Genetic Diversity Among Strains of Moraxella catarrhalis Cultured from the Nasopharynx of Young and Healthy Brazilian, Angolan and Dutch Children

 The present study describes the carriage patterns and genetic variability of Moraxella catarrhalis strains isolated from children living in different countries. Moraxella catarrhalis is genetically heterogeneous, but little is known about its geographic distribution and phenotypic and genetic diversity in warm-climate countries. A collection of 99 isolates from 30 Brazilian, 19 Angolan and 50 Dutch healthy children, all less than 5 years of age, was investigated for phenotypic and genotypic relatedness. The isolates from the three countries were similar where biochemical reactivity was concerned: 89 strains were β-lactamase-producing and 87 were complement-resistant as determined by phenotype. There was no geographical difference in the prevalence of β-lactamase-producing isolates, but the carriage rate of complement-resistant strains was significantly higher in Dutch than in Angolan children (P=0.004). Complement resistance of 66 randomly selected strains was genetically confirmed in a Southern hybridization assay by a novel DNA probe that is specific for complement-resistant strains and that demonstrated a sensitivity of 97% and a specificity of 100%. PCR amplification based on the probe sequence had a sensitivity of 98% and a specificity of 57% when compared to the outcome of a conventional culture spot test. PCR restriction fragment length polymorphism analysis of the MU 46 locus and pulsed-field gel electrophoresis of SpeI DNA macrorestriction fragments revealed genetic heterogeneity of strains from within and between the three countries, and no geographical clustering could be established. In conclusion, similar phenotypic characteristics but genotypic heterogeneity was found among Moraxella catarrhalis strains colonizing children in three different continents.     

Phenotypic and genotypic characterizations of rhizobia isolated from root nodules of peanut (Arachis hypogaea L.) grown in Moroccan soils

The phenotypic and genotypic characterization of sixty‐two rhizobial isolates obtained from nodules of Arachis hypogaea in north‐western Morocco was performed. Their physiological and biochemical properties revealed a great deal of diversity among them. Isolates were classified into two major groups based on the numerical analysis of their phenotypic and genotypic characteristics. Isolates in the first group were alkali‐ and salt‐sensitive, slow or extra‐slow growers; they did not use disaccharides as carbon source and varied in the use of amino acids. ARDRA analysis of the 16S rDNA region grouped them together with reference strains belonging to the genus Bradyrhizobium. In the second group, isolates were fast growers, acid‐sensitive, and alkali‐ and salt‐tolerant; they used both mono and disaccharides as carbon sources, and methionine was the only amino acid they could metabolize as a nitrogen source. ARDRA analysis grouped them with fast‐growing reference strains. Both groups exhibited a range of variability in tolerance to heavy metals. The Intergenic Spacer (IGS)‐PCR fingerprinting analysis confirmed a high genotypic diversity at the strain level. This characterization provides a basis for the selection of peanut‐nodulating rhizobia which may have applications in formulating appropriate inocula for improving peanut crop yield on Moroccan soils, including saline and acidic marginal areas. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Genotypic and Phenotypic Evidence of Different Drug-Resistance Mutation Patterns between B and Non-B Subtype Isolates of Human Immunodeficiency Virus Type 1 found in Brazilian Patients Failing HAART

We have investigated the phenotypic and genotypic susceptibility of 14 HIV-1 strains isolated from individuals failing HAART therapy to protease inhibitors (PI). Proviral and plasma viral pol gene fragment were amplified, sequenced and subtyped. Nine samples clustered with protease subtype B reference strains and the remaining samples were classified as non-B subtype corresponding to subtype F (n=4) and subtype A (n=1). Although all patients were treated with similar PI drug regimen, the non-B subtype isolates did not present the L90M and I84V mutations and used mainly G48V and V82A/F to achieve drug resistance. A strong cross-resistance phenotype among all four PI was associated with the mutation L90M in the subtype-B isolates, and with G48V and V82A/F in the non-B counterparts. This observation revealed that the non-B viruses tested had specific genotypic characteristics contrasting with the subtype-B isolates.     

