Prevalence, in-vitro secretory activity, and cytotoxicity of Aeromonas species associated with childhood gastroenteritis in Chennai (Madras), India

An investigation on the prevalence of Aeromonas in gastrointestinal illnesses of pediatric inpatients 1 month to 3 years of age was conducted from February 1997 through January 1998 in Madras. Sixteen Aeromonas spp. were isolated from 11 male and five female children among the 341 pediatric inpatients suffering from acute diarrhoea. A. caviae, which was isolated from nine cases, was found to be the most predominant isolate, followed by A. veronii biovar sobria, isolated from six cases, and A. hydrophila, isolated from one case. Shigella flexneri was recovered along with Aeromonas veronii biotype sobria serotype 035 from one 5-month-old female child. We did not notice any seasonal pattern in the association between Aeromonas and childhood gastroenteritis. None of the 147 stool samples obtained from age-matched non-diarrhoeic control children yielded Aeromonas spp. Isolation of Aeromonas spp. from patients suffering from gastroenteritis was found to be significant (chi 2 = 7.1312; P = 0.008, < 0.01). Among the 16 Aeromonas isolates, seven isolates of A. caviae and two isolates of A. veronii biovar sobria induced a secretory response in rabbit intestinal mucosa mounted in Ussing chambers as demonstrated by a significant increase in the short circuit current. Nine of the 16 Aeromonas isolates, including three isolates of A. caviae, five isolates of A. veronii biovar sobria, and the solitary isolate of A. hydrophila were also cytotoxic to CHO cells. Five of the six isolates of A. veronii biovar sobria and the A. hydrophila isolate produced hemolysin. The results of this study indicate that Aeromonas species are important causative agents of diarrhoea in childhood gastroenteritis and are prevalent throughout the year in Madras.     

Stimulatory effect of water stress on plant regeneration in aromatic Indica rice varieties

Frequency of regeneration of fertile plants from cell suspensions was significantly increased using water stress treatments in two commercially cultivated Indian aromatic rice varieties, Basmati 385 and Pusa Basmati 1. The water stress treatments included the use of 1.0% (w/v) agarose instead of 0.5% (w/v) for medium solidification, inclusion of mannitol (0.1, 0.2 and 0.4 M) in regeneration medium, or 24 h partial desiccation of calli. When the agarose concentration of the regeneration medium was increased from 0.5% to 1.0% (w/v), the frequency of shoot formation in Pusa Basmati 1 from cell suspension-derived calli increased by over eightfold, to 86%. Mannitol, at 0.1 to 0.2 M concentration, stimulated the frequency of shoot regeneration in Pusa Basmati 1 by fivefold but had no effect in Basmati 385. Mannitol at 0.4 M concentration completely inhibited shoot regeneration but promoted embryogenesis. These calli regenerated shoots with greater frequencies when transferred to mannitol-free medium. Partial desiccation of rice calli resulted in an up to threefold increase in the shoot regeneration frequency. Best regeneration frequencies (54–98%) were obtained when 24 hdesiccated calli were grown on regeneration medium with 1.0% (w/v) agarose. A similar stimulatory effect of water stress on plant regeneration was observed in another Indica rice variety, IR43, and a Japonica rice variety, Taipei 309.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -napthaleneacetic acid On leave from Department of Genetic, Haryana Agricultural University, Hisar, India     

Biochemical and molecular characterization of tetracycline-resistant Aeromonas veronii isolates from catfish

Eighty-one tetracycline-resistant Aeromonas sp. strains were isolated from farm-raised catfish. Morphological and biochemical characteristics indicated that 23 of the 81 aeromonads were Aeromonas hydrophila, 7 isolates were Aeromonas trota, 6 isolates were Aeromonas caviae, 42 isolates were Aeromonas veronii, and 3 isolates were Aeromonas jandaei. However, the AluI and MboI restriction fragment length polymorphism (RFLP) patterns of the PCR-amplified 1.4-kb 16S rRNA gene from all 81 tetracycline-resistant aeromonads from catfish were identical to the RFLP banding patterns of A. veronii ATCC 35626, indicating that all 81 isolates were strains of A. veronii. A multiplex PCR assay successfully amplified the 5 tetracycline-resistant genes (tetA to E) from the genomic DNA of all 81 isolates. The assay determined that tetE was the dominant gene occurring in 73/81 (90.0%) of the aeromonads. Plasmids (2.0 to 20 kb) were isolated from 33 of the 81 isolates. Dendrogram analysis of the SpeI pulsed-field gel electrophoresis identified 15 distinct macrorestriction patterns among the isolates. Our results indicate the need for use of 16S rRNA in the identification of Aeromonas spp. and the prevalence of catfish as a reservoir of tet genes.     

