Identification Of Anaerobic Bacteria Research Articles

Rapid, Sensitive, and Specific Detection of Clostridium tetani by Loop-Mediated Isothermal Amplification Assay

Tetanus is a specific infectious disease, which is often associated with catastrophic events such as earthquakes, traumas, and war wounds. The obligate anaerobe Clostridium tetani is the pathogen that causes tetanus. Once the infection of tetanus progresses to an advanced stage within the wounds of limbs, the rates of amputation and mortality increase manifold. Therefore, it is necessary to devise a rapid and sensitive point-of-care detection method for C. tetani so as to ensure an early diagnosis and clinical treatment of tetanus. In this study, we developed a detection method for C. tetani using loop-mediated isothermal amplification (LAMP) assay, wherein the C. tetani tetanus toxin gene was used as the target gene. The method was highly specific and sensitive, with a detection limit of 10 colony forming units (CFU)/ml, and allowed quantitative analysis. While detecting C. tetani in clinical samples, it was found that the LAMP results completely agreed with those of the traditional API 20A anaerobic bacteria identification test. As compared with the traditional API test and PCR assay, LAMP detection of C. tetani is simple and rapid, and the results can be identified through naked-eye observation. Therefore, it is an ideal and rapid point-of-care testing method for tetanus.

Identification of Anaerobic Bacteria by Bruker Biotyper Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry with On-Plate Formic Acid Preparation

Identification of anaerobic bacteria using phenotypic methods is often time-consuming; methods such as 16S rRNA gene sequencing are costly and may not be readily available. We evaluated 253 clinical isolates of anaerobic bacteria using the Bruker MALDI Biotyper (Bruker Daltonics, Billerica, MA) matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system with a user-supplemented database and an on-plate formic acid-based preparation method and compared results to those of conventional identification using biochemical testing or 16S rRNA gene sequencing. A total of 179 (70.8%) and 232 (91.7%) isolates were correctly identified to the species and genus levels, respectively, using manufacturer-recommended score cutoffs. MALDI-TOF MS offers a rapid, inexpensive method for identification of anaerobic bacteria.

A comparison of Api 20A vs MALDI-TOF MS for routine identification of clinically significant anaerobic bacterial strains to the species level

Adequate identification of anaerobic bacteria still presents a challenge for laboratories conducting microbiological diagnostics. The aim of this study was to compare the use of Api 20A and MALDI-TOF MS techniques for identification of obligate anaerobes. The results indicate that MALDI-TOF MS ensures a rapid and accurate identification of the species isolated from patients.

Eficacia de la espectrometría de masas MALDI-TOF en la identificación de bacterias anaerobias

Aim of the studyMALDI-TOF mass spectrometry (MS) is becoming a major resource in the Clinical Microbiology laboratory. Results on some groups of microorganisms are still controversial. We have studied the reliability of MALDI-TOF MS for the identification of anaerobic clinical isolates was studied compared to conventional biochemical methods, with rRNA 16S sequencing being used as a reference when discrepancies arose. Material and methodsA total of 126 anaerobic bacteria clinical isolates were studied by using API20A kits (bioMérieux, Marcy l’Étoile, France) and MALDI-TOF MS (Autoflex II, Bruker Daltonics, Germany), and using the data library BioTyper 2.0 (Bruker Daltonics, Germany). When discrepancies arose, or MALDI-TOF MS was not able to identify any microorganism, rRNA 16S sequencing was used as the reference standard. ResultsThe biochemical method and MALDI-TOF MS agreed in identifying 60.9% of isolates at species level, and 20.3% of isolates at genus level. Among the 48 discrepancies observed, rRNA 16S sequencing supported MALDI-TOF MS identification, at species level, in 32 isolates (66.7%), and in 8 isolates (16.7%) at genus level. rRNA 16S sequencing supported biochemical identification in only two isolates (4.2%) at species level, and in 26 isolates (54.2%) at genus level. The eight isolates for which MALDI-TOF MS did not manage to identify, or the identification obtained was rejected by sequencing, belonged to species that are still not added to the BioTyper II data library. ConclusionsResults obtained in this study show that, overall, MALDI-TOF MS identification of anaerobic bacteria is more reliable than identification obtained by conventional biochemical methods (24% more correct identifications at species level). The number of major errors (incorrect identification at the genus level) is also 2.5-times lower. Moreover, all the major errors obtained by MALDI-TOF MS were due to the absence of some species in the data library. Thus, when data libraries are more complete, reliability differences between both methods will probably be even higher.

