A probability matrix for the identification of gram-negative, aerobic, non-fermentative bacteria that grow on nutrient agar

Results of the identification of 621 strains of Gram-negative, aerobic, non-fermentative bacteria by a computer-based probabilistic method are given. Although many of the strains were atypical and have caused difficulty in identification in the medical diagnostic laboratory, the identification rate on this matrix was 91.5%.     

STAPHYOGRAM, a new rapid identification kit for the aerobic, gram-positive, catalase-positive cocci--application of fluorometric microplate hybridization for the pre-identification of 386 isolates used

A new simplified test kit, STAPHYOGRAM plate, was developed for 4-hr identification of aerobic, Gram-positive and catalase-positive cocci. The plate has 18 wells, in which different dehydrated substrates and nutrients are fixed. An 18-hr agar-culture suspension of a test strain with a turbidity of McFarland No. 4 was distributed into all wells in 50-microliters quantities. After 4-hr incubation at 37C, the profile number was obtained by summarizing positive reactions. The ability of the plate to differentiate the type strains of the 30 species of the three genera in the family Micrococcaceae was confirmed. These three genera are Staphylococcus, Micrococcus and Stomatococcus. The applicability of the fluorometric microplate hybridization technique to identification of aerobic, Gram-positive and catalase-positive cocci was confirmed by homologous hybridization among the type strains of the 30 species. Thus, 386 isolates of human and animal origin were pre-identified by microplate hybridization and used for evaluating the STAPHYOGRAM plate. Of the 236 profile numbers thus obtained with the 386 isolates, 218 (92.4%) were species-proper each and all for the 15 species of Staphylococcus and Stomatococcus mucilaginosus. A total of 342 (88.6%) of the 386 isolates were given such profile numbers, and were identified without any additional test. Among the 15 species identified primarily by the results of STAPHYOGRAM plate culture, S. caprae, S. lugdunensis, S. gallinarum and S. delphini were validly published after Approved Lists of Bacterial Names. The identified strains of S. caprae (48), S. haemolyticus (46), S. capitis (35) numbered between those of S. epidermidis (67) and S. saprophyticus (31). Profile numbers common to two species were seven (27 strains) and that to four species was one (17 strains). These 44 strains were identified with one to three additional tests. From these results, we were convinced that the STAPHYOGRAM test plate is useful for the rapid identification of members of family Micrococcaceae. By compiling STAPHYOGRAM plate data on genetically identified strains, an exclusive list of profile numbers will soon be prepared for perfection of the kit.     

Electrophoretic forms of glucose-6-phosphate dehydrogenase, acid phosphatase and esterase in Amoeba species amoebas

Glucose-6-phosphate dehydrogenase (G6PD), acid phosphatase and esterases in free-living amoebae of 7 Amoeba species were investigated with the use of disc-electrophoresis in polyacrylamide gel. The evidence provided is suggestive that the electrophoretic isoenzyme patterns of acid phosphatase and esterases (and G6PD in some cases), in addition to a few morphological characters, can serve as a taxonomic criterion for species identification within this genus, as well as for revealing erroneously classified species and strains. It is suggested that A. indica is an independent species whose preliminary diagnosis has been given in this paper. It is concluded that A. discoides and A. lescherae are strains of A. proteus, rather than two independent species. A and As-102 amoebian strains, kept in the collection of protozoan strains and species of the Institute of Cytology RAS and referred to as strains of A. proteus, belong in reality to another Amoeba species and even to another genus within the family Amoebidae. This conclusion has been documented by results of our analysis of electrophoretic patterns of acid phosphatase and esterases in these strains.     

