RAPD Genotype Research Articles

Virulence Gene Properties and Phylogenetic Relationship of Salmonella Serotypes Isolated from Different Sources in North of Iran

Background: Salmonella species can cause various infections in humans and animals. The presence of certain genes determines the virulence of a Salmonella serotype. Objectives: The current research endeavor was undertaken to assess the virulence characteristics and genotypic traits of Salmonella serotypes extracted from various sources within the geographical boundaries of Iran. Methods: Salmonella isolates, previously retrieved and preserved in the veterinary microbiology laboratory, underwent serotyping and polymerase chain reaction (PCR) identification of nine virulence-associated genes. Genotyping was carried out using random amplified polymorphic DNA-PCR (RAPD-PCR). Results: All Salmonella isolates showed the presence of invA, sdiA, hilA, and iroB virulence genes. There were a total of 17 different virulence gene patterns among Salmonella serotypes. The presence of fliC and sefA genes and their related patterns were significant among S. typhimurium and S. enteritidis serotypes, respectively (P < 0.05). In the RAPD-PCR fingerprinting, 11 distinct clusters were obtained, and 16 isolates (26.66%) were classified as untypeable strains. There was a significant association between RAPD genotypes and Salmonella serotypes (P < 0.05), while the association between these RAPD patterns and the source of the isolates was not significant (P > 0.05). Conclusions: According to the results, Salmonella serotypes from non-human sources carry significant virulence determinants and show similar genotypic patterns with human isolates. These findings provide valuable insights into the virulence properties and genetic diversity of Salmonella serotypes in Iran, which could inform the development of effective control and prevention strategies for salmonellosis in the region.

Characteristics of <i>Bacillus cereus</i> complex Group Strains Isolated from Permafrost in Yakutia for Assessment of Microbiological Risks During Climate Change

Strains of Bacillus genus were isolated from soil samples in the permafrost region (Yakutia, Russia). The phenotypic characteristics of the strains are given. The analysis of the obtained data made it possible to assign them to the group Bacillus cereus complex. PCR analysis made it possible to determine the profile of B. cereus toxin synthesis genes in the genomes of the studied strains. Genetic characterization was obtained by RAPD genotyping and using MLVA loci used for genotyping of the anthrax pathogen. The results of genotyping at different levels of resolution made it possible to differentiate the studied strains from the B. anthracis species, to show their intraspecific genetic differences and the degree of relationship. Whole genome sequencing was carried out, based on the data of which MLST genotyping was carried out, which revealed 2 known sequence types and one new one, described for the first time in this work. The results obtained are of practical importance and are extremely interesting from the point of view of the evolution and phylogeography of the B. cereus complex group, since the fact that strains were isolated from permafrost suggests that their age may be much older than expected.

Understanding Age-Related Longitudinal Dynamics in Abundance and Diversity of Dominant Culturable Gut Lactic Acid Bacteria in Pastured Goats.

Understanding gut lactic acid bacteria (LAB) in healthy hosts is an important first step in selecting potential probiotic species. To understand the dynamics of LAB in healthy goats, a cohort of thirty-seven healthy new-born goat kids was studied over a ten-month period. Total LAB was quantified using SYBR green qPCR. Seven hundred LAB isolates were characterized using microscopy, M13 RAPD genotyping and 16S rDNA sequencing. The highest and lowest LAB counts were detected at one week and ten months of age, respectively. Diverse LAB species were detected, whose identity and prevalence varied with age. The main isolates belonged to Limosilactobacillus reuteri, Limosilactibacillus fermentum, Lactobacillus johnsonni, Ligilactobacillus murinus, Ligilactobacillus salivarius, Limosilactobacillus mucosae, Lactiplantibacillus plantarum, Ligilactobacillus agilis, Lactobacillus acidophilus/amyolovolus, Pediococcus spp. and Enterococcus spp. Uniquely, L. reuteri and Pediococcus spp. were most common in pre- and peri-weaned goats, while Lactobacillus mucosae and Enterococcus spp. were predominant in goats one month and older. Based on RAPD genotyping, L. reuteri had the highest genotypic diversity, with age being a factor on the genotypes detected. This data may be relevant in the selection of age-specific probiotics for goats. The findings may also have broader implications by highlighting age as a factor for consideration in probiotic bacteria selection in other animal hosts.

