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Clinical and bacteriological characteristics of Helicobacter cinaedi infection

Open AccessPublished:July 09, 2014DOI:https://doi.org/10.1016/j.jiac.2014.06.007

      Abstract

      Helicobacter cinaedi was first isolated from rectal cultures from homosexual men in 1984. In the 1980s to mid 1990s, the microorganism was mainly isolated from samples from homosexual men or immunocompromised patients; however, during the last two decades, H. cinaedi has been isolated from immunocompromised and from immunocompetent individuals worldwide. In Japan, the isolation of this microorganism was first reported in 2003. Since then, many cases have been reported in hospitals across the country. Despite many reports, the etiological properties and pathogenicity of H. cinaedi remain elusive; however, we are increasingly able to recognize some of the features and the clinical relevance of infection. In particular, a long incubation period is essential for detection in an automatic blood culture system and many of the recent isolates are resistant to both macrolides and quinolones. Furthermore, there is an association between infection and severe or chronic illnesses, such as meningitis or arteriosclerosis, in addition to mild diseases such as fever, abdominal pain, gastroenteritis, proctitis, diarrhea, erysipelas, cellulitis, arthritis, and bacteremia.
      In this review, we introduce the current knowledge and our latest findings relating to H. cinaedi.

      Keywords

      1. Introduction

      The genus Helicobacter is a gram-negative spiral bacterium, belonging to the family Helicobacteriaceae of the order Campylobacterales within the class Epsilonproteobacteria.
      Almost all members of genus Helicobacter show curved spiral (S-shape) or fusiform rods that are 0.2–1.2 × 1.5–10 μm. Spiral cells may be tightly or loosely wound depending on the species and on the culture age and condition. Cells in old cultures or those exposed to air become coccoid. Periplasmic fibers may be observed on the cell surface in certain species (Fig. 1).
      Figure thumbnail gr1
      Fig. 1SEM image of Helicobacter species. a) H. cinaedi (PAGU611; a whole genome determined strain) showing S-shaped and bipolar flagella. b) H. bilis (PAGU599T) showing a fusiform cell body encircled by tightly wound periplasmic fibers and multiple sheathed flagella (×15,000, Bar = 1.0 μm).
      Helicobacter cinaedi was first reported as a Campylobacter-like organism type-1 (CLO-1) in 1984 by Fennell et al. [
      • Fennell C.L.
      • Totten P.A.
      • Quinn T.C.
      • Patton D.L.
      • Holmes K.K.
      • Stamm W.E.
      Characterization of Campylobacter-like organisms isolated from homosexual men.
      ] They described three different types of CLOs—CLO-1, CLO-2 (later named “Campylobacter fennelliae”), and CLO-3 (still unnamed)—based on biochemical traits (nitrate reduction and odor-producing ability) and membrane spot DNA–DNA hybridization results. The following year, Totten et al. [
      • Totten P.A.
      • Fennell C.L.
      • Tenover F.C.
      • Wezenberg J.M.
      • Perine P.L.
      • Stamm W.E.
      • et al.
      Campylobacter cinaedi (sp. nov.) and Campylobacter fennelliae (sp. nov.): two new Campylobacter species associated with enteric disease in homosexual men.
      ] proposed the name “Campylobacter cinaedi” for CLO-1 organisms, although they demonstrated that there are two genetic groups within CLO-1 type, namely, CLO-1a and CLO-1b, with DNA–DNA hybridization values of 42–51%. Comparable values between CLO-2 and CLO-3 strains were far lower (less than 7%). These data and the lack of biochemical differences between CLO-1a and CLO-1b groups allowed the authors to include both within a single species. Thus, “C. cinaedi” is genetically diverse, involving at least two genomospecies. In 1991, “C. cinaedi” and also “C. fennelliae” were moved into the genus Helicobacter [
      • Goodwin C.S.
      • Armstrong J.A.
      • Chilvers T.
      • Peters M.
      • Collins M.D.
      • Sly L.
      • et al.
      Transfer of Campylobacter pylori and Campylobacter mustelae to Helicobacter gen nov as Helicobacter pylon comb nov and Helicobacter mustelae comb nov, respectively.
      ] as H. cinaedi and Helicobacter fennelliae [
      • Vandamme P.
      • Falsen E.
      • Rossau R.
      • Hoste B.
      • Segers P.
      • Tytgat R.
      • et al.
      Revision of Campylobacter, Helicobacter, and Wolinella taxonomy: emendation of generic descriptions and proposal of Arcobacter gen. nov.
      ].
      To date, the validation of 33 species in genus Helicobacter has been proposed, but only seven species have been isolated from human clinical specimens (Table 1, Fig. 2). Helicobacter pylori, classified as a “gastric-Helicobacter species” [
      • Solnick J.V.
      • Schauer D.B.
      Emergence of diverse Helicobacter species in the pathogenesis of gastric and enterohepatic diseases.
      ], is the most well known species of the genus Helicobacter, although H. cinaedi, Helicobacter bilis, Helicobacter canadensis, Helicobacter canis, H. fennelliae, and Helicobacter pullorum, classed as “enterohepatic Helicobacter species” [
      • Solnick J.V.
      • Schauer D.B.
      Emergence of diverse Helicobacter species in the pathogenesis of gastric and enterohepatic diseases.
      ], have also been isolated from human clinical specimens.
      Table 1All validity proposed members of the genus Helicobacter.
      SpeciesHost animalValid yearSpeciesHost animalValid year
      Enterohepatic Helicobacter
      “Enterohepaic-Gastric" categories were basically according with Solnick et al. [5].
      Gastric Helicobacter
      1H. cinaedi
      Only seven species has been isolated from human specimen. Some reports described the detection of H. suis, H. felis, H. hepaticus, or H. ganmani using genetic methods but there is no report, to our knowledge, has isolated the organism.
      Human, Hamster199121H. pylori
      Only seven species has been isolated from human specimen. Some reports described the detection of H. suis, H. felis, H. hepaticus, or H. ganmani using genetic methods but there is no report, to our knowledge, has isolated the organism.
      Human, Monkey1989
      2H. canis
