Genotypic Diversity of Anaerobic Isolates from Bloodstream Infections

Accurate species determination for anaerobes from blood culturebottles has become increasingly important with the reemergenceof anaerobic bacteremia and prevalence of multiple-drug-resistantmicroorganisms. Our knowledge of the taxonomical diversity ofanaerobes that cause bloodstream infections is extremely limited,because identification historically has relied on conventionalmethods. Over a 5-year period, we profiled anaerobic bacteremiaat a large tertiary care hospital with 16S rRNA gene sequencingto gain a better understanding of the taxonomical diversityof the bacteria. Of 316 isolates, 16S rRNA gene sequencing andphylogenetic analysis identified 316 (100%) to the genus ortaxonomical group level and 289 (91%) to the species level.Conventional methods identified 279 (88%) to the genus leveland 208 (66%) to the species level; 75 (24%) were misidentifiedat the species level, and 33 (10%) results were inconclusive.High intragenus variability was observed for Bacteroides andClostridium species, and high intraspecies variability was observedfor Bacteroides thetaiotaomicron and Fusobacterium nucleatum.Sequence-based identification has potential benefits in comparisonto conventional methods, because it more accurately characterizesanaerobes within taxonomically related clusters and therebymay enable better correlation with specific clinical syndromesand antibiotic resistance patterns.

INTRODUCTION

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INTRODUCTION

Anaerobic microorganisms remain an important cause of bloodstreaminfections and account for 1 to 17% of positive blood culturesin the United States (3, 4, 8, 9, 11). The most commonly isolatedpathogens are the Bacteroides fragilis group, other speciesof Bacteroides, Peptostreptococcus species, and Clostridiumspecies (9). Most laboratories rely on conventional methodsfor identification of these common microorganisms and use algorithmsbased on key differential biochemical tests (16). However, DNAtarget sequencing has emerged as an attractive alternative,because identification is faster, more accurate, and independentof a microorganism's growth characteristics (10, 15, 17, 18).Sequence-based identification has enhanced our knowledge aboutthe taxonomical diversity among anaerobic bacteria and has affordedthe opportunity to better define the epidemiology of anaerobe-associateddiseases. For example, some anaerobes have been associated withspecific clinical syndromes, such as Clostridium sordellii withabortion (1), Clostridium tertium with neutropenia (12), andFusobacterium necrophorum with hypercoagulability (5). Additionally,national surveys have demonstrated increasing antimicrobialresistance for several anaerobic pathogens (2, 6, 14), and definitivespecies identification can be extremely useful for guiding selectionof empirical antibacterial therapy.

Our knowledge of the taxonomical diversity of anaerobes associatedwith clinically important bloodstream infections is limited.Most series of anaerobic bacteremia have been based on conventionalmethods of identification (3, 8, 9, 11), with little or no attentionto the genetic diversity within and among genera. Similarly,a systematic approach for identifying the emergence of potentiallynovel or unusual sequence variants of anaerobes that cause bloodstreaminfection has not been applied over a 5-year period. We retrospectivelystudied all anaerobic microorganisms that were recovered fromblood cultures at a large, tertiary care hospital. Our aim wasto define the spectrum of anaerobes causing bloodstream infectionsby 16S rRNA gene sequencing and to identify unusual speciesbelonging to taxonomically related groups. Aware that assessingtaxonomical diversity relies on representative sequence databases,we specifically used two reference databases and phylogeneticanalyses to assess intraspecies, intragenus, and intergenusvariability.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Anaerobic microorganisms recovered from blood cultures betweenJanuary 2000 and December 2004 at Duke University Hospital,Durham, NC, that were deemed clinically significant (19) wereretrospectively identified. During the study period, all threemajor blood culture systems were used: Bactec 9240 (BD Diagnostics,Sparks, MD), BacT/ALERT (classic and 3D) (bioMérieux,Inc., Durham, NC), and VersaTREK (Trek Diagnostic Systems, Cleveland,OH). Duke University Hospital is a large, 924-bed tertiary andquarternary care facility. Phenotypic identifications were performedby standard laboratory protocols that included a combinationof manual biochemical testing, use of the API 20A system (bioMérieux,Marcy l'Etoile, France), and/or use of the Sherlock microbialidentification system (MIDI, Inc., Newark, DE).

