Reiner Schaumann

Challenging the problem of clostridial identification with matrix-assisted laser desorption and ionization–time-of-flight mass spectrometry (MALDI–TOF MS)

Diverse techniques were applied to effect the identification and classification of isolated clostridial strains. Nevertheless, the correct identification of clostridial strains remains a laborious, time-consuming task which entails a not inconsiderable degree of expertise. In addition to this, traditional methods based on the metabolic properties of the bacteria require rigorously standardized media and growth conditions to assure the attainment of reproducible results. Although DNA-based methods, like the PCR of a species specific gene, are known to yield precise and reproducible results, their degree of effectivity is circumscribed by the fact that even the incidence of a toxin encoding gene is not necessarily linked to nor consequently indicative of the presence of an infectious disease. Moreover, most of these methods postulate an initial assumption concerning the expected bacterial species involved before the choice of PCR primer for use can be made. Consequently, the scope of these methods is restricted to that of targeted analyses. The 16S rDNA sequencing which is assumed to be the gold standard for bacterial classification having the unequivocal advantage of being capable of determining even uncultivable bacteria is nonetheless a time-consuming and costly technique. In the present study we describe the utilization of matrix-assisted laser desorption and ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for whole cell fingerprinting in combination with a dedicated bioinformatic software tool to distinguish between various clostridial species. Total 64 clostridial strains of 31 different species each displayed a mass spectrum unique to the strain involved, to the effect that it was also possible not only to differentiate between the strains examined, but also to establish to which species the individual strains belonged to. Starting with a single colony it was possible to correctly identify a Clostridium species within minutes. It was even possible to identify species which are normally difficult to differentiate by traditional methods, such as C. chauvoei and C. septicum. With the results obtained we were able to assemble a dendrogram of the Clostridium species which showed considerable similarities to dendrograms based upon 16S rDNA sequencing data. To conclude, our findings indicate that, inasmuch as the MALDI-TOF MS technology employed is based on a high-quality reference database, it may serve as an effective tool for the swift and reliable identification and classification of Clostridia.

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.

Periodontitis is associated with a loss of colonization by Streptococcus sanguinis

The aim of this study was to estimate differences in the prevalence of oral streptococcal species in the subgingival biofilm of patients with aggressive periodontitis and of healthy controls. Thirty-three patients with clinical and radiological proof of aggressive periodontitis and 20 healthy subjects were enrolled in this study. Clinical indices were recorded in a six-point measurement per tooth. Samples of the subgingival biofilm were taken with paper points from four teeth of each individual. Alpha- and non-haemolytic, small and catalase-negative colonies were biochemically identified using a rapid ID 32 STREP system and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A total of 118 strains of oral streptococci (11 species) were identified and Streptococcus sanguinis was found significantly more often in healthy subjects (P=0.001). Conversely, the absence of S. sanguinis was associated with high values of clinical indices (P=0.001-0.002). Aggressive periodontitis seems to be associated with a loss of colonization of S. sanguinis. Whether or not S. sanguinis offers protection against aggressive periodontitis needs to be determined. Otherwise, there were no significant differences in the distribution of oral streptococcal species in patients and healthy subjects.

Rapid identification of oral anaerobic bacteria cultivated from subgingival biofilm by MALDI-TOF-MS

INTRODUCTION To facilitate the identification of anaerobes cultivated from periodontal disease, whole cell bacterial identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was evaluated. METHODS A total of 84 strains (nine reference strains and 75 recent clinical isolates from 33 patients with aggressive periodontitis) previously identified with phenotypic methods were used. All the references and 10 clinical isolates belonging to the same species as the reference strains were genotypically identified by sequence analysis of the 16S ribosomal RNA gene. All the strains were then analyzed using MALDI-TOF-MS. RESULTS The reference strains of anaerobic bacteria used showed characteristic MALDI-TOF-MS spectra with peaks between m/z 2000 and up to about m/z 13,000. On visual inspection, the similarity of spectra produced by strains of a single genus could be recognized. Obvious differences between spectra produced by strains of different species were also easily noticed. The reproducibility of the method was proved by the similarity of spectra belonging to the same species. The spectra of the Prevotella intermedia strains identified with MALDI clustered together and clustered separately from the spectra of Prevotella nigrescens, proving that MALDI-TOF-MS is an accurate method that is capable of separating these two species. The quality of clustering was characterized by calculating an inconsistency coefficient (Mathworks:/Matlab Reference Manual v2007a/, Statistical toolbox). CONCLUSION Our results suggest that MALDI-TOF-MS might become a useful method for the identification of anaerobic bacteria, especially for those that cannot be readily identified by biochemical analysis. It may become an attractive system even for the routine identification of clinical isolates.