Insights into the taxonomy, genetics and physiology of bifidobacteria

Despite the generally accepted importance of bifidobacteria as probiotic components of the human intestinal microflora and their use in health promoting foods, there is only limited information about their phylogenetic position, physiology and underlying genetics. In the last few years numerous molecular approaches have emerged for the identification and characterization of bifidobacterial strains. Their use, in conjunction with traditional culturing methods, has led to a polyphasic taxonomy which has significantly enhanced our knowledge of the role played by these bacteria in the human intestinal ecosystem. The recent adaptation of culture-independent molecular tools to the fingerprinting of intestinal and food communities offers an exciting opportunity for revealing a more detailed picture of the true complexity of these environments. Furthermore, the availability of bifidobacterial genome sequences has advanced knowledge on the genetics of bifidobacteria and the effects of their metabolic activities on the intestinal ecosystem. The release of a complete Bifidobacterium longum genome sequence and the recent initiative to sequence additional strains are expected to open up a new era of comparative genomics in bifidobacterial biology. Moreover, the use of genomotyping allows a global comparative analysis of gene content between different bifidobacterial isolates of a given species without the necessity of sequencing many strains. Genomotyping provides useful information about the degree of relatedness among various strains of Bifidobacterium species and consequently can be used in a polyphasic identification approach. This review will deal mainly with the molecular tools described for bifidobacterial identification and the first insights into the underlying genetics involved in bifidobacterial physiology as well as genome variability.     

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.     

A Side by Side Comparison of Bruker Biotyper and VITEK MS: Utility of MALDI-TOF MS Technology for Microorganism Identification in a Public Health Reference Laboratory

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid, highly accurate, and cost-effective method for routine identification of a wide range of microorganisms. We carried out a side by side comparative evaluation of the performance of Bruker Biotyper versus VITEK MS for identification of a large and diverse collection of microorganisms. Most difficult and/or unusual microorganisms, as well as commonly encountered microorganisms were selected, including Gram-positive and negative bacteria, mycobacteria, actinomycetes, yeasts and filamentous fungi. Six hundred forty two strains representing 159 genera and 441 species from clinical specimens previously identified at the Laboratoire de santé publique du Québec (LSPQ) by reference methods were retrospectively chosen for the study. They included 254 Gram-positive bacteria, 167 Gram-negative bacteria, 109 mycobacteria and aerobic actinomycetes and 112 yeasts and moulds. MALDI-TOF MS analyses were performed on both systems according to the manufacturer’s instructions. Of the 642 strains tested, the name of the genus and / or species of 572 strains were referenced in the Bruker database while 406 were present in the VITEK MS IVD database. The Biotyper correctly identified 494 (86.4%) of the strains, while the VITEK MS correctly identified 362 (92.3%) of the strains (excluding 14 mycobacteria that were not tested). Of the 70 strains not present in the Bruker database at the species level, the Biotyper correctly identified 10 (14.3%) to the genus level and 2 (2.9%) to the complex/group level. For 52 (74.2%) strains, we obtained no identification, and an incorrect identification was given for 6 (8.6%) strains. Of the 178 strains not present in the VITEK MS IVD database at the species level (excluding 71 untested mycobacteria and actinomycetes), the VITEK MS correctly identified 12 (6.8%) of the strains each to the genus and to the complex/group level. For 97 (54.5%) strains, no identification was given and for 69 (38.7%) strains, an incorrect identification was obtained. Our study demonstrates that both systems gave a high level (above 85%) of correct identification for a wide range of microorganisms. However, VITEK MS gave more misidentification when the microorganism analysed was not present in the database, compared to Bruker Biotyper. This should be taken into account when this technology is used alone for microorganism identification in a public health laboratory, where isolates received are often difficult to identify and/or unusual microorganisms.     

