The authors have declared that no competing interests exist.
Conceived and designed the experiments: TL PC MRM JZ MRG. Performed the experiments: TL PC JZ DRB RH TD. Analyzed the data: TL MRM DRB RH TD PC MRG. Contributed reagents/materials/analysis tools: TL MRG MRM DRB TFB PK MM DG CNISP. Wrote the paper: TL.
¶ Membership of the Canadian Nosocomial Infection Surveillance Program (CNISP) is provided in the Acknowledgments.
Genotypic and phenotypic characterization was performed on a metronidazole resistant clinical isolate of
This is the first characterization of stable, metronidazole resistance in a
Metronidazole is the recommended treatment for mild to moderate CDI, while vancomycin is reserved for more severe cases owing to cost and concerns of vancomycin-resistant nosocomial infections
Recent studies support the general assumption that most
Metronidazole resistance mechanisms have been extensively investigated in other pathogenic bacteria such as
In 2009, our laboratory isolated a
Strain CD26A54 was isolated from a stool sample collected under the Canadian Nosocomial Surveillance Program (CNISP) which continuously monitors health-care acquired infections across Canada. Data collection was observational and considered a routine component of institutional infection prevention and control practices under provincial legislation, therefore informed consent was not required
The stool sample was processed as previously described
The NAP1
DNA for PCR analysis was prepared using InstaGene Matrix (Bio-Rad, Richmond, CA, USA). Multipex PCR was employed to detect the Toxin A (
Pulsed-field gel electrophoresis (PFGE) typing was performed with
All experiments were performed on strains that had been passaged three times on BAKHS agar after being removed from −80°C storage to maintain consistency between technical replicates. Growth curves were performed in Brain Heart Infusion (BHI) broth (BD, Mississauga, ON, Canada) at 37°C in an anaerobic chamber (Coy Laboratory Products Inc, Grass Lake, MI). The turbidity for all overnight starter cultures was measured with a spectrophotometer (Eppendorf, Mississauga, ON, Canada) at OD 600 nm and normalized before inoculating fresh growth curve tubes to minimize variation at time point 0. Quantitation of vegetative cells relative to spores was performed following a protocol described elsewhere
All samples were filtered using 13 mm diameter SPI–pore polycarbonate track etch filters with 100 nm pores (SPI supplies, West Chester, Pennsylvania, USA), held in 13 mm Swinnex® filter holders (Millipore, Billerica, Massachusetts, USA). All syringe filtering was performed using a Legato 200 syringe pump (KD Scientific, Holliston, Massachusetts, USA) operated with a flow rate of 1 ml/minute to ensure reproducible filtration conditions. When starting with fresh bacterial samples grown an agar plates, two loopfuls of bacteria were mixed in 800 µl of fixative (1% paraformaldehyde, 2% glutaraldehyde) generating a turbid suspension. The subsequent filter preparation of all the specimens was done in a fume hood, using Lure-Lok® syringes to pass all fluids through the filter assembly. Sample processing was conducted as follows: using a 2 ml syringe, 2 ml of PBS was passed through the apparatus to wet the filter. Next, 0.2 ml of the bacterial suspension was applied to the filter using a 1 ml syringe, followed by a 5 ml wash with PBS done using a 5 ml syringe. Then using 2 ml syringes, the filter was washed with 2 ml 50% ethanol, 2 ml 70% ethanol, 2 ml 85% ethanol, 2 ml 95% ethanol, and 2 ml 100% ethanol. The filter apparatus was then disassembled and the filter was allowed to air dry.
Once dry, the filter was cut and mounted onto an SEM stub. First, a double-sided adhesive carbon disc was stuck to the metal stub, and then the filter was mounted to the carbon disc, bacteria side up. Silver flash paint was then used to create a conductive contact between the stub and the filter paper. 1 cm of 0.2 mm diameter gold wire was measured, cut and wound on the filament of an Agar 208 Turbo Carbon Coater evaporator unit (Agar Scientific, Stansted, England). The vacuum was turned on, and once a vacuum was generated the low tension was gradually increased to evaporate the gold onto the sample.
Specimens were imaged in a Scanning Electron Microscope (JEOL CarryScope, JEOL Ltd., Tokyo, Japan) operated at 6 kV, with a spot size of 20, a 9 mm working distance, and at nominal instrument magnification of ×3,000. Digital images were acquired using the secondary electron detector.
