Traditional Biochemical Tests Research Articles

Characterization of human clinical isolates of Dietzia species previously misidentified as Rhodococcus equi

In this study, 16 human clinical isolates of Dietzia species previously misidentified as Rhodococcus equi were evaluated using phenotypic methods, including traditional and commercial (API Coryne) biochemical tests, antimicrobial susceptibility testing, and 16S rRNA gene and gyrB gene sequencing. Positive results for both the hydrolysis of adenine and Christie-Atkins-Munch-Petersen (CAMP) reaction allowed for differentiation between the Dietzia isolates and the type strain of Rhodococcus equi; however, traditional and commercial phenotypic profiles could not be used to reliably identify Dietzia species. The analysis of 16S rRNA gene and gyrB gene sequences could discriminate all Dietzia strains from the type strain of R. equi. Most Dietzia species had distinct 16S rRNA gene and gyrB gene sequences; however, the 16S rRNA gene sequences of the type strains of D. schimae and D. cercidiphylli were identical to D. maris and D. natronolimnaea, respectively. Based on comparative sequence analysis, five clinical isolates clustered with D. maris/D. schimae and nine with D. natronolimnaea/D. cercidiphylli. The two remaining isolates were found to be most closely related to the D. cinnamea/D. papillomatosis clade. Even though molecular analyses were not sufficiently discriminative to accurately identify all Dietzia species, the method was able to reliably identify isolates that were previously misidentified by phenotypic methods to the genus level.

Comparative Analysis of PCR–Electrospray Ionization/Mass Spectrometry (MS) and MALDI-TOF/MS for the Identification of Bacteria and Yeast from Positive Blood Culture Bottles

BACKGROUNDEmerging technologies for rapid identification of microbes demonstrate a shift from traditional biochemical and molecular testing algorithms toward methods using mass spectrometry (MS) for the semiquantitative analysis of microbial proteins and genetic elements. This study was performed to assess the diagnostic accuracy of 2 such technologies, PCR–electrospray ionization (ESI)/MS and MALDI-TOF/MS, with respect to phenotypic and biochemical profiling as a reference standard method. A positive challenge set of blood culture bottles was used to compare PCR-ESI/MS and MALDI-TOF/MS performance on a matched set of samples.METHODSWe performed characterization of bloodstream infections from blood cultures using the Ibis T5000 PCR-ESI/MS and the Bruker MALDI Biotyper 2.0 (MALDI-TOF/MS) platforms for microbial identification. Diagnostic accuracy was determined by independent comparison of each method to phenotypic and biochemical characterization with Vitek2 analysis as the reference standard identification.RESULTSThe diagnostic accuracy, represented as positive agreement, at the genus level was 0.965 (0.930–0.984) for PCR-ESI/MS and 0.969 (0.935–0.987) for MALDI-TOF/MS, and at the species level was 0.952 (0.912–0.974) with PCR-ESI/MS and 0.943 (0.902–0.968) for MALDI-TOF/MS. No statistically significant difference was found between PCR-ESI/MS and MALDI-TOF/MS in the ability to rapidly identify microorganisms isolated from blood culture.CONCLUSIONSOur results demonstrate that PCR-ESI/MS and MALDI-TOF/MS are equivalent in their ability to characterize bloodstream infections with respect to the reference standard, and highlight key differences in the methods that allow for each method to have a unique niche as a tool for rapid identification of microbes in blood cultures.

Biochemical characters and antibiotic susceptibility of Staphylococcus aureus isolates

Objective To observe the biochemical characters and antibiotic susceptibility of isolated Staphylococcus aureus (S. auerus) strains against some conventional and traditional antibiotics. Methods Thirty post operative pathogenic isolated S. aureus strains were used in this study. Bacterial culture was done in Mueller-Hinton broth at 37 °C. Characters of these strains were determined by traditional biochemical tests such as hydrolysis test of gelatin, urea, galactose, starch and protein, and fermentation of lactose and sucrose. Antibiotic susceptibility were carried out by minimum inhibitory concentration test, minium bactericidal concentration test, disc agar diffusion test and brain heart infusion oxacillin screening agar. Results From this study, it was observed that 100% S. aureus isolates showed positive results in gelatin, urea and galactose hydrolysis test, 50% isolates were positive in starch hydrolysis test, 35% in protein hydrolysis test, 100% isolates in lactose fermenting test, but no isolate was positive in sucrose fermenting test. Antibiotic susceptibility testing suggested that 20% of isolates were resistant to kanamycin and 46.67% were resistant to oxacillin. Conclusions These findings show that all these isolates have gelatin, urea, galactose hydrolysis and lactose fermenting activity. 20% of these isolates were resistant to kanamycin and 46.67% were resistant to oxacillin.

