Marina C. Claros

16S-23S rDNA internal transcribed spacer sequences for analysis of the phylogenetic relationships among species of the genus Fusobacterium.

The 16S-23S rDNA internal transcribed spacer (ITS) regions of all currently defined Fusobacterium species and related taxa such as Leptotrichia buccalis, Sebaldella termitidis and Streptobacillus moniliformans, were analysed to examine inter- and intraspecies as well as subspecies relationships. For the ITS-amplification, a new eubacterial universal primer pair was designed and used. The majority of the Fusobacterium strains, along with L. buccalis showed one major, and two to three weaker, distinct bands (short and long versions) with lengths of 800-830 bp and 1000-1100 bp. Nevertheless, six other patterns were also found within the genus Fusobacterium, demonstrating its heterogeneity. The ITS region was sequenced and found to consist both of conserved motifs, which functioned as a framework for alignment, and of variable sites, which provided high phylogenetic resolution. Analyses of the ITS-DNA sequences and ITS relative length (short version) allowed species and subspecies differentiation in most cases. The results confirmed the strikingly distant relationship between Fusobacterium prausnitzii and the genus Fusobacterium. Fusobacterium nucleatum subspecies, along with Fusobacterium naviforme, Fusobacterium simiae and Fusobacterium periodonticum, formed a cluster with an inherently high potential for diversification. Other clusters were formed by Fusobacterium necrophorum subspecies with Fusobacterium gonidaformans and by Fusobacterium varium with Fusobacterium mortiferum and Fusobacterium ulcerans. Fusobacterium russii as well as Fusobacterium perfoetens formed separate branches. Fusobacterium necrophorum subspp. necrophorum and funduliforme on the one hand, and Fusobacterium varium and Fusobacterium mortiferum on the other, were found to be very similar, even at the high-resolution ITS level.

Comparative Activity of Moxifloxacin in Vitro Against Obligately Anaerobic Bacteria

Abstract The antimicrobial activity of moxifloxacin and seven other antibiotics (four of them quinolones) against 292 strains of obligately anaerobic bacteria was assessed employing a broth microdilution technique performed in Wilkens-Chalgren broth. MIC50/MIC90 values (mg/l) for moxifloxacin were as follows: Bacteroides fragilis (n=62) 0.25/2, Bacteroides ovatus (n=70) 1/4, Bacteroides vulgatus (n=29) 0.25/1, Bacteroides thetaiotaomicron (n=17) 2/2, Bacteroides caccae (n=11) 1/2, Prevotella spp. (n=11) 0.25/2, Fusobacterium spp. (n=17) 1/4, Bilophila wadsworthia (n=29) 0.5/1, and Clostridium spp. (n=29) 0.125/0.5, respectively. MIC50 values (mg/l) for Bacteroides distasonis (n=8) and Peptostreptococcus spp. (n=9) were 0.25. The results indicated that moxifloxacin was almost as active as trovafloxacin, as active as gatifloxacin, and more active than levofloxacin and ciprofloxacin against the anaerobes tested.

Pasteurella multocida subsp.multocida and P. multocida subsp.septica Differentiation by PCR Fingerprinting and α-Glucosidase Activity

ABSTRACT Pasteurella multocida is composed of three subspecies that are often differentiated by fermentation of sorbitol and dulcitol. We studied 35 dulcitol-negative P. multocida isolates from infected dog and cat bite wounds, 16 of which yielded weak and/or conflicting fermentation reactions in Andrades sorbitol, thus making it difficult to distinguish between the two dulcitol-negative subspecies of P. multocida, i.e.,P. multocida subsp. multocida andP. multocida subsp. septica. All isolates and two control strains were further analyzed using a PCR fingerprinting technique with a single primer (M13 core) and assessed for α-glucosidase (α-Glu) activity. Although the PCR fingerprint patterns and α-Glu activity did not correlate well with the sorbitol fermentation reactions, they did correlate well with each other. All strains identified as P. multocida subsp.septica were positive for α-Glu activity and exhibited the group I PCR fingerprint profile. All strains categorized asP. multocida subsp. multocida displayed either the group II or group III PCR fingerprint profile; 9 of 11 of these isolates were α-Glu negative. These data suggest that both PCR fingerprinting and α-Glu activity provide reliable means for differentiating P. multocida subsp.multocida from P. multocida subsp.septica, particularly in strains that produce weak and/or discrepant sorbitol fermentation reactions.

In vitro activities of fourteen antimicrobial agents against obligately anaerobic bacteria

The in vitro activities of fourteen antimicrobial agents were tested against 292 clinical isolates of obligately anaerobic bacteria using the broth microdilution technique. Taking all strains as a group the MIC(50/90) (mg/l) values were metronidazole and imipenem 0.25/1, meropenem 0.25/0.5, trovafloxacin 0.25/1, gatifloxacin and moxifloxacin 0.5/2, levofloxacin 2/16, ciprofloxacin 4/32, clindamycin 0.5/8, amoxycillin/clavulanate 1/4, doxycycline and chloramphenicol 2/4, erythromycin 4/>32 and penicillin G 16/>32.

Use of Polymerase Chain Reaction Fingerprinting to Compare Clinical Isolates of Bacteroides fragilis and Bacteroides thetaiotaomicron from Germany and the United States

Accurate identification of Bacteroides species is often problematic. Therefore, we used a polymerase chain reaction (PCR) fingerprinting technique with either a single nonspecific primer derived from tDNA intergenic spacer or a single primer that anneals to mini- and microsatellite DNA sequences to compare 34 clinical isolates of B. fragilis and 21 clinical isolates of B. thetaiotaomicron from Southern California with 32 clinical isolates of B. fragilis and 10 isolates of B. thetaiotaomicron from Germany. All German B. fragilis isolates (32 of 32) formed one PCR fingerprint group that matched the PCR profile of the B, fragilis reference strain ATCC (American Type Culture Collection) 25285, representative of DNA homology group I. In contrast, the isolates from Southern California formed two PCR fingerprint groups. Although most of these strains (29 of 34) also matched B. fragilis ATCC 25285, some strains (4 of 34) matched the DNA homology group II reference strain VPI (Virginia Polytechnic Institute) 2393. One of the 34 strains showed a unique profile. German B. thetaiotaomicron strains (10 of 10) formed one PCR fingerprint group, matching the reference strain B. thetaiotaomicron ATCC 29742, whereas the B. thetaiotaomicron isolates from Southern California showed heterogenous profiles.

Oral and Intestinal Bacteroidetes

Bacteroidetes are a phylum of bacteria including the class Bacteroides which consists of the genera Bacteroides, Porphyromonas, and Prevotella. Whereas the genus Bacteroides is discussed and subjected with infections originating from the intestinal tract, Porphyromonas is clearly more associated with oral or vaginal infections. With both genera (and related such as Bilophila or Prevotella) different methods are described and discussed exemplarily. Critical to this development, however, is a proper understanding and application of the methodologies and knowledge of their limitations. In this chapter, molecular tools based on ITS (Internal Transcribed Spacer) amplification and sequencing as well as PCR fingerprint techniques are described along with examples showing ways to analyze the datasets. Both methods allow the identification of almost any given bacterial species or strain in pure culture or even directly in clinical samples in a sensitive and reproducible way. This chapter is complemented by discussing potential pitfalls that should be taken into consideration for producing proper results along with referring the reader to pertinent literature that will allow an individual deepening into the concept of molecular-typing in clinical bacteriology.