Evaluation of three rapid assays for detection of Clostridium difficile toxin A and toxin B in stool specimens.

Diagnosis of Clostridium difficile-associated disease continues to be difficult for clinical microbiology laboratories. The aim of this study was to evaluate the performance of three enzyme immunoassays for detection of C. difficile toxins A and B: the recently marketed rapid enzyme immunoassay Ridascreen Clostridium difficile Toxin A/B (R-Biopharm, Darmstadt, Germany) and two established enzyme immunoassays, the C. difficile Tox A/B II Assay (TechLab, Blacksburg, VA, USA) and the ProSpecT C. difficile Toxin A/B Microplate Assay (Remel, Lenexa, KS, USA). Stool specimens (n = 383) from patients with a clinical diagnosis of antibiotic-associated diarrhea were examined by these three enzyme immunoassays and were additionally cultured for C. difficile on selective agar. Samples giving discordant enzyme immunoassay results underwent confirmatory testing by tissue culture cytotoxin B assay and by PCR for toxin A (tcdA) and toxin B (tcdB) genes from C. difficile. Using the criteria adopted for this study, 60 (15.7%) samples tested positive for toxins A and/or B. Sensitivity and specificity of the enzyme immunoassays were, respectively, 88.3 and 100% for the TechLab enzyme immunoassay, 91.7 and 100% for the R-Biopharm enzyme immunoassay, and 93.3 and 100% for the Remel enzyme immunoassay. The differences between these results are statistically not significant (p > 0.05). The results show that all three enzyme immunoassays are acceptable tests for the detection of C. difficile toxins A and B directly in fecal specimens or in toxigenic cultures.

Infections Caused by Stenotrophomonas maltophilia– A Prospective Study

AbstractBackground:Stenotrophomonas maltophilia is an opportunistic microorganism, often highly resistant to routinely tested antibiotics. This microorganism is isolated in specimens from patients with nosocomial infections with increasing frequency. Patients and Methods: During a 1-year period (1998/1999) S. maltophilia was isolated from 137 specimens (0.26% of all investigated specimens) from 80 patients who were treated in a 1,500 bed major tertiary care teaching hospital in Leipzig. The data of 76 patients (133 specimens) could be collected and analyzed completely. Results: The pathogen was most frequently detected in specimens from the respiratory tract (54%). In five patients (six cases) S. maltophilia was isolated from blood cultures (0.3% of all positive blood cultures; 1.4% of all gram-negative isolates from blood cultures). 70 of the infected patients were inpatients and 32 (42%) of them were treated on the internal medicine wards. Of these 32 patients only six (19%) were pretreated with imipenem. The length of stay at the hospital resulted in an independent increased risk of infection with S. maltophilia. In addition, this organism was detected in six infected outpatients. Conclusion:S. maltophilia is not only a nosocomial pathogen. Pretreatment with a carbapemnem is no longer an unequivocal risk factor for an infection with S. maltophilia.

A step towards the discrimination of beta-lactamase-producing clinical isolates of Enterobacteriaceae and Pseudomonas aeruginosa by MALDI-TOF mass spectrometry.

Summary Background Matrix-Assisted Laser-Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) has already proven to be a powerful tool for species identification in microbiological laboratories. As adequate and rapid screening methods for antibiotic resistance are crucially needed, the present study investigated the discrimination potential of MALDI-TOF MS among extended-spectrum-beta-lactamase (ESBL) or metallo-beta-lactamases- (MBL) producing and the nonproducing strains of Escherichia coli (n=19), Klebsiella pneumoniae (n=19), and Pseudomonas aeruginosa (n=38), respectively. Material/Methods We used a MALDI-TOF MS protocol, usually applied for species identification, in order to integrate a screening method for beta-lactamases into the routine species identification workflow. The acquired spectra were analyzed by visual inspection, statistical similarity analysis and support vector machine (SVM) classification algorithms. Results Neither visual inspection nor mathematical similarity analysis allowed discrimination between spectra of beta-lactamase-producing and the nonproducing strains, but classification within a species by SVM-based algorithms could achieve a correct classification rate of up to 70%. Conclusions This shows that MALDI-TOF MS has definite potential to discriminate antibiotic-resistant strains due to ESBL and MBL production from nonproducing strains, but this performance is not yet sufficiently reliable for routine microbiological diagnostics.

