The current ICE age: biology and evolution of SXT-related integrating conjugative elements

SXT is an integrating conjugative element (ICE) that was initially isolated from a 1992 Vibrio cholerae O139 clinical isolate from India. This approximately 100-kb ICE encodes resistance to multiple antibiotics. SXT or closely related ICEs are now present in most clinical and some environmental V. cholerae isolates from Asia and Africa. SXT-related ICEs are not limited to V. cholerae. It is now clear that so-called IncJ elements such as R391 are closely related to SXT. More than 25 members of the SXT/R391 family of ICEs have now been identified in environmental and clinical isolates of diverse species of gamma-proteobacteria worldwide. In this review, we discuss the diversity, evolution and biology of this family of ICEs.     

Incidence of toxigenic vibrios in foods available in Taiwan

A total of 1088 vibrios and related species were isolated from seafood and aquacultured foods available in Taiwan. They were identified as Vibrio alginolyticus, V. cholerae, V. fluvialis I, V. fluvialis II, V. parahaemolyticus, V. mimicus, Aeromonas caviae, A. hydrophila, A. sobria and other species. Incidence of these Vibrio and Aeromonas species in these foods was high. Vibrio parahaemolyticus was frequently found in seawater and in foods of freshwater origin. The Vibrio isolates were examined for enzymatic and toxigenic activities. Most of them showed strong lipase or protease activities. Haemolytic activities of V. cholerae, V. fluvialis I and V. fluvialis II isolates were mostly strong. About 49% showed cytotoxic activity and 5% cytotonic activity in Chinese hamster ovary cell culture assay. Nevertheless, only three non-1 V. cholerae (2.07%) and two V. parahaemolyticus isolates (1.65%) produced cholera toxin and thermostable direct haemolysin activity, respectively. Various toxigenic vibrios may be important food-borne pathogens in this region because of their high incidence in foods.     

Incidence of toxigenic vibrios in foods available in Taiwan.

A total of 1088 vibrios and related species were isolated from seafood and aquacultured foods available in Taiwan. They were identified as Vibrio alginolyticus, V. cholerae, V. fluvialis I, V. fluvialis II, V. parahaemolyticus, V. mimicus, Aeromonas caviae, A. hydrophila, A. sobria and other species. Incidence of these Vibrio and Aeromonas species in these foods was high. Vibrio parahaemolyticus was frequently found in seawater and in foods of freshwater origin. The Vibrio isolates were examined for enzymatic and toxigenic activities. Most of them showed strong lipase or protease activities. Haemolytic activities of V. cholerae, V. fluvialis I and V. fluvialis II isolates were mostly strong. About 49% showed cytotoxic activity and 5% cytotonic activity in Chinese hamster ovary cell culture assay. Nevertheless, only three non-O1 V. cholerae (2.07%) and two V. parahaemolyticus isolates (1.65%) produced cholera toxin and thermostable direct haemolysin activity, respectively. Various toxigenic vibrios may be important food-borne pathogens in this region because of their high incidence in foods.     

Virulence factors in Vibrios and Aeromonads isolated from seafood

Thirty-one isolates from seafood, identified as Aeromonas hydrophila (7), Aeromonas caviae (11), Vibrio parahaemolyticus (3), Vibrio fluvialis (5), Vibrio alginolytictus (3), Vibrio metschnikovii (1) and Vibrio damsela (1), were tested for possible virulence factors including extracellular hydrolytic enzymes, haemolysins, cytotoxins (VERO and HEp-2 cells) and adherence ability (HEp-2 cells). All the A. hydrophila strains were beta-haemolytic and produced cytotoxins as well as one strain of V. fluvialis. A. hydrophila and A. caviae strains, frequently adhesive, showed both aggregative and diffusive patterns, while five Vibrio strains only (three V. fluvialis, one V. parahaemolyticus and one V. alginolyticus) were adhesive with an aggregative pattern.     

Comparison of the Heme Iron Utilization Systems of Pathogenic Vibrios

Vibrio alginolyticus, Vibrio fluvialis, and Vibrio parahaemolyticus utilized heme and hemoglobin as iron sources and contained chromosomal DNA similar to several Vibrio cholerae heme iron utilization genes. A V. parahaemolyticus gene that performed the function of V. cholerae hutA was isolated. A portion of the tonB1 locus of V. parahaemolyticus was sequenced and found to encode proteins similar in amino acid sequence to V. cholerae HutW, TonB1, and ExbB1. A recombinant plasmid containing the V. cholerae tonB1 and exbB1D1 genes complemented a V. alginolyticus heme utilization mutant. These data suggest that the heme iron utilization systems of the pathogenic vibrios tested, particularly V. parahaemolyticus and V. alginolyticus, are similar at the DNA level, the functional level, and, in the case of V. parahaemolyticus, the amino acid sequence or protein level to that of V. cholerae.     