      Only seven species has been isolated from human specimen. Some reports described the detection of H. suis, H. felis, H. hepaticus, or H. ganmani using genetic methods but there is no report, to our knowledge, has isolated the organism.
      Dog, Human199422H. acinonychisCheetah1993
      3H. bilis
      Only seven species has been isolated from human specimen. Some reports described the detection of H. suis, H. felis, H. hepaticus, or H. ganmani using genetic methods but there is no report, to our knowledge, has isolated the organism.
      Mouse, Dog, Human199723H. auratiHamster2002
      4H. canadensis
      Only seven species has been isolated from human specimen. Some reports described the detection of H. suis, H. felis, H. hepaticus, or H. ganmani using genetic methods but there is no report, to our knowledge, has isolated the organism.
      Human200224H. baculiformisCat2008
      5H. fennelliae
      Only seven species has been isolated from human specimen. Some reports described the detection of H. suis, H. felis, H. hepaticus, or H. ganmani using genetic methods but there is no report, to our knowledge, has isolated the organism.
      Human199125H. bizzozeroniiDog1996
      6H. pullorum
      Only seven species has been isolated from human specimen. Some reports described the detection of H. suis, H. felis, H. hepaticus, or H. ganmani using genetic methods but there is no report, to our knowledge, has isolated the organism.
      Chicken, Human199526H. cetorumDolphin, Whales2006
      7H. cholecystusHamster199727H. cynogastricusDog2006
      8H. ganmaniMouse200128H. felisCat, Dog1991
      9H. hepaticusMouse199429H. heilmanniiMonkey2012
      10H. mesocricetorumHamster200030H. mustelaeFerret1989
      11H. muridarumMouse, Rat199231H. nemestrinaeMonkey1991
      12H. pametensisBird, Swine199432H. salomonisDog1997
      13H. rodentiumMouse199733H. suisPig2008
      14H. trogontum
      Two isolation cases of H. trogontum from human clinical specimen have been reported on the Japanese Society for Clinical Microbiology Meeting (Journal report has not been yet). Besides these species, “Candidatus Helicobacter bovis" (gastric helicobacter) has been proposed in 1999 [95].
      Rat1996
      15H. typhloniusMouse2002
      16H. anserisGoose2006
      17H. brantaeGoose2006
      18H. marmotaeWoodchuck, cat2006
      19H. mastomyrinusRodent2006
      20H. equorumHorse2007
      a “Enterohepaic-Gastric" categories were basically according with Solnick et al.
      • Solnick J.V.
      • Schauer D.B.
      Emergence of diverse Helicobacter species in the pathogenesis of gastric and enterohepatic diseases.
      .
      b Only seven species has been isolated from human specimen. Some reports described the detection of H. suis, H. felis, H. hepaticus, or H. ganmani using genetic methods but there is no report, to our knowledge, has isolated the organism.
      c Two isolation cases of H. trogontum from human clinical specimen have been reported on the Japanese Society for Clinical Microbiology Meeting (Journal report has not been yet).Besides these species, “Candidatus Helicobacter bovis" (gastric helicobacter) has been proposed in 1999
      • De Groote D.
      • van Doorn L.J.
      • Ducatelle R.
      • Verschuuren A.
      • Tilmant K.
      • Quint W.G.
      • et al.
      Phylogenetic characterization of 'Candidatus Helicobacter bovis', a new gastric helicobacter in cattle.
      .
      Figure thumbnail gr2
      Fig. 2Phylogenetic relationships among all members of the genus Helicobacter, based on 16S rRNA gene sequence. Distances were calculated by the neighbor-joining method. The numbers at the branching points are bootstrap values. C. fetus subsp. fetus was used as the out group. The numbers in parenthesis are the accession numbers of the gene sequences. The “gastric-Helicobacter species” (indicated by shaded text) formed one well-separated cluster, excluding H. mustelae and H. aurati.
      There is an invalid species name related to the genus Helicobacter known as “Flexispira rappini” (sometime referred as “Helicobacter rappini”). “F. rappini” was first proposed by Bryner et al. [
      • Bryner J.H.
      • Littleton J.
      • Gates C.
      • Kirkbride C.A.
      • Richie A.E.
      Flexispira rappini gen. nov., sp. nov., a gram-negative rod from mammalian fetus and feces.
      ,
      • Bryner J.H.
      Flexispira rappini, gen. nov., sp. nov. A motile, urease-producing rod similar to Campylobacter pyloridis.
      ] for a strain group of ultrastructurally distinct, urease-producing strains isolated from lambs, dogs, canine, ovine, and humans. Since this first study, strains have been identified as “F. rappini” by 16S rRNA gene sequence comparison, despite notable morphological and phenotypic differences [
      • Tee W.
      • Leder K.
      • Karroum E.
      • Dyall-Smith M.
      “Flexispira rappini” bacteremia in a child with pneumonia.
      ,
      • Sorlin P.
      • Vandamme P.
      • Nortier J.
      • Hoste B.
      • Rossi C.
      • Pavlof S.
      • et al.
      Recurrent “Flexispira rappini” bacteremia in an adult patient undergoing hemodialysis: case report.
      ,
      • Tee W.
      • Jenney A.
      • McPhee A.
      • Mijch A.
      • Dyall-Smith M.
      “Helicobacter rappini” isolates from 2 homosexual men.
      ]. In 2000, Dewhirst et al. [
      • Dewhirst F.E.
      • Fox J.G.
      • Mendes E.N.
      • Paster B.J.
      • Gates C.E.
      • Kirkbride C.A.
      • et al.
      'Flexispira rappini' strains represent at least 10 Helicobacter taxa.
      ] reported that “F. rappini” strains represent at least 10 Helicobacter taxa, and then Hänninen et al. [
      • Hänninen M.L.
      • Utriainen M.
      • Happonen I.
      • Dewhirst F.E.
      Helicobacter sp. flexispira 16S rDNA taxa 1, 4 and 5 and finnish porcine Helicobacter isolates are members of the species Helicobacter trogontum (taxon 6).
      ,
      • Hänninen M.L.
      • Kärenlampi R.I.
      • Koort J.M.
      • Mikkonen T.
      • Björkroth K.J.
      Extension of the species Helicobacter bilis to include the reference strains of Helicobacter sp. flexispira taxa 2, 3 and 8 and finnish canine and feline flexispira strains.