Ornithine decarboxylating strains of Klebsiella pneumoniae demonstrated by DNA-DNA hybridization

Genotypic relatedness was assessed to clarify the taxonomic position of strains phenotypically behaving like K. pneumoniae, but for the ornithine reaction. Using DNA-DNA hybridization it could be shown that 25 non-motile ornithine decarboxylating strains showed high genotypic relatedness to the type strain of K. pneumoniae. Thus, it is proposed that they be considered as ornithine decarboxylating strains of the species K. pneumoniae. The API 20E system was used for phenotypic characterization, but the API code obtained by these strains was not registered in the API Profile Index. However, except for the ornithine reaction the isolates behaved as typical K. pneumoniae. Three ornithine negative strains of E. aerogenes were identified as K. pneumoniae by the API 20E System, but they showed high genotypic relatedness to the type strain of E. aerogenes.     

Identification of clinically relevant viridans group streptococci by phenotypic and genotypic analysis

Two phenotypic and three molecular methods were assessed for their ability to identify viridans group streptococci (VGS) to the species level. A panel of 23 clinical isolates, comprising strains isolated from infective endocarditis, blood cultures, pleural and peritoneal fluid, and 19 type/reference strains were analyzed. Identification was performed using two conventional phenotypic methods: API? rapid ID 32 Strep and the VITEK? 2 system, and genotypic analysis of the nucleotide sequence of the housekeeping gene sodA, restriction patterns generated by restriction fragment length polymorphism (RFLP) of the 16S rRNA gene and multilocus sequence analysis (MLSA) of seven housekeeping genes. The API? rapid ID 32 Strep accurately speciated 79% of the strains assessed, while the VITEK? 2 generated a successful identification for 55%, presenting limitations particularly with regard to species belonging to the mitis group. RFLP of the 16S rRNA gene correctly speciated 24% of the strains, having failed to allocate a species for 36% of the isolates examined. In contrast, sequence analysis of the sodA gene provided a correct identification for 95% of the strains assessed, while identification using the MLSA technique was unsuccessful due to practical limitations. The results generated herein indicate that no single methodology can be used to provide an accurate identification to the species level of all VGS, although nucleotide sequence analysis of the sodA gene proved to be useful in providing reliable speciation.     

The cultivation and rapid enzyme identification of DF-2

The investigation of two clinical isolates and two reference strains of DF-2 showed that supplementary cysteine and incubation in a humid atmosphere were important growth-promoting factors for these fastidious, gram-negative bacteria. Broth-base media with phenol red indicator were proven to be satisfactory for carbohydrate fermentation tests. Two four-hour enzyme assays (API ZYM and Rosco Diagnostic Tablets) were used to compare the enzymatic activity of DF-2 with that of 27 species of other non-enterobacterial organisms. The Rosco assay revealed that only the DF-2 strains had a positive alpha-fucosidase reaction, suggesting that this character may provide the means for rapid characterization and identification of these bacteria and also be of value for taxonomic classification. The incongruent results of the API ZYM assay seem to be due to the different substrates of the two assay systems.     

Isolation and preliminary probiotic selection of lactobacilli from koumiss in Inner Mongolia

From 16 samples of traditional fermented koumiss collected in Inner Mongolia Autonomous Region of China, forty‐eight lactobacilli strains were isolated and phenotypically characterized by their abilities to ferment different carbohydrates and by additional biochemical tests. The dominant lactobacilli species were identified as L. casei (17 strains), L. helveticus (10 strains) and L. plantarum (8 strains), with a lower frequency of isolation for L. coryniformis subsp. coryniformis (5 strains), L. paracasei (3 strains), L. kefiranofaciens (2 strains), L. curvatus (1 strain), L. fermentum (1 strain) and W. kandleri (1 strain). The pH values of all these samples were ranging from 3.37 to 3.94. In isolates, L. casei Zhang, L. helveticus ZL12‐1, and L. plantarum BX6‐6 were selected as potentially probiotic strains through the preliminary tests including resistance to low acid, abilities to grow in MRS with bile salts, antimicrobial activities and the viabilities during prolonged cold storage in fermented milk. Moreover 16S rDNA was conducted to confirm the identification. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Phenotypic and genotypic differentiation of Campylobacter spp. isolated from Austrian broiler farms: A comparison