Susceptibility to metronidazole, clindamycin, vancomycin, rifampicin, moxifloxacin and tigecycline were performed using Etest® strips (bioMérieux, Solina, Sweden) with standard methods described elsewhere
To test for heteroresistance towards metronidazole, Etest® assays were again set up according to the manufacturer's instructions, however they were incubated for an extended period of time to observe the presence or absence of slower growing satellite colonies within the clear ellipse. A similar protocol has been described previously
Draft genomes were acquired for the CD26A54_R and CD26A54_S isolates via combined use of single-end shotgun pyrosequencing reads (GS FLX-Titanium; Roche Diagnostics, Indianapolis, IN, USA) and paired-end, 100 bp Illumina reads (GAIIe; Illumina, San Diego, CA, USA; Sequenced at Eurofins MWG Operon, Huntsville, AL, USA). Pyrosequencing yielded an average 22X and 24X coverage while the Illumina data increased the coverage to 228X and 323X average coverage of the CD26A54_S and CD26A54_R genomes, respectively. The combined sequence data provided an estimated >99% coverage of each genome. Publically available NAP1 genomes were employed for comparative genomic analyses with the CD26A54_R and CD26A54_S strains
Strain | Year | Location | Source | Assembly | Genome size (nt) | No. of contigs | Reference | NCBI accession |
CD26A54_R | 2009 | B.C., Canada | Human | draft | 4,166,900 | 4 | this study | PRJNA172829 |
CD26A54_S | 2009 | B.C., Canada | Human | draft | 4,166,979 | 6 | this study | PRJNA172828 |
QCD-66c26 | 2007 | QC, Canada | Human | draft | 4,094,363 | 32 | 44 | NZ_CM000441 |
R20291 | 2006 | UK | Human | complete | 4,191,339 | 1 | 45 | NC_013316 |
QCD-37×79 | 2005 | ON, Canada | Human | draft | 4,092,698 | 45 | 44 | NZ_CM000658 |
QCD-97b34 | 2004 | Nfld, Canada | Human | draft | 3,998,408 | 60 | 44 | NZ_CM000657 |
QCD-32g58 | 2004 | QC, Canada | Human | draft | 4,109,689 | 16 | 44 | NZ_CM000287 |
BI-1 | 1988 | USA | Human | draft | 4,118,573 | 66 | 44 | NC_017179 |
CD196 | 1985 | France | Human | complete | 4,110,554 | 1 | 45 | NC_013315 |
GS-FLX reads were assembled
CD26A54_R and CD26A54_S were compared with other publically available NAP1 strains by generating a BLAST atlas (listed in
Growth curves were analyzed with a two-way ANOVA and Tukey posttest. The Kruskal-Wallis test for variance with Dunn's posttest was used to analyze the SEM length measurement and metronidazole MIC data as normality could not be assumed for these distributions. All analyses were conducted in PRISM 5 statistical software (GraphPad Software, La Jolla, CA, USA).
All three strains in the study, CD26A54_R, CD26A54_S and VLOO13 were typed as NAP1, pattern 001 by pulse-field gel electrophoresis (PFGE). The PCR toxin analyses were typical of NAP1 strains, being positive for all toxins (
CD26A54_R was observed to have a significantly reduced cell density in BHI broth compared to VLOO13 at all time points from 5–24 hours growth (
CD26A54_R, the metronidazole resistant strain (□) demonstrated aberrant growth compared to VLOO13 (□) at all time points between 5 and 24 hours (
When grown on standard BAKHS agar plates, all three
The quantitation of spores relative to vegetative cells within BHI broth was performed at 12, 24 and 48 hours post inoculation. Experimental optimization of the spore enumeration protocol was attempted using different shocking methods to select for spores and various media formulations for colony recovery. The cell quantities were inconsistent using these different methods, however a consistent trend was observed. Specifically, the relative percentage of spores increased for CD26A54_S and VLOO13, but remained at or near zero for the metronidazole resistant strain, CD26A54_R. The relative percentage of spores increased over time for both CD26A54_S (1.4%, 12.0%, 42.2%) and VLOO13 (3.2%, 21.7%, 87.2%) at 12, 24 and 48 hours, respectively. Conversely, the percentage of spores in the CD26A54_R culture remained at or near zero (0%, 0.0025%, 0%) for all three time points, respectively.
