Anomalous but helpful findings from the BBL Crystal ID kit with Haemophilus spp

Fourteen strains of Haemophilus (12 H. influenzae , 1 H. parainfluenzae and 1 H. aphrophilus ) were processed in BBL Crystal ID Enteric/Nonfermenter, API 20E and API 20NE kits, to determine whether the BBL kit misidentifies, as API kits may do, Haemophilus spp. as Pasteurella spp. The 13 H. influenzae and H. parainfluenzae strains produced uninterpretable colour reactions in the Crystal kit, thus signalling that an inappropriate species had been tested. On the other hand, the API kits (especially 20NE) often confidently 'identified' Haemophilus spp. as Pasteurella spp., giving no warning that this was a misidentification.     

Comparison of API 20NE and Biolog GN identification systems assessed by techniques of multivariate analyses.

The increasing use of commercial multitest systems for identification of environmental bacteria creates the problem of how to compare the identification results obtained from different systems. The limited use of species designations in such comparisons is caused by low usage of environmental bacteria in the development of commercial identification schemes. Two multivariate statistical methods, the Mantel's test and the co-inertia analysis, were applied to analyze data derived from the Biolog GN and the API 20NE systems of identification for 50 environmental bacterial strains. We found these two methods to be useful for revealing the relationship between the two sets of numerical taxonomic traits. Both of these methods showed that the distances according to the Biolog GN results between the studied strains were related to those derived from the API 20NE results, despite the differences in the test sets of the two systems. In addition, the co-inertia analysis allowed us to visualise the relationships between classifications of strains derived from the two identification systems and, simultaneously, to estimate the contribution of particular tests to the differentiation of bacterial strains.     

Comparison of two commercial methods with PCR restriction fragment length polymorphism of the tuf gene in the identification of coagulase-negative staphylococci

AIMS: Two commercial methods for the identification of coagulase-negative staphylococci (CNS) were compared with the restriction fragment length polymorphism (RFLP) of the amplified tuf gene, which served as the reference method. METHODS AND RESULTS: One hundred and forty-five CNS were evaluated using the API 32 Staph ID and the Crystal GP/ID BBL systems. The PCR-RFLP of the tuf gene served as the reference method. The APIStaph and the GP/ID BBL had an overall rate of agreement with the molecular method of 58.6% and 46.2% respectively, with the inability of the GP/ID BBL to characterize 11.7% of the isolates. The APIStaph showed higher sensitivity and better agreement than the GP/ID BBL with the PCR-RFLP, except for Staphylococcus hominis and Staphylococcus capitis. CONCLUSIONS: Neither of the commercial systems was as reliable as the PCR-RFLP method for identifying isolates of CNS. Overall the APIStaph had better agreement with the PCR-RFLP than the GP/ID system. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that the PCR-RFLP method is more reliable than the two commercial systems tested, suggesting that it is more reliable for routinely identifying CNS.     

Enzymatic/biochemical analysis of Actinomyces with commercial test kits with an emphasis on newly described species

In clinical microbiology laboratories, the identification of Actinomyces-like bacteria can be very laborious and problematic. In the present study, we focused on reactivity patterns of 4 commercial test kits, RapID ANA II, RapID 32A, RapID CB Plus, and BBL Crystal ANR ID, that could be used for rapid preliminary identification of Actinomyces isolates belonging to newly described Actinomyces and closely related species. Out of the 54 strains tested, 25 strains (46%) were correctly identified to the genus/group level by BBL Crystal ANR ID system, 16 strains (30%) by RapID 32 A, 11 strains (20%) by RapID CB Plus, and 7 strains (13%) by RapID ANA II. The main problems with these kits were due to occasional weak enzymatic and sugar fermentation reactions. In conclusion, chromogenic substrate sensitivity and specificity need to be enhanced in order to improve the reliability of the test results of these kits, and the present database updated in order to more precisely identify newly described Actinomyces and closely related species.     