      ] fell into each taxon as a valid Helicobacter species (Table 2). Only taxon 7 (one strain isolated from dog stomach) and taxon 10 (three strains isolated from cotton-top tamarin) have not been clear their taxonomic positions. Indeed, the title of the 16S rRNA gene sequence information under the DDBJ/NCBI/EBI accession number M88138 (ATCC43879) is still ‘Helicobacter sp. “Flexispira taxon 8” 16S ribosomal RNA gene’. This information may be a cause of misunderstanding and the researcher should carefully read both the title and the annotated text. Other provisional names, “Helicobacter westmeadii” [
      • Trivett-Moore N.L.
      • Rawlinson W.D.
      • Yuen M.
      • Gilbert G.L.
      Helicobacter westmeadii sp. nov., a new species isolated from blood cultures of two AIDS patients.
      ] and “Helicobacter sp. strain Mainz,” have been assigned to H. cinaedi [
      • On S.L.W.
      International Committee on Systematics of Prokaryotes: subcommittee on the taxonomy of Campylobacter and related bacteria.
      ].
      Table 2Taxonomical ascription of “Flexispira rappini".
      TaxonValid nameRepresentative strain
      1H. trogontumATCC 43968
      2H. bilisATCC 49314
      3H. bilisATCC 49320
      4H. trogontumATCC 49310
      5H. trogontumATCC 43966
      6H. trogontum8581T (=type strain of H. trogontum)
      7Not determined1302/Dog-4
      8H. bilisATCC 43879
      9H. bilisATCC 51630
      10Not determined97-6149-5
      H. cinaedi was first isolated from rectal swabs obtained from homosexual men with proctitis, proctocolitis, and enteritis [
      • Fennell C.L.
      • Totten P.A.
      • Quinn T.C.
      • Patton D.L.
      • Holmes K.K.
      • Stamm W.E.
      Characterization of Campylobacter-like organisms isolated from homosexual men.
      ], but the number of reports of H. cinaedi infection has been steadily growing throughout the last two decades. Because early reports mainly described the isolation of these microorganisms from homosexual men or immunocompromised patients, and their presence was attributed to human immunodeficiency virus infection, agammaglobulinemia, or some other underlying disease [
      • Gaudreau C.
      • Lachance N.
      Infection with Helicobacter cinaedi resistant to ciprofloxacin in men with AIDS.
      ,
      • Mammen Jr., M.P.
      • Aronson N.E.
      • Edenfield W.J.
      • Endy T.P.
      Recurrent Helicobacter cinaedi bacteremia in a patient infected with human immunodeficiency virus: case report.
      ,
      • Tee W.
      • Street A.C.
      • Spelman D.
      • Munckhof W.
      • Mijch A.
      Helicobacter cinaedi bacteraemia: varied clinical manifestations in three homosexual males.
      ,
      • Simons E.
      • Spacek L.A.
      • Lederman H.M.
      • Winkelstein J.A.
      Helicobacter cinaedi bacteremia presenting as macules in an afebrile patient with X-linked agammaglobulinemia.
      ,
      • Pasternak J.
      • Bolivar R.
      • Hopfer R.L.
      • Fainstein V.
      • Mills K.
      • Rios A.
      • et al.
      Bacteremia caused by Campylobacter-like organisms in two male homosexuals.
      ,
      • Burman W.J.
      • Cohn D.L.
      • Reves R.R.
      • Wilson M.L.
      Multifocal cellulitis and monoarticular arthritis as manifestations of Helicobacter cinaedi bacteremia.
      ], the organisms were thought to be related to specific hosts. Recently, however, given that increasing numbers of infections have also been reported in immunocompetent patients [
      • Lasry S.
      • Simon J.
      • Marais A.
      • Pouchot J.
      • Vinceneux P.
      • Boussougant Y.
      Helicobacter cinaedi septic arthritis and bacteremia in an immunocompetent patient.
      ,
      • Van Genderen P.J.
      • Goessens W.H.
      • Petit P.L.
      Helicobacter cinaedi-associated bacteraemia and erysipelas in an immunocompetent host: a diagnostic challenge.
      ,
      • Kitamura T.
      • Kawamura Y.
      • Ohkusu K.
      • Masaki T.
      • Iwashita H.
      • Sawa T.
      • et al.
      Helicobacter cinaedi cellulitis and bacteremia in immunocompetent hosts after orthopedic surgery.
      ,
      • Holst H.
      • Andresen K.
      • Blom J.
      • Højlyng N.
      • Kemp M.
      • Krogfelt K.A.
      • et al.
      A case of Helicobacter cinaedi bacteraemia in a previously healthy person with cellulitis.
      ], the patient group affected by H. cinaedi is larger than originally thought.
      In Japan, the first report describing the isolation of H. cinaedi was published in 2003 [
      • Murakami H.
      • Goto M.
      • Ono E.
      • Sawabe E.
      • Iwata M.
      • Okuzumi K.
      • et al.
      Isolation of Helicobacter cinaedi from blood of an immunocompromised patient in Japan.
      ]. Since then, isolation of this microorganism has been reported in patients regardless of gender and within a wide age range, from newborns to the elderly, by many hospitals throughout the country. Matsumoto et al. [
      • Matsumoto T.
      • Goto M.
      • Murakami H.
      • Tanaka T.
      • Nishiyama H.
      • Ono E.
      • et al.
      Multicenter study to evaluate bloodstream infection by Helicobacter cinaedi in Japan.
      ] reported that the H. cinaedi positive rate in blood cultures was 0.06% (6/16,743 samples) of total blood samples and 0.22% (6/2718 samples) of blood samples with any positive culture, based on a prospective multicenter analysis in 13 hospitals over 6 months in Tokyo. This microorganism is not a clinically scarcity. Indeed, we have encountered many cases of H. cinaedi cellulitis and bacteremia that occurred continuously in both immunocompromised and immunocompetent subjects in hospitals. Now, we recognize that this microorganism should be considered a causative agent of nosocomial infection [
      • Kitamura T.
      • Kawamura Y.
      • Ohkusu K.
      • Masaki T.
      • Iwashita H.
      • Sawa T.
      • et al.
      Helicobacter cinaedi cellulitis and bacteremia in immunocompetent hosts after orthopedic surgery.
      ,
      • Rimbara E.
      • Mori S.
      • Matsui M.
      • Suzuki S.
      • Wachino J.
      • Kawamura Y.
      • et al.
      Molecular epidemiologic analysis and antimicrobial resistance of Helicobacter cinaedi isolated from seven hospitals in Japan.
      ,
      • Minauchi K.
      • Takahashi S.
      • Sakai T.
      • Kondo M.
      • Shibayama K.
      • Arakawa Y.
      • et al.
      The nosocomial transmission of Helicobacter cinaedi infections in immunocompromised patients.
      ]. The association of this microorganism with a variety of human infections is receiving a growing amount of attention.