An identification scheme based on restriction fragment length polymorphism of polymerase chain reaction products (PCR-RFLP) was developed to differentiate isolates of the genera Campylobacter, Arcobacter and Helicobacter. Based on the 16S rRNA gene of these genera, PCR amplified a 1216-bp fragment. The amplicons were digested with the restriction enzymes RsaI and EcoRV. Additional differentiation was obtained using a PCR-assay based on the hippuricase gene. Genotyping was performed on several reference strains from the National Collection of Typing Culture (NCTC), London, and on 130 field isolates. In parallel, a phenotypic differentiation was performed, in order to compare the results. In 119 cases (91.5%) the results obtained from the genotypic characterization were concordant with those from phenotypic testing. Co-infections with Campylobacter jejuni and Campylobacter coli in two samples and seven hippurate-negative C. jejuni-strains were identified by the genotypic method. Furthermore, PCR-RFLP assays identified an atypical isolate as Campylobacter fetus/hyointestinalis.     

Comparison of use of phenotypic and genotypic characteristics for identification of species of the anamorph genus Candida and related teleomorph yeast species.

A total of 49 type and neotype isolates and 32 clinical isolates of the anamorph genus Candida and related teleomorph genera were obtained from different culture collections and clinical laboratories. Isolates were subjected to two phenotypic methods of identification, Vitek yeast biochemical card (YBC) and API ID 32C, both based on carbohydrate assimilation, and one genotypic method, PCR fingerprinting, based on the detection of DNA polymorphisms between minisatellite-specific sequences with the primer M13 (5' GAGGGTGGCGGTTCT 3'). The correct identification of a strain at the Centraalbureau voor Schimmelcultures was used as the gold standard for the identification of an isolate. When the study was restricted to species included in the respective biochemical databases, the Vitek YBC and API ID 32C systems performed adequately with positive identification rates of 87.3 and 76.8%, respectively. When uncommon species were added to the study, several of which are not included in the databases, the identification efficiencies were 76.5 and 77.5%, respectively. By comparison, all isolates were correctly identified by PCR fingerprinting, with 63 reference species profiles in the databank. Sufficient polymorphisms among the total set of banding patterns were observed, with adequate similarity in the major patterns obtained from a given species, to allow each isolate to be assigned unambiguously to a particular species. In addition, variations in minor bands allowed for differentiation to the strain level. PCR fingerprinting was found to be rapid, reproducible, and more cost-effective than either biochemical approach. Our results provide reference laboratories with an improved identification method for yeasts based on genotypic rather than phenotypic markers.     

Phenotypic and genotypic characterization of VanA Enterococcus isolated during the first nosocomial outbreak in Brazil

We report the phenotypic and genotypic characterization of 50 VanA Enterococcus clinical isolates from infected patients and 97 isolates from colonized patients obtained during a nosocomial outbreak in a single hospital in S?o Paulo, Brazil during 1998. The identification of strains to the species level by conventional biochemical and phenotypic tests and by multiplex PCR assay had 100% agreement. Both E. faecalis and E. faecium were isolated from patients during this outbreak. The vanA genotype was confirmed by PCR. Antibiotic susceptibility testing showed that E. faecium isolates are generally less susceptible to antibiotics than E. faecalis. By PCR, 24 of 26 VRE strains tested carried the Tn1546 element. Pulsed-field gel electrophoresis identified five distinct patterns for E. faecalis (A, B, C, D, E) and three for E. faecium (M, N, and O). A single PFGE pattern was identified in the majority of strains of each species and does not discriminate between case and carrier isolates.     

