Sherlock Microbial Identification System Research Articles

First Outbreak of Bacterial Black Rot on Cabbage, Broccoli, and Brussels Sprouts Caused by Xanthomonas campestris pv. campestris in the Mediterranean Region of Turkey

During warm and humid periods in the winters of 2004 to 2006, severe leaf necrosis and vein rot symptoms were observed on cabbage (Brassica oleracea var. capitata L.), broccoli (Brassica oleracea var. italica Plenck.), and Brussels sprouts (Brassica oleracea var. gemmifera D.C.) in the Mediterranean Region of Turkey. Symptoms were characterized by yellow, V-shaped areas of the leaf margin, with the internal tissue turning from brown to black. Infected seedlings were also observed in commercial nurseries in Adana with a disease incidence of nearly 10 to 25%. Isolations made from leaves and veins of the affected plants on yeast dextrose calcium carbonate agar yielded yellow, mucoid, and convex colonies. Twenty isolates recovered from diseased leaf samples were selected at random to identify the causal organism. All isolates were nonspore forming, gram negative, rod shaped, motile, aerobic, oxidase-negative, catalase-positive, and amylolytic-positive (3). All isolates induced hypersensitive responses on tobacco (Nicotiana tabacum cv. Samsun). The isolates were identified as Xanthomonas campestris pv. campestris on the basis of fatty acid methyl ester (FAME) profiles determined by Sherlock Microbial Identification System software (Microbial ID, Newark, DE) and indirect ELISA. The similarity indices for the FAME analysis ranged from 80 to 94% (2). Indirect ELISA with a polyclonal antibody (Agdia, Elkhart IN; BRA 97000/0500) further confirmed the identity of the pathogen in both pure culture and infected plant. The mean absorbance values for three replications of indirect ELISA tests ranged from 1.411 to 3.508 at a wavelength of A405 (1). Pathogenicity of the isolates was tested on 5-week-old cabbage plants by spray inoculation using bacterial suspensions (107 CFU/ml) prepared in saline buffer (0.85% NaCl). Sterile saline buffer was sprayed on negative control plants. Inoculated and control plants were maintained for 5 days at 25°C and 70% relative humidity to observe symptom development. No symptoms developed on negative control plants. The bacterium was reisolated from inoculated cabbage plants and identified as X. campestris pv. campestris by FAME and an ELISA test. To our knowledge, this is the first report of the occurrence and outbreak of X. campestris pv. campestris in the Mediterranean Region of Turkey.

First Report of Soft Rot Caused by Pectobacterium carotovorum on Pinellia ternata in China.

Pinellia ternata (Thumb.) Breit. (Araceae) is a traditional herb used as an antivomit, anticough, analgesic, and sedative in China for more than 1,000 years. From the summer through fall of 2003 to 2005, a high incidence (approximately 10 to 25%) of disease outbreaks characterized by water-soaked lesions and soft rot with a stinky odor were observed in the cultivated P. ternata in Shanxi Province. Water-soaked lesions were first observed on the stem base and then the plant collapsed, although the upper portion remained asymptomatic. Subsequently, the lesions expanded rapidly over the entire plant. The macerated tuber was usually reduced to a whitish, mushy, and foul-smelling pulp surrounded by undecayed periderm. A Pectobacterium species was consistently recovered from the diseased tubers on nutrient agar media. Koch's postulates were completed by stab inoculating 6-week-old peach- and willow-leaved P. ternata cultivars with the bacterial suspensions (1 × 108 CFU/ml) (1). Ten control plants for each cultivar were inoculated with sterile water. After inoculation, plants were maintained in a growth chamber at 25°C with relative humidity ranging from 80 to 90%. After 2 to 3 days, typical soft rot symptoms were observed on the inoculated plants. A Pectobacterium species was reisolated from the symptomatic tubers while control plants remained healthy. This experiment was repeated in May, July, and September. The pathogenic isolates were identified as typical Pectobacterium carotovorum on the basis of morphological, physiological, and biochemical characteristics (2). The Microlog system (version 3.5; Biolog, Hayward, CA) and Sherlock Microbial Identification System (version 4.5; MIDI Newark, DE) also identified them as Pectobacterium carotovorum on the basis of similarity indices more than 66.9 and 78.2%, respectively. Their identity was then confirmed by sequencing the gene encoding the 16S rRNA (GenBank Accession No DQ785511). To our knowledge, this is the first report of Pectobacterium carotovorum as the cause of soft rot of P. ternata.