RAPD-PCR-Based Fingerprinting Method as a Tool for Epidemiological Analysis of Trueperella pyogenes Infections.

In this study, a Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) method for genetic typing of Trueperella pyogenes, an opportunistic bacterial pathogen, was designed. The method optimization was performed for 37 clinical T. pyogenes strains isolated from various infections in different animal species. Optimal conditions for reliable and reproducible DNA fingerprinting were determined according to the modified Taguchi method. The developed method was assessed regarding its typeability, reproducibility, and discriminatory power using the Hunter’s and Gatsons’ index of discrimination. A high degree of genetic diversity was shown between the studied strains, which represented 31 genotypes. The generated RAPD profiles were relatively complex and simultaneously easy to interpret due to the wide size range of amplicons. The discriminatory index of the designed method was sufficiently high; thus, only strains epidemiologically related displayed identical RAPD genotypes. In conclusion, the DNA fingerprinting of T. pyogenes by the developed RAPD-PCR method is a reliable typing tool that may allow a better understanding of the epidemiology as well as pathogenesis of infections caused by this pathogen.

Antibiotic resistance pattern and molecular typing by pcr-rapd analysis in clinical isolates of pseudomonas aeruginosa from motahari hospital, tehran, iran

Background: Pseudomonas aeruginosa infection is one of the major challenges in burn patients. This organism is resistant to a wide range of antibiotics. The aim of this study was to investigate the antibiotic susceptibility and genetic relatedness in Pseudomonas aeruginosa isolated from patients admitted to Motahari Burn Center, Tehran, Iran. Methods: This study was conducted on 186 burn patients with Pseudomonas aeruginosa colonization following admission in Motahari Burn Center during one year. Antibiotic resistance test was performed by disc diffusion method and genetic relatedness was evaluated by PCR-RAPD analysis. Results: The highest and lowest percentage of resistance was observed against ceftizoxime and ciprofloxacin. The genotyping study by RAPD PCR technique revealed 57 different genotypes, among which RAPD 5, RAPD 8, and RAPD 9 were the most prevalent patterns and produced by 14%, 9%, and 7% of the isolates, respectively. Conclusion: In total, no association was found between RAPD genotypes and antibiotic resistance patterns, and death rate. © 2021, Kerman University of Medical Sciences. All rights reserved

Molecular epidemiology and carbapenem resistance of Pseudomonas aeruginosa isolated from patients with burns.

The aim of this study was to investigate the molecular epidemiology and carbapenem resistance mechanisms of Pseudomonas aeruginosa isolated from patients with burns in Azerbaijan, Iran. Pseudomonas aeruginosa was isolated from 38 patients with burns. Disk diffusion and agar dilution methods were used to determine antibiotic susceptibility patterns. The overproduction of AmpC β-lactamase and efflux pumps were detected by phenotypic methods. The presence of carbapenemase-encoding genes was detected by multiplex polymerase chain reaction (PCR). Expression of the OprD gene and MexAB efflux pumps were also evaluated with real-time PCR. Random amplified polymorphic DNA typing (RAPD-PCR) was used for genotyping of carbapenem-resistant Pseudomonas aeruginosa (CRPA). Minimum inhibitory concentration (MIC) assays demonstrated high levels of resistance to all classes of antibiotics except colistin and polymyxin B. The initial screening by carbapenem disks indicated 24 isolates (63.15%) as CRPA. Different mechanisms of carbapenem resistance were observed, including carbapenemase production (8.4%), overexpression of AmpC (25%) and decreased expression of OprD (75%). The overexpression of MexAB efflux pumps was detected in 19 (79.1%) isolates by phenotypic assay or real-time PCR. The resistance to carbapenem was multifactorial in most cases (58.3%). The RAPD genotyping revealed different patterns with nine clusters. According to our results, the prevalence of CRPA is at an alarming level. Our results did not demonstrate an epidemic clone. The most common mechanism of carbapenem resistance was decreased expression of OprD. Therefore, we suggest a reconsideration in the management of CRPA infections of patients in our burn care hospital in Azerbaijan, Iran.

