Screening among nocardioform bacteria for strains able to degrade aniline and monochloroanilines

A total of 106 strains of nocardioform bacteria were chosen from the IMET Culture Collection and checked for their ability to degrade aniline. Among them only four Rhodococcus strains proved to be capable of removing aniline from a liquid mineral salts medium devoid of any other carbon and nitrogen source. This capability was found to be due to total degradation of the aromatic substrate via the β-ketoadipate pathway which is initiated by the action of inducible oxygenating enzymes. Without any exception, the ring-cleaving enzymes induced during cell precultivation with aniline were pyrocatechases of the “ordinary” type. None of the four aniline-assimilating Rhodococcus strains could utilize any of the monochloroaniline isomers as the sole carbon and energy source for growth. However, resting aniline-grown cells of these strains mediated the turnover of 2- and 3-chloroaniline in the presence of chloramphenicol. The respective rates were substantially higher (31 to 59% as compared with the unsubstituted aniline) when glucose was provided as the additional carbon substrate. In contrast, 4-chloroaniline turnover occurred with low rates (3–6%), even in the presence of glucose. With all of the four Rhodococcus strains under study, 3-chloroaniline turnover resulted in chloride levels which corresponded to an almost stoichiometric dechlorination of the substrate. 2-Chloroaniline, however was only partially dechlorinated even in the presence of glucose indicating that certain chlorinated metabolite(s) was (were) accumulated in the respective incubation media.     

Isolation and characterization of bacteria from crude petroleum oil contaminated soil and their potential to degrade diesel fuel

Potential hydrocarbon degrader bacteria were isolated from soil samples that have been exposed to crude petroleum oil spills. Bacterial population of these polluted soils showed counts ranging between 9.5 x 10(5) and 237.5 x 10(5) CFU/g soil with 2 different colony types of bacterial strains which have been recovered on the agar plates. Results indicated that longer aged contamination exhibited a greater number of microorganisms. Phenotypic examination of the recovered bacteria revealed that they belong mainly to the genus Pseudomonas, Enterobacter and Acinetobacter. Turbidity, dry weight and physical appearance were used as an indication for the ability of these bacteria to grow on diesel. Action of three different Pseudomonas species, Acinetobacter lowffi, Enterobacter cloacae and Rhodococcus erythropolis on 0.1 % (v/v) diesel was followed at 1, 2, 6 and 12 h. Pseudomonas putida and P. mallei and Enterobacter cloacae indicated a positive reaction; however, Pseudomonas maltophilia and Acinetobacter lowffi showed no effect.     

Molecular genetic characterization of multidrug-resistant Acinetobacter baumannii strains and assessment of their sensitivity to phage AP22

Molecular genetic analysis of 130 multidrug-resistant nosocomial Acinetobacter baumannii strains was performed. The strains were obtained from patients admitted to different hospitals in large Russian cities (Chelyabinsk, Moscow, Nizhny Novgorod, and St. Petersburg) in 2005–2010. Species identification was performed by the amplified 16S rRNA gene restriction analysis and by determining the bla _OXA-51-like genes intrinsic for A. baumannii using PCR. Genetic typing of the strains was performed by RAPD-PCR. All strains fell into two clusters, A and B, with the dominant RAPD groups A1 and B1, respectively, including 82% (107 out of 130) of all strains under study. Susceptibility of the strains to bacteriophage AP22 was determined. The phage was shown to infect specifically and to lyse 69% of 130 strains and 82% (88 out of 107) of A. baumannii strains from the dominant RAPD groups. The ability of bacteriophage AP22 to lyse a broad range of clinically relevant A. baumannii strains makes it an attractive candidate for designing phage cocktails intended to control the A. baumannii-associated nosocomial infections. Moreover, the phage can be used for identifying A. baumannii in the bacteriological tests of clinical samples.     