Bacterial Populations of Broiler Carcasses Washed in Mixtures of Potassium Hydroxide and Lauric Acid

The bactericidal effect of mixtures of KOH and lauric acid (LA) on the microflora of broiler carcasses was examined. Carcasses were washed by shaking in KOH-LA for 1 min on a mechanical shaker. In one set of experiments, the population of bacteria recovered from carcasses following each of 3 successive washes in 1.0% KOH-2.0% LA or in distilled water (control) was enumerated. The number of total plate count bacteria, Campylobacter, and Escherichia coli in aliquots of whole-carcass-rinses of the washed carcasses was determined. Results indicated that fewer bacteria were generally recovered from carcasses after each successive wash in KOH-LA or distilled water, but significantly fewer bacteria were recovered from carcasses washed in KOH-LA than from carcasses washed in distilled water. Bacteria recovered from carcasses after the first and third wash in KOH-LA were identified using the MIDI Sherlock Microbial Identification System. Gram-positive and gram-negative bacteria were identified in the bacterial flora of carcasses washed once in KOH-LA; however, only gram-positive cocci were identified in the bacterial flora of carcasses washed 3 times in KOH-LA. Additional experiments were performed to compare the number of bacteria recovered from carcasses washed 2 times in 0.25% KOH-0.5% LA, 0.5% KOH-1.0% LA, or 1.0% KOH-2.0% LA or in distilled water (control). Results indicated that significantly fewer bacteria were recovered from carcasses washed in higher concentrations of KOH-LA than from carcasses washed in lower concentrations of KOH-LA. Findings from these experiments show that washing carcasses in KOH-LA can reduce carcass contamination by bacteria responsible for human foodborne diseases and spoilage of fresh poultry.

Utility of gas chromatography for rapid identification of mycobacterial species frequently encountered in clinical laboratory

Over the last years, the clinical importance of mycobacteria has been raised. In this regard, it is important their identification in order to establish either the clinical significance or the appropriate therapy of the disease. Biochemical tests are usually time consuming until the report of results, that is why more rapid techniques are needed. As an alternative identification method, we have used a commercially available system for microbial identification based on whole cellular fatty acids analysis using gas-chromatography (GC). Sixty-eight strains of Mycobacterium tuberculosis, Mycobacterium gordonae, Mycobacterium xenopi, Mycobacterium kansasii, Mycobacterium fortuitum, and Mycobacterium avium-intracellulare were clearly identified by their unique fatty acid profile using the Sherlock Microbial Identification System (MIS). The results were in agreement with those obtained with traditional methods. This method is highly automated, rapid, easy to perform with a sample preparation for lipid analysis which is neither time consuming nor requiring a particular expertise. On this basis the MIS-GC method for the identification of some clinically important mycobacteria appears to be suitable for routine clinical use.

Evaluation of the BD Phoenix Automated Microbiology System for Identification and Antimicrobial Susceptibility Testing of Staphylococci and Enterococci