Isolation and characterization E. coli O157:H7 from meat &cheese and detection of Stx1, Stx2, hlyA, eaeA by using multiplex PCR

total of 189 samples were collected from 74 raw uncooked minced beef meat, 115 local white cheeses from 3 different areas in Baghdad. All samples were surveyed and examined for the presence of the Escherichia coli O157:H7 and differentiate it from other Non -Sorbitol Fermenting Escherichia coli (NSF E. coli). The Bacterial isolates were identified by using morphological diagnostic methods; Samples were cultured on liquid enrichment medium, incubated at 41.5Cº for 6 hrs, and then cultured on Cefixime Tellurite -Sorbitol MacConkey Agar (CT- SMAC). 66 non-sorbitol fermenting bacterial isolates were obtained of which 13 were identified as Escherichia coli from (6 meat and 7 cheese samples). By using traditional biochemical tests and Api20E diagnostic system without differentiation between serotype O157:H7 and other NSF E. coli isolates. Four specific biochemical tests (Cellobiose fermentation, β-Glocuronidase production, KCN and Enterohemolysin production) were done to differentiate serotype O157 differentiation from other NSF bacteria. Only 2 isolates belonging to the serotype O157 were obtained of which one isolate from meat and other isolate from cheese. Latex agglutination test for O157 and H7 showed that the 5 isolates gave positive results with both kits. The Bacterial isolates were identified by using Multiplex Polymerase Chain Reaction (MPCR) technology for the presence or absence of 4 genes (Stx1, Stx2, hlyA and eaeA) that encode for main virulence factors to diagnose E. coli O157:H7 isolated from various sources by using specific primers in mPCR. The result showed that gene content variety in two E. coli O157:H7 isolates, 1 from meat contain all 4 genes and other isolate from cheese contains 2 genes: Stx1 and hlyA .

Complete genome sequence of Weeksella virosa type strain (9751T)

Weeksella virosa Holmes et al. 1987 is the sole member and type species of the genus Weeksella which belongs to the family Flavobacteriaceae of the phylum Bacteroidetes. Twenty-nine isolates, collected from clinical specimens provided the basis for the taxon description. While the species seems to be a saprophyte of the mucous membranes of healthy man and warm-blooded animals a causal relationship with disease has been reported in a few instances. Except for the ability to produce indole and to hydrolyze Tween and proteins such as casein and gelatin, this aerobic, non-motile, non-pigmented bacterial species is metabolically inert in most traditional biochemical tests. The 2,272,954 bp long genome with its 2,105 protein-coding and 76 RNA genes consists of one circular chromosome and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

Pyrosequencing for rapid detection of resistance to rifampin, isoniazid, ofloxacin and amikacin in Mycobacterium tuberculosis