Comparison of Parental and Transgenic Alfalfa Rhizosphere Bacterial Communities Using Biolog GN Metabolic Fingerprinting and Enterobacterial Repetitive Intergenic Consensus Sequence-PCR (ERIC-PCR)

Abstract Rhizosphere bacterial communities of parental and two transgenic alfalfa (Medicago sativa L.) of isogenic background were compared based on metabolic fingerprinting using Biolog GN microplates and DNA fingerprinting of bacterial communities present in Biolog GN substrate wells by enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR). The two transgenic alfalfa expressed either bacterial (Bacillus licheniformis) genes for alpha-amylase or fungal (Phanerochaete chrysosporium) genes for Mn-dependent lignin peroxidase (Austin S, Bingham ET, Matthews DE, Shahan MN, Will J, Burgess RR, Euphytica 85:381–393). Cluster analysis and principal components analysis (PCA) of the Biolog GN metabolic fingerprints indicated consistent differences in substrate utilization between the parental and lignin peroxidase transgenic alfalfa rhizosphere bacterial communities. Cluster analysis of ERIC-PCR fingerprints of the bacterial communities in Biolog GN substrate wells revealed consistent differences in the types of bacteria (substrate-specific populations) enriched from the rhizospheres of each alfalfa genotype. Comparison of ERIC-PCR fingerprints of bacterial strains obtained from substrate wells to substrate community ERIC-PCR fingerprints suggested that a limited number of populations were responsible for substrate oxidation in these wells. Results of this study suggest that transgenic plant genotype may affect rhizosphere microorganisms and that the methodology used in this study may prove a useful approach for the comparison of bacterial communities. Received: 1 June 1998; Accepted: 20 October 1998     

Growth of <Emphasis Type="Italic">Paenarthrobacter aurescens</Emphasis> strain TC1 on atrazine and isopropylamine during osmotic stress

Paenarthrobacter aurescens strain TC1 can use the herbicide atrazine and its degradation product isopropylamine as nutrients. Because osmotic stress can change the morphology of arthrobacters and decrease their metabolism of some carbon compounds, the effects of increasing NaCl concentrations on strain TC1 and its ability to utilize atrazine and isopropylamine were determined. Strain TC1 was cultured in minimal media with different NaCl concentrations and varying combinations of d-glucose, ammonium sulfate, atrazine, or isopropylamine. Growth was measured quantitatively as an increase in turbidity. Physiological effects were assessed using Biolog? GP test plates and BD BBL Crystal GP or bioMérieux API 20E test systems. The effects of osmoprotective compounds were determined in liquid media and on agar plates. Strain TC1 formed multicellular myceloids and its growth rate slowed as the salt concentration increased, but the culture yields were similar up to 0.6 mol l^?1 NaCl. The bacteria metabolized about half the carbon sources in Biolog? GP test plates, but their use of some compounds and several hydrolytic activities decreased with high salt concentrations. However, strain TC1 grew well with atrazine and isopropylamine as the nitrogen source in media containing up to 0.6 mol l^?1 NaCl. Growth in 0.8 mol l^?1 NaCl was more limited but could be enhanced by glycine betaine, L-proline, and L-glutamate. P. aurescens strain TC1 can continue to use atrazine and isopropylamine as nutrients during osmotic stress and so may be particularly useful for remediation of contaminated soils with low water activity.     