Molecular and phenotypic characterization of ring chromosome 22

We performed a phenotype study of 35 individuals (19 males, 16 females) with ring chromosome 22 or r(22) with a mean age of 10 years. In common with other studies, a phenotype of moderate-to-profound learning difficulties and delay or absence of speech affected all individuals with the exception of the case with the smallest deletion. Autistic traits were significantly associated with r(22), as shown by an autism screening questionnaire. Mild and variable dysmorphic features, predominantly craniofacial and distal limb, were observed. Internal organ involvement was uncommon. Even though ring chromosomes are reportedly associated with growth abnormalities, only 2 out of 24 individuals showed evidence of growth failure, while 2 showed accelerated growth. Chromosome 22 long arm deletions, as determined by hemizygosity for informative microsatellite markers, varied from <67 kb to 10.2 Mb in size (or <0.15 to 21% of total chromosome length), with no significant differences in the parental origin of the ring chromosome. Few phenotypic features correlated with deletion size suggesting a critical gene, or genes, of major effect lies close to the telomere. Loss of the SHANK3/PROSAP2 gene has been proposed to be responsible for the main neurological developmental deficits observed in 22q13 monosomies. This study supports this candidate gene by identifying a phenotypically normal r(22) individual whose ring chromosome does not disrupt SHANK3. All other r(22) individuals were hemizygous for SHANK3, and we propose it to be a candidate gene for autism or abnormal brain development.     

Comparison of outbreak and nonoutbreak Acinetobacter baumannii strains by genotypic and phenotypic methods.

Thirty-one Acinetobacter baumannii strains, comprising 14 strains from 14 outbreaks in different northwestern European cities and 17 sporadic strains, were compared by investigating various properties of the strains including biotype, antibiogram, cell envelope protein electrophoretic profile, ribotype pattern, and the band pattern generated by a novel genomic fingerprinting method, named AFLP, which is based on the selective amplification of restriction fragments. Results showed that 12 strains from unrelated outbreaks were linked together in two clusters according to their similarities by these typing methods, whereas sporadic strains were more heterogeneous. Outbreak strains appeared to be markedly more resistant to antibiotics than nonoutbreak strains. The uniformity of typing characters in two sets of outbreak strains suggests that strains in each cluster have a common clonal origin.     

Molecular and phenotypic characterization of invasive group B streptococcus strains from infants in Norway 2006–2007

Multilocus sequence typing of an almost complete collection of invasive group B streptococcus (GBS) strains from infants in Norway, conducted in 2006–2007, revealed 27 sequence types (ST), of which 23 clustered into five clonal complexes. The case fatality rate of invasive GBS disease in infants was 16/98 (16.3%). Type V strains were predominant among strains resistant to erythromycin and clindamycin (11/18; 61.1%). All type V strains from fatal cases (5/16) were ST1, resistant to erythromycin and clindamycin, and belonged to three pulsed-field gel electrophoresis-clusters. Further analysis of virulence characteristics of these apparently highly virulent subtypes of type V, ST1 GBS strains is warranted.     

Genetic and phenotypic characterization of sylvatic dengue virus type 4 strains

Four serotypes of dengue virus (DENV 1-4) currently circulate between humans and domestic/peridomestic Aedes mosquitoes, resulting in 100 million infections per year. All four serotypes emerged, independently, from sylvatic progenitors transmitted among non-human primates by arboreal Aedes mosquitoes. This study investigated the genetic and phenotypic changes associated with emergence of human DENV-4 from its sylvatic ancestors. Analysis of complete genomes of 3 sylvatic and 4 human strains revealed high conservation of both the 5′- and 3′-untranslated regions but considerable divergence within the open reading frame. Additionally, the two ecotypes did not differ significantly in replication dynamics in cultured human liver (Huh-7), monkey kidney (Vero) or mosquito (C6/36) cells, although significant inter-strain variation within ecotypes was detected. These findings are in partial agreement with previous studies of DENV-2, where human strains produced a larger number of progeny than sylvatic strains in human liver cells but not in monkey or mosquito cells.     