Chemical extraction versus direct smear for MALDI-TOF mass spectrometry identification of anaerobic bacteria

In the present study, two pre-analytic processes for mass spectrometric bacterial identification were compared: the time-consuming reference method, chemical extraction, and the direct smear technique directly using cultured colonies without any further preparation. These pre-analytic processes were compared in the identification of a total of 238 strains of anaerobic bacteria representing 34 species. The results showed that 218/238 strains were identified following chemical extraction, 185 identifications (77.7%) were secured to both genus and species [log(score) > 2.0] whereas 33 identifications (14%) were secured to genus only [log(score) between 1.7 and 2.0]. Following direct smear, 207/238 anaerobic bacteria were identified, 158 identifications (66.4%) were secured to both genus and species [log(score) > 2.0] whereas 49 identifications were secured to genus only [log(score) between 1.7 and 2.0]. Twenty strains were not identified [log(score) < 1.7] by MALDI-TOF MS following chemical extraction whereas 31 strains were not identified with the direct smear technique. Although direct smear led to a significant decrease of the log(score) values for the Clostridium genus and the Gram positive anaerobic bacteria (GPAC) group (p < 0.0001, Wilcoxon test), identification to both species and genus were not changed. However these differences were not statistically significant (p = 0.1, Chi square). Therefore, MALDI-TOF MS identification following the direct smear technique appears to both non-inferior to the reference method and relevant for anaerobic bacteria identification.

Identification of clinical isolates of anaerobic bacteria using matrix-assisted laser desorption ionization-time of flight mass spectrometry

We evaluated the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) for the rapid identification of anaerobic bacteria that had been isolated from clinical specimens and previously identified by 16s rRNA sequencing. The Bruker Microflex MALDI-TOF instrument with the Biotyper Software was used. We tested 152 isolates of anaerobic bacteria from 24 different genera and 75 different species. A total of 125 isolates (82%) had Biotyper software scores greater than 2.0 and the correct identification to genus and species was made by MALDI-TOF for 120 (79%) of isolates. Of the 12 isolates with a score between 1.8 and 2.0, 2 (17%) organisms were incorrectly identified by MALDI-TOF. Only 15 (10%) isolates had a score less than 1.8 and MALDI-TOF gave the wrong genus and species for four isolates, the correct genus for two isolates, and the correct genus and species for nine isolates. Therefore, we found the Bruker MALDI-TOF MicroFlex LT with an expanded database and the use of bacteria extracts rather than whole organisms correctly identified 130 of 152 (86%) isolates to genus and species when the cut-off for an acceptable identification was a spectrum score ≥1.8.

Comparison of two matrix-assisted laser desorption ionisation-time of flight mass spectrometry methods for the identification of clinically relevant anaerobic bacteria

Two commercially available MALDI-TOF MS systems, Bruker MS and Shimadzu MS, were compared for the identification of clinically relevant anaerobic bacteria. A selection of 79 clinical isolates, representing 19 different genera, were tested and compared with identification obtained by 16S rRNA gene sequencing. Correct genus identification was achieved for 71% of isolates by Shimadzu MS and for 61% by Bruker MS. Correct identification at the species level occurred in 61% and 51%, respectively. Shimadzu showed markedly better results for identification of Gram-positive anaerobic cocci. In contrast, the Bruker system performed better than Shimadzu for the Bacteroides fragilis group. When strains not present in the database were excluded from the analyses for each database, both systems performed equally well, with 76.7% and 75.0% correct genus identification for Shimadzu and Bruker, respectively. Similarly, when the most recently updated Bruker database was applied, no difference was observed. We conclude that the composition and quality of the database is crucial for a correct identification. The databases currently available for both systems need to be optimized before MS can be implemented for routine identification of anaerobic bacteria.

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

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. 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. 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). 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.

Protected telescopic catheter also allows anaerobic bacteria isolation in patients with ventilatory-acquired pneumonia

To analyze the possibility of isolating anaerobic bacteria using protected telescopic catheter (PTC) in patients with ventilatory-associated pneumonia. A prospective epidemiological study in a 12-bed intensive care unit. 104 patients with suspected ventilatory-associated pneumonia undergoing PTC for microbiological determination including specific methods for anaerobic bacteria identification (mean age 59.3, range 17-82; SAPS II 44.6+/-17.9). We performed 156 PTC procedures. Of the 14 cases ventilatory-associated pneumonia occurred early in 6 and in 8 late. In 52 samples at least one bacteria was isolated (33.3%). Of the 156 samples 14 yielded with anaerobic bacteria, representing 26.9% of the positive samples and 9% of all the samples. Considering the potential pathogenic role of anaerobic bacteria in patients with ventilatory-associated pneumonia, the possibility of isolating these bacteria using PTC and taking into account its easiness and limited side effects should be underlined.