Objective To develop an assay to determine Mycobacterium tuberculosis resistance to rifampin, isoniazid, ofloxacin and amikacin by pyrosequencing and evaluate the value of this method in clinical application. Methods Eighty-nine clinical isolates of Mycobacterium tuberculosis from tuberculosis patients were collected from Shanghai Pulmonary Hospital during 2008 to 2009. All strains were isolated from decontaminated sputum, cultured on Lowenstein-Jensen media and identified by traditional biochemical tests with standard methods. Ten Mycobacterium tuberculosis were selected from the strain bank of Shanghai Pulmonary Hospital. The optimal conditions of detection of rpoB, katG, gyrA and rrs gene by pyroseuencing were determined, using the 10 Mycobacterium tuberculosis strains whose drug susceptibility of Bactec 960 and sequence of rpoB, katG, gyrA, rrs gene were known. Then the drug susceptibility of 89 Mycobacterium tuberculosis clinical isolate strains were detected by pyrosequencing using this conditions and the results were compared with that of the Bactec 960 methods. Results The pyrosequencing program of sequence analysis was suitable for the detection of the mutations of rpoB and gyrA genes, and the program of single nucleotide polymorphism was suitable for katG and rrs genes. Among the 89 Mycobacterium tuberculosis clinical isolates,when using the drug susceptibility of Bactec 960 as the standard, the sensitivity of rifampin, isoniazid,ofloxacin and amikacin is 98.0%, 64. 1%, 79.5%, 78. 3% respectively, the specificity is 97.5%,100. 0%, 90. 0%, 100. 0% respectively, the accuracy is 97.8%, 77. 5%, 85.4%, 94. 4% respectively,tested by pyrosequencing. The results of the detection of resistance to rifampin, isoniazid, ofloxacin and amikacin in Mycobacterium tuberculosis using pyrosequencing technique were almost the same with that of Bactec 960, and Kappa ≥0. 7 in each detection. Conclusion Pyrosequencing is thus a rapid, accurate and high throughput method for detecting Mycobacterium tuberculosis resistance to these four drugs. Key words: Mycobacterium tuberculosis; Antitubercular agents; Sequence analysis, DNA; Microbial sensitivity tests

A study on the drug resistance to p-nitrobenzoic acid in Mycobacterium tuberculosis

To investigate the resistance of Mycobacterium tuberculosis (MTB) to p-nitrobenzoic acid (PNB), in order to provide the scientific basis for reliable identification of laboratory strains of mycobacterium. Strains of mycobacterium grown in PNB media were identified with additional traditional biochemical tests, according to the standard protocols of laboratory diagnostics for tuberculosis by the Chinese Antituberculosis Association. For mycobacteria grown in the PNB media but highly suspected as MTB by traditional biochemical tests, MPB64 monoclonal antibody was used for the differentiation between MTB and non-tuberculosis mycobacteria group (NTM). Minimal inhibitory concentrations (MIC) of PNB was further determined for culture-confirmed MTB, 10 strains of clinically isolated MTB (control), H(37)Rv standard strain, mycobacterium avium standard strain, and 5 strains of clinically isolated NTM. A total of 1114 strains of MTB were confirmed, among which 58 PNB manifested resistance. The rate of resistance was 5.21% (58/1114), with an MIC ranging for 1.0 - 1.5 g/L. The MICs of control MTB and H(37)Rv standard strain were 0.25 - 0.5 g/L and 0.25 g/L, respectively. Both mycobacterium avium standard strain and clinically isolated NTM showed an MIC of > 2.0 g/L. Differences between groups were statistically significant (t = 4.87, 5.09, 6.68, respectively, P < 0.01). In order to avoid laboratory misdiagnosis, for primary screening with NTB with PNB culture, the presence of MTB characteristics, including cream-colored broccoli-like colony morphologies, as well as clinical response to first-line anti-tuberculosis medications, despite PNB tolerance, warrants further investigations of traditional biochemical tests, differentiation with MPB64 monoclonal antibodies, or simply by the use of temperature manipulation or drug-sensitivity test results.

Blood Culture Contamination in a Neonatal Intensive Care Unit in Shiraz, Southwest-Central Iran

Objective: To measure bacterial contamination rates in blood culture specimens and distinguish sepsis from blood culture contamination in newborn hospitalized patients in a neonatal intensive care unit and to recognize the most commonly isolated bacteria. Materials and Methods: Blood samples of 578 neonates were collected and cultured throughout the year of study (March 2006 to February 2007). Isolated bacteria were identified by traditional biochemical tests. Clinical criteria combined with laboratory data were used to differentiate the contaminated cultures from clinically significant cultures. Results: Of the 578 neonatal blood culture samples, 78 (13.49%) were positive for bacteria, and 49 isolates (8.47%) were classified as contaminants. Pseudomonas aeruginosa and Staphylococcus aureus were the most common isolates from true bacteremia, and Staphylococcus epidermidis and diphtheroids were the most common contaminants. Conclusion: The blood culture contamination rate in our studied neonatal intensive care unit was high. A variety of measures are recommended for reducing the rate of blood culture contamination to avoid undesirable outcomes associated with blood culture contamination.