Numerical taxonomy and identification of lactic acid bacteria from spoiled, vacuum-packaged vienna sausages

Sixty-one lactic acid bacteria from spoiled vacuum-packaged vienna sausages and 15 reference strains were tested for 72 phenotypic characteristics. An identification key and a computer data base, both specific for lactic acid bacteria from meat sources, were used for identification and the results were compared. There was a high correlation (86.9%) between the two procedures in the identification of strains to genus level. However, only a 54.8% correlation was obtained in identifying strains to the species level. With numerical taxonomy ( S _sm matching coefficient with average linkage clustering) 60 strains were recovered in six clusters at the 89% similarity level. While most Leuconostoc strains clustered separately from the Lactobacillus strains, the identity of many leuconostocs was not clarified. The presence of a heterogeneous cluster containing typical and 'atypical' strains of the Lactobacillus saké/curvatus group and a separate homogeneous Lact. curvatus cluster was noted. Closer examination of the data suggested that the 'atypical' lactobacilli were all strains of Lact. saké.     

Identification of Azospirillum strains by restriction fragment length polymorphism of the 16S rDNA and of the histidine operon

Abstract DNA fingerprints of several Azospirillum strains, belonging to the five known species A. amazonense, A. brasilense, A. halopraeferens, A. irakense and A. lipoferum , were obtained by restriction analysis of the amplified 16S rDNA and by restriction fragment length polymorphism of the histidine biosynthetic genes. Data obtained showed that amplified rDNA restriction analysis is an easy, fast, reproducible and reliable tool for identification of Azospirillum strains, mainly at the species level, whereas restriction fragment length polymorphism could, in some cases, differentiate strains belonging to the same species. Moreover, both analyses gave congruent results in grouping strains and in the assignment of new strains to a given species.     

Discriminant analysis of volatile fatty acids produced in culture medium: a novel approach to the identification of Pseudomonas species

The volatile fatty acids produced in culture medium by 357 Pseudomonas strains belonging to eight species were determined quantitatively by GLC. The resultant chromatograms were submitted to discriminant analysis. Stable discriminant functions were computed and included in a computerized identification system which also involved some distinctive volatile fatty acids regarded as two-state qualitative characters (presence or absence characters). Using a test group of 249 strains belonging to the studied species, more than 89% of the identifications made by this system agreed with those made by conventional biochemical methods despite the relatively poor differentiation between P. putida and P. fluorescens. When the individual species within the matrices were weighted with prior probabilities reflecting results given by two simple biochemical tests, 96% of the 249 strains were correctly identified.     

Multilocus Sequence Typing, Pulsed-Field Gel Electrophoresis, and fla Short Variable Region Typing of Clonal Complexes of Campylobacter jejuni Strains of Human, Bovine, and Poultry Origins in Luxembourg

Campylobacter jejuni is the most common cause of bacterial gastroenteritis in Luxembourg, with a marked seasonal peak during summer. The majority of these infections are thought to be sporadic, and the relative contribution of potential sources and reservoirs is still poorly understood. We monitored human cases from June to September 2006 (n = 124) by molecular characterization of isolates with the aim of rapidly detecting temporally related cases. In addition, isolates from poultry meat (n = 36) and cattle cecal contents (n = 48) were genotyped for comparison and identification of common clusters between veterinary and human C. jejuni populations. A total of 208 isolates were typed by sequencing the fla short variable region, macrorestriction analysis resolved by pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). We observed a high diversity of human strains during a given summer season. Poultry and human isolates had a higher diversity of sequence types than isolates of bovine origin, for which clonal complexes CC21 (41.6%) and CC61 (18.7%) were predominant. CC21 was also the most common complex found among human isolates (21.8%). The substantial concordance between PFGE and MLST results for this last group of strains suggests that they are clonally related. Our study indicates that while poultry remains an important source, cattle could be an underestimated reservoir of human C. jejuni cases. Transmission mechanisms of cattle-specific strains warrant further investigation.     

Differentiation of Clostridium difficile,Clostridium bifermentans,Clostridium sordellii,and Clostridium perfringens from Diarrheal Stool by API ZYM and API LRA Oxidase Test

A simple, rapid and reliable outline for identification of clostridia isolates from human infections was developed. It consists of a combination of API ZYM and API LRA Oxidase tests. The enzymatic activities were performed with strains sub-cultured onto carbohydrate-free medium (Columbia blood agar). Fifty-five strains of Clostridium difficile, C. bifermentans, C. sordellii, and C. perfringens from clinical specimens and eight reference standard strains representing different species of the same genus were analyzed. The accuracy of the new method was evaluated by comparison with the results obtained by DNA/DNA analysis.     