香港养殖海鲷弧菌致病菌药物敏感性及耐药质粒研究

从发病海鲷(Sparus sarba)中共分离到51株弧菌(\%Vibrio)\%,经API20E细菌快速鉴定系统及Alsina和Blanch关键生理生化特性分析鉴定为7个种,它们分别是：溶藻胶弧菌(\%V.alginolyticus)(24株),创伤弧菌(V.vulnificus)(12株)和副溶血弧菌(V.parahaemolyticus)(7株),火神弧菌(V.logei)(4株),远洋弧菌Ⅱ菌(V.pelagius Ⅱ)(2株),河弧菌(V.fluvialis)(1株)和地中海弧菌(V.mediterranei)(1株)\%。其中3种优势菌溶藻胶弧菌创伤弧菌和副溶血弧菌证实对海鲷有致病性。另外采用平板稀释法检测了51株菌对16种抗菌素的敏感性。发现所有菌株对ceftriaxone,链霉素,萘啶酮酸和利福霉素敏感,几乎所有菌株对ceftazidime, netilimicin,氯霉素和sulfamethoxazole敏感.大部分菌株对氨苄青霉素 (60.8%),cefuroxime(667%),丁胺卡那霉素(55%),卡那霉素(588%)和三甲氧苄氨嘧啶(765%)等具有较强的耐药性。通过对菌株中所含有的耐药质粒进行分析,发现15株菌株含有1～4个质粒,分子量范围为9～123kb之间,对12株既含有较大分子量质粒又具有耐药性的菌株进行了质粒转化试验,结果其中9株菌的质粒具有转化能力,转化率为１０－１１～１０－９,表明所分离的菌株的抗药性是由于细菌染色体相关突变造成的。     

Distribution of Vibrio cholerae virulence genes among different Vibrio species isolated in Sardinia, Italy

The members of the genus Vibrio include harmless aquatic strains as well as strains capable of causing epidemics of cholera. Diarrhoea caused by Vibrio cholerae is attributed to cholerae enterotoxin (CT) codified by the ctx operon and regulated by a number of virulence genes such as toxT, toxR and toxS. Fifty-two Vibrio strains were isolated from different aquatic environments in and around Sardinia and searched by PCR for the presence of ctxA, zot, ace, toxR, toxS, toxT, tcpA and vpi virulence genes in the genomes of the isolates. The toxR operon was found in 27 Vibrio alginolyticus strains out of 42 analysed, in three out of four V. cholerae non-O1 strains and in three Vibrio parahaemolyticus isolates. A positive amplification for the virulence pathogenic island (vpi) was produced by five V. alginolyticus strains. Finally, the ace expected amplification fragment was found in two V. alginolyticus isolates whereas the amplification with zot primers produced the expected fragment in one V. alginolyticus isolate. Differentiation of these strains with a PCR fingerprinting technique revealed no association between the presence of virulence genes and a particular fingerprinting pattern. Although most Vibrio species are considered non-pathogenic or only potentially harmful to humans, the finding of V. cholerae virulence genes in other members of the genus Vibrio, and the recent reports of the creation and evolution of pandemic strains of V. cholerae, may give a new perspective to the significance of these results.     

2009-2011年北京通州区分离的副溶血性弧菌的耐药谱分析

目的：了解北京市通州区副溶血性弧菌的耐药情况,用以指导临床治疗用药及防控措施的制定。方法：选取通州区2009~2011年在食品监测和门诊病人中分离收集的60株副溶血性弧菌,采用微量肉汤稀释法,测定菌株对23种抗生素的最小抑菌浓度,并用碱裂解法和脉冲场凝胶电泳检测菌株的质粒谱,用PCR方法检测菌株中整合子及SXT元件的携带情况。结果：菌株对阿莫西林、氨苄西林、盘尼西林和多粘菌素等4种抗生素的敏感性低于40%,对妥布霉素有5%的中度耐药,对其余18种抗生素100%敏感;质粒携带率低,仅2株菌SXT整合酶阳性,Ⅰ、Ⅳ类整合子全部阴性。结论：北京市通州区副溶血性弧菌的耐药不严重,除2类抗生素外,其余抗生素在副溶血性弧菌感染的治疗中都有效。     

Genomic analysis of a novel integrative conjugative element in Vibrio cholerae

Integrative conjugative elements (ICEs) are a class of self-transmissible mobile elements that mediate horizontal gene transfer in bacteria, and play an important role in bacterial evolution. Since 1992, ICEs of the SXT/R391 family have been found to be widely distributed among Vibrio cholerae strains isolated in Asian countries. Here we describe ICEVchB33, an ICE found in the genomes of two V. cholerae O1 Eltor strains, one isolated in India, 1994, and the other from Mozambique, 2004. ICEVchB33 revealed a new genetic organization, different from other ICEs of the SXT/R391 family, demonstrating the genomic plasticity of these elements.     

