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.     

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.     

Evolutionary and diagnostic implications of intragenomic heterogeneity in the 16S rRNA gene in Aeromonas strains

Sequencing 16S rRNA genes (SSU) cloned from Aeromonas strains revealed that strains contained up to six copies differing by < or = 1.5%. The SSU copies from Aeromonas veronii LMG13695 clustered with sequences from four Aeromonas species. These results demonstrate intragenomic heterogeneity of SSU and suggest caution when using SSU to identify aeromonads.     

Virulence properties of motile aeromonads isolated from farmed frogs Rana tigerina and R. rugulosa

Virulence factors were compared in Aeromonas species isolated from clinically normal and septicaemic farmed frogs from Thailand. Haemolysin activities against frog erythrocytes were significantly different within the collection of aeromonads. Groups of high haemolytic activity (unspeciated Aeromonas, Au), moderate haemolytic activity (A. hydrophila), and low haemolytic activity (A. veronii biovar sobria, A. veronii biovar veronii, A. caviae, A. schubertii) were noted. DNA colony hybridisation studies revealed that Au isolates possessed a haemolysin gene (ASH1) which was not present in any of the other Thai aeromonads or type strains tested. Elastinolytic activity was demonstrated in 90% of the Au isolates, 60% of the A. hydrophila isolates and in none of the other motile aeromonads. The cytotoxic activity of the Aeromonas isolates varied according to the source of cells used in the assays. Cells from rainbow trout were extremely sensitive to Au toxins but less so to toxins produced by other species. In contrast mammalian cells showed very little sensitivity to Au toxins but were more sensitive to toxins produced by A. hydrophila. Selection of suitable assay substrates is therefore important.     

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.     

Analysis of 16S rRNA gene mutations in a subset of Aeromonas strains and their impact in species delineation

Characterization of 999 Aeromonas strains using a published 16S rDNA RFLP identification method showed that 8.1% of the strains produced unexpected (hereafter called "atypical") restriction patterns, making their identification uncertain. Atypical patterns were due to the presence of nucleotide polymorphisms among the rrn operons of the 16S rRNA gene (so-called microheterogeneities). Double sequencing signals at certain positions revealed the nucleotide composition was responsible for the microheterogeneities. Although the number of microheterogeneities was relatively low (0.06-0.66%), trees inferred from the 16S rRNA gene led either to a misidentification or to an inconclusive result for the majority of these strains. Strains with atypical patterns were, however, correctly identified using the rpoD gene sequences, as belonging to Aeromonas caviae, A. veronii, and A. media. All of them, but particularly the two former species, are associated with human disease. Microheterogeneities in 16S rRNA gene sequence were significantly (P 0.01) more prevalent in clinical than in environmental strains. This work also analyzed the effects of these microheterogeneities on the taxonomic position of the investigated strains. The results suggest the need for recording microheterogeneities in the 16S rRNA gene.     

Phenotypic and genotypic characterization of tetracycline- and oxytetracycline-resistant Aeromonas hydrophila from cultured channel catfish (Ictalurus punctatus) and their environments

Oxytetracycline-resistant (OTcr) and tetracycline-resistant (Tcr) Aeromonas hydrophila were isolated commonly from catfish intestinal contents and the water and sediment of catfish culture ponds, but less frequently in market catfish. Isolates demonstrated two resistance phenotypes, Tcr OTcr and Tcs OTcr, when plated directly on to MacConkey agar containing 30 micrograms of tetracycline or oxytetracycline per ml. Tcs OTcr isolates expressed Tcr after induction by 1 h of growth in tryptic soy broth containing 1 microgram of tetracycline per ml. Over 90% of the resistant aeromonads hybridized with DNA probes for class A or class E Tcr determinants; class E was twice as prevalent as class A. The distribution of class A and E Tcr determinants varied with the Tcr phenotypes. Prior to induction, 86% of isolates with class A determinants were Tcr as well as OTcr, while only 16% with class E determinant were Tcr. Three of 5 Tcr aeromonads without class A or class E determinants and 1 of 14 with class E determinants transferred Tcr to Escherichia coli.     