ERIC- PCR Typing, RAPD-PCR Fingerprinting and Quorum Sensing Gene Analysis of Pseudomonas aeruginosa Isolated from Different Clinical Sources

Recently, Pseudomonas aeruginosa infections proportions have increased significantly. Molecular typing and virulence analysis are good techniques, which can lead us to know P. aeruginosa infections. P. aeruginosa isolates were identified by using molecular method (16S rDNA gene) via PCR technique for accurate identification. The highest percent 41.26% of P. aeruginosa bacteria was found in the burn infections followed by 28.57% in wound swabs, 17.46% in ear discharge and lowest percentage were obtained from sputum samples. All isolates classified into six groups (A-F) according to classes of antibiotics. Of the 63 bacterial isolates, 100% were resistant to carbencillin, whereas 31.74% were resistant to ticarcillin and all isolates susceptible to imipenem. In addition all of clinical isolates indicated multidrug resistant (MDR) patterns, the highest rate of MDR was observed with pattern C these isolates were able to resist (9-12) antibiotics. All isolates were typed genotypically by using two methods of amplification, ERIC and RAPD-PCR. The results of the ERIC-PCR typing of P. aeruginosa bacteria that 96.82% showed amplification bands ERIC-PCR also revealed 17 groups of genotypes (A-R) and 4 unique isolates. The results of RAPD-PCR fingerprint revealed 12 groups of genotypes (A-M) of 40–90% similarity according to coefficient values and 4 unique isolates, except 7.93% were untypeble. QS genes (lasI, lasR, rhlI, rhlR), screen showed all isolates 100% were positive for one or more QS genes, in the other hand 82.53% carrying lasI, lasR, rhlI, and rhlR, while the 15.87% carrying lasI, rhlI, and rhlR and 1.58% carrying lasI, lasR, and rhlR genes. ERIC genotyping significantly correlated resistance patterns but not with virulence control QS genes. RAPD genotyping significantly correlated with source of infection, resistance patterns and virulence control QS genes. These results can help initial diagnosis MDR P. aeruginosa outbreaks associated with specific genotyping patterns.

Intestinal Candida parapsilosis isolates from Rett syndrome subjects bear potential virulent traits and capacity to persist within the host

BackgroundRett syndrome (RTT) is a neurological disorder mainly caused by mutations in MeCP2 gene. It has been shown that MeCP2 impairments can lead to cytokine dysregulation due to MeCP2 regulatory role in T-helper and T-reg mediated responses, thus contributing to the pro-inflammatory status associated with RTT. Furthermore, RTT subjects suffer from an intestinal dysbiosis characterized by an abnormal expansion of the Candida population, a known factor responsible for the hyper-activation of pro-inflammatory immune responses. Therefore, we asked whether the intestinal fungal population of RTT subjects might contribute the sub-inflammatory status triggered by MeCP2 deficiency.MethodsWe evaluated the cultivable gut mycobiota from a cohort of 50 RTT patients and 29 healthy controls characterizing the faecal fungal isolates for their virulence-related traits, antifungal resistance and immune reactivity in order to elucidate the role of fungi in RTT’s intestinal dysbiosis and gastrointestinal physiology.ResultsCandida parapsilosis, the most abundant yeast species in RTT subjects, showed distinct genotypic profiles if compared to healthy controls’ isolates as measured by hierarchical clustering analysis from RAPD genotyping. Their phenotypical analysis revealed that RTT’s isolates produced more biofilm and were significantly more resistant to azole antifungals compared to the isolates from the healthy controls. In addition, the high levels of IL-1β and IL-10 produced by peripheral blood mononuclear cells and the mixed Th1/Th17 cells population induced by RTT C. parapsilosis isolates suggest the capacity of these intestinal fungi to persist within the host, being potentially involved in chronic, pro-inflammatory responses.ConclusionsHere we demonstrated that intestinal C. parapsilosis isolates from RTT subjects hold phenotypic traits that might favour the previously observed low-grade intestinal inflammatory status associated with RTT. Therefore, the presence of putative virulent, pro-inflammatory C. parapsilosis strains in RTT could represent an additional factor in RTT’s gastrointestinal pathophysiology, whose mechanisms are not yet clearly understood.