16S rRNA gene sequencing. Bacterial DNA was extracted directly from frozen glycerol preparationsof bacteria. The tube contents were thawed for 30 min at roomtemperature, and 50 µl of stock was removed and placedinto molecular-grade water to a final volume of 200 µl.DNA was extracted with the QIAmp DNA minikit (Qiagen, Hilden,Germany) according to the manufacturer's instructions. PCRswere performed in a 20-µl volume containing 1x Taq buffer;0.25 U of TaKaRa Taq; 3.0 mM MgCl₂ (Takara Bio, Inc., Shiga,Japan); 200 µM each dATP, dGTP, and dCTP; 600 µMdUTP (Roche Diagnostics Corporation, Alameda, CA); 0.2 µMof each primer; and 2 µl of template. The primers usedfor amplification were 5F (5'-TTGGAGAGTTTGATCCTGGCTC-3') and1194R (5'-ACGTCATCCCCACCTTCCTC-3'). PCR mixtures were amplifiedby initial holding at 94°C for 5 min and then 30 cyclesof denaturing at 94°C for 30 seconds, annealing at 60°Cfor 30 seconds, and extension at 72°C for 2 min. The reactionended with a final extension at 72°C for 2 min and a holdat 4°C. The presence and sizes of amplicons were confirmedby gel electrophoresis. PCR products were purified with ExoSAP-ITreagent (USB Corporation, Cleveland, OH) per the manufacturer'sinstructions. PCR products were bidirectionally sequenced withthe original amplification primer, 5F, and a reverse primer,810R (5'GGCGTGGACTTCCAGGGTATCT-3'). Sequencing reactions wereperformed with Big Dye terminator reagents on an ABI Prism 310or 3730xl instrument (Applied Biosystems, Foster City, CA) bya standard automated sequencer protocol.

Sequence and phylogenetic analyses. Sequences were analyzed with MicroSeq ID software v2.0 (AppliedBiosystems). Sequence-based identifications were determinedindividually with the MicroSeq 16S rDNA 500 Library v2.0 andSmartGene IDNS-Bacteria (version 3.2.3r8) databases (SmartGene,Inc., Raleigh, NC). SmartGene is a web-based application forsequence comparison with a reference database based on GenBanksequences. Alignments and phylogenetic trees were constructedas previously described (13). Final genus- and species-levelidentifications were assigned by both phylogenetic analysisand the following general guidelines: $≥$ 99% identity to a referenceentry identified a microorganism to the species level, 95.0to 98.9% identity identified a microorganism to the genus level,and microorganisms with <95% identity to any reference sequencewere considered unable to be identified definitively. Multiplespecies were assigned to isolates when top matches were between99.0 and 99.9%. When five or more clinical isolates were identifiedwithin a group or species, interspecies or intergroup variabilitywas determined by measuring the percent identity and recordingthe value as the percent difference.

RESULTS

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RESULTS

The profile of anaerobes causing clinically significant bacteremiaover the 5-year period is delineated in Table 1.

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TABLE 1. Anaerobic bacteremia at Duke University Medical Center, January 2000 to December 2004

Sequence-based identification. Of 316 isolates, 16S rRNA gene sequencing with phylogeneticanalysis identified 316 (100%) to the genus or taxonomical grouplevel and 289 (91%) to the species level. Of those identifiedto the species level, two (0.6%) could not be resolved by sequencinganalysis and were assigned to multiple species, Clostridiumsporogenes/Clostridium botulinum and Clostridium aerotolerans/Clostridiumxylanolyticum.

Identification by conventional methods. For 316 isolates, conventional methods identified 279 (88%)to the genus level and 208 (66%) to the species level; 75 (24%)were misidentified at the species level, and 33 (10%) of resultswere inconclusive (Tables 2 and 3).

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TABLE 2. Level of identification for each method (n = 316 isolates)

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TABLE 3. Representative misclassifications by conventional methods

Analysis of microbial diversity. Figures 1 and 2 show radial dendrograms illustrating the geneticdiversity of clinical isolates causing bacteremia. Since Clostridiumand Bacteroides were the most represented groups, it is notsurprising that these genera had the highest intragenus variability,i.e., 27.5% and 15.4%, respectively. Intraspecies variabilityfor the most common species of Clostridium was low: 0.7% forClostridium perfringens, 0.2% for Clostridium tertium, and 0.0%for Clostridium ramosum. Intraspecies variabilities for B. fragilis,Bacteroides thetaiotaomicron and its relatedness group, andBacteroides ovatus were 3.3%, 4.0%, and 0.8%, respectively.Intraspecies variabilities of Eggerthella lenta and Fusobacteriumnucleatum were 1.7% and 2.7%, respectively. We observed unusualsequence variants that grouped within taxonomical clusters ofAnaerococcus sp., Bacteroides thetaiotaomicron, Parabacteroidesdistasonis, Bacteroides intestinalis, Clostridium subterminale,Eggerthella lenta, and Peptoniphilus sp. For example, we foundthree isolates with sequences that were distinctly differentfrom reference sequences of Eggerthella lenta and six sequencevariants for Bacteroides thetaiotaomicron (Fig. 1 and 2).