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。       

Phylogenetic affiliation of Aeromonas culicicola MTCC 3249(T) based on gyrB gene sequence and PCR-amplicon sequence analysis of cytolytic enterotoxin gene

We determined the gyrB gene sequences of all 17 hybridizations groups of Aeromonas. Phylogenetic trees showing the evolutionary relatedness of gyrB and 16S rRNA genes in the type strains of Aeromonas were compared. Using this approach, we determined the phylogenetic position of Aeromonas culicicola MTCC 3249(T), isolated from midgut of Culex quinquefasciatus. In the gyrB based-analysis A. culicicola MTCC 3249(T) grouped with A. veronii whereas, it grouped with A. jandaei in the 16S rRNA based tree. The number of nucleotide differences in 16S rRNA sequences was less than found with the gyrB sequence data. Most of the observed nucleotide differences in the gyrB gene were synonymous. The Cophenetic Correlation Coefficient (CCC) for gyrB sequences was 0.87 indicating this gene to be a better molecular chronometer compared to 16S rRNA for delineation of Aeromonas species. This strain was found to be positive for the cytolytic enterotoxin gene. PCR-Amplicon Sequence Analysis (PCR-ASA) of this gene showed that the isolate is affiliated to type I and is potentially pathogenic. These PCR-ASA results agreed in part with the gyrB sequence results.     

广东中山地区气单胞菌环境株的分离、鉴定及系统发生关系

从广东省中山市的池塘水样、底泥、健康鱼、肠道及稻田土样中用Aeromonas的选择培养基分离到10株气单胞菌。通过生理生化测试、16S rDNA序列测定、与气单胞菌典型菌株的16S rDNA序列进行比对和聚类分析,对它们进行了鉴定,并研究了它们之间的系统发生关系。结果显示该地区环境中气单胞菌的优势种除A.hydrophila(HG1组)外,还有A.caviae(HG4组)、A.jandaei(HG9组)和A.veronii(HG10组),其中后两种是国内新记录。这是国内首次对环境中气单胞菌多样性进行研究。     

Genetic diversity of root-nodulating bacteria isolated from pea (Pisum sativum) in subtropical regions of China

Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied using 16S rRNA gene RFLP patterns, 16S rRNA sequencing, 16S–23S rRNA inter-genic spacer (IGS) region RFLP patterns and G-C rich random amplified polymorphic DNA (RAPD). The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns. The 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns group I were very closely related (identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370. Group II consisting of WzP3 and WzP15 was closely related to Rhizobium etli CFN42. The analysis of the 16S–23S IGS RFLP pat-terns divided the isolates into 18 genotypes and four groups. Group I was clustered with R. legumino-sarum USDA2370. Group II consisted of YcP2, YcP3 and CqP7. The strains of group III were distributed abroad. Group IV consisted of WzP3, WzP15 and R. etli CFN42. RAPD divided the isolates into nine clusters in which group IV only consisted of YcP2 and the strains of group V and IX were from Wenzhou and Xiantao, respectively. This assay demonstrated the geographical effect on genetic diversity of pea rhizobia.     

Genetic diversity of root-nodulating bacteria isolated from pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>) in subtropical regions of China

Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied using 16S rRNA gene RFLP patterns, 16S rRNA sequencing, 16S–23S rRNA intergenic spacer (IGS) region RFLP patterns and G-C rich random amplified polymorphic DNA (RAPD). The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns. The 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns group I were very closely related (identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370. Group II consisting of WzP3 and WzP15 was closely related to Rhizobium etli CFN42. The analysis of the 16S-23S IGS RFLP patterns divided the isolates into 18 genotypes and four groups. Group I was clustered with R. leguminosarum USDA2370. Group II consisted of YcP2, YcP3 and CqP7. The strains of group III were distributed abroad. Group IV consisted of WzP3, WzP15 and R. etli CFN42. RAPD divided the isolates into nine clusters in which group IV only consisted of YcP2 and the strains of group V and IX were from Wenzhou and Xiantao, respectively. This assay demonstrated the geographical effect on genetic diversity of pea rhizobia.     