Identification of the optochin-resistant Streptococcus pneumoniae strains

So far the prevalent method of the identification of pneumococci in the routine diagnostic practice has included three tests: sensitivity to optoxin, solubility in biliary salts and reaction with omniserum. At present optoxin-resistant pneumococcal strains, as well as those giving dual reaction to solubility in biliary salts, occur sufficiently often thatconsiderably complicates the identification of this infective agent. We have summarized experience in the isolation of S. pneumoniae strains, resistant to optoxin and not soluble in biliary salts; in addition, the prospects of introducing the complex approach to the identification of a given culture with the use of molecular diagnostic methods into daily practice are evaluated.     

Evaluation of the use of the bioMerieux Rapid ID32 A for the identification of Clostridium botulinum

The neurotoxins produced by Clostridium botulinum are amongst the most potent known to man. Toxin production is detected by a mouse bioassay, which requires several days for a result and is not acceptable for routine use unless there is a high level of suspicion. The Rapid ID32 A kit produced by bioMerieux gives an identification of an isolate within 4 h. The aim of this study was to examine the efficiency of the identification of Cl. botulinum using the Rapid ID32 A. Forty-two strains of Cl. botulinum , one strain each of botulinum toxin-producing Cl. butyricum and Cl. baratii , and four strains of Cl. sporogenes , were tested. One strain of Group I Cl. botulinum gave a presumptive identification of Group II Cl. botulinum , six strains of Cl. botulinum were identified as 50–98% Cl. botulinum in some tests, and 17 strains of Cl. botulinum were identified as <50% Cl. botulinum. Thirteen strains of Cl. botulinum were identified as >99% Cl. sporogenes or 86% Cl. histolyticum , and five strains gave a combination of these results. All strains of Cl. sporogenes were correctly identified. These results show that some strains of Cl. botulinum may not be correctly identified using the Rapid ID32 A.     

Identification of Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus by rDNA-based techniques

Ribosomal DNA-based techniques including the analysis of profiles generated by ISR amplification, ISR restriction and ARDRA have been evaluated as molecular tools for identifying Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus. They have been applied for the molecular characterization of 91 strains with the following identities: eight Carnobacterium including the eight type species of the genus; 61 Lactobacillus including 40 type strains out of 45 species, 13 Leuconostoc, out of them 11 are type strains and three are subspecies of Lc. mesenteroides; and nine strains representing the six species of genus Pediococcus. The genetic relationship displayed between these species by rrn-based profiles is sustained by their phylogenetic relationships and can therefore be considered useful for taxonomic purposes. Profiles obtained by ISR amplification allowed identification at genus level of Carnobacterium and Leuconostoc, and even at species level in genus Carnobacterium. Genera Lactobacillus and Pediococcus could not be distinguished from each other by applying this technique. The Lactobacillus species analysed here (45) were differentiated using ARDRA-DdeI and ISR-DdeI profiles, sequentially, and Pediococcus species by ISR-DdeI profiles. It was necessary to combine profiles generated by restriction of ISR-DdeI, ARDRA-DdeI and ARDRA-HaeIII in order to complete the identification of Leuconostoc species.     