Speculations on the microbiology laboratory of the future.

Changes in the availability of skilled laboratory personnel, new technologies, and the financial environment will all influence the practice of diagnostic microbiology in the near and more distant future. Because of the special expertise needed for the accurate identification of anaerobic bacteria, the ability to diagnose anaerobic infections may decline as a consequence of these changes. Physicians should anticipate a difficult time in the years between the loss of expertise in traditional methods and development of reliable and accurate molecular assays.

Routine analysis of short-chain fatty acids for anaerobic bacteria identification using capillary electrophoresis and indirect ultraviolet detection

The diagnosis of anaerobes can be difficult to perform, using classical biochemical tests. Characterization of metabolic end-products such as short-chain fatty acids (SCFA) was often used because of their reproducible biosynthesis. Despite this, SCFA are difficult to study using gas chromatography, due to their high volatility. Furthermore, the treatment of the samples are long and fastidious. Capillary electrophoresis and indirect UV detection (CE–indirect UV) is a well-known analytical method to study inorganic or organic anions. In this work, we validate the analysis of SCFA using CE–indirect UV detection. To do this, we studied the culture media of 98 anaerobic strains for the detection and quantitation of the following acids: succinic, pyruvic, acetic, lactic, propionic, 2-hydroxybutyric, butyric, 2-hydroxyvaleric, isovaleric, isocaproic, and 3-phenylpropionic. We verified that the CE–indirect UV detection analysis of SCFA for taxonomical data can be used as a mean for rapid identification for the study of anaerobes.

Identification of Clostridium botulinum with API 20 A, Rapid ID 32 A and RapID ANA II.

Three commercially available test systems for the identification of anaerobic bacteria were evaluated for the identification of 18 proteolytic group I and 69 non-proteolytic group II Clostridium botulinum, four Clostridium sporogenes and 18 non-toxigenic group II C. botulinum-like strains. All proteolytic C. botulinum strains were misidentified by the Rapid ID 32 A and RapID ANA II, while 14 strains and all C. sporogenes strains were identified as C. botulinum or C. sporogenes by the API 20 A. Reversely, all non-proteolytic C. botulinum strains were misidentified by the API 20 A while the Rapid ID 32 A recognized 67 and RapID ANA II 68 strains. All C. sporogenes strains were recognized by the RapID ANA II, while the Rapid ID 32 A recognized one strain. All non-proteolytic non-toxigenic strains were identified as C. botulinum group II by the Rapid ID 32 A, 17 strains by the RapID ANA II, and one strain by the API 20 A. The results show that these test systems do not provide a reliable method for identification of C. botulinum.

Identification of Clostridium botulinum with API 20 A, Rapid ID 32 A and RapID ANA II

Three commercially available test systems for the identification of anaerobic bacteria were evaluated for the identification of 18 proteolytic group I and 69 non-proteolytic group II Clostridium botulinum, four Clostridium sporogenes and 18 non-toxigenic group II C. botulinum-like strains. All proteolytic C. botulinum strains were misidentified by the Rapid ID 32 A and RapID ANA II, while 14 strains and all C. sporogenes strains were identified as C. botulinum or C. sporogenes by the API 20 A. Reversely, all non-proteolytic C. botulinum strains were misidentified by the API 20 A while the Rapid ID 32 A recognized 67 and RapID ANA II 68 strains. All C. sporogenes strains were recognized by the RapID ANA II, while the Rapid ID 32 A recognized one strain. All non-proteolytic non-toxigenic strains were identified as C. botulinum group II by the Rapid ID 32 A, 17 strains by the RapID ANA II, and one strain by the API 20 A. The results show that these test systems do not provide a reliable method for identification of C. botulinum.

Update on Pan-American research on anaerobes: the situation in Argentina.