Restriction fragment length polymorphism of 16S-23S rDNA intergenic spacer of Aeromonas spp

We analyzed restriction fragment length polymorphism (RFLP) of 16S-23S rDNA intergenic spacer region (ISR) of Aeromonas species. A total of 69 isolates belonging to 18 DNA hybridization groups (HG; equivalent of genomic species) were used in this study. ISRs were amplified by PCR and the products were digested with four restriction endonucleases: Hin6I, Csp6I, TaqI, and TasI. The RFLP patterns obtained after digesting by particular enzymes revealed ISR polymorphism of isolates allocated to individual genomic species. The combined Hin6I, Csp6I, TaqI, and TasI restriction profiles were examined by Dice coefficient (SD) and unweighted pair group method of clustering (UPGMA). The isolates were allocated into 15 groups, three strains were unclustered. The strains belonging to the following genomic species: A. hydrophila, A. bestiarum, A. salmonicida, A. caviae, A. media, A. schubertii, A. allosaccharophila, A. popoffii, and A. culicicola formed distinct clusters. Strains belonging to HG 6, HG 7, HG 11, and HG 16 revealed similar combined RFLP patterns and constituted one group. Similarly, the strains of A. jandaei (HG 9) and the type strain of A. trota were allocated into one cluster. Two isolates of HG 14 formed distinct cluster. We noticed a genetic diversity among A. veronii isolates, the strains were clustered in two groups. Our study showed that combined ISR-RFLP analysis may be used for identification of some species of Aeromonas.     

我国主要生态区域绿豆慢生根瘤菌的遗传多样性和系统发育研究

利用16SrRNAPCR-RFLP、16SrRNA序列分析以及16S-23SrRNAIGS(IntergeneticSpacer)PCR-RFLP技术对分离自中国主要生态区域的44株慢生型绿豆根瘤菌和5株参比菌株进行了遗传多样性和系统发育研究。16SrRNAPCR-RFLP分析表明:在76%的相似水平上,所有供试菌株可分为三大类群:群I由LYG1等13株慢生根瘤菌组成,该群在系统发育上与B.japonicum和B.liaoningense的参比菌株存在一定的差异;群Ⅱ由XJ1等21株供试菌株、B.japonicum和B.liaoningense的代表菌株组成;群Ⅲ由10株来自广东和广西的菌株和B.elkanii的代表菌株组成。16S-23SrRNAIGSPCR-RFLP分析将供试菌株分为A、B两大群。群A由34株供试菌株、B.japonicum和B.liaoningense的代表菌株组成。在85%的相似性水平上,可再分为AⅠ、AⅡ和AⅢ3个亚群。群B由10株分离自广西和广东的菌株和B.elkanii的代表菌株组成。在85%的相似性水平上,可再分为BI和BⅡ两亚群,表现出一定的多样性。与16SrRNAPCR-RFLP相比,16S-23SrRNAIGSPCR-RFLP具有更高的解析度,供试菌株表现出更加丰富的遗传多样性。分离自中国新疆、广东和广西等地的菌株在分群上具有较为明显的地域特征。     

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.     