Characterisation of Pasteurella dagmatis-like isolates recovered from the feline oral cavity

Three Pasteurella dagmatis-like strains recovered from the feline oral cavity were analysed by traditional biochemical tests, the Biolog Microstation™ ID System, and 16S rRNA and sodA gene sequence analysis. The molecular biological methods revealed that these strains differ from P. dagmatis, forming a new genomospecies in the genus Pasteurella sensu stricto. Furthermore, sequence analysis and multiple alignments of 16S rRNA and the sodA gene established that the P. pneumotropica NCTC10827 and the P. dagmatis-like strains described here possess high genetic similarity.

Molecular diagnosis of E.coliO157:H7 Which Isolated from Children with Diarrhea by using Multiplex PCR

A total of 96 stool samples were collected from children with bloody diarrhea from two hospitals in Baghdad. All samples were surveyed and examined for the presence of the Escherichia coli O157:H7 and differentiate it from other Non -Sorbitol Fermenting Escherichia coli (NSF E. coli). The Bacterial isolates were identifed by using morphological diagnostic methods, Samples were cultured on liquid enrichment medium, incubated at 37C? for 24 hrs, and then cultured on Cefixime Tellurite -Sorbitol MacConkey Agar (CT- SMAC). 32 non-sorbitol fermenting bacterial isolates were obtained of which 11 were identified as Escherichia coli by using traditional biochemical tests and API20E diagnostic system without differentiation between serotype O157:H7 and other NSF E. coli isolates . Four special biochemical tests were done for serotype O157:H7 differentiation from other NSF bacteria. Only 3 isolates belonging to the serotype O157:H7 were obtained . Latex agglutination test for O157 and H7 showed that the 3 isolates gave positive results with both tests. The Bacterial isolates were identifed by using Multiplex Polymerase Chain Reaction (MPCR) technology for the presence or absence of 4 genes (Stx1, Stx2, hlyA and eaeA) that encode for main virulence factors to diagnose E. coli O157:H7 isolated By using specific primers in MPCR . The result showed that one E. coli O157:H7 isolates contain all 4 genes , other isolates contain 3 genes: Stx2, hlyA &amp; eaeA.

Transmission of a Klebsiella pneumoniae clone harbouring genes for CTX-M-15-like and SHV-112 enzymes in a neonatal intensive care unit of a Kuwaiti hospital

The spread of antibiotic-resistant bacteria has become a large problem in most countries including Kuwait. This antibiotic resistance is usually due to the production of extended-spectrum beta-lactamase (ESBL) enzymes such as SHV, TEM and CTX-M. This study reports the emergence and spread of an ESBL-producing Klebsiella pneumoniae clone in a neonatal intensive care unit (NICU) in a Kuwaiti hospital. Eight ESBL-producing K. pneumoniae isolates were from blood cultures of seven neonates, and two were from the fingers of two healthcare workers in a NICU in Al Jahra Hospital, Kuwait. All isolates were obtained in February-March 2006, except for one, which was obtained in August 2005. Identification of the bacteria was based on traditional bacteriological and biochemical tests using the Vitek system. Antibiotic susceptibility was tested by the disc diffusion method using 16 different antibiotics. ESBLs were detected using disc approximation and double-disc synergy methods and confirmed as ESBLs using Etest. PCR and DNA sequencing were performed to determine the genotypes and mutations in the beta-lactamase genes (blaTEM, blaSHV and blaCTX-M). Genetic relatedness was determined by PFGE. All isolates were confirmed to have ESBLs by the Vitek system, disc approximation test, double-disc diffusion test and Etest, being resistant to cefotaxime, ceftazidime, cefepime, gentamicin, tobramycin and ciprofloxacin but susceptible to tetracycline and trimethoprim-sulfamethoxazole. Molecular studies showed the isolates to have TEM-1 beta-lactamase, a CTX-M-15-like ESBL and the newly discovered SHV-112 ESBL. PFGE showed that all isolates had identical banding patterns. The results indicate that a single clone of ESBL-producing K. pneumoniae caused bloodstream infections among babies in a NICU of a Kuwaiti hospital, and may have emerged at least 5 years ago. This clone was also present on the hands of healthcare workers, suggesting that they may have been involved in its transmission. Further studies are recommended to determine whether this clone is also spreading in other Kuwaiti hospitals.