We evaluated the Phoenix automated microbiology system (BD Diagnostic Systems, Sparks, MD) for the identification (ID) and antimicrobial susceptibility testing (AST) of challenge and clinical staphylococci and enterococci recovered from patients in a tertiary-care medical center. In total, 424 isolates were tested: 90 enterococci; 232 Staphylococcus aureus isolates, including 14 vancomycin-intermediate S. aureus isolates; and 102 staphylococci other than S. aureus (non-S. aureus). The Phoenix panels were inoculated according to the manufacturer's instructions. The reference methods for ID comparisons were conventional biochemicals and cell wall fatty acid analysis with the Sherlock microbial identification system (v 3.1; MIDI, Inc. Newark, DE). Agar dilution was the reference AST method. The overall rates of agreement for identification to the genus and the species levels were 99.7% and 99.3%, respectively. All S. aureus isolates and enterococci were correctly identified by the Phoenix panels. For the non-S. aureus staphylococci, there was 98.0% agreement for the ID of 16 different species. The AST results were stratified by organism group. For S. aureus, the categorical agreement (CA) and essential agreement (EA) were 98.2% and 98.8%, respectively. Three of three very major errors (VMEs; 1.7%) were with oxacillin. For non-S. aureus staphylococci, the CA, EA, VME, major errors, and minor error rates were 95.7%, 96.8%, 0.7%, 1.7%, and 2.9%, respectively. The two VMEs were with oxacillin. For the enterococci, there was 100% CA and 99.3% EA. All 36 vancomycin-resistant enterococci were detected by the Phoenix system. The Phoenix system compares favorably to traditional methods for the ID and AST of staphylococci and enterococci.

Molecular characterisation of Xanthomonas strains isolated from aroids in Mauritius

Mauritius is one of the largest world producers of Anthurium cut flowers but outbreaks of bacterial blight have never been reported on the island. This work was about the characterisation and identification of bacterial strains isolated from Anthurium andreanum, Dieffenbachia maculata and Aglaonema simplex in Mauritius. Fifteen strains, that showed the morphological properties of Xanthomonas on conventional media, were tested on two semi-selective media (Esculin-trehalose and cellobiose-starch). ELISA tests using a panel of monoclonal antibodies were carried out and three out of 15 strains reacted with a Xanthomonas-specific monoclonal antibody (MAb XII). Analysis using four sets of ribosomal primers revealed that the same three Mauritius strains shared conserved PCR products with reference xanthomonads including virulent strains of Xanthomonas axonopodis pv. dieffenbachiae ( Xad). BIOLOG tests and the Sherlock Microbial Identification system (MIDI) identified these three new strains at the species level as X. axonopodis. The complementary tests that were carried out clearly confirmed that the three strains are xanthomonads and, moreover, a DNA probe which showed specificity to Xad strains suggested that the three Mauritius strains are non-virulent forms of the pathogen causing Anthurium blight.

Cellular Fatty-Acid Analysis of Bacillus thuringiensis Var. kurstaki Commercial Preparations

Cellular fatty acid (CFA) composition of Bacillus thuringiensis var. kurstaki (Btk) preparations was determined by use of the MIDI Sherlock microbial identification system on a Hewlett-Packard 5890 gas chromatograph. Four commercial preparations--one Btk sample obtained from the U.S. Forest Service, one Btk sample obtained from Dugway Proving Ground, and Btk and Bacillus thuringiensis var. israelensis (Bti) preparations obtained from American Type Culture Collection (ATCC)--were analyzed and evaluated. This study demonstrated the capability to detect the strain variation in the bacterial species Btk and to clearly differentiate strain variants on the basis of qualitative and quantitative differences in hydrolyzable whole CFA compositions in the preparations examined. We conclude that CFA analysis may be used to identify commercial products but that a more intensive study would be required to evaluate the potential of CFA to provide an inexpensive screening tool applicable to several levels of isolate or product evaluation, including how applied preparations might interact with natural populations over time.

Content and composition of fatty acids in normal and inflamed gingival tissues

The balance of essential fatty acid is important for good health and normal development. Essential fatty acids (EFA) are the precursors of prostaglandins (PGs), thromboxanes and leukotrienes (LT). The aim of this clinical study was to determine the total fatty acid level of polyunsaturated fatty acids (PUFA), monounsaturated fatty acids (MUFA), saturated fatty acids (SFA) and each fatty acids level of inflamed and normal gingival tissues. Twenty-seven subjects were included the present study. Nineteen samples of inflamed human gingival tissue (nine of fibrous hyperplasia (FH), ten of peripheral giant cell granuloma (PGCG) and eight samples of normal human gingival tissue were analyzed. The characteristics of inflammation were assessed histologically. Variance analyses were performed to assess the differences among tissues. The total cellular fatty acid profiles of the tissues in inflamed human gingival tissue and in eight samples of normal human gingival tissue were determined by gas chromatography using Sherlock microbial identification system (MIS) software (Microbial ID, Newark, DE, USA) with a database of FAME profiles for eukary. PUFAs, MUFAs, and SFAs were quantified by Sherlock microbial identification system (MIS) or database gas chromatography (DGC). There were statistically significant differences between the concentrations in inflamed (FH, PGCG) and healthy gingival tissues for PUFA and MUFA ( P < 0.001 , P < 0 , 011 , respectively). There were statistically significant differences among the concentrations in FH, PGCG, and healthy gingival tissues for SFA ( P < 0.0001 ). Arachidonic acid, docosahexaenoic acid, linoleic acid were increased in inflamed tissue. The results of this study showed that unsaturated fatty acids (PUFA and MUFA) significantly increased in inflamed gingival tissues.