      2. Diseases caused by H. cinaedi

      H. cinaedi infection causes many kinds of symptoms including fever, abdominal pain, gastroenteritis, proctitis, diarrhea, erysipelas, cellulitis, arthritis, neonatal meningitis, and bacteremia [
      • Lawson A.J.
      Helicobacter.
      ]. Recently, a case of meningitis in a healthy adult and that of an axillobifemoral bypass graft infection in an immunocompetent patient were also reported [
      • Sugiyama A.
      • Mori M.
      • Ishiwada N.
      • Himuro K.
      • Kuwabara S.
      First adult case of Helicobacter cinaedi meningitis.
      ,
      • Suematsu Y.
      • Morizumi S.
      • Okamura K.
      • Kawata M.
      A rare case of axillobifemoral bypass graft infection caused by Helicobacter cinaedi.
      ].
      Numerous reports have described bacteremia caused by H. cinaedi rather than by other Helicobacter species. This may be because this organism has a strong ability for vascular invasion resulting in bacterial translocation from the intestinal tract to the vascular system. Among the many cases of H. cinaedi bacteremia, the main symptom is fever. However, various symptoms are important to note. Fever is typically accompanied by arthritis and cellulitis at various sites in the body, which can be regarded either as the primary site of infection of bacteremia or a secondary focus of infection through the bacteremia.
      In our experience, some patients had a sudden onset of local flat cellulitis (salmon-pink in color) accompanied by fever and an increase in C-reactive protein levels at various times after orthopedic surgery (range, 8–113 days; mean, 29 days) (Fig. 3) [
      • Kitamura T.
      • Kawamura Y.
      • Ohkusu K.
      • Masaki T.
      • Iwashita H.
      • Sawa T.
      • et al.
      Helicobacter cinaedi cellulitis and bacteremia in immunocompetent hosts after orthopedic surgery.
      ]. Cellulitis was often multifocal with no wound infection. Many of these patients had been treated for fracture and were immunocompetent.
      Figure thumbnail gr3
      Fig. 3(A) A typical skin lesion of cellulitis caused by H. cinaedi. Cellulitis is typically salmon-pink in color and is accompanied by swelling (arrows). (B) Clinical course of a patient with cellulitis caused by H. cinaedi. At a same time as a sudden onset of cellulitis, body temperature (BT), white blood cell count (WBC), and C-reactive protein (CRP) levels were elevated. After taking Sulbactam/Ampicillin (SBT/ABPC), these symptoms quickly improved.
      Regarding a new disease relating to H. cinaedi infection, we recently found that H. cinaedi infection is involved in the progression of atherosclerosis. To investigate the relationship of H. cinaedi infection and atherosclerosis, we first analyzed H. cinaedi infection in the human atherosclerotic aorta by using immunohistochemistry with a specific anti-H. cinaedi antibody. Surprisingly, H. cinaedi antigen was clearly detected in atherosclerotic plaques in almost all postmortem human specimens [
      • Khan S.
      • Okamoto T.
      • Enomoto K.
      • Sakashita N.
      • Oyama K.
      • Fujii S.
      • et al.
      Potential association of Helicobacter cinaedi with atrial arrhythmias and atherosclerosis.
      ], where it was colocalized with macrophages. These observations strongly suggest that H. cinaedi may be closely associated with atherosclerosis in humans. We further investigated the effect of H. cinaedi infection on the development of atherosclerosis and its molecular mechanisms by using Apoeshl atherosclerosis model mice. Apoeshl mice orally infected with H. cinaedi for 8 weeks developed atherosclerosis in the aorta more extensively than uninfected control mice, as confirmed by lipid staining with Oil Red O for atherosclerosis plaques (Fig. 4(A)) [
      • Khan S.
      • Rahman H.N.A.
      • Okamoto T.
      • Matsunaga T.
      • Fujiwara Y.
      • Sawa T.
      • et al.
      Promotion of atherosclerosis by Helicobacter cinaedi infection that involves macrophage-driven proinflammatory responses.
      ]. To the best of our knowledge, this is first evidence of the involvement of H. cinaedi infection in the development of atherosclerosis.
      Figure thumbnail gr4
      Fig. 4(A) Promotion of atherosclerosis by H. cinaedi infection in Apoeshl mice aortas. Mice were killed at 8 weeks after oral infection and atherosclerotic lesions in the aortic sinus were stained with Oil Red O (red). Scale bars, 200 μm. Modified from Ref.
      [
      • Khan S.
      • Rahman H.N.A.
      • Okamoto T.
      • Matsunaga T.
      • Fujiwara Y.
      • Sawa T.
      • et al.
      Promotion of atherosclerosis by Helicobacter cinaedi infection that involves macrophage-driven proinflammatory responses.
      ]
      . (B) Foam cell formation induced by H. cinaedi infection in cultured mouse peritoneal macrophages. Cells were infected with H. cinaedi (MOI of 0.1 for 24 h) and stained with Oil Red O. Nuclei were stained with DAPI. Scale bars, 20 μm. Modified from Ref.
      [
      • Khan S.
      • Rahman H.N.A.
      • Okamoto T.
      • Matsunaga T.
      • Fujiwara Y.
      • Sawa T.
      • et al.
      Promotion of atherosclerosis by Helicobacter cinaedi infection that involves macrophage-driven proinflammatory responses.
      ]
      .
      The chronic inflammatory response is a widely accepted key mechanism in the progression of atherosclerosis [
      • Libby P.
      • Ridker P.M.
      • Hansson G.K.
      Inflammation in atherosclerosis: from pathophysiology to practice.
      ,
      • Tabas I.