Rapid identification of Aeromonas species using 16S rDNA targeted oligonucleotide primers: a molecular approach based on screening of environmental isolates

M. DORSCH, N.J. ASHBOLT, P.T. COX AND A.E. GOODMAN. 1994. Published 16S rDNA sequencing data for Aeromonas species were analysed and the validity of signature sequences derived from our investigations of these sequences was examined by sequencing the corresponding 16S rDNA regions of 67 environmental isolates from sewage effluents and receiving waters around Sydney and one clinical isolate, all previously classified as Aeromonas species. Species-specific probes for Aer. hydrophila and Aer. veronii were designed and tested in PCR assays and clearly discriminated these species from the other Aeromonas isolates as identified by 16S rDNA sequences.     

Introgression of Drought Tolerance into Elite Basmati Rice Variety through Marker-Assisted Backcrossing

Drought is one of the major abiotic threat to rice production in the context of climate change. Super Basmati is an elite, fine grain basmati rice variety grown in Punjab, Pakistan. Due to drought sensitive in nature, its yield has been facing an alarming situation in production because of gradual decrease in irrigated water for a couple of years. Three reported novel QTLs for drought tolerance were selected for incorporation into Super Basmati by employing marker assisted selection strategy. IR55419-04 with novel QTLs was used as a donor parent. Foreground selection was performed by applying PCR based QTL linked SSR markers followed by recombinant selection by using 2–4 flanking markers. Background selection was exercised by using polymorphic SSR markers for maximum genome recovery of the Super Basmati. The individuals homozygous at the target QTLs and with maximum background of Super Basmati at the rest of the non-target genome was selected for evaluation of drought tolerance. Under drought stress conditions, the yields of all introgressed lines (ILs) were 44.2%–125.7% higher than recurrent parent. Six superior ILs that are drought tolerant and very similar to Super Basmati in terms of agronomic and grain quality traits are marked for release as drought-tolerant varieties in arid regions or for use in breeding programs of high grain quality and drought-tolerant parents.     

Endophytic colonization and in planta nitrogen fixation by a Herbaspirillum sp. isolated from wild rice species.

Nitrogen-fixing bacteria were isolated from the stems of wild and cultivated rice on a modified Rennie medium. Based on 16S ribosomal DNA (rDNA) sequences, the diazotrophic isolates were phylogenetically close to four genera: Herbaspirillum, Ideonella, Enterobacter, and Azospirillum. Phenotypic properties and signature sequences of 16S rDNA indicated that three isolates (B65, B501, and B512) belong to the Herbaspirillum genus. To examine whether Herbaspirillum sp. strain B501 isolated from wild rice, Oryza officinalis, endophytically colonizes rice plants, the gfp gene encoding green fluorescent protein (GFP) was introduced into the bacteria. Observations by fluorescence stereomicroscopy showed that the GFP-tagged bacteria colonized shoots and seeds of aseptically grown seedlings of the original wild rice after inoculation of the seeds. Conversely, for cultivated rice Oryza sativa, no GFP fluorescence was observed for shoots and only weak signals were observed for seeds. Observations by fluorescence and electron microscopy revealed that Herbaspirillum sp. strain B501 colonized mainly intercellular spaces in the leaves of wild rice. Colony counts of surface-sterilized rice seedlings inoculated with the GFP-tagged bacteria indicated significantly more bacterial populations inside the original wild rice than in cultivated rice varieties. Moreover, after bacterial inoculation, in planta nitrogen fixation in young seedlings of wild rice, O. officinalis, was detected by the acetylene reduction and (15)N(2) gas incorporation assays. Therefore, we conclude that Herbaspirillum sp. strain B501 is a diazotrophic endophyte compatible with wild rice, particularly O. officinalis.     

Molecular characterization and distribution of virulence-associated genes amongst Aeromonas isolates from Libya

AIMS: The aim of the study was to type 52 Aeromonas spp. isolates from chicken carcasses, children with diarrhoea and a hospital environment in Libya, and to determine the distribution of putative virulence genes amongst them. METHODS AND RESULTS: Macrorestriction analysis using pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of 16S rRNA and aroA genes were used to type the isolates. Whereas 30 of 32 chicken isolates were identified as Aeromonas veronii, eight of 12 environmental isolates were Aer. caviae. Three species were identified amongst the eight isolates from children. Aeromonas veronii and Aer. caviae isolates could be divided into eight and five PFGE types, respectively. All species could be further subtyped into one of 21 aroA PCR-RFLP groups. Aerolysin-like haemolysin or enterotoxin gene sequences were detected in all the isolates. Overall carriage rates for hlyA and alt were 77 and 75%, respectively. CONCLUSIONS: Seven of eight isolates from children were of different subtypes, indicating a lack of any common source of acquisition. Isolates of common molecular type did not share identical distributions of putative virulence genes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the effectiveness of using molecular typing to identify and study genetic variation amongst Aeromonas isolates.     