Genetic and phenotypic characterization of sylvatic dengue virus type 2 strains

The four serotypes of endemic dengue viruses (DENV) circulate between humans and peridomestic Aedes mosquitoes. At present endemic DENV infect 100 million people per year, and a third of the global population is at risk. In contrast, sylvatic DENV strains are maintained in a transmission cycle between nonhuman primates and sylvatic Aedes species, and are evolutionarily and ecologically distinct from endemic DENV strains. Phylogenetic analyses place sylvatic strains basal to each of the endemic serotypes, supporting the hypothesis that each of the endemic DENV serotypes emerged independently from sylvatic ancestors. We utilized complete genome analyses of both sylvatic and endemic DENV serotype 2 (DENV-2) to expand our understanding of their genetic relationships. A high degree of conservation was observed in both the 5'- and 3'-untranslated genome regions, whereas considerable differences at the nucleotide and amino acid levels were observed within the open reading frame. Additionally, replication of the two genotypes was compared in cultured cells, where endemic DENV strains produced a significantly higher output of progeny in human liver cells, but not in monkey kidney or mosquito cells. Understanding the genetic relationships and phenotypic differences between endemic and sylvatic DENV genotypes may provide valuable insight into DENV emergence and guide monitoring of future outbreaks.     

Envelope glycosylation determined by lectins in microscopy sections of Acinetobacter venetianus induced by diesel fuel

It was suggested in a previous study that cells of Acinetobacter venetianus VE-C3 adhere to diesel fuel by synthesizing a capsular polysaccharide containing glucose and/or mannose. To study the fine structure of cells and localization of bacterial polysaccharide in the presence of diesel fuel, two lectins were used: ConA, an agglutinin from Canavalia ensiformis specific for mannose and/or glucose residues, and PNA, an agglutinin from Arachis hypogaea, for terminal galactose residues. The lectins were conjugated with electron dense ferritin for transmission electron microscopy (TEM) and with fluorescein isothiocyanate (FITC) for scanning confocal laser microscopy (SCLM). Samples were prepared by freeze substitution, which allows glycosylation to be determined in situ in thin sections of specimens. The distribution of glycosylation was imaged with and without treatment of specimens with their specific hapten (glucose and galactose). The glycosylation activity produced a polysaccharide capsule. Emulsified diesel fuel nanodroplets were observed at the cell envelope perimeter. Fine structure of vesicles consisted of polysaccharide and diesel fuel nanodroplets. Lectin blotting analysis showed ConA-positive glycoprotein with an apparent molecular mass of 22 kDa in the outer membrane. Its production was induced by diesel fuel. This glycoprotein was probably responsible for bioemulsifying activity at the cell envelope. Several other glycoproteins were positive for PNA lectin, the main constituent migrating with an apparent molecular weight of 17.8 kDa. However, they were all constitutive and probably involved in cell biofilm formation at the oil surface.     

Molecular and phenotypic characterization of potentially new Shigella dysenteriae serotype

From September 1997 to November 1998, the French National Center for Salmonella and Shigella received 22 Shigella isolates recovered from 22 different patients suffering from dysentery. None of these isolates reacted with any of the antisera used to identify established Shigella serotypes, but all of them agglutinated in the presence of antisera to a previously described potentially new Shigella dysenteriae serotype (represented by strain 96-204) primarily isolated from stool cultures of imported diarrheal cases in Japan. All French isolates, as well as strain 96-204, showed biochemical reactions typical of S. dysenteriae and gave positive results in a PCR assay for detection of the plasmid ipaH gene coding for invasiveness. No Shiga toxin gene was detected by PCR. These isolates were indistinguishable by molecular analysis of ribosomal DNA (ribotyping) and seemed to be related to S. dysenteriae serotypes 3 and 12. However, further characterization by restriction of the amplified O-antigen gene cluster clearly distinguished this new serotype from all other Shigella or Escherichia coli serotypes.     