Bacterial ultrastructure was examined by scanning electron microscopy (SEM). The results of this analysis are presented in
SEM images of VLOO13 (A), CD26A54_S (B) and CD26A54_R (C). (D) Histograms of calculated bacterial length are presented; they demonstrate the cell length variation that exists across the three
Following the isolation of
Antibiotic susceptibility for a panel of 6 antibiotics was performed on the CD26A54_R, CD26A54_S and VLOO13 strains by Etest® strip (
Strain | Metronidazole | Clindamycin | Vancomycin | Rifampicin | Moxifloxacin | Tigecycline |
CD26A54_R | 12–256 | 8 | 0.75 | 0.003 | >32 | 0.094 |
CD26A54_S | 2–12 | 6 | 0.75 | 0.004 | >32 | 0.125 |
VLOO13 | 0.38–1.5 | 2 | 0.75 | 0.004 | >32 | 0.125 |
0.19–0.25 | 1.5 |
|
|
0.38 |
|
|
|
|
4 | 1.5 |
|
0.75 | |
|
|
2 |
|
|
|
|
|
|
|
|
0.064 |
|
N/A = not applicable as a quality control strain for this specific antibiotic.
Susceptibility to metronidazole was repeatedly tested for a total of
We examined heteroresistance towards metronidazole using Etest® strips. At 48 hours, the CD26A54_R strain was classified above the resistant breakpoint (MIC = 32 mcg/ml). Under prolonged incubation for 96 hours, we observed further growth leading to reduction of the ellipse (MIC = 96 mcg/ml). The CD26A54_S isolate was susceptible to metronidazole at 48 hours (MIC = 3 mcg/ml), however slower growing colonies were observed within the ellipse increasing the MIC to 16 mcg/ml at 96 hours. The wild type VLOO13 strain, demonstrated MIC values within the same doubling dilution at both 48 and 96 hours, MIC = 0.38 mcg/ml and 0.5 mcg/ml respectively. The
The macrodiversity of the CD26A54_R and CD26A54_S strains were compared to publically available NAP1 genomes using BLASTn to generate a BLAST atlas (
BLASTn was used to compare NAP1 genomes listed in
There were 64 total variants identified for the CD26A54_R strain compared to only 45 total variants of the CD26A54_S strain, each in relation to the publically available reference R20291 (NC_013316.1) genome. There were 20 variants, unique to CD26A54_R within CDS (coding DNA sequence) including 17 SNP and 3 indel while CD26A54_S only contained 3 variants, 2 SNP and 1 indel (
R20291 Reference | CD26A54_R | CD26A54_S | Gene | Predicted product | PCR |
Coverage |
Frequency of | |||
Position | Sequence | Variant | AA | Variant | AA | name | variant (%) |
|||
920174 | A | ATG | Asn219fs | – | – | putative transcription antiterminator | 150 | 96 | ||
969792 | T | C | SYN | – | – | putative membrane protein | Y | 295 | 100 | |
1220387 | G | A | Val113Ile | – | – |
|
homoserine dehydrogenase | 373 | 93.3 | |
1268654 | C | A | Thr99Asn | – | – |
|
stage 0 sporulation protein A | Y | 170 | 87.1 |
1337528 | G | A | Ala30Thr | – | – |
|
DNA topoisomerase 1 | 225 | 87.1 | |
1353228 | G | A | Glu41Lys | – | – |
|
ferric uptake regulation protein | Y | 339 | 100 |
1481407 | T | C | SYN | – | – | putative oligopeptide transporter | 195 | 88.2 | ||
1487300 | G | A | Leu283Phe | – | – | GntR family transcription regulator | 85 | 100 | ||
1503742 | C | A | Asp254Tyr | – | – | putative signalling protein | 340 | 100 | ||
1783253 | G | A | Asp593Asn | – | – | sodium ABC extrusion transporter permease | 181 | 100 | ||
1890570 | C | T | Ser328Phe | – | – |
|
thiamine biosynthesis protein | Y | 325 | 94.5 |
2160266 | T | G | Ser308Ala | – | – | peptidase | 319 | 100 | ||
2516619 | T | G | Asp259Ala | – | – |
|
putative germination-specific protease | Y | 321 | 99.4 |
2759453 | T | TA | Tyr214fs | – | – |
|
oxygen-independent coproporphyrinogen III oxidase | Y | 179 | 85.5 |
2955260 | C | T | Ala229Thr | – | – |
|
glycerol-3-phosphate dehydrogenase (NAD(P)+) | 243 | 64.6 | |
2995946 | G | T | Ala334Glu | – | – | pseudogene | 486 | 52.7 | ||
3014935 | C | T | Gly423Glu | – | – |
|
pyruvate-flavodoxin oxidoreductase | Y | 362 | 100 |
3120023 | GT | G | Lys4fs | – | – | putative N-acetylmuramoyl-L-alanine amidase | 295 | 74.6 | ||
3277693 | T | C | SYN | – | – | altronate hydrolase | 431 | 95.1 | ||
4159729 | G | A | Leu646Phe | – | – | diguanylate phosphodiesterase | 125 | 100 | ||
2688508 | T | – | – | A | Leu701Ile | DNA topoisomerase | 338 | 99.7 | ||
3473445 | AT | – | – | A | Asp76fs | GntR family transcriptional regulator | 183 | 84.2 | ||
4147900 | A | – | – | G | SYN | hypothetical protein | 270 | 98.9 |
Wet lab confirmation of variant by sequenced PCR products using target specific primers.