Incidence of toxic Aeromonas isolated from food and human infection

One hundred and ninety four Aeromonas isolates (99 from food and 95 from clinical sources) were analyzed as to the species involved and the toxins produced. Of the clinical isolates of Aeromonas, 29.4% were enterotoxigenic, 43.1% were hemolytic and 89% were cytotoxigenic. Among the food isolates, 18.2% were enterotoxigenic, 17.1% were hemolytic and 72.7% were cytotoxigenic. Aeromonas sobria and Aeromonas veronii produced more enterotoxin and cytotoxin than the other isolates, whereas A. veronii and Aeromonas salmonicida produced cell-free hemolysin. Most of the isolates produced cytotoxins (81%) active on Vero (green monkey kidney) and Chinese hamster ovary cells, but only the culture supernatant of A. sobria produced vacuolation in these cell lines.     

Virulence markers in Aeromonas hydrophila and Aeromonas veronii biovar sobria isolates from freshwater fish and from a diarrhoea case

AIMS: To evaluate the public health significance of representative strains of two Aeromonas spp., mainly from freshwater fish, on the basis of production of virulence-associated factors and presence of the haemolytic genes aerA and hlyA. METHODS AND RESULTS: Eleven strains of Aer. hydrophila, three strains of Aer. veronii biovar sobria (all from freshwater fish) and one strain of Aer. hydrophila from human diarrhoea were tested for potential virulence traits and for the presence of the haemolytic genes aerA and hlyA. Ten Aer. hydrophila isolates were aerA(+)hlyA(+) and two aerA(+)hlyA(-). Aeromonas veronii biovar sobria isolates were aerA(-)hlyA(-). Strains from the three genotypes showed enterotoxic activity in the suckling mouse assay. At 28 degrees C, four Aer. hydrophila fish strains could be considered as potentially virulent (possessing at least two of these characteristics: haemolytic, cytotoxic and enterotoxic). One Aer. veronii biovar sobria strain and the clinical isolate were cytotoxic on Vero cells. When grown at 4 degrees C, these six isolates fulfilled virulence criterion, but at 37 degrees C, only one fish strain, an Aer. hydrophila, did. CONCLUSIONS: The potential health risk derived from the presence of Aer. hydrophila and Aer. veronii biovar sobria in ice-stored freshwater fish should not be underestimated. SIGNIFICANCE AND IMPACT OF THE STUDY: Expression of virulence factors is affected by temperature incubation and not always related to the presence of haemolytic genes.     

Distribution of Aeromonas Species in the Intestinal Tracts of River Fish

Aeromonas isolates were obtained from fish intestines, water, and sediments from an urban river and identified by the DNA-DNA microplate hybridization method. The isolates were Aeromonas veronii (22%), Aeromonas caviae (18%), Aeromonas hydrophila (13%), Aeromonas sobria (8%), Aeromonas jandaei (7%), and other Aeromonas spp. (33%). Aeromonas species occurred at high densities with high incidences, regardless of season. The results strongly suggest that aeromonads are indigenous in fish intestines, water, and sediments of rivers and have the potential to be predominant in aquatic environments.     

Virulence potential and genetic diversity of Aeromonas caviae, Aeromonas veronii, and Aeromonas hydrophila clinical isolates from Mexico and Spain: a comparative study

A comparative study of 109 Aeromonas clinical isolates belonging to the 3 species most frequently isolated from patients with diarrhea in Mexico and Spain was performed to investigate the distribution of 3 prominent toxin genes and the gene encoding flagellin of lateral flagella; 4 well-established virulence factors in the genus Aeromonas. The aerolysin-hemolysin toxin genes were the most prevalent, being present in 89% of the total isolates. The ast toxin gene was conspicuously absent from the Aeromonas caviae and Aeromonas veronii groups but was present in 91% of the Aeromonas hydrophila isolates. Both the alt toxin gene and the lafA flagellin gene also had a low incidence in A. caviae and A. veronii. Differences in the prevalence of alt and lafA were observed between isolates from Mexico and Spain, confirming genus heterogeneity according to geographic location. Carriage of multiple toxin genes was primarily restricted to A. hydrophila isolates, suggesting that A. caviae and A. veronii isolates circulating in Mexico and Spain possess a limited array of virulence genes. Enterobacterial repetitive intergenetic consensus - polymerase chain reaction showed that the Aeromonas populations sampled lack dominant clones and were genetically heterogeneous, with A. caviae being the most diverse species. Further surveys of virulence determinants in genetically heterogeneous populations of Aeromonas isolates circulating worldwide are required to enhance the understanding of their capacity to cause disease.     