Bacillus horneckiae sp. nov., isolated from a spacecraft-assembly clean room

Five Gram-stain-positive, motile, aerobic strains were isolated from a clean room of the Kennedy Space Center where the Phoenix spacecraft was assembled. All strains are rod-shaped, spore-forming bacteria, whose spores were resistant to UV radiation up to 1000 J m(-2). The spores were subterminally positioned and produced an external layer. A polyphasic taxonomic study including traditional biochemical tests, fatty acid analysis, cell-wall typing, lipid analyses, 16S rRNA gene sequencing and DNA-DNA hybridization studies was performed to characterize these novel strains. 16S rRNA gene sequencing and lipid analyses convincingly grouped these novel strains within the genus Bacillus as a cluster separate from already described species. The similarity of 16S rRNA gene sequences among the novel strains was >99 %, but the similarity was only about 97 % with their nearest neighbours Bacillus pocheonensis, Bacillus firmus and Bacillus bataviensis. DNA-DNA hybridization dissociation values were <24 % to the closest related type strains. The novel strains had a G+C content 35.6+/-0.5 mol% and could liquefy gelatin but did not utilize or produce acids from any of the carbon substrates tested. The major fatty acids were iso-C(15 : 0) and anteiso-C(15 : 0) and the cell-wall diamino acid was meso-diaminopimelic acid. Based on phylogenetic and phenotypic results, it is concluded that these strains represent a novel species of the genus Bacillus, for which the name Bacillus horneckiae sp. nov. is proposed. The type strain is 1P01SC(T) (=NRRL B-59162(T) =MTCC 9535(T)).

A novel multiplex PCR assay forSalmonellasubspecies identification

To develop a novel multiplex polymerase chain reaction (PCR) assay with six primer pairs for Salmonella subspecies identification. Five primer pairs were chosen to detect the genes (fljB, mdcA, gatD, stn and STM4057) responsible for several phenotypic traits or encoding (sub) species-specific regions. A primer pair for invA was added to simultaneously detect Salmonella. The combination of these primer pairs was expected to give unique results to all subspecies, including Salmonella bongori. The multiplex PCR assay was optimized and evaluated with 53 Salmonella strains representing all S. enterica subspecies, S. bongori and five non-Salmonella strains. The multiplex PCR assay revealed that the genotypes were well correlated with the phenotypes in the Salmonella strains tested. The unique band patterns to their subspecies were generated from 94.3% (50/53) of the Salmonella strains, and no product from other strains by the multiplex PCR assay. The multiplex PCR assay we developed was found to be a rapid, specific and easy to perform method compared with traditional biochemical tests for Salmonella subspecies identification, especially for rapid screening of large numbers of samples. The assay will be useful for characterizing Salmonella isolates from reptiles, which belong to various subspecies, and therefore add to the scientific understanding of reptile-associated Salmonellosis.

Primer estudio de Enterobacter sakazakii en alimentos en Cuba

Basándose en ejemplos concretos de sistemas de salud, se aportan elementos y argumentos al debate internacional sobre la importancia de los niveles de atención y de la relación óptima entre niveles. El primer nivel tiene tres funciones esenciales: ser el punto de entrada al sistema, facilitar y coordinar el itinerario de su paciente en el sistema, y asegurar la integración y la síntesis de la información de salud de cada paciente. Describiendo los diferentes obstáculos y problemas posibles, en primer lugar la lógica comercial que se ha ido introduciendo en muchos sistemas, los autores definen puntos de atención y proponen ajustes para asegurar el acceso universal, la equidad, la eficacia y eficiencia, y la sostenibilidad de un sistema de salud.