Apparent bias in river water inoculum following centrifugation

We collected four measures of viable bacterial concentration (heterotrophic plate count, total coliform, fecal coliform, and Escherichia coli) and three measures of well color development in Biolog GN2 microtiter plates from water samples that were collected on two or three separate occasions from a fixed site on 19 different streams throughout Oregon. Our goal was to determine whether concentrating the water sample with centrifugation prior to analysis would change the in situ composition of the culturable bacterial assemblage. Each sample was split and one subsample was centrifuged while the other subsample served as a control. A shift in the proportion of each group of culturable bacteria toward more fecal coliform bacteria was observed following centrifugation. In samples with the lowest initial heterotrophic bacterial densities (under 50CFU/100ml), the observed concentration following centrifugation was much lower than expected. However, samples that had high initial heterotrophic bacterial densities (over 1000CFU/100ml) had concentrations at or above expected values following centrifugation, but were biased toward a higher proportion of coliform bacteria. Bacteria in centrifuged subsamples utilized more sole-carbon substrates on Biolog GN2 microtiter plates and showed a shorter lag time prior to tetrazolium color development than their uncentrifuged counterparts. Future research that focuses on characterizing and accounting for the bias associated with centrifugation of water samples held for less than 24h is recommended.     

A rapid PCR based method to distinguish between Lactococcus and Enterococcus

Phenotypic characterisation of Lactococcus and Enterococcus species remains unreliable as strains of both genera have been isolated which do not conform to the traditional criteria for separation of these genera. A bank of 131 isolates was phenotypically characterised by three methods: (a) traditional broth tests, (b) API Rapid ID 32 Strep and (c) BBL Crystal ID kits. Differences in genus designation between commercial kits were evident for 12 strains (9%), while 7 strains (5%) remained unidentified by either kit. Published 16S rRNA sequences were aligned and used to design genus-specific primers which, when used in separate PCR reactions, were capable of distinguishing all type strains of Lactococcus and Enterococcus. These primers did not react with known species of Streptococcus, Pediococcus, Lactobacillus, Leuconostoc or Tetragenococcus. Isolates which could not be identified by phenotype were assigned to either genus on the basis of the gene primers.     

Enumeration of Obesumbacterium proteus in brewery yeasts and characterization of isolated strains using Biolog GN microplates and protein fingerprinting

Of a range of media tested for enumeration of Obesumbacterium proteus in brewers' yeast, Universal Beer agar and Wallerstein Laboratories Differential medium were most effective. MacConkey agar (several types) and Membrane Lauryl Sulphate agar were least effective. Other media (Wort agar, YM agar) were of intermediate efficacy. Nine O. proteus strains from commercial yeast samples were characterized using the API 20E test kit, the Biolog GN microplate (BGNM) and by SDS-PAGE of their total soluble proteins. Both the BGNM and SDS-PAGE techniques allowed the strains to be differentiated from one another: the API 20E kit did not. All strains isolated from UK breweries belonged to O. proteus biogroup II. Four of these strains displayed a branching cell morphology not hitherto described in any member of the Enterobacteriaceae.     

Microbial structural diversity estimated by dilution-extinction of phenotypic traits and T-RFLP analysis along a land-use intensification gradient

The present work tested whether the relationship between functional traits and inoculum density reflected structural diversity in bacterial communities from a land-use intensification gradient applying a mathematical model. Terminal restriction fragment length polymorphism (T-RFLP) analysis was also performed to provide an independent assessment of species richness. Successive 10-fold dilutions of a soil suspension were inoculated onto Biolog GN(R) microplates. Soil bacterial density was determined by total cell and plate counts. The relationship between phenotypic traits and inoculum density fit the model, allowing the estimation of maximal phenotypic potential (Rmax) and inoculum density (KI) at which Rmax will be half-reduced. Though Rmax decreased with time elapsed since clearing of native vegetation, KI remained high in two of the disturbed sites. The genetic pool of bacterial community did not experience a significant reduction, but the active fraction responding in the Biolog assay was adversely affected, suggesting a reduction in the functional potential.     