38株维氏气单胞菌分离株的AFLP基因分型研究

试验通过人工设计合成的通用接头以及与接头序列相匹配的专用引物, 对38株维氏气单胞菌分离株和1株维氏气单胞菌标准菌株进行AFLP基因分型研究。结果显示, 采用5对引物使39株维氏气单胞菌扩增出1080条可见条带, 片段长度在50-1000 bp之间, 其中多态性条带727条, 平均每对引物组合产生154条多态性条带, 平均多态性检出率为66.7%。按UPGMA方法对条带进行聚类分析, 建立聚类分支树状图, 将39株维氏气单胞菌分为9个基因型, 其中第四类群基因Ⅳ型包含了14株菌株, 约占总菌数的35.9%, 分布在5个不同的地区, 推测其可能是引起斑点叉尾 发病的主要流行性基因型。不同地区分离的维氏气单胞菌具有地域性差异, 而同一地区分离株既存在相同基因型现象, 也存在基因型多样性现象。       

Broad diversity of conjugative plasmids in integron-carrying bacteria from wastewater environments

In this study we assessed the occurrence, diversity and conjugative potential of plasmids in integron-carrying Aeromonas and Enterobacteriaceae from wastewaters. Sixty-six strains were included as donors in mating assays using rifampicin-resistant Escherichia coli and Pseudomonas putida recipient strains. The diversity of plasmids from donors and transconjugants (resistant to tetracycline or streptomycin) was evaluated by restriction analysis and replicon typing targeting 19 incompatibility groups. Restriction patterns revealed a diverse plasmid pool present in these strains. Plasmids were assigned to FrepB (Aeromonas salmonicida, Aeromonas veronii, Aeromonas sp., E.?coli, Enterobacter sp.), FIC (A.?salmonicida, Aeromonas sp.), FIA (Shigella sp.), I1 (A.?veronii, Aeromonas sp., E.?coli), HI1 (E.?coli) and U (Aeromonas media) replicons. Nevertheless, 50% of the plasmids could not be assigned to any replicon type. Among integron-positive transconjugants, FrepB, I1 and HI1 replicons were detected. Results showed that wastewaters enclose a rich plasmid pool associated with integron-carrying bacteria, capable of conjugating to different bacterial hosts. Moreover, replicons detected in this study in Aeromonas strains expand our current knowledge of plasmid diversity in this genus.     

Misidentification of Aeromonas veronii biovar sobria as Vibrio alginolyticus by the Vitek system

AIMS: To find the cause of misidentification of aeromonads when using the Vitek system. METHODS AND RESULTS: Two Aeromonas veronii biovar sobria isolates were misidentified as Vibrio alginolyticus by the Vitek system. Both strains' identification was confirmed by biochemical testing, API 20E/20NE kits and/or 16S RFLP analysis. Thirty-one known Aeromonas species were tested by the Vitek system using 0.45 and 0.85% saline in the suspension medium. It was not clear whether low salinity causes misidentification of Aeromonas species more frequently. CONCLUSIONS: The specified reaction time may be inappropriately short for some critical biochemical tests of some strains. An ingenious reading strategy regarding incubation time is necessary to improve identification of Aeromonas species by the Vitek system. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report of misidentification of A. veronii biovar sobria as V. alginolyticus in the Vitek system.     