Fold coverage of Illumina sequences at this position.
Frequency of the variant, presented as a percentage for comparison.
R20291 Reference | CD26A54_R & S | Gene | Predicted product | PCR |
Coverage |
Frequency | ||
Position | Sequence | Variant | AA | name | of variant (%) |
|||
6417 | G | T | Ala118Ser |
|
gyrase A | 457 | 100 | |
9661 | C | CA | Ala71fs |
|
anti-sigma factor protein | 269 | 91.5 | |
120932 | C | A | SYN |
|
DNA directed RNA polymerase subunit A | 411 | 99.8 | |
1116866 | G | T | Ala53Ser | putative membrane protein (pseudogene) | 207 | 100 | ||
1568676 | C | A | Gln138Lys | ruberythrin | 486 | 100 | ||
1876233 | A | C | Val390Gly | putative arsenical pump membrane protein | 288 | 100 | ||
2134201 | A | C | Glu70Ala | lipoprotein | 227 | 100 | ||
2297662 | T | C | Glu110Gly | putative lipid kinase | 259 | 99.2 | ||
2297977 | A | T | Leu5Stp | conserved hypothetical protein | 300 | 100 | ||
2531178 | A | G | SYN | PTS system, EIIc component | 323 | 100 | ||
2743315 | TA | T | Phe255fs | ATP-dependent helicase | 179 | 92.7 | ||
2840701 | C | A | Ser414Ile | membrane-associated 5′-nucleotidase | 286 | 100 | ||
2947240 | G | T | Ala217Glu | hypothetical protein | 355 | 100 | ||
2955330 | A | G | SYN |
|
glycerol-3-phosphate dehydrogenase | 546 | 100 | |
2976764 | G | A | Thr30Ile |
|
UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase | 250 | 100 | |
3304975 | C | T | Glu72Lys |
|
V-type ATP synthase subunit C | 88 | 100 | |
3437506 | T | G | SYN | PTS system transporter subunit IIC | 274 | 100 | ||
3489680 | C | A | Met193Ile | PTS system transporter subunit IIBC | 508 | 100 | ||
3531583 | C | T | Val389Ile | PTS system transporter subunit IIABC | 245 | 100 | ||
3814691 | TC | T | Glu136fs | putative nitroreductase | Y | 184 | 100 | |
3815543 | TG | T | Trp99fs | transcriptional regulator | 373 | 91.7 | ||
3832901 | T | C | SYN | RNA methyltransferase | 370 | 99.2 | ||
3913225 | A | C | SYN | transposase | 30 | 100 |
Wet lab confirmation of variant by sequenced PCR products using target specific primers.
Fold coverage of Illumina sequences at this position.
Frequency of the variant, presented as a percentage for comparison.
Distribution of all detected variants (within CDS and intergenic) relative to R20291 are illustrated in
The total SNP and indel variants for the CD26A54_R (red diamonds) and the CD26A54_S (green diamonds) are plotted along the R20291 reference genome. The reference sequence tracks include CDS (black bars), GC content (pink peaks) and GC skew (orange peaks).
We have presented four phenotypes associated with the stable, metronidazole-resistant CD26A54_R subpopulation: (i) aberrant growth in liquid media, (ii) attenuated cell wall separation (iii) lack of spore production by 48 hours and (iv) heteroresistance or more accurately, a slower growing subpopulation that increases the metronidazole MIC. By standard diagnostic methods CD26A54_S is considered susceptible to metronidazole, however this strain shares some characteristics with its resistant counterpart, specifically attenuated cell separation and heteroresistance against metronidazole thus also distinguishing it from the wild type VLOO13. We will discuss how whole genome sequence data may explain these phenotypes however further investigation is needed to understand the role of these changes in metronidazole resistance and how this impacts pathogenicity.