Carbapenemase and efflux pump genes in Acinetobacter calcoaceticus Acinetobacter baumannii complex strains from Singapore

Sir, We recently published a paper describing the distribution of OXA and IMP b-lactamases in Acinetobacter spp. from Singapore. Since that manuscript was submitted, there has been accumulating evidence suggesting that OXA-51-type b-lactamases are ubiquitous in Acinetobacter baumannii sensu stricto. To resolve the identity of isolates from our original study that were identified using biochemical tests as A. baumannii, but were negative for blaOXA-51-type genes, we used PCR to re-test all 12 strains using a new set of universal blaOXA-51-type primers. We also sequenced the 16S rDNA gene (first 500 bp) using primer 8FPL described by Relman and in-house primer 515R (50-TTA CCG CGG CAG CTG GCA C-30), and the 16– 23S rRNA gene intergenic spacer using the primers described by Chang et al. In addition, we tested for the presence of the efflux genes adeB, adeE and adeY using the primers described by Chu et al., since it has been shown that the AdeABC multidrug efflux pumps are intrinsic to A. baumannii, whereas the AdeDE and AdeXY pumps are found predominantly in Acinetobacter genomospecies 3. The final definition of strains to species level was based primarily on the 16–23S rRNA gene intergenic spacer sequences but also took into consideration the results of all the other tests (Table 1). Five strains were positive for blaOXA-51-type genes with the new primer set and remain identified as A. baumannii. Another five strains were re-identified as either Acinetobacter genomospecies 3 or Acinetobacter genomospecies 13TU. Conflicting results did not allow us to discriminate within the Acinetobacter calcoaceticus–Acinetobacter baumannii complex for two isolates. The segregation of carbapenemase genes between the different Acinetobacter genomospecies is striking (Table 1). Of particular note, the IMP-4 metallo-b-lactamase is found in either Acinetobacter genomospecies 3 or Acinetobacter genomospecies 13TU but not in A. baumannii. Our data do support the hypothesis that blaOXA-51-type genes are intrinsic to A. baumannii. The distribution of efflux genes is also consistent with that reported by Chu et al. The A. calcoaceticus–A. baumannii complex is composed of four closely related species (A. calcoaceticus, A. baumannii, Acinetobacter genomospecies 3 and Acinetobacter genomospecies 13TU), which are difficult to differentiate phenotypically using traditional laboratory biochemical tests and commercial kits. In routine clinical practice, this distinction between the

Environmental parameters and antimicrobial susceptibility of enterobacteriaceae isolated from estuarine waters of São Vicente, São Paulo state, Brazil

Diseases transmitted by water consists a serious public health problem and enterobacteria are the main group of microorganisms responsible for outbreaks in humans. Such pathogenic bacteria proliferate in water polluted by domestic and industrial sewage and reach the population through seawater contact. The aim of the present work was to study environmental parameters as well as to identify Enterobacteriaceae species and their antimicrobial susceptibility in water samples collected from the estuarine area of Sao Vicente city (Sao Paulo State, Brazil). Strains were identified by using traditional biochemical tests described in literature and antimicrobial susceptibility tests were carried out using the disk diffusion method. Out of 26 samples, Escherichia coli was the most frequent species (40.1%), followed by Citrobacter, Enterobacter and Klebsiella. The most effective drugs against the tested microorganisms were gentamycin, netilmicin, ciprofloxacin and cefepime. Since these bacteria are commonly found in seashore contaminated by sewage effluents, it can be concluded that estuarine waters of Sao Vicente are polluted and potentially capable of causing diseases and spreading pathogenic bacteria to human communities.

Phenotypic characterization and molecular taxonomic studies on Bacillus and related isolates from Phragmites australis periphyton

Reeds may play important role in the self-purification of aquatic habitats due to the filtration capacity and their periphyton communities developing on the underwater plant surfaces. The efficiency of this process and the transformation of organic substances can be influenced by the species composition and activity of microbial communities, including Bacillus and related species. For cultivation based bacteriological examinations reed periphyton samples were collected from 30 cm beneath the water surface of Lake Velencei and the Soroksár Danube branch (Hungary). After a primary selection 40 Bacillus and related strains were investigated by traditional morphological, biochemical tests, API 20E, API 50CHB and BIOLOG GP2 systems, and identified by 16S rDNA sequence comparison. The isolated strains were characterized by wide biochemical activity spectrum (i.e., the metabolism of carbohydrates and biopolymers) as well as widespread ecological tolerance based on their NaCl and pH range investigations. Species with facultative alkaliphilic features ( Marinibacillus marinus, Bacillus firmus) were detected only from the reed biofilm of Lake Velencei, while alkalitolerant Bacillus and related species from both sampling sites. 22 endospore-forming strains were identified as members of species B. cereus, B. firmus, B. flexus, B. licheniformis, B. megaterium, B. muralis, B. pumilus, B. subtilis and Marinibacillus marinus. Only one species, with 95–96% sequence similarities to B. pumilus was found to be common among the strains from Lake Velencei and the Soroksár Danube branch. Altogether, 18 strains could not be identified as known species of Bacillus, Brevibacillus and Paenibacillus, hence they may represent new bacterial taxa.