Bacterial Canker Caused by Clavibacter michiganensis subsp. michiganensis on Greenhouse-Grown Tomato in the Western Mediterranean Region of Turkey.

During the 2003 winter growing season, a disease occurred on tomato (Lycopersicon esculentum L.) cvs. Selin, Astona, 198 RN, Malike, and Tulin that were growing in almost all greenhouses of Serik, Aksu, Kumluca, Demre, and Kinik in the Antalya Province, and Keçiborlu, Çandir, and Şeyhler in the Isparta Province in the western Mediterranean Region of Turkey. Disease incidence ranged from 26 to 65%, resulting in economically heavy losses. Symptoms were water-soaked, dark brown-to-black lesions on the leaf margins and asymmetrical wilting of the leaflets. In advanced stages of disease, vascular tissue had a light brown discoloration. A gram-positive bacterium was consistently isolated and formed light gray colonies with internal light gray flecks approximately 2 to 3 mm in diameter on the modified semiselective medium (SCM) (2). Thirty isolates were identified as Clavibacter michiganensis subsp. michiganensis using polymerase chain reaction (PCR) with primers CMM 5 and CMM 6 on the basis of consistent amplification of a 614-bp DNA fragment (1). The isolates were also identified as C. michiganensis subsp. michiganensis (similarity 0.810 to 0.888%) using fatty acid methyl ester (FAME) analysis and Sherlock Microbial Identification System software (Microbial ID, Newark, DE). A pathogenicity test was done by spraying a 6-week-old tomato seedling (cv. Selin) with the bacterial concentration of 108 CFU/ml in sterile water. Control tomato seedlings were only sprayed with sterile water. The inoculated plants were covered with polyethylene bags for 48 h and kept in a growth chamber at 25°C. Symptoms developed on the inoculated plants within approximately 10 days and were similar to those observed in the greenhouse. No symptoms developed on control plants. The bacterium was reisolated from inoculated plants, and its identity was confirmed by colony type on SCM, PCR, and FAME analysis. Although it has been known to be present previously in Aegean, eastern Mediterranean, and the eastern Anatolia regions of Turkey, to our knowledge, this is the first report of bacterial canker on greenhouse tomatoes in the western Mediterranean Region of Turkey. Occurrence of the disease in almost all greenhouses may be the result of the high relative humidity present and the lack of protective bactericide applications in many greenhouses of the region.

Use of MIDI–Fatty Acid Methyl Ester Analysis To Monitor the Transmission of Campylobacter during Commercial Poultry Processing