      Macrophage death and defective inflammation resolution in atherosclerosis.
      ]. Gene expression analysis by real-time reverse transcription-PCR revealed significantly increased expression of inflammation-related genes, such as inducible nitric oxide synthase, interleukin-1, and Toll-like receptor 4, in aortic tissues of H. cinaedi-infected Apoeshl mice compared with those in uninfected control mice [
      • Khan S.
      • Rahman H.N.A.
      • Okamoto T.
      • Matsunaga T.
      • Fujiwara Y.
      • Sawa T.
      • et al.
      Promotion of atherosclerosis by Helicobacter cinaedi infection that involves macrophage-driven proinflammatory responses.
      ]. Mediators responsible for leukocyte adhesion and recruitment in the vascular wall, such as C–C motif chemokine 2 and intercellular adhesion molecule-1, were also upregulated in infected mice. Moreover, nested PCR analysis, which is a highly specific and sensitive detection method for H. cinaedi that we recently developed [
      • Oyama K.
      • Khan S.
      • Okamoto T.
      • Fujii S.
      • Ono K.
      • Matsunaga T.
      • et al.
      Identification of and screening for human Helicobacter cinaedi infections and carriers via nested PCR.
      ], clearly showed that H. cinaedi DNA and RNA existed in the aorta of infected mice [
      • Khan S.
      • Rahman H.N.A.
      • Okamoto T.
      • Matsunaga T.
      • Fujiwara Y.
      • Sawa T.
      • et al.
      Promotion of atherosclerosis by Helicobacter cinaedi infection that involves macrophage-driven proinflammatory responses.
      ]. These findings suggested that oral infection by H. cinaedi might translocate to vascular tissue and induce chronic inflammation in the aorta, subsequently leading to atherosclerotic plaque formation.
      We further investigated the mechanism of atherosclerosis promotion by H. cinaedi infection by using in vitro experiments. The accumulation of lipids in macrophages, which is known as foam cell formation, is thought to be a critical step in the development of atherosclerosis. Thus, we examined the effect of H. cinaedi infection on foam cell formation in cultured macrophages derived from mouse and human [
      • Khan S.
      • Rahman H.N.A.
      • Okamoto T.
      • Matsunaga T.
      • Fujiwara Y.
      • Sawa T.
      • et al.
      Promotion of atherosclerosis by Helicobacter cinaedi infection that involves macrophage-driven proinflammatory responses.
      ]. Twenty-four hours after H. cinaedi was added to the culture medium, macrophages had markedly accumulated lipid droplets, which is the hallmark of foam cell formation (Fig. 4(B)). Uninfected cells and H. pylori-infected cells did not show such accumulation of lipid droplets, suggesting that H. cinaedi specifically induced foam cell formation. Further examination of the mechanism of foam cell formation induced by H. cinaedi infection revealed that a change in the metabolism of cholesterol induced by infection may be involved. Specifically, H. cinaedi infection of macrophages increased the expression of LDL receptor, known to be involved in cholesterol intake, in macrophages. Also, H. cinaedi infection of macrophages decreased the expression of the ATP-binding cassette transporter G1 (ABCG1), which is thought to be involved in the excretion of cholesterol to outside of the macrophages. Although the detailed molecular mechanisms of the changes in expression of the LDL receptor and ABCG1 during H. cinaedi infection remain unclear, these changes would affect intracellular cholesterol metabolism and cause an accumulation of cholesterol. These results suggest that H. cinaedi infection promotes the development of atherosclerosis through chronic vascular inflammation, macrophage activation, and subsequent foam cell formation (Fig. 5).
      Figure thumbnail gr5
      Fig. 5Schematic representation of a possible mechanism of atherosclerosis promotion by H. cinaedi. Based on the data in Ref.
      [
      • Khan S.
      • Rahman H.N.A.
      • Okamoto T.
      • Matsunaga T.
      • Fujiwara Y.
      • Sawa T.
      • et al.
      Promotion of atherosclerosis by Helicobacter cinaedi infection that involves macrophage-driven proinflammatory responses.
      ]
      .
      Some reports have suggested that infection by specific microbes may be involved in the pathogenesis of atherosclerosis. These microbes include various pathogens, such as Chlamydia pneumoniae and Porphyromonas gingivalis [
      • Moazed T.C.
      • Kuo C.
      • Grayston J.T.
      • Campbell L.A.
      Murine models of Chlamydia pneumoniae infection and atherosclerosis.
      ,
      • Li L.
      • Messas E.
      • Batista Jr., E.L.
      • Levine R.A.
      • Amar S.
      Porphyromonas gingivalis infection accelerates the progression of atherosclerosis in a heterozygous apolipoprotein E-deficient murine model.
      ]. However, the exact mechanisms involved in the promotion of atherosclerosis and to what extent they are related to human atherosclerosis is not fully understood. Our recent findings presented above showed a new mechanism of atherosclerosis development promoted by bacterial infection. These may be related to the vascular tropism of H. cinaedi and frequent recurrences. Further investigation is needed to clarify the involvement of H. cinaedi infection in human atherosclerosis and the detailed molecular mechanisms involved in H. cinaedi promotion of foam cell formation in macrophages. Additional investigations to determine the etiological role of H. cinaedi in the development of atherosclerotic cardiovascular diseases are also warranted.