Molecular characterization of three rabbit rotavirus strains

Summary We report biochemical (RNA and protein patterns and gene-coding assignments) and serologic (serotype and subgroup) properties of three strains of rabbit rotaviruses — Ala, C11 and R2. The RNA electropherotypes were a standard short pattern for R2, an unusual short pattern for Ala, and an unusual long pattern for C11. Serologic studies indicated that these viruses were all group A serotype 3 rotaviruses. In addition, the Ala and C11 viruses were found to possess subgroup I specificity, whereas the R2 virus possessed subgroup II specificity. In contrast to their distinctive RNA patterns, the polypeptide patterns of the rabbit viruses were similar to those of SA11. To identify cognate genes and determine gene-coding assignments for the rabbit rotaviruses, cDNA probes of individual SA11 genes were hybridized to rabbit rotaviral genomic RNA segments that had been separated by polyacrylamide gel electrophoresis and transferred to filters (Northern blots). The order of genome segments 7–11 for each of the rabbit rotaviruses was unique and differed from that of SA11 genes. These differences were possibly due to rearrangements of the RNA sequences within these individual genome segments. Sequence analysis of the individual RNA segments will confirm whether genome rearrangements are the molecular basis for these novel migration patterns.On leave from the Department of Laboratory Medicine, Kinan General Hospital, Tanabe, Japan.     

Molecular and phenotypic characterization of Phialemonium and Lecythophora isolates from clinical samples

Several members of the fungal genera Phialemonium and Lecythophora are occasional agents of severe human and animal infections. These species are difficult to identify, and relatively little is known about their frequency in the clinical setting. The objective of this study was to characterize morphologically and molecularly, on the basis of the analysis of large-subunit ribosomal DNA sequences, a set of 68 clinical isolates presumed to belong to these genera. A total of 59 isolates were determined to be Phialemonium species (n = 32) or a related Cephalotheca species (n = 6) or Lecythophora species (n = 20) or a related Coniochaeta species (n = 1). Nine isolates identified to be Acremonium spp. or Phaeoacremonium spp. were excluded from further study. The most common species were Phialemonium obovatum and Phialemonium curvatum, followed by Lecythophora hoffmannii, Cephalotheca foveolata, and Lecythophora mutabilis.     

Molecular characterization of Echinococcus granulosus strains in Sardinia

Investigations were undertaken to determine the genotypes of the parasite Echinococcus granulosus that were present in livestock animals on the island of Sardinia. Liver, lung, and spleen samples were obtained from 770 sheep, 229 cattle, and 277 pigs slaughtered in Sardinia between January 2003 and April 2005, and the number and fertility of hydatid cysts were determined. Protoscoleces and/or germinal layer were collected from individual cysts, DNA was extracted from 91 samples, and polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) methods were used for identification of the strain genotype for each sample (G1, G5, G6/G7). Fragments of the mitochondrial cytochrome c oxidase subunit 1 and NADH dehydrogenase I were sequenced. Hydatid disease prevalence of 75.3, 41.5, and 9.4% were found in the organs collected from sheep, cattle, and pigs, respectively. Molecular analysis showed that 89 of 91 ovine, bovine, and swine cysts belonged to the G1 genotype (common sheep strain) of E. granulosus. Parasite isolates from two pigs were identified to belong to the G7 genotype (pig strain). Our results confirm the high prevalence of E. granulosus infection in livestock animals in Sardinia and reveal the presence of at least two parasite genotypes in Sardinia.     

Molecular characterization of two strains of Shope fibroma virus

An isolate of Shope fibroma virus (SFV), designated Indiana (SFV-I), was previously described to be tumorigenic in vivo as SFV, cytocidal in vitro as the orthopoxviruses (vaccinia, rabbitpox, etc.), and to share antigenic determinants with SFV and vaccinia. The genetic relatedness of SFV-I to SFV and vaccinia was studied by means of Southern blotting and hybridization. The results indicated that SFV-I shares extensive DNA homology with vaccinia, but few common sequences with SFV. By contrast, SFV and vaccinia show no sequence homology. These findings suggest that SFV-I is an orthopoxvirus which carries some genetic information from the leporipoxviruses. Recombinants between two genera of poxviruses have not been reported before.     

