence of the IS elements confer increased expression of the genes [8,21]. The “direction” of the IS element is defined by the coding strand orientation of the reading frame encoding the transposase, but some IS elements are known to hold an outward oriented promoter oriented in the opposite direction of the transposase gene [8]. The outward facing promoter is located in the inverted regions, making it possible that the promoter motifs are also present on the opposite/complement strand [22]. Indeed, in strain O42, the promoter motif TAnnTTTG can be found upstream of nimA, where ISBf13, is on the complement strand of the nimA upstream region. The same phenomenon has also been observed by Husain and colleagues while describing a conjugative transposon harboring nimJ [23]. The consequence of an IS element presence on the opposite strand of the resistance genes needs to be explored experimentally. In most cases, the identification of IS elements was hampered by the termination of contigs relatively close to identified antimicrobial resistance genes. Partial hits to known IS elements were observed in these short upstream regions, however partial IS element hits were also recorded upstream of cfiA in two cases where the contigs were not terminated just upstream of the genes. Unfortunately, partial IS element hits upstream of a gene must therefore be interpreted with caution and cannot necessarily be attributed to actual presence of an IS element. It is known that repetitive sequences impede de novo genome assembly using short reads and result in fractured assemblies [24]. In testing assemblies from simulated reads derived from a finished reference genome 25%e77% of non-reconstructable genes were tagged as transposase- or IS-related, depending on the read length simulated [25]. Taking this into account, a terminated contig at a position where a potential IS element has been identified by BLAST, could be taken as further indication that an IS element is indeed present at that position. The identification of new alleles in B. fragilis with approximately 90 %ID to tetQ and bexB, the possible blaOXA gene, and the ermF gene disrupted by a transposase are notable findings resulting from simple analysis of the WGS assemblies. It is possible that these genes would not have been identified using specific primer based PCR screening, as the primer bindings sites could have changed in the new tetQ and bexB alleles, and a PCR product of the disrupted ermF gene would differ from the known ermF product being notably longer and might go unnoticed. Interestingly, the isolate with the disrupted version of ermF was still clindamycin resistant, but at a much lower level than isolates carrying full length erm genes. This raises the question whether the protein product of the disrupted ermF gene has lower, but still functional, ribosomal RNA methylation activity than the full length version. Several authors predict that whole genome sequencing and prediction of antimicrobial resistance from the genetic sequences will be implemented as a routine test in the near future in clinical microbiology [26e29]. However, a better understanding of the genetic determinants for resistance is required before correct prediction of antimicrobial resistance properties in B. fragilis from WGS data will be feasible. ResFinder and the custom BLAST database constructed for this study only identifies known acquired resistance genes and IS elements. Point mutations, inversions, deletions and genomic rearrangements that could alter antimicrobial susceptibility phenotypes are not identified using this approach. Fluoroquinolone and rifampicin resistance has been shown to be influenced by point mutations in B. fragilis [30,31], but we did not

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include phenotypic susceptibility testing for these antibiotics as they are not currently used for treatment of B. fragilis infections. Studies on E. coli show complicated relationships between resistance determinants with some resistance mutations conferring increased sensitivity to other antibiotics and cross-resistance determinants influencing the resistance to multiple antibiotics [32]. An even more detailed “genetic antiobiogram” for each isolate could be generated from a method designed to also incorporate these possible mechanisms and inter-dependencies once they are discovered for B. fragilis. cfiA mediated carbapenem-resistance is one of the most studied areas of antimicrobial resistance in B. fragilis, but no explanation of the resistant phenotypes without the presence of IS elements upstream of the gene has yet been found. IS elements upstream of a resistance gene are almost always associated with antimicrobial resistance, but as we have found in this study, there is an inherent confounding issue concerning the termination of contigs due to probable IS sequences. Therefore, to accurately predict resistance for B. fragilis, it is likely that complete, or almost complete, genome sequences are required. The results presented in this study are based on 2*250 bp paired-end sequencing on the Illumina MiSeq platform, offering fragmented draft assemblies with hundreds of contigs per genome at a fairly low cost (approximately 220 USD) and generated in a reasonable time frame. Short read paired-end sequencing on the Illumina platform was chosen as this approach was used in previous proof-of-concept studies comparing WGS results to phenotypic resistance testing in various aerobic bacteria [12,13]. If WGS is to be used for rapid clinical diagnostics and antiobiogram generation, genome finishing requiring multitudes of Sanger sequencing experiments is too laborious and costly to be applicable. Sequencing mate-pair libraries on the Illumina platform and utilizing these with the paired-end sequences in the de novo assembly would result in a “higher quality” assemblies with significant reduction in the contig numbers [33]. This would possibly result in upstream regions encompassing the IS elements in the final assemblies. However, high quality mate-pair libraries are up to seven times more expensive than paired-end libraries and can be difficult to produce. The preparation and sequencing of an additional library would significantly increase the costs as well as the processing time per isolate, which makes this method unlikely to be the first choice in a clinical setting. Other technologies such as single molecule, real-time DNA sequencing, as offered by Pacific Biosciences [34], or the emerging nanopore based technologies [35] offer the prospect of finished single chromosome microbial genomes, but using the Pacific Bioscience platforms is more expensive and in some instances error prone than the Illumina platform and nanopore based sequencing is still under development. 5. Conclusions Genes encoding proteins with antimicrobial resistance properties and IS elements could be identified using WGS data from six clinical blood isolates, the easy to use web-based tool ResFinder and custom BLAST database. Complete IS elements were identified upstream of four genes, but in most cases, only short upstream sequences were available due to contig termination. However, partial IS elements could be identified at these sites, and the termination of the contigs at these sites is a possible indication of IS element presence. As the fragmented assemblies resulting from paired end data inhibits certain identification of IS elements upstream of identified resistance genes, complete, finished genome sequences are most likely required for full “genetic antiobiograms” for B. fragilis and the required technologies to easily achieve this are still under development.

Please cite this article in press as: Sydenham TV, et al., Identification of antimicrobial resistance genes in multidrug-resistant clinical Bacteroides fragilis isolates by whole genome shotgun sequencing, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.10.009

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Please cite this article in press as: Sydenham TV, et al., Identification of antimicrobial resistance genes in multidrug-resistant clinical Bacteroides fragilis isolates by whole genome shotgun sequencing, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.10.009