      3. Virulence factors

      Only two virulence factors have been reported in the literature: cytolethal distending toxin (Cdt) and alkyl hydroperoxide reductase (AhpC). Cdt is a lethal toxin to eukaryotic cells as a result of apoptosis induction and cell cycle arrest in host eukaryotic cells [
      • Taylor N.S.
      • Ge Z.
      • Shen Z.
      • Dewhirst F.E.
      • Fox J.G.
      Cytolethal distending toxin: a potential virulence factor for Helicobacter cinaedi.
      ,
      • Jinadasa R.N.
      • Bloom S.E.
      • Weiss R.S.
      • Duhamel G.E.
      Cytolethal distending toxin: a conserved bacterial genotoxin that blocks cell cycle progression, leading to apoptosis of a broad range of mammalian cell lineages.
      ]. AhpC is an enzyme that converts various alkyl hydroperoxides to their corresponding alcohols, and can change hydrogen peroxide to water. This enzyme contributes to microorganism survival in host conditions via the protection of the cells from oxidative stress [
      • Charoenlap N.
      • Shen Z.
      • McBee M.E.
      • Muthupalani S.
      • Wogan G.N.
      • Fox J.G.
      • et al.
      Alkyl hydroperoxide reductase is required for Helicobacter cinaedi intestinal colonization and survival under oxidative stress in BALB/c and BALB/c interleukin-10-/- mice.
      ].
      During our investigation to determine the complete genome sequence of a human clinical isolate (PAGU 611) from the blood sample of a cellulitis subject, we revealed that the microorganism holds a Type VI secretion system (T6SS) which is thought to be related to its virulence [
      • Goto T.
      • Ogura Y.
      • Hirakawa H.
      • Tomida J.
      • Morita Y.
      • Akaike T.
      • et al.
      Complete genome sequence of Helicobacter cinaedi strain PAGU611, isolated in a case of human bacteremia.
      ]. T6SS is a kind of complex multi-component secretion machine, often called a “needle” or “molecular syringe”. In many cases, T6SS delivers bacteriolytic effectors to target cells, such as other bacteria or eukaryotic hosts, and in some cases is involved in symbiotic interactions with eukaryotic hosts [
      • Filloux A.
      • Hachani A.
      • Bleves S.
      The bacterial type VI secretion machine: yet another player for protein transport across membranes.
      ,
      • Jani A.J.
      • Cotter P.A.
      Type VI secretion: not just for pathogenesis anymore.
      ].
      In the case of Helicobacter hepaticus, another enterohepatic species that is harbored in mouse intestines, T6SS was reported to play an important role in persistent colonization to promote a balanced relationship with the host via the T6SS directed anti-inflammatory gene expression profiles in intestinal epithelial cells and CD4+ T cells [
      • Chow J.
      • Mazmanian S.K.
      A pathobiont of the microbiota balances host colonization and intestinal inflammation.
      ]. Another report described an association between VgrG1, a secreted protein of T6SS, and bacterial colitogenic potential [
      • Bartonickova L.
      • Sterzenbach T.
      • Nell S.
      • Kops F.
      • Schulze J.
      • Venzke A.
      • et al.
      Hcp and VgrG1 are secreted components of the Helicobacter hepaticus type VI secretion system and VgrG1 increases the bacterial colitogenic potential.
      ]. The role of the T6SS in H. cinaedi infection is not clear; however, there might be a similar virulence function.
      The PAGU 611 chromosome encodes two known virulence factors, described above, cdt and ahpC genes, and also several putative virulence-related proteins, such as fibronectin- and fibrinogen-binding proteins, neutrophil activation protein, and Campylobacter jejuni invasion antigen B [
      • Goto T.
      • Ogura Y.
      • Hirakawa H.
      • Tomida J.
      • Morita Y.
      • Akaike T.
      • et al.
      Complete genome sequence of Helicobacter cinaedi strain PAGU611, isolated in a case of human bacteremia.
      ]. The type strain of H. cinaedi, another complete genome determined strain [
      • Miyoshi-Akiyama T.
      • Takeshita N.
      • Ohmagari N.
      • Kirikae T.
      Complete genome sequence of Helicobacter cinaedi type strain ATCC BAA-847.
      ], has all of the above-mentioned (putative) virulence factors; thus, these factors might be commonly harbored within the human isolates.
      Further investigation is needed to clarify the virulence of this microorganism.

      4. Clinical laboratory tests

      It is well known that H. cinaedi is a fastidious and slow-growing organism and that detection and cultivation are extremely difficult. In this section, methods of detection, culture, and identification are described, as well as the description of new taxon for the genus Helicobacter.