Relationship between luminous fish and symbiosis. I. Comparative studies of lipopolysaccharides isolated from symbiotic luminous bacteria of the luminous marine fish, Physiculus japonicus

In order to investigate the relationship between host and symbiosis in the luminous marine fish, Physiculus japonicus, the bacterial lipopolysaccharides (LPS) of symbiotic luminous bacteria were compared serologically and electrophoretically. Five symbiotic luminous bacteria (PJ strains) were separately isolated from five individuals of this fish species caught at three points, off the coasts of Chiba, Nakaminato, and Oharai. LPS preparations were made from these bacteria by Westphal's phenol-water method and highly purified by repeated ultracentrifugation. These LPSs contained little or no 2-keto-3-deoxyoctonate and had powerful mitogenic activity. In sodium dodecylsulfate polyacrylamide gel electrophoresis, these PJ-1 to -5 LPSs were separated by their electrophoretic patterns into three groups; the first group included PJ-1 and PJ-4, the second group PJ-2 and PJ-3, and the third group PJ-5 alone. The results agreed with those of the double immunodiffusion test; precipitin lines completely coalesced within each group but not with other groups. In immunoelectrophoresis, one precipitin line was observed between anti PJ-2 LPS serum and PJ-5 LPS but the electrophoretic mobility of PJ-5 LPS was clearly different from that of the PJ-2 LPS group. Furthermore, in a 50% inhibition test with PJ-2 LPS by the passive hemolysis system, the doses of PJ-2 LPS, PJ-3 LPS, and PJ-5 LPS required for 50% inhibition (ID50) in this system were 0.25, 0.25, and 21.6 micrograms/ml for each alkali-treated LPS, respectively, and the ID50's of both PJ-1 LPS and PJ-4 LPS were above 1,000 micrograms/ml. These results indicate that PJ-5 LPS has an antigenic determinant partially in common with LPS from the PJ-2 group but not with LPS from the PJ-1 group and that the symbiotic luminous bacterium PJ-5 is more closely related to the PJ-2 group than to the PJ-1 group. These results show that the species Physiculus japonicus is symbiotically associated with at least three immunologically different strains of luminous marine bacteria in its specialized light organ.     

Viability and metabolic features of bacteria indigenous to a contaminated deep aquifer

The quantitation and characterization of indigenous bacteria of a deep aquifer, located in the southwestern United States and contaminated with halogenated aliphatic compounds, was undertaken. Water samples were obtained aseptically from depths of 45 to 151 m from four sites that ranged from 260 to 1,800 m in distance from the location of contaminant release. Sediment samples were also obtained from the proximal and distal sites for analyses. Results for aerobic and anaerobic colony-forming units were obtained on four agar media that were used to retrieve heterotrophs, oligotrophs, and pseudomonads. Most probable number estimates were obtained from a liquid medium favorable for oligotrophs. Representative isolates were tested against Biolog plates (Biolog, Inc., Hayward, Calif.) for patterns of carbon source utilization. Of 103 Gram-negative (GN) isolates, 48 could not be identified and the others were only tentatively identified via the Biolog database, and none of the 35 Gram-positive (GP) isolates were identifiable. However, the metabolic patterns were subjected to average cluster linkage analyses; the GN and GP bacteria were separable into eight and four groups, respectively. The oligotroph group comprised one-third of the GN and one-half of the GP isolates. The consensus carbon source utilization pattern for each group was determined and will be useful in future characterization of additional aquifer bacterial isolates. Although predominantly aerobic and oligotrophic, the microbial community of this aquifer was highly diverse with discernible viability and metabolic features of the microbiota distinctive to each of the four water and two sediment samples. Correspondence to: C.M. McCarthy.     

Anomalies in species identification of enterococci from veterinary sources using a commercial biochemical identification system

AIMS: A commercial biochemical panel ID kit was used to identify presumptive enterococci isolates of veterinary or agricultural origin obtained during different steps of culture. METHODS AND RESULTS: Fifty isolates identified as enterococci using a genus PCR assay were tested for genus and species identification using the BBL Crystal Identification Gram-Positive ID kit (Becton Dickinson, Sparks, MD, USA). Following sub-culture of the isolates three times, 59% agreement with the original panel ID was obtained. After four and six sub-cultures, percentage agreement increased to 61 and 64%, respectively. Nineteen of the 50 cultures were identified as both Enterococcus faecalis and E. faecium. CONCLUSIONS: As a result of the variability between speciation of isolates following re-culture, additional methods for speciation are warranted. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that the identification of the genus and species of non-human enterococcal isolates can vary greatly during successive passages when using this kit.     