Biofilm formation, antibiotic susceptibility and RAPD genotypes in Pseudomonas aeruginosa clinical strains isolated from single centre intensive care unit patients.

The aim of this study was to analyse genotypes, antimicrobial susceptibility patterns and serotypes in Pseudomonas aeruginosa clinical strains, including the clonal dissemination of particular strains throughout various intensive care units in one medical centre. Using random amplified polymorphic DNA (RAPD-PCR) and P. aeruginosa antisera, 22 different genotypes and 8 serotypes were defined among 103 isolates from 48 patients. No direct association between P. aeruginosa strain genotypes and serotypes was observed. RAPD typing in strains with the same serotype revealed different genotypes and, on the contrary, most strains with a different serotype displayed the same amplification pattern. The resulting banding patterns showed a high degree of genetic heterogeneity among all isolates from the patients examined, suggesting a non-clonal relationship between isolates from these patients. A higher degree of antibiotic resistance and stronger biofilm production in common genotypes compared to rare ones and genetic homogeneity of the most resistant strains indicated the role of antibiotic pressure in acquiring resistant and more virulent strains in our hospital. In conclusion, genetic characterisation of P. aeruginosa strains using RAPD method was shown to be more accurate in epidemiological analyses than phenotyping.

Pseudomonas aeruginosa in Dairy Goats: Genotypic and Phenotypic Comparison of Intramammary and Environmental Isolates

Following the identification of a case of severe clinical mastitis in a Saanen dairy goat (goat A), an average of 26 lactating goats in the herd was monitored over a period of 11 months. Milk microbiological analysis revealed the presence of Pseudomonas aeruginosa in 7 of the goats. Among these 7 does, only goat A showed clinical signs of mastitis. The 7 P. aeruginosa isolates from the goat milk and 26 P. aeruginosa isolates from environmental samples were clustered by RAPD-PCR and PFGE analyses in 3 genotypes (G1, G2, G3) and 4 clusters (A, B, C, D), respectively. PFGE clusters A and B correlated with the G1 genotype and included the 7 milk isolates. Although it was not possible to identify the infection source, these results strongly suggest a spreading of the infection from goat A. Clusters C and D overlapped with genotypes G2 and G3, respectively, and included only environmental isolates. The outcome of the antimicrobial susceptibility test performed on the isolates revealed 2 main patterns of multiple resistance to beta-lactam antibiotics and macrolides. Virulence related phenotypes were analyzed, such as swarming and swimming motility, production of biofilm and production of secreted virulence factors. The isolates had distinct phenotypic profiles, corresponding to genotypes G1, G2 and G3. Overall, correlation analysis showed a strong correlation between sampling source, RAPD genotype, PFGE clusters, and phenotypic clusters. The comparison of the levels of virulence related phenotypes did not indicate a higher pathogenic potential in the milk isolates as compared to the environmental isolates.

Late-onset neonatal group B streptococcal disease associated with breast milk transmission: molecular typing using RAPD-PCR

Group B Streptococcus (GBS) is considered to be the major cause of neonatal sepsis and meningitis of bacterial origin. Late-onset GBS infection is infrequent and occurs between 1 week and 3 months of age. The transmission of GBS through the ingestion of breast milk is reported in the literature, but only a few of these cases have been confirmed by molecular techniques. In this article we report five cases of late-onset GBS disease: transmission through maternal milk was confirmed in four cases, using the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) typing assay. In addition, the RAPD-PCR assay showed that each of the isolated clones belonged to a different RAPD genotype, thus revealing that the late-onset GBS infections were not epidemiologically related.