      4.1 Detection on blood culture system

      Isolates of H. cinaedi are mainly obtained from blood and, to a lesser extent, fecal samples. In fact, H. cinaedi is in many cases first detected from blood culture using an automatic blood culture system. Nowadays, many hospital laboratories employ an automatic blood culture system, such as the BACTEC or BacT/ALERT systems. Recently, another blood culture system, VersaTREK, has been introduced in Japan.
      Because H. cinaedi are slow-growing organisms, a relatively prolonged incubation time is generally required. In many reports, and in our experience, 4–10 days are needed for a positive result in the culture bottle of an automatic blood culture system, such as the BACTEC system using an aerobic bottle. Therefore, once the culture test is finished within 3–4 days, the possibility of detection can become critically low due to not reaching the detection limit. Detection reports of H. cinaedi using the BacT/ALERT system are very limited. In the case of the BacT/ALERT system, H. cinaedi has been detected using both aerobic and anaerobic bottles [
      • Lasry S.
      • Simon J.
      • Marais A.
      • Pouchot J.
      • Vinceneux P.
      • Boussougant Y.
      Helicobacter cinaedi septic arthritis and bacteremia in an immunocompetent patient.
      ,
      • Suzuki T.
      • Kawamura C.
      • Yoshimura H.
      • Moriguchi M.
      • Okusu K.
      • Honma M.
      • et al.
      A study of Helicobacter cinaedi detection case reports using BacT/ALERT in 4 clinical facilities.
      ,
      • Orlicek S.L.
      • Welch D.F.
      • Kuhls T.L.
      Septicemia and meningitis caused by Helicobacter cinaedi in a neonate.
      ]. In our experience, the VersaTREK system is superior for the detection of this microorganism. Because the VersaTREK system provides excellent growth ability and is highly sensitive, H. cinaedi isolates can be detected very quickly. Some clinical laboratory technologists have reported being able to detect H. cinaedi within 3 days [
      • Hosoda T.
      • Hayakawa S.
      • Wakuda M.
      • Higashimoto Y.
      • Furukawa H.
      • Ishikawa T.
      • et al.
      Potential for an early stage detection of Helicobacter cinaedi on automated blood culture systems.
      ]. In our preliminary experiment using five H. cinaedi isolates, the VersaTREK system detected all isolates within 3 days, whereas other systems needed more incubation time or detection failed [
      • Tomida J.
      • Tsurunaga M.
      • Hosoda T.
      • Hayakawa S.
      • Suematsu H.
      • Sawamura H.
      • et al.
      Evaluation of automated blood culture systems about the detection capability for Helicobacter cinaedi.
      ].

      4.2 Culture

      H. cinaedi isolates essentially required microaerobic conditions (5–10% O2) and high humidity. Blood agar plates stored in a refrigerator for a few days often do not support the growth of H. cinaedi because the water content may be reduced; therefore, the use of fresh medium is strongly recommended. It is well known that the growth of H. cinaedi is accelerated by adding hydrogen gas (5–10%) to the microaerobic conditions. It is preferable to use such gas conditions (e.g. 6% O2, 7% H2, 7% CO2, and 80% N2) in the initial culture step of the clinical specimen or in the culture bottle to increase the culture success rate. Unfortunately, many commercially available microaerobic gas generating packs, such as the Gas-Pak system, can deoxidize and generate CO2 but not supply hydrogen gas; therefore, H. cinaedi growth sometimes fails or is insufficient. H. cinaedi cultured on an agar plate may appear as a swarming thin film, which is difficult to identify visually. Therefore, the culture should be carefully checked on the plate.
      Many selective media are suitable for the isolation of H. cinaedi. Baba et al. [
      • Baba M.
      Infectious diseases case conference seminar–from clinical questions to research insights.
      ] reported that many different selective media for Campylobacter or Helicobacter, such as Skirrow and Butzler Blaser, can be used, with the exception of CCDA (charcoal-cefazolin-sodium deoxycholate agar), which failed to grow the H. cinaedi isolates [
      • Engberg J.
      • On S.T.W.
      • Harrington C.S.
      • Gerner-Smidt P.
      Prevalence of Campylobacter, Arcobacter, Helicobacter, and Sutterella spp. in human fecal samples as estimated by a reevaluation of isolation methods for Campylobacters.
      ,
      • Tanaka T.
      • Goto M.
      • Okuzumi K.
      • Yoneyama A.
      • Matsumoto T.
      • Yamaguchi K.
      • et al.
      Isolation and identification of Helicobacter cinaedi-like organisms isolated from blood culture in practical laboratory procedures.
      ].
      Tomida et al. [
      • Tomida J.
      • Kashida M.
      • Oinishi K.
      • Endo R.
      • Morita Y.
      • Kawamura Y.
      Evaluation of various media for rapid detection of Helicobacter spp.
      ] reported that “Helicobacter medium” (Nissui Pharm. Co. Ltd) is excellent, because it has good potential for supporting the growth of H. cinaedi isolates. Furthermore, because the medium contains a serum (not erythrocytes) and reduction-reactive dyes, the growing bacteria are easy to observe, even in film form, due to their purple color against the translucent medium.
      These selective media would be useful for isolating H. cinaedi from specimens such as feces or environmental samples. Another approach using filtration method is described in “Cape Town Campylobacter and Helicobacter Protocol”, a document issued by the South Central Association for Clinical Microbiology, a not-for-profit organization (http://www.scacm.org/index.htm).