Intraspecific metabolic diversity among strains ofBurkholderia cepacia isolated from decayed onions,soils, and the clinical environment

A collection of 218 strains ofBurkholderia cepacia (including 18% strain replicates) was assembled from organic soils, decayed onions, and clinical sources. Each strain was characterized for virulence to onion, catabolic ability using the Biolog GN microtiter plate, and several other behaviors. Overall test reproducibility was estimated at 98%. The results obtained using the Biolog GN system corresponded well to those obtained using standard methods. Three coefficients of resemblance (Gower similarity, pattern difference, and Jaccard similarity) were calculated and clustered by the group-average method. The sorted matrices and phenograms, while giving evidence of an underlying phenetic structure to theB. cepacia nomenspecies, gave little evidence of sorting by broad source of isolation. Strains isolated from within fields or samples were frequently found to be similar, however, strains isolated from fields with similar cropping histories were not. The Gower-transformed centroids of ordained clusters were projected in a principal coordinate system and estimates of disjunction were calculated. Strains ofB. cepacia were shown to be non-uniformly distributed in taxonomic space. Strains isolated by serial dilution on onion slices formed a tight phenetic cluster which includes the type strain of the nomenspecies and that of a synonymous group (Pseudomonas multivorans); the strains in this phenon were generally virulent to onion and were partially differentiated from others by pectolytic behavior and by the production of diffusible pigment on King's medium A. Further characterization should better resolve the taxonomy of the nomenspecies.     

Molecular and phenotypic characterization of Pseudomonas spp. isolated from milk

Putative Pseudomonas spp. isolated predominantly from raw and processed milk were characterized by automated ribotyping and by biochemical reactions. Isolates were biochemically profiled using the Biolog system and API 20 NE and by determining the production of proteases, lipases, and lecithinases for each isolate. Isolates grouped into five coherent clusters, predominated by the species P. putida (cluster A), P. fluorescens (cluster B), P. fragi (as identified by Biolog) or P. fluorescens (as identified by API 20 NE) (cluster C), P. fragi (as identified by Biolog) or P. putida (as identified by API 20 NE) (cluster D), and P. fluorescens (cluster E). Isolates within each cluster also displayed similar enzyme activities. Isolates in clusters A, C, and D were generally negative for all three enzyme activities; isolates in cluster B were predominantly positive for all three enzyme activities; and isolates in cluster E were negative for lecithinase but predominantly positive for protease and lipase activities. Thus, only isolates from clusters B and E produced enzyme activities associated with dairy product flavor defects. Thirty-eight ribogroups were differentiated among the 70 isolates. Ribotyping was highly discriminatory for dairy Pseudomonas isolates, with a Simpson's index of discrimination of 0.955. Isolates of the same ribotype were never classified into different clusters, and ribotypes within a given cluster generally showed similar ribotype patterns; thus, specific ribotype fragments may be useful markers for tracking the sources of pseudomonads in dairy production systems. Our results suggest that ribogroups are generally homogeneous with respect to nomenspecies and biovars, confirming the identification potential of ribotyping for Pseudomonas spp.     

Bioluminescence intensity difference observed in luminous bacteria groups with different motility

Aims:  The aim of this study was to compare the luminescent intensity of bioluminescence from marine luminous bacteria with different motility.
Methods and Results:  Luminescent bacteria were separated according to their motility using a microfluidic device. The cell densities of the separated samples were measured using a counting plate. The luminescent intensity of the separated samples was measured using a luminometer. The luminescent intensity per cell was calculated, and the values from the mobile (swimmers) and the nonmobile cells (nonswimmers) per single cell were compared; as a result, the former were proved to be larger than the latter.
Conclusions:  Microfluidics were shown to be effective for the separation of bioluminescent bacteria and the bioluminescent intensity difference per cell was recognized with this experiment.
Significance and Impact of the Study:  This study introduced for the first time a method to examine the individual cell function of Photobacterium kishitanii .     