Discrimination of the bumble bee species Bombus occidentalis Greene and B. terricola Kirby by morphometric, colour and RAPD variation

The taxonomic status of B. terricola Kirby and B. occidentalis Greene has long been questioned. However recent COI gene sequence data suggests that B. occidentalis and B. terricola do represent good biological species. In this paper we test the hypothesis that B. terricola and B. occidentalis are conspecific by using independent morphometric and genetic (RAPD) data. For comparison we also analyzed one consubgeneric species, B. moderatus (now B. crytptarum), and one non-consubgeneric species B. (Pyrobombus) perplexus. Discriminant function analysis of wing morphometric data correctly classified over 85% of the specimens of B. occidentalis and B. terricola. Analysis of molecular variance of the RAPD data showed a significant difference (P < 0.0001) between B. occidentalis and B. terricola. Colour variation from laboratory reared colonies of B. occidentalis suggests that probably two gene locus inheritance is likely but that hybridization as the sole basis for the colour variation seen in the nominate taxon B. occidentalis can be excluded. We conclude that B. terricola and B. occidentalis should be regarded as distinct species which have recently diverged, and that they can be distinguished by wing morphometrics and RAPD genotypes. Where they are sympatric (e.g. in Alberta) colour pattern variation is confined to B. occidentalis.

Characterization of Klebsiella pneumoniae Strains Isolated from Urinary Tract Infection: Detection of ESBL Characteristics, Antibiotic Susceptibility and RAPD Genotyping

In this study, a hundred Klebsiella pneumoniae strains isolated from urinary tract infections were evaluated in terms of genotyping, susceptibility to certain antibiotics and detection of extended spectrum of beta lactamase (ESBL) production. The random amplified polymorphic DNA (RAPD-PCR) method was used to identify the genetic differentiation of K. pneumoniae isolates. A total of 26 different DNA bands ranging between 334 bp and 28033 bp were detected among the strains. It was found that 100 K. pneumoniae strains revealed 11 different RAPD profiles. Antibiotic susceptibility tests were conducted using a disc diffusion method against 16 antibiotics. Fifty-five different resistance profiles were determined among the strains. ESBL-productions of the strains were determined by the double disc synergy test (DDST) and ESBL E-test methods. ESBL production rates among the strains were found to be 55% by E-test method and 45% by DDST method. While ESBL-producing K. pneumoniae strains showed the greatest resistance to penicillin G (100%), followed by piperacillin (92.7%) and erythromycin (85.4%),the resistance rates of non ESBLproducing strains to those antibiotics were determined as 97.8%, 88.8% and 88.8%, respectively. Both groups of strains showed the highest sensitivity to meropenem. Based on the results obtained from the study, it was concluded that the detection of ESBL-producing strains by the E-test method was more sensitive than by the DDST method. Phenotypic and genotypic identification methods should be used together to detect ESBL presence. The RAPD-PCR method alone will not be adequate in the genotyping of the strains and alternative DNA-based methods should be used.

In Vivo Emergence of Aspergillus terreus with Reduced Azole Susceptibility and a Cyp51a M217I Alteration

Azole resistance in Aspergillus terreus isolates was explored. Twenty related (MB) and 6 unrelated A. terreus isolates were included. CYP51A sequencing and RAPD genotyping was performed. Five MB isolates were itraconazole susceptible, whereas the minimum inhibitory concentrations (MICs) for 15 MB isolates were elevated (1-2 mg/L). Voriconazole and posaconazole MICs were 0.5-4 and 0.06-0.5 mg/L, respectively, for MB isolates but 0.25-0.5 and <0.03-0.06 mg/L, respectively, for controls. Sequencing identified a Cyp51Ap M217I alteration in all 15 isolates with elevated itraconazole MICs. Genotyping showed that 18 of 20 MB isolates were identical and unique, suggesting endogenous origin. In conclusion, itraconazole resistance in A. terreus was linked to an M217I Cyp51A alteration.