In BHI broth, CD26A54_R appeared to have a prolonged lag phase followed by reduced cell density, particularly during the late-log to early stationary phases. Even at 24 hours post-inoculation, CD26A54_R never reached the same cell density as either CD26A54_S or VLOO13. This may suggest CD26A54_R encountered a nutrient limitation in the media that was not exogenously required by the other strains, enabling the other two to reach a higher cell density. Genetically, there are two variants that may have contributed to the aberrant growth observed, namely the frameshift mutation in the
Deficiencies leading to disruptions in electron transport have been previously attributed to small colony variants (SCV) in other bacterial genera. Some general characteristics of SCV include smaller colonies on agar, slower growth, decreased respiration, attenuated cell separation and antibiotic resistance; additionally, they are often associated with persistent or recurrent infections
Resistance to additional antibiotics was tested to assess possible disruptions in metabolic pathways such as protein or cell wall biosynthesis that could contribute to altered cell morphology and aberrant growth however the MIC values of CD26A54_R were similar to one or both of the metronidazole-susceptible strains. A high prevalence of moxifloxacin and clindamycin resistance among ribotype 027 strains has been reported previously (97.5% and 47.5% respectively), although no observations of metronidazole resistance or altered cell morphology were reported
In addition to the phenotypic and genotypic changes described above, we identified genetic mutations similar to those of other bacterial genera which have been shown to confer metronidazole resistance. The CD26A54_R strain possesses a point mutation in the
As discussed above, there are numerous possible mechanisms to explain the metronidazole resistance observed in the current study. In the literature there are multiple mechanisms for metronidazole resistance described within a species suggesting resistance can occur via different pathways and can be a multifactorial process. In the present study we presented phenotypic observations and genomic analyses of a stable, metronidazole resistant isolate of
We would like to acknowledge the National Science and Engineering Research Council of Canada's Visiting Fellowship in Canadian Government Laboratories Program; the Genomics, Bioinformatics and Media Core Facilities at the National Microbiology Laboratory for their expertise, hard work and technical support. Members of the Canadian Nosocomial Infection Surveillance Program are: David Boyd, National Microbiology Laboratory, Public Health Agency of Canada; Natalie Bridger, Eastern Health-HSC, St. John's, Nfld.; Elizabeth Bryce, Vancouver General Hospital, Vancouver, BC; John Conly, Foothills Medical Centre, Calgary, Alta.; André Dascal, SMBD-Jewish General Hospital, Montreal, Que.; Janice de Heer, Interior Health Authority, Kelowna, BC; John Embil, Health Sciences Centre, Winnipeg, Man.; Joanne Embree, Health Sciences Centre, Winnipeg, Man.; Gerald Evans, Kingston General Hospital, Kingston, Ont; Sarah Forgie, Stollery Children's Hospital, Edmonton, AB; Charles Frenette, McGill University Health Centre, Montreal, Que; David Haldane, Queen Elizabeth II Health Sciences Centre, Halifax, Nova ScotiaGregory German, Queen Elizabeth Hospital, Charlottetown, PEI; George Golding, National Microbiology Laboratory, Public Health Agency of Canada; Denise Gravel, Centre for Communicable Diseases and Infection Control, Public Health Agency of Canada; Deanna Hembroff, University Hospital of Northern BC, Prince George, BC; Elizabeth Henderson, Alberta Health Services, Calgary, Alta.; Michael John, London Health Sciences Centre, London, Ont.; Lynn Johnston, Queen Elizabeth II Health Sciences Centre, Halifax, NS; Kevin Katz, North York General Hospital, Toronto, ON; Pamela Kibsey, Victoria General Hospital, Victoria, BC; Magdalena Kuhn, South East Regional Health Authority, Moncton, NB; Joanne Langley, IWK.Health Centre, Halifax, NS; Camille Lemieux, University Health Network, Toronto, Ont.; Nicole Le Saux, Children's Hospital of Eastern Ontario, Ottawa, ON; Mark Loeb, Hamilton Health Sciences Corporation, Hamilton, Ont.; Susan Richardson, Hospital for Sick Children, Toronto, Ont.; Allison McGeer, Mount Sinai Hospital, Toronto, Ont.; Dominik Mertz, Hamilton Health Sciences Corporation, Hamilton, Ont; Mark Miller, SMBD-Jewish General Hospital, Montreal, Que.; Robyn Mitchell, Centre for Communicable Diseases and Infection Control, Public Health Agency of Canada; Dorothy Moore, Montreal Children's Hospital, McGill University Health Centre, Montreal, Que.; Aboubakar Mounchili, Centre for Communicable Diseases and Infection Control, Public Health Agency of Canada; Michael Mulvey, National Microbiology Laboratory, Public Health Agency of Canada; Suzanne Pelletier, Health Sciences North, Sudbury, ON; Linda Pelude, Centre for Communicable Diseases and Infection Control, Public Health Agency of Canada; Caroline Quach, Montreal Children's Hospital, McGill University Health Centre, Montreal, Quebec Virginia Roth, The Ottawa Hospital, Ottawa, Ont.