The presence of Campylobacter spp. on broiler carcasses and in scald water taken from a commercial poultry processing facility was monitored on a monthly basis from January through June. Campylobacter agar, Blaser, was used to enumerate Campylobacter in water samples from a multiple-tank scalder; on prescalded, picked, eviscerated, and chilled carcasses; and on processed carcasses stored at 4°C for 7 or 14 days. The MIDI Sherlock microbial identification system was used to identify Campylobacter-like isolates based on the fatty acid methyl ester profile of the bacteria. The dendrogram program of the Sherlock microbial identification system was used to compare the fatty acid methyl ester profiles of the bacteria and determine the degree of relatedness between the isolates. Findings indicated that no Campylobacter were recovered from carcasses or scald tank water samples collected in January or February, but the pathogen was recovered from samples collected in March, April, May, and June. Processing generally produced a significant (P < 0.05) decrease in the number of Campylobacter recovered from broiler carcasses, and the number of Campylobacter recovered from refrigerated carcasses generally decreased during storage. Significantly (P < 0.05) fewer Campylobacter were recovered from the final tank of the multiple-tank scald system than from the first tank. MIDI similarity index values ranged from 0.104 to 0.928 based on MIDI–fatty acid methyl ester analysis of Campylobacter jejuni and Campylobacter coli isolates. Dendrograms of the fatty acid methyl ester profile of the isolates indicated that poultry flocks may introduce several strains of C. jejuni and C. coli into processing plants. Different populations of the pathogen may be carried into the processing plant by successive broiler flocks, and the same Campylobacter strain may be recovered from different poultry processing operations. However, Campylobacter apparently is unable to colonize equipment in the processing facility and contaminate broilers from flocks processed at later dates in the facility.

Spread of Campylobacter spp. During Poultry Processing in Different Seasons

The presence of Campylobacter spp. on broiler carcasses and in scald tank water in a commercial poultry processing facility was monitored at monthly intervals from July through December. The spread of the pathogen had previously been monitored in the same facility from January through June of the same year. Campylobacter were enumerated on prescalded, picked, eviscerated, and chilled broiler carcasses; on processed carcasses stored at 4 C for 7 or 14 days, and in scald tank water samples. The fatty acid methyl o ester (FAME) profile of the Campylobacter isolates and the degree of relatedness between the Campylobacter isolates was determined using the MIDI Sherlock Microbial Identification System (MIS). Findings indicated that Campylobacter jejuni was present on carcasses and in scald tank water samples collected from July through December. Processing significantly (P < 0.05) decreased the number of Campylobacter recovered from broiler carcasses, however. Furthermore, significantly (P < 0.05) fewer C. jejuni were consistently recovered from the third tank of the multiple tank scald system than from the first tank. Findings indicated that poultry flocks may introduce several strains of C. jejuni into processing facilities. Additionally, different populations of the pathogen may be carried into the processing plant by successive broiler flocks, and some strains of C. jejuni may reappear in the same processing facility during different times of the year.

First report of bacterial blight of anthurium caused by Xanthomonas axonopodis pv. dieffenbachiae in Turkey

In the spring of 2001, a serious outbreak of a suspected bacterial blight disease was observed on anthurium plants (Anthurium andreanum) grown for potted plant production in commercial glasshouses of the Mediterranean region in Turkey. Disease incidence was recorded in the range 20–25%. Bacterial blight symptoms evident on lower surface leaves were small, yellowish water-soaked spots that enlarged and coalesced. Eventually, brown to black blighted areas were often observed together with a striking yellow zone along leaf margins. A gummy bacterial exudate sometimes flowed down from the infected plant parts. Stems appeared black, wet and slimy from the bacterial growth in the pith area. A yellow-pigmented bacterium was consistently isolated from diseased plants onto ET Agar (Norman & Alvarez, 1989). Eighteen representative strains were isolated and all were Gram-negative, rod-shaped, motile, aerobic, oxidase-negative, catalase-positive and amylolytic-positive. They all produced positive hypersensitive reactions on tobacco (Nicotiana tabacum cv. Samsun). All of the strains were identified as Xanthomonas axonopodis pv. dieffenbachiae (Xad) based on fatty acid methyl ester (FAME) profiles determined by Sherlock Microbial Identification System software (Microbial ID, Newark, DE, USA) (Bouzar et al., 1994; Jones et al., 1998), and pathogenicity studies on anthurium plants. This is the first report of the observation of Xad in Turkey.

Prevalence and in-vitro antimicrobial susceptibility patterns of Acinetobacter strains isolated from patients in intensive care units.