Comparison of Three Methods for Identification of Streptococcus milleri Group Isolates to Species Level

 A collection of 180 clinical isolates of Streptococcus milleri group were identified to species level using two phenotypic methods (a commercial system and the reference method based on differential phenotypic reactions) and a genotypic method (hybridisation of the 16S rRNA gene with species-specific probes) in order to evaluate the performance of the respective methods. A high level of agreement (80%) was observed between the results of the reference method and the genotypic method. The highest level of agreement was found for the species Streptococcus anginosus (83%), a high level of agreement (76%) also being achieved for Streptococcus constellatus and Streptococcus intermedius. The sensitivity of the commercial system compared to the genotypic method was 76% overall, but it was low (57.5%) for Streptococcus intermedius. Twenty-five strains belonged to the recently described CI strains.     

Production of α-galactosidase by Lactobacillus plantarum isolated from diverse sources

α-Galactosidase activity was observed in six strains of Lactobacillus plantarum isolated from fermented cereal products, human intestinal flora and fermented tea. The cultural conditions under which the enzyme activity was detected suggest that the enzyme is inducible. Development of mutants in four out of the six strains was observed and the mutants recorded high enzyme activity than the parent strains. Effect of different carbohydrates on enzyme activity showed glucose and raffinose being repressive in the parent strains. Although all the carbohydrate sources supported growth, highest amount of enzyme activity was recorded on lactose and glucose. The enzymatic potential of L plantarum in reducing flatulence properties of the raw material base of some West African fermented foods is suggested.     

Characterization and identification of Geobacillus spp. isolated from Soldhar hot spring site of Garhwal Himalaya,India

13 morphologically distinct strains of thermophilic bacteria isolated from a hot spring site in Garhwal region of Indian Himalaya have been characterized and identified using phenotypic and genotypic characters. All the strains developed circular to irregular colonies between 2–3 mm on Tryptone Yeast extract (TY) agar plates at 65 °C following 24–36 h incubation. In TY broth, facultative bacterial growth was observed within 12–16 h of incubation at 65 °C. The bacterial strains could tolerate a temperature range between 40–45 °C to 85–90 °C (optimum 65–70 °C) and pH between 4–11 (optimum 6–8). The cell morphology varied from short to long rods arranged in single, diplobacilli (in V or L shape) or short or long spiral chains with coiling. The bacterial strains varied in respect of their biochemical tests conducted for various enzymes, fermentation of sugars, tolerance to antibiotics and salt. Based on the 16S rRNA analysis, 11 strains showed maximum similarity with Geobacillus stereothermophilus, one strain with G. kaustophilus and one with Geobacillus sp. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Effect of acid and alkali stress on extracellular metabolite profile of Lactobacillus plantarum ATCC 14917

As a multifunctional lactic acid bacterium, Lactobacillus plantarum has been proved to survive in the human gastrointestinal tract, and it can also colonize this tract. In this study, the effects of L. plantarum ATCC 14917 metabolic profile caused by initial acid–base (pH 5.5 and 8.5) stress were investigated using ¹H nuclear magnetic resonance spectroscopy and multivariate data analysis. The results showed that the metabolome mainly consisted of 14 metabolites, including the components like amino acids, sugars, organic acids, and alkaloids. According to the nontargeted principal component analysis, there was a decrease in most of the metabolites in the alkali-treated group (mainly change in PC1) except acetate, whereas the production of lactate and glycine was increased in the acid-treated group (mainly change in PC2). Furthermore, the initial alkali stress inhibits the secretion of lactic acid, as a decrease was observed in the activity of lactate dehydrogenase and acetic dehydrogenase of L. plantarum ATCC 14917 in the alkali group. All these findings revealed that alkali stress could limit the acid environment formation of L. plantarum 14917 in the fermentation process; however, low acid pH is more suitable for the growth of L. plantarum.     