Sharing of Bacterial Strains Between Breast Milk and Infant Feces

In previous years, it has been shown that human milk is a potential source of bacteria for the infant gut. The results of this work confirm the presence of the same specific bacterial strains of Bifidobacterium, Lactobacillus, and Staphylococcus in breast milk and infant fecal samples. The identity of bacteria isolated from breast milk and infant feces from 20 mother-infant pairs was investigated at the strain level. DNA from Staphylococcus, Lactobacillus, and Bifidobacterium was detected by qRTi-PCR in nearly all samples analyzed. These samples were cultured on different agar media. One colony representative of each morphology was selected and identified at the species level combining classical tests and molecular techniques (PCR, RAPD, PFGE, and/or MLST genotyping). Breast milk and infant feces from 19 mother-infant pairs shared different Staphylococcus, Lactobacillus, and/or Bifidobacterium species and strains. Significantly, 2 mother-infant pairs shared 4 bacterial strains although most pairs shared 2. These results confirm that breast milk and infant feces from mother-infant pairs share the same strain(s), indicating that breastfeeding could contribute to the bacterial transfer from the mother to the infant and, therefore, to the infant gut colonization.

Asymmetric somatic hybridization between Bupleurum scorzonerifolium Willd. and Taxus chinensis var. mairei

In an attempt to transfer the genetic components needed for taxol synthesis into a more tractable plant, protoplasts of Taxus chinensis var. mairei were UV irradiated for various times prior to their fusion with protoplasts of Bupleurum scorzonerifolium. Of the 60 presumptive hybrid calli obtained, selections of the 9 most rapidly growing were recognized as hybrid clones based on a combination of their callus morphology, esterase profile, chromosome number, and RAPD genotype. The RAPD data showed that 82.4-96.8% of the hybrid genome was inherited from the recipient (B. scorzonerifolium) and 4.6-13.9% from the donor (T. chinensis), with 1.6-6.9% of the RAPD fragments being not present in either parent. None of the hybrid clones expressed a detectable quantity of taxol, but four produced more of the triterpenoid oleanolic acid than did calli of the recipient. With the use of semi-quantitative reverse transcriptase PCR, it was possible to show variation in the expression of several triterpenoid biosynthetic pathway related genes between B. scorzonerifolium and the hybrids. Increasing the concentration of oleanolic acid requires that the expression levels of the genes encoding IPP isomerase, squalene synthase, squalene epoxidase and β-amyrin synthase are increased, while at the same time, those of the genes encoding the branching enzymes cycloartenol synthase and lupeol synthase need to be reduced.

POD ISOENZYME AND RAPD ANALYSIS ON LITCHI IN FUQING

The paper discussed the genetic relationship between superior individual late-maturity Yurongwanli and other cultivars. To do so POD isoenzyme and RAPD technology were used to analyze litchi germplasm resources of Fuqing city. There were 4 bands produced by POD isoenzyme electrophoresis. According to the differences of these bands, Shishengshu1, Shishengshu2, Xiafanzhi and Dongliuyihao were put in the frist category, while Shangfanzhi and Yuanhong were placed in the second category. RAPD-PCR analysis showed that seven primers produce a total of 59 bands, with 8.43 fragments per prime. Among these 59 bands 40 were polymorphic, with a 67% polymorphism rate. The DPS2000 software was used to analyze the RAPD genotype date matrix. The result showed that the 7 cultivars can be divided into two categories when the genetic distance D value is 0.70 and four categories when the D value is 0.60. These two results indicated that Yurongwanli has the closest genetic relationship with Hongxiuqiu.

Morphological and genomic characterization of Filobasidiella depauperata: a homothallic sibling species of the pathogenic cryptococcus species complex.