Habitats and Distribution Patterns of Marine Luminous Bacteria in the Western Baltic Sea

Numbers of luminous bacteria were counted at three stations of the brackish water ecosystem of the western Baltic Sea from July 1985 to July 1986. Additional samples were taken during three cruises from stations at the North Atlantic Ocean, the Norwegian Sea and adjacent marine areas. — In Kiel Bight (western Baltic) values varied between 0 and 68,000 luminous cfu 1⁻¹. With exception of the coastal station a distinct seasonal distribution pattern was shown in a water depth of 20 m: high numbers found in summer were opposed to low numbers in winter, the peaks being rather high in comparison to those of other areas. Statistical analysis showed that the results of 20 m were significantly different from those of 0 and 10 m depth; however, there was no correlation with temperature and salinity. Taxonomic studies revealed that the population consisted primarily of the genus Photobacterium. — The optimum of salinity was not a brackish but a marine one and was about 30% for the majority of the strains tested. A smaller number of strains grew best at a salinity between 10 and 15%. Optima of temperature ranged from 15 to 20 °C for most of the test strains. — Taxonomic analysis was also performed with luminous strains from marine areas adjacent to the western Baltic Sea, Photobacterium being the dominant genus here, too. Luminous bacteria were also enriched from the external surface and the gut contents of whitings (Merlangius merlangus) and cods (Gadus morhua). A model is proposed which explains the distribution pattern found. According to this, the gut-dwelling luminous bacteria are transported by their hosts from the North Sea into the western Baltic Sea. Here they are released into the environment, thus inhabiting another niche.     

Comparison of two kinds of Biolog microplates (GN and ECO) in their ability to distinguish among aquatic microbial communities.

We compared the abilities of Biolog's GN and ECO plates to distinguish among aerobic and heterotrophic bacterial communities in samples from six aquatic environments. The Biolog system is based on interpreting patterns of sole-carbon substrate utilization indicated by color development in a 96-well microtiter plate. Whether of fresh or saltwater origin, bacterial communities utilized > 95% of substrates in both types of plates. Samples from any one environment exhibited similar time courses of average well color development (AWCD) in both GN and ECO plates. Principal component analysis was performed on data sets resulting from combinations of algorithms (AWCD and curve-integration methods) and levels of color development (end-point and set-point approaches). In all cases, the two types of plates demonstrated an equal capacity to discriminate among the heterotrophic expressions of the six microbial communities. Substantial deviation from an anticipated 1:1 correspondence occurred when color development of 25 substrates common to both types of plates was compared. The discrepancies likely are related to the different formulations of low-nutrient media in GN and ECO plates.     

Contribution by symbiotically luminous fishes to the occurrence and bioluminescence of luminous bacteria in seawater

Seawater samples from a variety of locations contained viable luminous bacteria, but luminescence was not detectable although the system used to measure light was sensitive enough to measure light from a single, fully induced luminous bacterial cell. When the symbiotically luminous fishCleidopus gloriamaris was placed in a sterile aquarium, plate counts of water samples showed an increase in luminous colony-forming units. Luminescence also increased, decreasing when the fish was removed. Light measurements of water samples from a sterile aquarium containingPhotoblepharon palpebratus, another symbiotically luminous fish, whose bacterial symbionts have not been cultured, showed a similar pattern of increasing light which rapidly decreased upon removal of the fish. These experiments suggest that symbiotically luminous fishes release brightly luminous bacteria from light organs into their environment and may be a source of planktonic luminous bacteria. Although planktonic luminous bacteria are generally not bright when found in seawater, water samples from environments with populations of symbiotically luminous fish may show detectable levels of light.