Investigation of seven Vibrio virulence genes among Vibrio alginolyticus and Vibrio parahaemolyticus strains from the coastal mariculture systems in Guangdong, China

AIMS: To investigate the distribution of the virulence of two Vibrio species among different strains obtained from the mariculture systems on the coast of Guangdong in China and the correlation between the virulence strains and the virulence genes among Vibrio alginolyticus. METHODS: Besides three strains, 72 V. alginolyticus strains and seven Vibrio parahaemolyticus strains were examined by PCR or semi-nested PCR for the virulence genes (tlh, trh, tdh, toxR, toxRS, ctxA, VPI). Additionally, the virulence of 18 V. alginolyticus strains was tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Virulence genes homologous to those in the V. parahaemolyticus and Vibrio cholerae are widely distributed among V. alginolyticus and V. parahaemolyticus in the coastal mariculture systems in Guangdong, China. Some of the V. alginolyticus strains are pathogenic to aquatic animals, and might have derived their virulence genes from V. parahaemolyticus or V. cholerae, representing a possible reservoir of these genes. However, there is no correlation between presence and absence of the virulence genes used to investigate V. alginolyticus and its virulent strains. In this report, we also show that tlh is distributed among V. alginolyticus.     

A variant type of Vibrio cholerae SXT element in a multidrug-resistant strain of Vibrio fluvialis

Vibrio fluvialis strain H-08942 was isolated from an infant aged 6 months who was suffering from cholera-like diarrhea in India. This strain showed the typical multidrug-resistance phenotype of an SXT element. It was resistant to sulfamethoxazole (Su), trimethoprim (Tm), chloramphenicol (Cm) and streptomycin (Sm), in addition to other antibiotics such as ampicillin (Am), furazolidone (Fz), nalidixic acid (Na), and gentamicin (Gm). The SXT element is a Vibrio cholerae-derived integrative and conjugative element (ICE) that has also been referred to as a conjugative transposon. Our goal was to find a relationship between these resistant phenotypes and the presence of the SXT element in this unique strain. By using PCR, we detected the antibiotic resistance genes, the integrase gene and the attP attachment site of SXT element. Cloning and DNA sequencing results showed that both the SXT integrase gene and its attP site of V. fluvialis were similar but not identical to those of V. cholerae. The SXT integrase gene of V. fluvialis has a 99% identity to that of V. cholerae, and the attP site of SXT of V. fluvialis is variant and shorter (641 bp) than that of V. cholerae (785 bp). It was possible for the SXT of V. fluvialis to be transferred by conjugation to a laboratory strain of Escherichia coli. Here, we report the detection of a variant SXT element in species other than V. cholerae, with molecular characterization and analysis of its integrase gene and its attP site.     

Genomic analysis of ICEVchBan8: An atypical genetic element in Vibrio cholerae

Genomic islands (GIs) and integrative conjugative elements (ICEs) are major players in bacterial evolution since they encode genes involved in adaptive functions of medical or environmental importance. Here we performed the genomic analysis of ICEVchBan8, an unusual ICE found in the genome of a clinical non-toxigenic Vibrio cholerae O37 isolate. ICEVchBan8 shares most of its genetic structure with SXT/R391 ICEs. However, this ICE codes for a different integration/excision module is located at a different insertion site, and part of its genetic cargo shows homology to other pathogenicity islands of V. cholerae.     