Fifty-six Acinetobacter species strains (49 Acinetobacter baumanii, 5 Acinetobacter calcoaceticus, 2 Acinetobacter iwoffii) were detected using both conventional methods and gas chromatography of bacterial fatty acids with the MIDI Sherlock Microbial Identification System. The susceptibilities of these strains to 16 antimicrobial agents were investigated by the disc-diffusion method according to the National Committee for Clinical Laboratory Standards. The production of extended-spectrum beta-lactamases (ESBLs) and inducible beta-lactamases (IBLs) by the strains were investigated by the double-disc-synergy and disc-approximation methods, respectively. Imipenem was the most effective agent for Acinetobacter baumanii strains (95.9% of strains were sensitive), while meropenem and netilmicin showed moderate activity (87.7% and 79.6% of strains, respectively, responded). Acinetobacter baumanii strains were less sensitive to cefoperazone-sulbactam (53.1%), ofloxacin (51.0%), ciprofloxacin (42.8%), and amikacin (36.7%). Acinetobacter calcoaceticus and Acinetobacter iwoffii strains were sensitive to imipenem, meropenem and netilmicin. IBLs and ESBLs were produced, respectively, by 8.9% and 7.1% of all bacterial strains. The strains isolated were sufficiently sensitive to imipenem, but not to ofloxacin or ciprofloxacin, and were very resistant to amikacin.

Enumeration and Identification of Yeasts Associated with Commercial Poultry Processing and Spoilage of Refrigerated Broiler Carcasses

Yeasts associated with broiler carcasses taken from various stages of commercial poultry processing operations and broiler carcasses stored at refrigerated temperatures were enumerated and identified. Whole carcass rinses were performed to recover yeasts from carcasses taken from a processing facility and processed carcasses stored at 4°C for up to 14 days. Yeasts in the carcass rinsates were enumerated on acidified potato dextrose agar and identified with the MIDI Sherlock Microbial Identification System. Dendrograms of fatty acid profiles of yeast were prepared to determine the degree of relatedness of the yeast isolates. Findings indicated that as the carcasses are moved through the processing line, significant decreases in the number of yeasts associated with broiler carcasses usually occur, and the composition of the yeast flora of the carcasses is altered. Significant (P < 0.05) increases in the yeast population of the carcasses generally occur during storage at 4°C, however. Furthermore, it was determined that the same strain of yeast may be recovered from different carcasses at different points in the processing line and that the same strain of yeast may be isolated from carcasses processed on different days in the same processing facility.

Bacterial canker, caused by Clavibacter michiganensis ssp. michiganensis, on tomatoes in eastern Anatolia region of Turkey

In 2001, a disease occurred on tomato (Lycopersicon esculentum) cv. Target growing in six different commercial fields in the production areas of Oltu, Ispir and Yusufeli in the eastern Anatolia region of Turkey. Disease incidence was almost 100%, resulting in heavy losses. Initial symptoms were small (1–3 mm in diameter), water-soaked, dark brown to black lesions on the leaf margins and stems, and white, raised ‘bird’s eye’ spots on fruits. Defoliation, one-sided wilting, tip die-back, open stem cankers and vascular discoloration were observed on plants in advanced stages of the disease. These symptoms were consistent with those of bacterial canker described by Vidaver & Davis (1988) and were found only on cv. Target. A slow-growing, Gram-positive, pale-yellow bacterium was consistently isolated on yeast dextrose carbonate agar medium (Lelliot & Stead, 1987). Sixteen representative isolates were identified as Clavibacter michiganensis ssp. michiganensis (similarity 47–89%) using fatty acid methyl ester (FAME) analysis and Sherlock Microbial Identification System software (Microbial ID, Newark, DE, USA) (Gitaitis & Beaver, 1990). Pathogenicity was tested by spraying 5-week-old-tomato seedlings (cv. Easy Harvest) with bacteria suspended (108 cfu mL−1) in sterile water. Control plants were sprayed with sterile water. After inoculation plants were covered with polyethylene bags for 48 h and maintained in a greenhouse at 25°C. Symptoms similar to those observed in the field developed on the inoculated plants within 7–13 days. No symptoms developed on control plants. The bacterium was re-isolated from inoculated plants and its identity confirmed by FAME analysis. Although it has been reported previously in Aegean and Mediterranean regions (Özaktan, 1991), this is the first record of bacterial canker on tomatoes in the eastern Anatolia region of Turkey. Occurrence of the disease only on plants of cv. Target suggests that contaminated seeds and/or transplants of cv. Target may have been the source of the pathogen.