The fungal species Cryptococcus neoformans and Cryptococcus gattii cause respiratory and neurological disease in animals and humans following inhalation of basidiospores or desiccated yeast cells from the environment. Sexual reproduction in C. neoformans and C. gattii is controlled by a bipolar system in which a single mating type locus (MAT) specifies compatibility. These two species are dimorphic, growing as yeast in the asexual stage, and producing hyphae, basidia, and basidiospores during the sexual stage. In contrast, Filobasidiella depauperata, one of the closest related species, grows exclusively as hyphae and it is found in association with decaying insects. Examination of two available strains of F. depauperata showed that the life cycle of this fungal species shares features associated with the unisexual or same-sex mating cycle in C. neoformans. Therefore, F. depauperata may represent a homothallic and possibly an obligately sexual fungal species. RAPD genotyping of 39 randomly isolated progeny from isolate CBS7855 revealed a new genotype pattern in one of the isolated basidiospores progeny, therefore suggesting that the homothallic cycle in F. depauperata could lead to the emergence of new genotypes. Phylogenetic analyses of genes linked to MAT in C. neoformans indicated that two of these genes in F. depauperata, MYO2 and STE20, appear to form a monophyletic clade with the MAT a alleles of C. neoformans and C. gattii, and thus these genes may have been recruited to the MAT locus before F. depauperata diverged. Furthermore, the ancestral MAT a locus may have undergone accelerated evolution prior to the divergence of the pathogenic Cryptococcus species since several of the genes linked to the MAT a locus appear to have a higher number of changes and substitutions than their MATα counterparts. Synteny analyses between C. neoformans and F. depauperata showed that genomic regions on other chromosomes displayed conserved gene order. In contrast, the genes linked to the MAT locus of C. neoformans showed a higher number of chromosomal translocations in the genome of F. depauperata. We therefore propose that chromosomal rearrangements appear to be a major force driving speciation and sexual divergence in these closely related pathogenic and saprobic species.

Pseudomonas aeruginosa Cystic Fibrosis isolates of similar RAPD genotype exhibit diversity in biofilm forming ability in vitro

BackgroundPseudomonas aeruginosa is considered to grow in a biofilm in cystic fibrosis (CF) chronic lung infections. Bacterial cell motility is one of the main factors that have been connected with P. aeruginosa adherence to both biotic and abiotic surfaces. In this investigation, we employed molecular and microscopic methods to determine the presence or absence of motility in P. aeruginosa CF isolates, and statistically correlated this with their biofilm forming ability in vitro.ResultsOur investigations revealed a wide diversity in the production, architecture and control of biofilm formation. Of 96 isolates, 49% possessed swimming motility, 27% twitching and 52% swarming motility, while 47% were non-motile. Microtitre plate assays for biofilm formation showed a range of biofilm formation ability from biofilm deficient phenotypes to those that formed very thick biofilms. A comparison of the motility and adherence properties of individual strains demonstrated that the presence of swimming and twitching motility positively affected biofilm biomass. Crucially, however, motility was not an absolute requirement for biofilm formation, as 30 non-motile isolates actually formed thick biofilms, and three motile isolates that had both flagella and type IV pili attached only weakly. In addition, CLSM analysis showed that biofilm-forming strains of P. aeruginosa were in fact capable of entrapping non-biofilm forming strains, such that these 'non-biofilm forming' cells could be observed as part of the mature biofilm architecture.ConclusionsClinical isolates that do not produce biofilms in the laboratory must have the ability to survive in the patient lung. We propose that a synergy exists between isolates in vivo, which allows "non biofilm-forming" isolates to be incorporated into the biofilm. Therefore, there is the potential for strains that are apparently non-biofilm forming in vitro to participate in biofilm-mediated pathogenesis in the CF lung.

Clonal Identification Based on Quantitative, Codominant, and Dominant Marker Data: A Comparative Analysis of Selected Willow (SalixL.) Clones

Clonal identification in forestry may employ different means, each with unique advantages. A comparative evaluation of different approaches is reported. Nine quantitative leaf morphometric parameters, 15 variable codominant (isoenzyme) and 15 variable dominant (RAPD) loci, were used. All clones presented unique multilocus isoenzyme genotypes and 86% presented unique multilocus RAPD genotypes. Quantitative, isoenzyme and molecular data were subjected to principal component analysis, the latter two data sets after vector transformation. Most of the variability (quantitative 99%, isoenzyme 72.5%, RAPD 89%) was accounted for in the first three axes. This study has shown: (1) individual quantitative parameters were inefficient for clonal identification, (2) multilocus clonal identification was successful, (3) dominant markers were more polymorphic than codominant ones: 1.5 variable loci per enzyme system, 7.5 variable RAPD loci per primer, (4) 15 codominant marker loci could identify about 2.8 times more individuals than 15 dominant ones, but this advantage is surpassed when 42 dominant loci are employed, (5) multivariate analysis of morphological, codominant and dominant genetic data could not discriminate at the clonal level. It was concluded that due to their higher number of loci available dominant markers perform better than codominant ones, despite the higher informativeness of the latter.