Detection and identification of Vibrio species using whole-cell protein pattern analysis

Outbreaks of foodborne diseases associated with Vibrio species such as V. parahaemolyticus, V. vulnificus, and V. cholerae frequently occur in countries having a dietary habit of raw seafood consumption. For rapid identification of different Vibrio species involved in foodborne diseases, whole-cell protein pattern analysis for 13 type strains of 12 Vibrio species was performed using SDS-PAGE analysis. Pathogenic Vibrio species such as V. parahaemolyticus, V. vulnificus, V. cholerae, V. alginolyticus, V. fluvialis, and V. mimicus were included in the 12 Vibrio species used in this study. Each of the 12 Vibrio species showed clearly specific band patterns of its own. Two different strains of V. parahaemolyticus showed two different SDS-PAGE wholecell protein patterns, giving the possibility of categorizing isolated strains in the same V. parahaemolyticus species into two subgroups. The 36 Vibrio isolates collected from sushi restaurants in Busan were all identified as V. parahaemolyticus by comparing their protein patterns with those of Vibrio type strains. The identified isolates were categorized into two different subgroups of V. parahaemolyticus. The whole-cell protein pattern analysis by SDS-PAGE can be used as a specific, rapid, and simple identification method for Vibrio spp. involved in foodborne diseases at the subspecies level.     

Formation of SXT tandem arrays and SXT-R391 hybrids

SXT is an integrative and conjugative element (ICE) isolated from Vibrio cholerae. This approximately 100-kb ICE encodes resistance to multiple antibiotics and integrates site specifically into the chromosome. SXT excises from the chromosome to form a circular but nonreplicative extrachromosomal molecule that is required for its transfer. Here we found that a significant fraction of freshly isolated SXT exconjugants contained tandem SXT arrays. There was heterogeneity in the size of the SXT arrays detected in single exconjugant colonies. Some arrays consisted of more than five SXTs arranged in tandem. These extended arrays were unstable and did not persist during serial passages. The mechanism accounting for the generation of SXT arrays is unknown; however, array formation was not dependent upon recA and appeared to depend on conjugative transfer. While such arrays did not alter the transfer frequency of wild-type SXT, they partially complemented the transfer deficiency of a Deltaxis SXT mutant, which is ordinarily unable to generate the extrachromosomal intermediate required for SXT transfer. Exconjugants derived from donor strains that harbored tandem arrays of SXT and R391, an SXT-related element, contained functional hybrid elements that arose from recA-independent recombination between the two ICEs. Thus, arrays of SXT-related elements promote the creation of novel ICEs.     

An Electrophoretic Analysis of Variation in the Glucose-6-Phosphate Dehydrogenase and Malate Dehydrogenase of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio fluvialis

A total of 59 isolates of Vibrio cholerae, V. parahaemolyticus and V. fluvialis were studied using the techniques of enzyme electrophoresis. The enzymes used were malate dehydrogenase (E.C.I. 1.1.37) and glucose-6-phospate dehydrogenase (E.C.I. 1.1.49). The results in general confirmed classical methods of vibrio taxonomy. The three species could be separated from each other and identified by their enzyme variant types.
The term zymovar, a group of strains having similar enzyme variants, is introduced. In the V. cholerae isolates six zymovars were found. All V. cholerae serovar 01 strains were classified in the same zymovar except for some strains of environmental origin, which occurred in another zymovar. In V. fluvialis two zymovars were detected corresponding with biovars 1 and 2 of this organism. All isolates of V. parahaemolyticus gave the same enzyme type.
The technique of enzyme electrophoresis appears to be a useful tool in vibrio taxonomy and used in conjunction with other methods may aid in the elucidation of the systematics of the genus.     

Isolation of medically significant Vibrio species from riverine sources in south east Queensland

Vibrio and Vibrio-like bacteria were isolated from water, sediments, plants and faeces from eight riverine sites in South East Queensland, Australia, using filtrations, enrichments and selective growth on thiosulphate-citrate-bile salts-sucrose (TCBS) and Simidu agars. Isolates (402 in toto) were classified by numerical analysis of phenotypic characteristics and comparison with fifteen reference cultures. Of the isolates 41 (10.2%) were identified as Vibrio cholerae, 33 (8.2%) as V. fluvialis and 118 (29.4%) as motile Aeromonas species. Other isolates resembled V. parahaemolyticus, V. alginolyticus, V. vulnificus and V. hollisae. No isolates were positively identified as V. damsela or Plesiomonas shigelloides. Vibrio species tolerant of low salt levels and aeromonads were widely distributed in riverine locations but more halophilic species were restricted to more saline areas. Simidu agar failed to select for vibrios as effectively as TCBS agar. Enrichment for 18 h produced more isolates than for 6 h.     