Occurrence of Bacterial Stripe of Pearl Millet in Georgia.

Bacterial stripe, caused by Acidovorax avenae subsp. avenae, was observed on breeding lines of pearl millet (Pennisetum glaucum (L.) R. Br.) in Georgia in 1999 and 2001. A gram-negative, oxidase-positive, rod-shaped bacterium that produced circular, cream-colored, nonfluorescent, butyrous colonies with entire margins on King's medium B was consistently isolated from leaf lesions. The bacterium was identified as A. avenae subsp. avenae by gas-chromatography of extracted, whole-cell, fatty acid methyl esters using the Sherlock Microbial Identification System (MIDI, Newark, DE) and by substrate utilization patterns using the Biolog Identification System (Biolog Inc., Hayward, CA). Isolates from pearl millet produced amplicons of expected size (360 bp) from 16S rDNA after conducting polymerase chain reaction (PCR) with primers WFB1 and WFB2, which are specific for A. avenae. When bacterial suspensions of 1 × 108 CFU/ml were infiltrated into the intercellular spaces of leaves of pearl millet seedlings in the greenhouse, typical water-soaked, reddish-brown stripes developed and were identical to those observed in the field. In contrast to previous reports (1), the pearl millet strains produced atypical symptoms on sweet corn (cvs. Merit and Primetime). Necroses were restricted, lacked customary water-soaking, and were similar to symptoms produced by the watermelon pathogen, A. avenae subsp. citrulli, which was used as a negative control. In contrast, three strains of A. avenae subsp. avenae previously isolated from corn in Georgia produced typical water-soaked stripes in both millet and the sweet corn 'Merit'. However, like the millet strains, A. avenae subsp. avenae strains from corn produced atypical symptoms on the sweet corn 'Primetime'. Using immunomagnetic separation and PCR (2), A. avenae subsp. avenae was detected in remaining samples of pearl millet seed planted in Georgia in 2001, as well as in remnant samples of seed sent to Puerto Rico for increase in 2000. The A. avenae subsp. avenae strain recovered from seed was identified by the methods listed above, and in the greenhouse it was identified by the production of typical water-soaked stripes after inoculation of pearl millet. This is the first report of A. avenae subsp. avenae infecting pearl millet in the United States. The detection and distribution of seedborne inoculum in breeding lines is significant since the program at Tifton represents a major effort by the U.S. Department of Agriculture to develop higher-yielding, disease-resistant pearl millet hybrids. Furthermore, the strains from pearl millet appear to be different from previous A. avenae subsp. avenae strains isolated from corn in Georgia, because they did not produce typical disease symptoms when infiltrated in corn leaves.

Differentiation between Candida dubliniensis and Candida albicans by fatty acid methyl ester analysis using gas-liquid chromatography.

Candida dubliniensis is often found in mixed culture with C. albicans, but its recognition is hampered as the color of its colonies in primary culture on CHROMagar Candida varies. Furthermore, definite identification of C. dubliniensis is difficult to achieve, time-consuming, and expensive. Therefore, a method to discriminate between these two closely related yeast species by fatty acid methyl ester (FAME) analysis using gas-liquid chromatography (Sherlock Microbial Identification System [MIS]; MIDI, Inc., Newark, Del.) was developed. Although the chromatograms of these two species revealed no obvious differences when applying FAME analysis, a new library (CADLIB) was successfully created using Sherlock Library Generation Software (MIDI). The amount and frequency of FAME was analyzed using library training files (n = 10 for each species), preferentially those comprising reference strains. For testing the performance of the CADLIB, clinical isolates genetically assigned to the respective species (C. albicans, n = 32; C. dubliniensis, n = 28) were chromatographically analyzed. For each isolate tested, MIS computed a similarity index (SI) indicating a hierarchy of possible strain fits. When using the newly created library CADLIB, the SIs for C. albicans and C. dubliniensis ranged from 0.11 to 0.96 and 0.53 to 0. 93 (for all but one), respectively. Only three isolates of C. albicans (9.4%) were misidentified as C. dubliniensis, whereas all isolates of C. dubliniensis were correctly identified. Resulting differentiation accuracy was 90.6% for C. albicans and 100% for C. dubliniensis. Cluster analysis and principal component analysis of the resulting FAME profiles showed two clearly distinguishable clusters matching up with two assigned species for the strains tested. Thus, the created library proved to be well suited to discriminate between these two species.