The study of anaerobic bacteria became important in Argentina when the first outbreak of botulism was reported in Mendoza in 1922. In the 1970s, the emphasis was on the development of methods to obtain suitable samples for isolation and identification of anaerobic bacteria. In 1985, a working group belonging to the Argentine Association of Microbiology was organized to study anaerobic bacteria and infections. Last year, I became involved with the immunologic, microbiological, and epidemiological features of periodontitis. Other investigators in our country are studying Clostridium difficile, Campylobacter species, and Helicobacter species as well as lactobacilli, Propionibacterium species (as probiotics to protect the gastrointestinal tracts of children), anaerobic veterinary infections, food contamination by anaerobes, and biotechnological applications of anaerobes.

A COMPARISON STUDY BETWEEN OXYRASE ANAEROBIC AGAR PLATES AND CONVENTIONAL ANAEROBIC CHAMBER FOR THE ISOLATION AND IDENTIFICATION OF ANAEROBIC BACTERIA FROM CLINICAL INFECTIONS

Misplaced confidence in the broad‐spectrum antibiotics, increased resistance among previously predictable anaerobic antibiograms, and the push to maximize productivity of available space and downsizing trends has created a need for a simplified cost‐effective, and superior method for the isolation and identification of anaerobic bacteria. In this study, the Oxyrase anaerobic plate system which requires no extraneous apparatus to create an anaerobic environment was compared to an anaerobic chamber in the isolation of anaerobic bacteria from 212 consecutive wound specimens. Brucella blood agar and KVL agar plates were used in this comparison study. RapID ANA II, AP120A, special potency disks, and GLC were used for identification. Of the 212 specimens cultured, 87 yielded anaerobic bacteria comprising 182 strains. Thirty‐nine strains failed to grow in the anaerobic chamber but grew on the OxyPlates(TM). These strains were predominantly Peptostreptococcus species (28%), Eubacterium species (20%), and Propionibacterium species (20%). Fourteen strains failed to grow on the OxyPlates, but grew in the anaerobic chamber. No trend was noted and all organisms in this category grew on the OxyPlates from other specimens. In conclusion, the Oxyrases anaerobic plate system appears to be an excellent alternative to the conventional anaerobic chamber in the isolation and identification of clinically significant anaerobes found in human samples, obviating the need for separate anaerobic‐aerobic workstations, expensive anaerobic apparatus, and additional incubator space.

Comparison of BBL Crystal® ANR ID Kit and API rapid ID 32 A for Identification of Anaerobic Bacteria

BBL Crystal ANR ID Kit and the API System rapid ID 32 A are miniaturized identification systems for anaerobes using enzymatic tests. The incubation period of both systems is 4 hours. A comparative evaluation of the BBL Crystal Identification System Anaerobe ID Kit (Becton Dickinson Microbiology Systems, Cockeysville, USA) with anaerobes grown on Columbia and Schaedler agar plates (Becton Dickinson Microbiology Systems, Cockeysville, USA) and the API System rapid ID 32 A (BioMérieux SA, Lyon, France) with bacteria grown on Columbia agar (Becton Dickinson Microbiology Systems, Cockeysville, USA) which is recommended by the manufacturer as cultivation medium, was performed with 207 mostly fresh clinical anaerobe isolates, including 104 gram-negative bacilli, 12 gram-negative cocci, 15 gram-positive cocci, 14 gram-positive sporeforming bacilli and 62 representatives of gram-positive non-sporeforming bacilli. With supplemental testing the Crystal system with inocula from Columbia and Schaedler agar and API inoculates from Columbia agar identified to genus level 144 (69.6%), 152 (73.4%) and 109 (52.7%) isolates, respectively. Misidentification to genus level was found by Crystal from Columbia and Schaedler agar and by API from Columbia agar in 17 (8.2%), 15 (7.3%) and 12 (5.8%) isolates, respectively. 36 isolates were not determined to species level by classical anaerobic methods or the systems only identified to genus level. 26 anaerobes were not included in the database of the Crystal or API system. From the remaining 145 clinical isolates with supplemental testing, Crystal from Columbia and Schaedler agar plates correctly identified 91 (62.8%) and 102 (70.3%), respectively, and API, 69 (47.6%) isolates. For the correct identification to genus and species level of the 207 clinical isolates tested, the Crystal system from Columbia and Schaedler agar and API system from Columbia agar required supplemental testing, as specified by the manufacturer, for 39 (27.1%), 34 (22.4%) and 14 (12.8%) isolates, respectively. Among the 207 clinical isolates tested, 27 had been frozen and 26 had been lyophilized. In a comparative evaluation, the fresh isolates showed a slightly to significantly better identification rate than the frozen or lyophilized specimens in all three tests. The individual reproducibility of the Crystal ANR ID, which had been tested before the accuracy study was performed, ranged from 90.8% to 100%. The overall reproducibility was determined to be 97.3%. Time consumption studies and cost analysis did not show a significant difference between both systems, but Crystal ANR ID was found to be easier to use than API rapid ID 32.