Characterization of a Vibrio cholerae virulence factor homologous to the family of TonB-dependent proteins

IrgA is an iron-regulated virulence factor for infection in an animal model with classical Vibrio cholerae strain 0395. We detected gene sequences hybridizing to irgA at high stringency in clinical isolates in addition to 0395, including another classical strain of V. cholerae, three V. cholerae strains of the El Tor biotype, three non-O1 isolates of V. cholerae, and individual isolates of Vibrio parahaemolyticus, Vibrio fluvialis, and Vibrio alginolyticus. No hybridization to irgA was seen with chromosomal DNA from Vibrio vulnificus or Aeromonas hydrophila. To verify that irgA is the structural gene for the major iron-regulated outer membrane protein of V. cholerae, we determined the amino-terminal sequence of this protein recovered after gel electrophoresis and demonstrated that it corresponds to the amino acid sequence of IrgA deduced from the nucleotide sequence. Gel electrophoresis showed that two El Tor strains of V. cholerae had a major iron-regulated outer membrane protein identical in size and appearance to IrgA in strain 0395, consistent with the findings of DNA hybridization. We have previously suggested that IrgA might be the outer membrane receptor for the V. cholerae siderophore, vibriobactin. Biological data presented here, however, show that a mutation in irgA had no effect on the transport of vibriobactin and produced no defect in the utilization of iron from ferrichrome, ferric citrate, haemin or haemoglobin. The complete deduced amino acid sequence of IrgA demonstrated homology to the entire class of Escherichia coli TonB-dependent proteins, particularly Cir. Unlike the situation with Cir, however, we were unable to demonstrate a role for IrgA as a receptor for catechol-substituted cephalosporins. The role of IrgA in the pathogenesis of V. cholerae infection, its function as an outer membrane receptor, and its potential interaction with a TonB-like protein in V. cholerae remain to be determined.     

Development of monoclonal antibodies for simple identification of Vibrio alginolyticus

AIMS: The present study was aimed to produce monoclonal antibodies (MAbs) for simple and specific identification of Vibrio alginolyticus infection in shrimp. METHODS AND RESULTS: Mice were immunized with heat killed V. alginolyticus four times at 2-week intervals. The best response mouse was used for spleen donor in hybridoma production. Screening of hybridoma clones producing desired antibodies was performed by dot blotting against V. alginolyticus and other bacterial species, Western blotting and immunohistochemistry of infected shrimp tissues. Four groups of MAbs were obtained; the first group of MAbs demonstrated their limited specificity only to V. alginolyticus used for immunization, while the second and the third groups recognized all three isolates of V. alginolyticus used for testing. The fourth group of MAbs bound to all three isolates of V. alginolyticus and also recognized Vibrio parahaemolyticus, Vibrio harveyi, Vibrio fluvialis and Vibrio vulnificus but did not bind to Vibrio mimicus, Vibrio cholerae, Vibrio penaeicida and other bacterial species tested. MAbs in groups 1, 2 and 3 were able to use for the detection of bacterial infection in the tissues by means of immunohistochemistry. CONCLUSIONS: MAbs specific to V. alginolyticus was produced. These MAbs can be used for specific identification of the bacteria by simple 'dot blotting' method and immunohistochemistry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated an immunological tool that can be used for simple and accurate identification of V. alginolyticus as well as for the diagnosis of V. alginolyticus infection in animals. This immunological tool can replace costly and laborious biochemical tests.     

Potentially pathogenic vibrios associated with mussels from a tropical region on the Atlantic coast of Brazil

Mussels ( Perna perna ) harvested on the coast of Ubatuba, in three different stations in the State of São Paulo, Brazil, were examined for Vibrio spp. over a 1 year period. The ranges of most probable number (MPN 100 g^-1) were: Vibrio alginolyticus (<3–24000), V. parahaemolyticus (<3–24000), V. fluvialis (<3–1100), V. cholerae non-O1 (<3–23), V. furnissi (< 3–30), V. mimicus (< 3–9) and V. vulnificus (< 3–3). The highest incidence was observed for V. alginolyticus (92–100%), followed by V. parahaemolyticus (67–92%), V. fluvialis (34–67%), V. vulnificus (8–17%), V. furnissii (0–17%), V. mimicus (0–17%) and V. cholerae non-O1 (0–8%). Tests for virulence factors were positive in 34.1% of the vibrios in the rabbit ileal loop and 31.7% in the Dean test. Positive results in the Kanagawa test were obtained with 0.51% of V. parahaemolyticus strains. The mean values (MPN 100 g^-1) of faecal coliforms in mussels from the three regions varied from 1100 to 44000, and seawater collected at the same stations gave average values for faecal coliforms in the range 18–3300 MPN 100 ml^-1. These results highlight the potential risks of food poisoning associated with raw or undercooked seafood.