Genotypic and phenotypic characterization of Lactobacillus plantarum strains isolated from Thai fermented fruits and vegetables

Ten Lactobacillus strains originally isolated from Thai fruits and vegetables fermentation were characterized by various phenotypic and genotypic methods. The phenotypic analysis using the method of carbohydrate fermentation patterns (API50CHL) revealed that the isolates belonged to the L. plantarum species. This was further confirmed by 16S rRNA gene sequencing. Multilocus sequence typing (MLST) revealed a strongly clonal population structure and a low genotypic diversity in this collection. However, the analyzed L. plantarum population demonstrated a higher level of diversification after API50CHL that reflects the role of available carbohydrate sources in bacterial evolution. Our results support the postulate that a combination of conventional biochemical and genotyping methods allows a thorough characterization and identification of isolates. We propose that genotypic characterization could be complemented by biochemical characterization to discriminate L. plantarum strains. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Molecular characterization of novel G5 bovine rotavirus strains

Group A rotaviruses are a major cause of acute gastroenteritis in young children as well as many domestic animals. The rotavirus genome is composed of 11 segments of double-stranded RNA and can undergo genetic reassortment during mixed infections, leading to progeny viruses with novel or atypical phenotypes. The aim of this study was to determine if the bovine group A rotavirus strains KJ44 and KJ75, isolated from clinically infected calves, share genetic features with viruses obtained from heterologous species. All 11 genes sequences of the KJ44 and KJ75 strains were sequenced and analyzed. The KJ44 VP4 had 91.7% to 96.3% deduced amino acid identity to the bovine related P[1] strain, whereas the KJ75 strain was most closely related to the bovine related P[5] strain (91.9% to 96.9% amino acid identity). Both KJ44 and KJ75 strains also contained the bovine related VP3 gene. The remaining 9 segments were closely related to porcine group A rotaviruses. The KJ44 and KJ75 strains showed high amino acid identity to the G5 rotaviruses, sharing 90.4% to 99.0% identity. In addition, these strains belonged to the NSP4 genotype B, which is typical of porcine rotaviruses and subgroup I, with the closest relationship to the porcine JL-94 strain. These results strongly suggest that bovine rotavirus strains with the G5 genotype occur in nature as a novel G genotype in cattle as a result of a natural reassortment between bovine and porcine strains.     

Molecular characterization of Brucella strains isolated from marine mammals

Recently, gram-negative bacteria isolated from a variety of marine mammals have been identified as Brucella species by conventional phenotypic analysis. This study found the 16S rRNA gene from one representative isolate was identical to the homologous sequences of Brucella abortus, B. melitensis, B. canis, and B. suis. IS711-based DNA fingerprinting of 23 isolates from marine mammals showed all the isolates differed from the classical Brucella species. In general, fingerprint patterns grouped by host species. The data suggest that the marine mammal isolates are distinct types of Brucella and not one of the classical species or biovars invading new host species. In keeping with historical precedent, the designation of several new Brucella species may be appropriate.     

Molecular characterization of norovirus GII strains identified in Albania

Noroviruses (NoVs) are considered as the leading cause of diarrheal diseases in all groups of age. In the last decade the number of NoV outbreaks worldwide is increasing. Data published by the systems of NoV surveillance show the GII.4 strain as the human predominant genotype circulating worldwide and new genetic variants of GII.4 were associated with epidemic events. In Albania the economy transformation has damaged significantly the environment and a large circulation of enteric viruses was reported in the past with the presence of NoV among the genotyped strains. This study aimed to characterize, by molecular analysis, the NoV GII strains detected in Albania during two time periods: in 2010 from the outbreak occurred in Ballsh and in 2002–2003 from sporadic cases of diarrhoea. A total of 21 Nov GII strains were characterized. The NoV GII.4 was genotyped more frequently and it was related closely to the pandemic variants recorded in GenBank. During 2002–2003, six NoV GII recombinant strains have been characterized. J. Med. Virol. 85:731–736, 2013. © 2013 Wiley Periodicals, Inc.