Bacteroides fragilis group : Trends in resistance

Representing the major part of the human colon microflora, members of the Bacteroides fragilis group are frequently involved in mixed aerobic and anaerobic infections. Recent studies show an increased resistance of the B. fragilis group against several antimicrobial agents. The aim of the present study was to determine the susceptibility of 87 B. fragilis group strains isolated in 2003/2004 in Western Austria against eight antimicrobial agents by Etest®. Furthermore, the resistance patterns were compared with those of 45 B. fragilis group strains isolated in 1992 and referred to the world wide trend towards increased resistance. In 1992 as well as in 2003/2004, all strains were susceptible against metronidazole and imipenem. However, comparing the MIC-values of the B. fragilis group strains collected 1992 with data from 2003/2004, a significant increase in resistance was found for clindamycin (p < 0.01). Regarding cefoxitin, a similar trend could be observed. However, this difference was not yet significant (p = 0.144). Our findings underline the emerging resistance of the B. fragilis group against antimicrobial agents and underscore the importance of susceptibility testing of anaerobes even in routine laboratories.

Inducible Metronidazole Resistance in nim–Positive and nim–Negative Bacteroides fragilis Group Strains after Several Passages Metronidazole Containing Columbia Agar Plates

Background:Recent data show an emergence of resistance in the Bacteroides fragilis group against several antimicrobial agents and inducible resistance against metronidazole in nim–positive strains. The aim of the present study was to investigate inducible metronidazole resistance in nim–positive as well as in nim–negative B. fragilis group strains.Materials and Methods:Of 18 B. fragilis strains (including four nim–positive reference strains and one ATCC strain), two Bacteroides ovatus strains, and one Bacteroides thetaiotaomicron DSM strain minimum inhibitory concentration (MIC) values for metronidazole were determined by Etest® and analyzed for nim genes (nimA to –G) by PCR. For this purpose bacterial suspensions were incubated on supplemented Columbia agar plates containing metronidazole at twice the MIC value of the specific strain and incubated under anaerobic conditions for 48 hours. After incubation, growing bacteria were harvested and thereafter incubated at four times the original MIC. This procedure was repeated with increasing antibiotic concentrations. The resulting MIC values were confirmed by Etest®.Results:The MIC values for metronidazole of the four nim–positive reference strains ranged from 3 to 8 mg/l. The B. fragilis ATCC 25285 strain and the B. thetaiotaomicron DSM 2255 strain were nim negative with MIC values of 0.19 mg/l and 0.75 mg/l, respectively. Three clinical isolates of B. fragilis strains showed MIC values of > 256 mg/l. In all three strains, nim genes were detected by PCR. The other clinical isolates were nim negative. In these strains, MIC values ranged from 0.19 to 0.75 mg/l. After several passages on metronidazole containing agar, all B. fragilis group strains exhibited MIC values of > 256 mg/l determined by Etest®.Conclusion:Metronidazole resistance can be selected not only in nim–positive strains but also in nim–negative strains, suggesting that mechanisms other than nim genes are involved. These findings and the emerging resistance of the B. fragilis group against several antimicrobial agents underscore the importance of susceptibility testing of anaerobes even in routine laboratories.

Comparative In Vitro Activities of Gemifloxacin, Other Quinolones, and Nonquinolone Antimicrobials against Obligately Anaerobic Bacteria

ABSTRACT The in vitro activity of gemifloxacin was compared to that of other quinolone and nonquinolone antimicrobials against 204 anaerobes by the agar dilution technique. The data indicate that gemifloxacin has a rather selective anaerobic activity. Most Peptostreptococcus, Porphyromonas, and Fusobacterium species are susceptible, while gemifloxacins activity against other gram-negative anaerobes appears to be variable.