Genotypic characterization of thermophilic bacilli: A study on new soil isolates and several reference strains

A genotypic study using amplified ribosomal DNA restriction analysis (ARDRA), random amplified polymorphic DNA fingerprinting (RAPD) and ribosomal spacer analysis (RSA) in comparison with DNA-DNA reassociation experiments was carried out with 85 thermophilic Bacillus isolates from uncultivated soil of 14 different geographical areas and seventeen reference strains representing defined thermophilic Bacillus species. This approach permitted the attribution of 51 % of the new isolates to the Bacillus thermoleovorans group and the identification of 40% of the new isolates as B. “thermodenitrificans”. Moreover, 2 strains were assigned to B. pallidus species and 1 isolate to B. thermosphaericus species. The remaining 6% of our thermophilic isolates from soil, constituting 2 DNA-DNA homology groups, are still unidentified. A detailed genotypic characterization of the heterogeneous species of B. thermoleovorans and B. stearothermophilus was also presented.     

Revisited distribution of nonfermenting Gram-negative bacilli clinical isolates

Nonfermenting Gram-negative bacilli (NF-GNB) are ubiquitous environmental opportunistic bacteria frequently misidentified by conventional phenotypic methods. The aim of this study was to determine the distribution of NF-GNB species by 16?S rRNA gene sequencing (used as reference method) and to compare performances of biochemical tests and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). From nine French hospitals, 188 NF-GNB isolates (except P. aeruginosa and A. baumannii) were prospectively collected from 187 clinical samples between December 2008 and May 2009. By using the genotypic approach, 173 (92%) and 188 (100%) isolates were identified to the species and genus level, respectively. They covered 35 species and 20 genera, with a predominance of Stenotrophomonas maltophilia, Achromobacter xylosoxidans, and Pseudomonas putida group bacteria. Of the 173 species-level identified strains, concordant identification to the species-level was obtained for 75.1%, 83% and 88.9% of isolates with API 20 NE strip, the VITEK-2 (ID-GN card) system and MALDI-TOF-MS, respectively. By excluding S. maltophilia isolates accurately identified by the three methods, genus-level identification was much higher for MALDI-TOF-MS (92.9%), compared with API 20 NE and VITEK-2 (76.2% and 80.8%, respectively). In conclusion, MALDI-TOF-MS represents a rapid, inexpensive, and accurate tool for routine identification of NF-GNB in human clinical samples.     

Comparison of genotypic and phenotypic methods for species-level identification of clinical isolates of coagulase-negative staphylococci

To compare commonly used phenotypic methods with genotypic identification methods 47 clinical isolates of coagulase-negative staphylococci (CONS), 10 CONS ATCC strains, and a Staphylococcus aureus clinical isolate were identified using the API Staph ID test, BD Phoenix Automated Microbiology System, and 16S rRNA gene and tuf gene sequencing. When necessary part of the sodA gene was sequenced for definitive identification. The results show that tuf gene sequencing is the best method for identification of CONS, but the API Staph ID test is a reasonably reliable phenotypic alternative. The performance of the BD Phoenix Automated Microbiology System for identification of CONS is poor. The present study also showed that although genotypic methods are clearly superior to phenotypic identifications, a drawback of sequence-based genotypic methods may be a lack of quality of deposited sequences in data banks. In particular, 16S rRNA gene sequencing suffers from the lack of high quality among sequences deposited in GenBank. Furthermore, genotypic identification based on 16S rRNA sequences has limited discriminating power for closely related Staphylococcus species. We propose partial sequencing of the tuf gene as a reliable and reproducible method for identification of CONS species.     