Fourier-transform infrared microspectroscopy,a novel and rapid tool for identification of yeasts

Fourier-transform infrared (FT-IR) microspectroscopy was used in this study to identify yeasts. Cells were grown to microcolonies of 70 to 250 micro m in diameter and transferred from the agar plate by replica stamping to an IR-transparent ZnSe carrier. IR spectra of the replicas on the carrier were recorded using an IR microscope coupled to an IR spectrometer, and identification was performed by comparison to reference spectra. The method was tested by using small model libraries comprising reference spectra of 45 strains from 9 genera and 13 species, recorded with both FT-IR microspectroscopy and FT-IR macrospectroscopy. The results show that identification by FT-IR microspectroscopy is equivalent to that achieved by FT-IR macrospectroscopy but the time-consuming isolation of the organisms prior to identification is not necessary. Therefore, this method also provides a rapid tool to analyze mixed populations. Furthermore, identification of 21 Debaryomyces hansenii and 9 Saccharomyces cerevisiae strains resulted in 92% correct identification at the strain level for S. cerevisiae and 91% for D. hansenii, which demonstrates that the resolution power of FT-IR microspectroscopy may also be used for yeast typing at the strain level.     

Usefulness of rpoB gene sequencing for identification of Afipia and Bosea species, including a strategy for choosing discriminative partial sequences

Bacteria belonging to the genera Afipia and Bosea are amoeba-resisting bacteria that have been recently reported to colonize hospital water supplies and are suspected of being responsible for intensive care unit-acquired pneumonia. Identification of these bacteria is now based on determination of the 16S ribosomal DNA sequence. However, the 16S rRNA gene is not polymorphic enough to ensure discrimination of species defined by DNA-DNA relatedness. The complete rpoB sequences of 20 strains were first determined by both PCR and genome walking methods. The percentage of homology between different species ranged from 83 to 97% and was in all cases lower than that observed with the 16S rRNA gene; this was true even for species that differed in only one position. The taxonomy of Bosea and Afipia is discussed in light of these results. For strain identification that does not require the complete rpoB sequence (4,113 to 4,137 bp), we propose a simple computerized method that allows determination of nucleotide positions of high variability in the sequence that are bordered by conserved sequences and that could be useful for design of universal primers. A fragment of 740 to 752 bp that contained the most highly variable area (positions 408 to 420) was amplified and sequenced with these universal primers for 47 strains. The variability of this sequence allowed identification of all strains and correlated well with results of DNA-DNA relatedness. In the future, this method could be also used for the determination of variability "hot spots" in sets of housekeeping genes, not only for identification purposes but also for increasing the discriminatory power of sequence typing techniques such as multilocus sequence typing.     

Sequence Analysis of 96 Genomic Regions Identifies Distinct Evolutionary Lineages within CC156, the Largest Streptococcus pneumoniae Clonal Complex in the MLST Database

Multi-Locus Sequence Typing (MLST) of Streptococcus pneumoniae is based on the sequence of seven housekeeping gene fragments. The analysis of MLST allelic profiles by eBURST allows the grouping of genetically related strains into Clonal Complexes (CCs) including those genotypes with a common descent from a predicted ancestor. However, the increasing use of MLST to characterize S. pneumoniae strains has led to the identification of a large number of new Sequence Types (STs) causing the merger of formerly distinct lineages into larger CCs. An example of this is the CC156, displaying a high level of complexity and including strains with allelic profiles differing in all seven of the MLST loci, capsular type and the presence of the Pilus Islet-1 (PI-1). Detailed analysis of the CC156 indicates that the identification of new STs, such as ST4945, induced the merging of formerly distinct clonal complexes. In order to discriminate the strain diversity within CC156, a recently developed typing schema, 96-MLST, was used to analyse 66 strains representative of 41 different STs. Analysis of allelic profiles by hierarchical clustering and a minimum spanning tree identified ten genetically distinct evolutionary lineages. Similar results were obtained by phylogenetic analysis on the concatenated sequences with different methods. The identified lineages are homogenous in capsular type and PI-1 presence. ST4945 strains were unequivocally assigned to one of the lineages. In conclusion, the identification of new STs through an exhaustive analysis of pneumococcal strains from various laboratories has highlighted that potentially unrelated subgroups can be grouped into a single CC by eBURST. The analysis of additional loci, such as those included in the 96-MLST schema, will be necessary to accurately discriminate the clonal evolution of the pneumococcal population.