Polyphasic characterization of indigenous lactobacilli and lactococci from PDO Canestrato Pugliese cheese

A polyphasic approach, involving both genotypic and phenotypic analyses was used to characterize 33 isolates of lactic acid bacteria from the raw milk Protected Designation of Origin (PDO) Canestrato Pugliese cheese, in order to select candidate strains that can be used as autochthonous starter cultures in the dairy industry. Randomly amplified polymorphic DNA (RAPD) analysis and clustering by the unweighted pair-group method with arithmetic averages (UPGMA) were used to evaluate the genotypic diversity, while phenotypic characterization was performed through miniaturized assays and traditional biochemical tests. Technological properties of major interest for cheese-making (acidification, tendency to lysis, proteolytic and peptidase activity) were also evaluated, and selected subset of data was statistically examined by the one-way ANOVA and the Tukey–Kramer HSD test, to highlight strain-specific and species-specific differences among the isolates. A high degree of diversity appeared in the phenotypic and technological traits in opposition to a relatively low genotypic diversity. Although none of the isolates showed the best performances in all the activities, an appropriate mixture of strains could be selected for providing an efficacious autochthonous starter culture.

Explorations de la fonction musculaire par spectrométrie et imagerie de résonance magnétique

31P MRS and 1H MRI of skeletal muscle have become major new tools allowing a complete non invasive investigation of muscle function both in the clinical setting and in basic research. The comparative analysis of normal and diseased muscle remains a major requirement to further define metabolic events surrounding muscle contraction and the metabolic anomalies underlying pathologies. Also, standardized rest-exercise-recovery protocols for the exploration of muscle metabolism by P-31 MRS in healthy volunteers as well as in patients with intolerance to exercise have been developed. The CRMBM protocol is based on a short-term intense exercise, which is very informative and well accepted by volunteers and patients. Invariant metabolic parameters have been defined to characterize the normal metabolic response to the protocol. Deviations from normality can be directly interpreted in terms of specific pathologies in some favorable cases. This protocol has been applied to more than 4,000 patients and healthy volunteers over a period of 15 years. On the other hand, MRI investigations provide anatomical and functional information from resting and exercising muscle. From a diagnostic point of view, dedicated pulse sequences can be used in order to detect and quantify muscle inflammation, fatty replacement, muscle hyper and hypotrophy. In most cases, MR techniques provide valuable information which has to be processed in conjunction with traditional invasive biochemical, electrophysiological and histoenzymological tests. P-31 MRS has proved particularly useful in the therapeutic follow-up of palliative therapies (coenzyme Q treatment of mitochondriopathies) and in family investigations. It is now an accepted diagnostic tool in the array of tests which are used to characterize muscle disorders in clinical routine. As a research tool, it will keep bringing new information on the physiopathology of muscle diseases in animal models and in humans and should play a role in the metabolic characterization of gene and cell therapy.

Comparison of culture and biochemical tests with PCR for detection of Brachyspira hyodysenteriae and Brachyspira pilosicoli

Traditional culture and biochemical tests (CBT) were compared with PCR for sensitivity and detection of Brachyspira hyodysenteriae and Brachyspira pilosicoli in seeded faeces and clinical samples from diarrhoeic pigs. A duplex PCR system was developed based on primers detecting the tlyA-gene of B. hyodysenteriae and the 16S rRNA-gene of B. pilosicoli. Sensitivities for the PCR system were determined on seeded faeces, using DNA that had been recovered from primary cultures or extracted directly from faeces. Compared to CBT, PCR applied to DNA extracted directly from faeces lowered the sensitivity by a factor of 1000 to 10,000. B. hyodysenteriae and B. pilosicoli detection was compared for CBT and PCR using 200 clinical samples. CBT detected more B. hyodysenteriae isolates in the clinical samples than PCR, but fewer B. pilosicoli positive samples. An atypical strongly haemolytic isolate was detected only by CBT.