Discordance between genotypic and phenotypic drug resistance profiles in human immunodeficiency virus type 1 strains isolated from peripheral blood mononuclear cells

The aim of the study was to analyze the relationship between genotypic and phenotypic drug resistance profiles of human immunodeficiency virus type 1 (HIV-1) strains isolated from patients during double-analogue nucleoside therapy. A drug-resistant HIV strain was isolated from 20 out of 25 patients, with 16 (64%) subjects carrying a virus with multiple drug resistance mutations. The most frequent resistance mutations were M184V (18 isolates) and M41L (7 isolates). Discordance between the genotypic and phenotypic profile for at least one drug was detected in 16 out of 25 strains. Particularly, eight isolates had a discordant genotypic-phenotypic resistance pattern for two drugs and one isolate had such a pattern for three drugs. A genotypic resistance pattern with a phenotypic sensitivity profile was detected in six isolates (four resistant to zidovudine and two resistant to lamivudine). On the other hand for several strains a genotypic pattern of sensitivity pattern to abacavir (10 strains), didanosine (7 strains), stavudine (3 strains), zidovudine (2 strains), and lamivudine (1 strain) with a phenotypic resistance profile was detected. After a follow-up period of 8 months, an impairment of virological and immunological parameters was detected only in subjects with an HIV-1 isolate with a phenotypic resistance profile in despite of the genotypic results. Predicting resistance phenotype from genotypic data has important limitations. Despite the low number of patients and the short follow-up period, this study suggests that during failing therapy with analogue nucleosides, a phenotypic analysis could be performed in spite of an HIV genotypic sensitivity pattern.     

Genotypic characterization of non starter lactic acid bacteria involved in the ripening of artisanal Bitto PDO cheese

Bitto of Valchiavenna, an artisanal Italian cheese produced without the addition of any starter cultures, has been attributed a protected designation origin (PDO) cheese, but the strain composition of the natural microbial population colonizing this traditional dairy product is still unknown. To obtain preliminary information on the non starter lactic acid bacteria involved in its ripening, a total of 136 NSLAB isolates, randomly selected from MRS and M17 agar plates, were collected from three different cheese samples after 120 days of ripening. The new isolates were identified by combining PCR 16S–23S rDNA spacer analyses, partial 16S rRNA gene sequencing, species‐specific probes and colony hybridization. Eighty‐two isolates, representing 60% of the total strains selected, were homofermentative cocci: 83% of them were enterococci, with Enterococcus durans being the predominant species found. Pediococcus spp. were also isolated, together with strains of Streptococcus thermophilus. Within lactobacilli, 57% of the isolates were identified as Lactobacillus paracasei; Lact. curvatus, Lact. plantarum, Lact. fermentum, were present in a lower amount. The isolates were differentiated at strain‐level by Rep‐PCR analysis. This is the first effort to microbiological characterization of Valchiavenna's Bitto; the results suggest the possibility of preserving the wild bacterial population in order to protect the typical organoleptic characteristics of this traditional raw milk cheese and to select new strains for the dairy industry. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Comparative analysis of the restriction endonuclease profiles of the Dumas and Singapore strains of varicella-zoster virus

The incidence of varicella in Singapore has been increasing since 1984. In 1991,17,930 cases were reported in a population of about 3 million. A serological survey completed in 1990 demonstrated that only 43% of the cohort had antibodies to varicella-zoster virus (VZV), indicating inadequate herd immunity. To exclude novel VZV strains, representative VZV isolates from 9 chicken pox and 4 zoster patients were characterised by restriction endonuclease analysis. DNAs were extracted from viral isolates propagated in MRC5 human embryo lung cells and were digested separately with Bg/ll, EcoRI, Pstl, Sall, and Xbal enzymes. The cleavage profiles of these VZV strains derived from both chicken pox and zoster lesions revealed no distinct differences. This observation implies that the current upsurge of chicken pox most likely stems from closely related VZV genotypes infecting a susceptible population with insufficient herd immunity. Comparison of the restriction fragments of the Singapore and the Dumas strains revealed polymorphisms of the Sal/I-D, SaI/l-E, and Xbal-l fragment lengths, which correlated with variable regions I, II, and Ill of the VZV genome, thereby representing geographically distinct genotypic variants of VZV. © 1993 Wiley-Liss, Inc.