Analysis of cellular fatty acids and phenotypic relationships of Agrobacterium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium species using the Sherlock Microbial Identification System.

Previous studies have demonstrated that cellular fatty acid analysis is a useful tool for identifying unknown strains of rhizobia and establishing taxonomic relationships between the species. In this study, the fatty acid profiles of over 600 strains belonging to the genera Agrobacterium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium were evaluated using the gaschromatography-based Sherlock Microbial Identification System (MIS). Data collected with the MIS showed that the three phylogenetically defined biovars of the genus Agrobacterium formed discrete clusters, whilst species belonging to the genus Mesorhizobium formed three subclusters which were easily distinguished. These three subclusters contained Mesorhizobium ciceri and Mesorhizobium mediterraneum, Mesorhizobium tianshanense fatty acid group I and Mesorhizobium plurifarium, and Mesorhizobium huakuii and Mesorhizobium loti. The genus Sinorhizobium was composed of an individual position for Sinorhizobium meliloti and a large cluster comprising Sinorhizobium fredii, Sinorhizobium saheli, Sinorhizobium terangae, Sinorhizobium kostiense and Sinorhizobium arboris. S. meliloti contained significantly higher levels of the fatty acid 19:0 cyclo omega 8 cis and clustered with Rhizobium sp. (Hedysarum coronarium). However, discrimination between the species of genera Sinorhizobium and Rhizobium was a function of the concentration of 16:0 3-OH. The genus Rhizobium contained a single cluster containing Rhizobium sp. (Hedysarum coronarium), Rhizobium gallicum, Rhizobium leguminosarum and Rhizobium etli, along with individual positions for Rhizobium giardinii, Rhizobium tropici, Rhizobium galegae and Rhizobium hainanense. R. tropici and R. hainanense exhibited similarity to Agrobacterium biovar 2, whilst R. galegae was similar to Agrobacterium biovar 1. R. giardinii appeared unique, with comparatively little similarity to the other species. Analysis of the genus Bradyrhizobium revealed large differences from the other genera studied. Two subgroups of Bradyrhizobium elkanii were detected and easily distinguished from Bradyrhizobium japonicum. Bradyrhizobium liaoningense and Bradyrhizobium sp. (Arachis hypogaea), a group isolated from Chinese peanut plants, showed similarities to B. japonicum, whilst a subgroup of M. tianshanense appeared identical to Bradyrhizobium sp. (Arachis hypogaea).

Characterization of the periodontal microflora by the fatty acid profile of the broth-grown microbial population

The applicability of fatty acid analysis to the characterization of periodontal microflora was investigated using gas-liquid chromatography (GLC) and the software of the Sherlock Microbial Identification System (MIS) from MIDI Inc. Sulcus fluid was collected with paper points and anaerobically cultured in broth at 35 degrees C for four days. The broth-grown microbial population was extracted and the fatty acid methyl esters (FAME) were separated by GLC. The investigation of 67 specimens from asymptomatic sulci and of 32 specimens from inflamed sulci showed that the patterns of FAME profiles, the clustering of FAME profiles by computerized 2-D plot procedure, and the determination of the peak area index (PAI) of the FAME profiles differentiate between normal and pathological sulcus flora. Comparison of the clinical sulcus rating and the FAME data indicated that a pathological FAME profile may precede manifest periodontitis, and the normalization of the FAME profile may precede healing. It is concluded that the FAME analysis of sulcus fluid is a diagnostic aid for periodontological surveillance, for the initiation of preventive treatment of periodontitis, and for controlling the antimicrobial efficiency of therapeutic measures.