Occurrence, diversity and pathogenicity of mesophilic Aeromonas in estuarine waters of the Italian coast of the Adriatic Sea

A total of 208 strains of Aeromonas were isolated by monthly sampling from two estuaries (one provided with, and the other devoid of a waste-water treatment system) on the Italian coast of the Adriatic sea between September 1994 and August 1995. Biotyping at the species level allowed the identification of 96 strains (46%) as Aer. caviae , 46 (22%) as Aer. sobria , 33 (16%) as Aer. hydrophila and 25 (12%) as Aer. veronii . Eight strains (4%) were regarded as unnamed aeromonads. Aeromonas caviae was the most prevalent species in water with a high degree of pollution, while Aer. hydrophila strains were more commonly isolated from cleaner water. Aeromonas sobria and Aer. veronii were equally distributed in both estuaries. There was no correlation between temperature and numbers of aeromonads in either estuary. Using a biochemical fingerprinting method, strains were divided into similarity groups (PhP-types) based on their biochemical phenotypes. Several different PhP-types were found in each estuary, yielding a high diversity for these strains. However, some identical PhP-types were also found in both estuaries and at different times of the year, indicating that certain Aeromonas strains can survive more widely varying physico-chemical conditions. The production of toxins capable of causing cytoskeletal-dependent changes in the morphology of Chinese hamster ovary (CHO) cells was detected in 14 strains and appeared to be dependent on the season.     

Development of a rapid identification method for Aeromonas species by multiplex-PCR

Existing biochemical methods cannot distinguish among some species of Aeromonads, while genetic methods are labor intensive. In this study, primers were developed to three genes of Aeromonas: lipase, elastase, and DNA gyraseB. In addition, six previously described primer sets, five corresponding to species-specific signature regions of the 16S rRNA gene from A. veronii, A. popoffii, A. caviae, A. jandaei, and A. schubertii, respectively, and one corresponding to A. hydrophila specific lipase (hydrolipase), were chosen. The primer sets were combined in a series of multiplex-PCR (mPCR) assays against 38 previously characterized strains. Following PCR, each species was distinguished by the production of a unique combination of amplicons. When the assays were tested using 63 drinking water isolates, there was complete agreement in the species identification (ID) for 59 isolates, with ID established by biochemical assays. Sequencing the gyrB and the 16S rRNA gene from the remaining four strains established that the ID obtained by mPCR was correct for three strains. For only one strain, no consensus ID could be obtained. A rapid and reliable method for identification of different Aeromonas species is proposed that does not require restriction enzyme digestions, thus simplifying and speeding up the process.     

Isolation of tetracycline-resistant clinical Helicobacter pylori without mutations in 16S rRNA gene in Bangladesh

The occurrence of 16S rRNA gene mutations associated with resistance to tetracycline in H. pylori isolated in Bangladesh was investigated. Tetracycline susceptibility was determined by the agar dilution method. The 16S rRNA genes of these isolates were sequenced and analyzed. A tetracycline accumulation assay was performed. DNA sequence and transformation tests of nine tetracycline-resistant (MIC = 2 microg/ml) Bangladeshi H. pylori clinical isolates showed that in no case was the resistance due to mutations in the 16S rRNA gene, the only known cause of tetracycline resistance in this pathogen. Tetracycline accumulation assays implicated altered uptake or efflux.     

Culturable Actinobacteria from the Marine Sponge Hymeniacidon perleve: Isolation and Phylogenetic Diversity by 16S rRNA gene-RFLP Analysis

A total of 106 actinobacteria associated with the marine sponge Hymeniacidon perleve collected from the Yellow Sea, China were isolated using eight different media. The number of species and genera of actinobacteria recovered from the different media varied significantly, underlining the importance of optimizing the isolation conditions. The phylogenetic diversity of the actinobacteria isolates was assessed using 16S rRNA gene amplification–restriction fragment length polymorphism (RFLP) analysis of the 106 strains with different morphologies. The RFLP fingerprinting of selected strains by HhaI-digestion of the 16S rRNA genes resulted in 11 different patterns. The HhaI-RFLP analysis gave good resolution for the identification of the actinobacteria isolates at the genus level. A phylogenetic analysis using 16S rRNA gene sequences revealed that the isolates belonged to seven genera of culturable actinobacteria including Actinoalloteichus, Micromonospora, Nocardia, Nocardiopsis, Pseudonocardia, Rhodococcus, and Streptomyces. The dominant genus was Streptomyces, which represented 74% of the isolates. Three of the strains identified are candidates for new species.