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FIG. 1. Neighbor-joining radial dendrogram for all gram-positive anaerobes and a cluster of gram-negative anaerobes. Only unique sequences are illustrated, with numbers of isolates for each unique sequence in parentheses.

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FIG. 2. Neighbor-joining radial dendrogram for gram-negative anaerobes not shown in Fig. 1. Only unique sequences are illustrated, with numbers of isolates for each unique sequence in parentheses.

DISCUSSION

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DISCUSSION

Accurate species determination for anaerobes from blood cultureshas become increasingly important, because anaerobic bacteremiawith multiple-drug-resistant organisms has emerged as a significanthealth care problem as there are more patients at risk fromimmunosuppression and multiple comorbidities (6-9). To our knowledge,this study is the first longitudinal survey of anaerobic bacteremiaat a large tertiary care hospital that identified anaerobesby 16S rRNA gene sequencing. We corroborate previous observationsthat the most common anaerobes that cause bloodstream infection,in decreasing order of frequency, are Bacteroides fragilis,other Bacteroides species, Clostridium species, anaerobic gram-positivecocci, Fusobacterium nucleatum, and Prevotella spp. Unlike priorreports that were limited by conventional methods, we observedwith sequence-based identification a significant proportionof bloodstream infections from less common members of the Bacteroidesand Clostridium taxonomical groups. We also document the firstcases of anaerobic bacteremia from Bacteroides dorei, Bacteroidesfinegoldii, Parabacteroides merdae, Clostridium argentinense,Clostridium celerecrescens, Clostridium colicanis, Ruminococcusgnavus, and Tissierella praeacuta. Conventional identificationmisclassified or inconclusively identified approximately 25%of isolates, thereby missing a potential opportunity to definethe epidemiology of or susceptibility patterns for these clinicallysignificant anaerobic bloodstream infections. Of importance,conventional methods misclassified the Gram reaction and generafor several isolates and misidentified Parabacteroides distasonis,Bacteroides caccae, and Bacteroides vulgatus, three speciesknown to have resistance to multiple antibacterials (14). Clinicaldecision-making based on erroneous conventional identificationscould adversely affect patient care if a suboptimal empiricalantibacterial regimen was selected or if misidentification beliedthe underlying source of infection.

We acknowledge that many laboratories cannot routinely employpartial 16S rRNA gene sequencing for anaerobic identificationdue to a lack of technical expertise and to cost. However, overthe past several years, various commercial platforms and referencedatabases have become available for DNA target sequencing, enablingless experienced, nonmolecular bench technologists to determineand analyze DNA sequences. Laboratories should develop algorithmsto screen for those isolates that can be adequately identifiedby conventional methods and should refer only a subset of isolatesfor 16S rRNA gene sequencing. Additionally, implementation ofDNA target sequencing reduces the need for highly experiencedpersonnel, a well-documented diminishing resource, and can resultin a labor savings of least one full-time equivalent certifiedmedical technologist (13).

Sequence data are a more valuable tool than identification byconventional methods, because they are objective and can beeasily exchanged between different laboratories for comparison.Sequence-based identification enables us to appreciate the degreeof heterogeneity within taxa, which can be represented by eitherhigh intraspecies variability or unusual sequence variants withintaxonomically related clusters. The clinical relevance of reclassifyingunusual sequence variants as new species cannot be reliablydetermined with a single institutional data set. Additionally,phylogeny may vary by the type of DNA target sequenced, withsequences potentially clustering into different groups using16S rRNA, rpoB, or tuf targets. We propose that investigatorsmaintain viable culture collections of unusual anaerobes anddeposit their sequences into public databases, but we cautionagainst the impulse to describe them as unique species. A consensushas not been reached within the microbiology community aboutdrawing finer distinctions between species in a meaningful way,and the concept of species has not been clearly delineated.Instead, we recommend that investigators deposit unusual sequencesas "variants within taxonomical relatedness groups," affordingthe opportunity to carefully evaluate their taxonomical andclinical significance longitudinally and then determine theneed for unique species designations. Improved disease surveillanceusing DNA target sequencing will provide us with the abilityto correlate certain anaerobes with specific clinical syndromesand better understand the development of antibiotic resistancewithin individual taxonomical groups.

FOOTNOTES

* Corresponding author. Mailing address: ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108. Phone: (801) 583-2787. Fax: (801) 584-5207. E-mail: cathy.petti{at}aruplab.com

Published ahead of print on 5 March 2008.

REFERENCES

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