致病性维罗纳气单胞菌检测方法的建立

发掘维罗纳气单胞菌特异性更强的检测靶点和毒力相关基因靶点,建立能够检测致病性维罗纳气单胞菌的PCR检测方法.通过序列比对分析气单胞菌的16S rRNA基因序列,筛选对维罗纳气单胞菌特异的引物,用于检测种特异性,利用气单胞菌气溶素基因保守引物,检测菌株的致病性,并进行反应条件和反应体系的优化,灵敏度试验和特异性试验.发掘并设计的维罗纳气单胞菌16S rRNA特异性引物结合气单胞菌气溶素基因保守引物建立的检测方法,对12株气单胞菌和10株非气单胞菌的检测结果显示,所有致病性维罗纳气单胞菌都能扩增到大小分别为343 bp和232 bp的特异性条带,而非维罗纳气单胞菌的致病性气单胞菌只能扩增到232 bp的气溶素基因特异性条带,其它菌株都不能扩增到目的条带.灵敏度试验表明,该反应体系的检测灵敏度为1.35×10-3 mg/L.我们建立的致病性维罗纳气单胞菌检测方法能特异地检测致病性维罗纳气单胞菌,并具有高度灵敏性.     

草鱼肠道纤维素降解细菌的分离与鉴定

为更好地弄清草鱼（Ctenopharyngodon idella）肠道纤维素降解细菌的种类,采用羧甲基纤维素（CMC）作为唯一碳源的选择性培养基,分别从草鱼肠道内容物和肠道黏膜中分离到了40株产纤维素酶细菌。16S rRNA基因序列的分析结果显示,大多数产纤维素酶细菌为气单胞菌属（Aeromonas）的种类,其次为肠杆菌属（Enterobacter）的细菌以及未经分离纯培养的细菌（Uncultured bacterium）。进一步研究细菌产纤维素酶能力发现,纤维素酶活性显著性高于其他菌株的分别是A. veronii MC2、A. veronii BC6、肠杆菌科（Enterobacteriaceae）中一种未经分离纯培养的细菌BM3（Uncultured bacterium BM3）和A. jandaei HC9。草鱼肠道中简答气单胞菌（A. jandaei）、类志贺邻单胞菌（Plesiomonas shigelloides）、阴沟肠杆菌（E. cloacae）以及产气肠杆菌（E. aerogenes）是被作为产纤维素酶细菌的首次报道。       

青虾“软壳综合症”病原及其特性

从患软壳综合症的濒死青虾体内分离到一株细菌QXL0711B, 经人工感染试验, 其对青虾的半数致死浓度(LC50)为1.47×106 CFU/mL, 具有较强毒力。API 32E系统鉴定及16S rRNA序列分析, 该病原菌为维罗纳气单胞菌温和生物变种(Aeromonas veronii biovar sobria, 登录号: FJ808727)。其系统发育分析表明, 菌株QXL0711B与维罗纳气单胞菌(登录号: X71120)和维罗纳气单胞菌温和生物变种(登录号: AY987729)的亲缘关系最近,     

Typing of clinical and environmental Aeromonas veronii strains based on the 16S-23S rDNA spacers

Genetic relationships of Aeromonas veronii strains isolated from human and environmental sources were investigated by restriction fragment length polymorphism (RFLP) of the polymerase chain reaction-amplified intergenic spacer region (ISR) flanked by the 16S and 23S rRNA genes. When using endonucleases AluI, HinfI and CfoI the 16S-23S rDNA-RFLP patterns showed considerable overall similarity, although most strains yielded specific profiles. Several intra-specific lines of descent comprised clinical strains linked to isolates from environmental sources. Strains having identical patterns may be individuals derived from highly similar, if not the same, microorganism. Results suggest that the ISR sequence-based method can be used to demonstrate colonization of a public water supply with a particular microorganism. In addition it could be very useful for tracing recurrent episodes of diarrhea and Aeromonas infection outbreaks.     

Aggressiveness analysis of foliar fungi to leaves of wheat and rice crops

Abstract

The investigations were based on two surveys of wheat and one survey of rice. Alternaria alternata, Curvularia lunata, Helminthosporium tetramera and Bipolaris sorokiniana were isolated and identified from foliage and soil of both wheat and rice crops and their aggressiveness was studied by aggressiveness analysis screened out into different aggressiveness classes. The aggressiveness of isolated fungi was studied on wheat varieties (Inqalab-91 and Chakwal-86) and rice varieties (Basmati-385 and IRRI-6) under controlled conditions. In the foliar aggressiveness test of A. alternata, the overall number of aggressive isolates was higher on wheat varieties than rice. Bipolaris sorokiniana isolates showed foliar blight symptoms on wheat but not on rice varieties. In C. lunata, the overall number of aggressive isolates was higher on wheat. In the foliar aggressiveness test of H. tetramera, the number of non-aggressive isolates was almost the same on wheat and rice varieties. In the present study it became clear that A. alternate, B. sorokiniana, C. lunata and H. tetramera are common foliar pathogens in rice and wheat crops and can cause soil-borne and foliar diseases.