Infection Caused by Propionibacterium in Children

Three hundred eighty-six isolates of Propionibacterium sp. were recovered from 2,003 specimens studied for the identification of anaerobic bacteria in children during a 15-year period. Three hundred forty-three (89%) of these were Propionibacterium acnes. A total of 50 (13%) Propionibacterium isolates identified from 45 patients were considered to cause infection. Clinically significant infections caused by Propionibacterium sp. were associated with bacteremia in 10 children; ear infection in eight; abscesses in seven; adenitis and central nervous system infection in five each; burns in four; wounds in three; tumors and bone in two each; and cysts, eye, sinus, and mastoid in one each. Predisposing or underlying conditions were present in 33 children (73%). These included the presence of a foreign body (17), immunodeficiency (six), malignancy (five), diabetes (five), previous surgery (four), and steroid therapy (two). Antimicrobial therapy was given to 41 (91%) children. Surgical drainage was concomitantly performed in 22 (49%). Four patients died. This study highlights the importance of Propionibacterium sp. as an unusual, but potentially important, pathogen in children.

Evaluation of an automated system for identification of anaerobic bacteria.

A fully automated computer-assisted system (ATB system, bioMérieux, France) which uses disposable microenzymatic panels was evaluated for its ability to identify 215 strains of anaerobic bacteria (clinical isolates and reference strains). All strains were examined using conventional identification protocols and by gas chromatographic analysis of short-chain fatty acids. Automated reading of Rapid ID32A test kits (bioMérieux, France) by the ATB system gave correct identification for 195 strains (90.7%): 92.25% of gram-negative anaerobes (116 strains) and 89% of gram-positive anaerobes (99 strains) were correctly identified. Twelve strains (5.6%) were incorrectly identified and 8 strains (3.7%) were not identified by the system. For some strains in the Bacteroides fragilis group, for Clostridium difficile and for the Fusobacterium genus, additional tests suggested by the ATB software were necessary to reach a final identification at the species or genus level. On the basis of the high incidence of correct identifications and the comparison of these results with those obtained previously using other commercially available kits, the ATB system was found to be a reliable method for identification of anaerobic bacteria in clinical laboratories.

Comparative Evaluation of the Rapid Id 32A Kit System, Miniaturized Standard Procedure and a Rapid Fermentation Procedure for the Identification of Anaerobic Bacteria

The RAPID ID 32A and a rapid fermentation procedure were compared with a miniaturized standard procedure for the identification of 102 isolates of anaerobic bacteria belonging to 11 genera. The miniaturized standard procedure identified all 102 isolates at the genus level and 90 at the species level. The rapid fermentation procedure failed to identify 12 and 24 isolates at the genus and species levels respectively and is unsatisfactory. The RAPID ID 32A system misidentified 4 isolates (4%) at the genus level, 3 of which could have been avoided if the results of the Gram stain, the fundamental procedure in medical microbiology, had been taken into account. The system correctly identified 89 isolates at the species level. Since it does not produce species identifications within the genera Mobiluncus and Veillonella of which 7 and 2 isolates respectively were included in the study, the percentage of correct identifications at the species level is 96.7% (90/93). The RAPID ID 32A system is about 25 times more expensive than the miniaturized standard procedure if the investment for a gas chromatograph is not taken into account.

A Modified Procedure for the Identification of Anaerobic Bacteria by High Performance Liquid Chromatography — Quantitative Analysis of Short-Chain Fatty Acids

We have developed a new rapid method for analysing volatile and non-volatile short-chain fatty acids using high-performance liquid chromatography. Within 50 min, 22 fatty acids in a standard mixture could be detected in a single chromatographic run. The fatty acids released by anaerobic bacteria in the culture media were ether-extracted and analysed with an Aminex HPX-87H column. Using a microprocessor-controlled chromatography unit, a quantitative analysis of the fatty acids produced in bacterial cultures was possible. Resolution, rapidity and sensitivity were improved as compared to previous methods by using an eluent of 5% acetonitrile in 0.01 N H2SO4 and changing the column temperature to 35 degrees C.