      4.3 Identification

      The biochemical identification of this organism is problematic due to unstable phenotypic reactions. For example, results of the 42 °C (Celsius temperature) growth test led to disagreement between researchers; Lawson [
      • Lawson A.J.
      Helicobacter.
      ] described a negative result but Kiehlbauch et al. [
      • Kiehlbauch J.A.
      • Brenner D.J.
      • Cameron D.N.
      • Steigerwalt A.G.
      • Makowski J.M.
      • Baker C.N.
      • et al.
      Genotypic and phenotypic characterization of Helicobacter cinaedi and Helicobacter fennelliae strains isolated from humans and animals.
      ] reported a positive result. The results of the alkaline-phosphatase test are difficult to read because the gradual color changes are dependent on the incubation time and certain strains give only the faintest hint of color [
      • On S.L.
      • Holmes B.
      Assessment of enzyme detection test useful in identification of campylobacteria.
      ].
      Due to these unstable phenotypic reactions and a lack of substantial data sets, commercially available identification kits do not produce reliable results. Therefore, identification has been based on nucleotide sequence or species-specific polymerase chain reaction (PCR). We have developed a nested PCR system with high specificity and sensitivity (c.a. 102 CFU/ml) for detecting H. cinaedi based on the sequence of the known virulence factor gene, cdtB [
      • Oyama K.
      • Khan S.
      • Okamoto T.
      • Fujii S.
      • Ono K.
      • Matsunaga T.
      • et al.
      Identification of and screening for human Helicobacter cinaedi infections and carriers via nested PCR.
      ]. By using this cdtB gene-based PCR detection system, we identified more than 200 isolates received from various hospitals across the country. Another advantage of using PCR techniques is that culture is unnecessary. Since the culture of H. cinaedi isolates is very difficult and sometimes, as mentioned above, cells fail to even grow, the present DNA detection test is convenient, as it can be directly performed even in these cases from the contents of a culture bottle using PCR.
      Analysis of 16S rRNA gene sequences is one of the most common approaches for investigating the phylogenetic positions of bacterial strains; however, Vandamme et al. [
      • Vandamme P.
      • Clare S.
      • Harrington C.S.
      • Jalava K.
      • On S.L.W.
      Misidentifying Helicobacters: the Helicobacter cinaedi example.
      ] reported a problem due to misidentification of H. cinaedi using 16S rRNA gene sequences. The isolate believed to be H. cinaedi was located some distance from the phylogenetic cluster of the type strain, it is required careful consideration. Yet almost all isolates that we found were located within or very close to the type strain's cluster, and were correctly identified using 16S rRNA gene phylogenetic analysis. As described above, the species H. cinaedi includes at least two genetically diverse microorganisms, and Vandamme et al. [
      • Vandamme P.
      • Clare S.
      • Harrington C.S.
      • Jalava K.
      • On S.L.W.
      Misidentifying Helicobacters: the Helicobacter cinaedi example.
      ] used certain strains such as the previously named “Helicobacter sp. strain Mainz”, or certain canine isolates; therefore, the antecedents of the strains should be clarified.
      Kuhnert and Burnens [
      • Kuhnert P.
      • Burnens A.
      Misidentifying Helicobacter cinaedi.
      ] highlight another potential source of error in the identification of H. cinaedi. ATCC 35863 was designated and distributed as a type strain of H. cinaedi but is actually H. fennelliae. Identification operations involve matching data sets obtained from unknown isolates with those of previously described taxa, so any mislabeling of the latter can result in unknown isolates being misidentified [
      • Kuhnert P.
      • Burnens A.
      Misidentifying Helicobacter cinaedi.
      ]. Fortunately, this problem has already been fixed (ATCC 35863 is now labeled as H. fennelliae).
      PCR-DGGE (denature gradient gel electrophoresis) method using amplified 16S rRNA gene for the identification of Helicobacter species has also been reported [
      • Al-Soud W.A.
      • Bennedsen M.
      • On S.L.W.
      • Ouis I.S.
      • Vandamme P.
      • Nilsson H.O.
      • et al.
      Assessment of PCR-DGGE for the identification of diverse Helicobacter species, and application to faecal samples from zoo animals to determine Helicobacter prevalence.
      ]. Other gene sequences, such as RNA polymerase-β subunit (rpoB) and β′-subunits (rpoC) genes [
      • Zakharova N.
      • Paster B.J.
      • Wesley I.
      • Dewhirst F.E.
      • Berg D.E.
      • Severinov K.V.
      Fused and overlapping rpoB and rpoC genes in Helicobacters, Campylobacters, and related bacteria.
      ], DNA gyrase protein B-subunit (gyrB) gene [
      • Hannula M.
      • Hänninen M.L.
      Phylogenetic analysis of Helicobacter species based on partial gyrB gene sequences.
      ], 60 kDa heat shock protein gene (hsp60) [
      • Mikkonen T.P.
      • Kärenlampi R.I.
      • Hänninen M.L.
      Phylogenetic analysis of gastric and enterohepatic Helicobacter species based on partial HSP60 gene sequences.
      ], 23S rRNA gene [
      • Dewhirst F.E.
      • Shen Z.
      • Scimeca M.S.
      • Stokes L.N.
      • Boumenna T.
      • Chen T.
      • et al.
      Discordant 16S and 23S rRNA gene phylogenies for the genus Helicobacter: implications for phylogenetic inference and systematics.
      ], and urease protein B-subunit (ureB) gene [
      • Hänninen M.L.
      • Kärenlampi R.I.
      • Koort J.M.
      • Mikkonen T.
      • Björkroth K.J.
      Extension of the species Helicobacter bilis to include the reference strains of Helicobacter sp. flexispira taxa 2, 3 and 8 and finnish canine and feline flexispira strains.
      ] have also been used in phylogenetic studies of the genus Helicobacter. These analysis methods are certainly thought to be useful, but each method has particular strengths and limitations relating to the accumulation of sequence data and the distinguishing powers. Therefore, researchers must carefully consider the advantages and disadvantages of each analysis method.

      4.4 Description of a new taxon within the genus helicobacter

      “Recommended minimal standards for describing new species of the genus Helicobacter” was published in 2000 by the International Committee of Systematic Bacteriology subcommittee on the taxonomy of Campylobacter and related bacteria [
      • Dewhirst F.E.
      • Fox J.G.
      • On S.L.W.
      Recommended minimal standards for describing new species of the genus Helicobacter.
      ,
      • Vandamme P.
      • On S.L.W.
      Recommendations of the subcommittee on the taxonomy of Campylobacter and related bacteria.
      ]. The recommendation stated that at least five strains should be used and both phenotypic and molecular data collected. Some basic biochemical testing procedures and the medium formulas for Helicobacter and Campylobacter are described by On and Holmes [
      • On S.L.
      • Holmes B.
      Assessment of enzyme detection test useful in identification of campylobacteria.
      ,
      • On S.L.W.
      • Holmes B.
      Effect of inoculum size on the phenotypic characterization of Campylobacter species.
      ,
      • On S.L.W.
      • Holmes B.
      Reproducibility of tolerance tests that are useful in the identification of campylobacteria.
      ]. The molecular data described in the recommendation includes DNA G + Cmol%, almost full-length 16S rRNA gene sequences (more than 1450 bp) including intervening sequences (if any), DNA–DNA hybridization data, and others. To propose a new species or subspecies, researchers should include these data.

      5. Antimicrobial susceptibility testing

      There are no recommended guidelines for susceptibility testing and the treatment of diagnosed infections with H. cinaedi.
      In 1991, one report clearly stated that H. cinaedi failed to grow during testing for antimicrobial susceptibility by a broth microdilution method [
      • Sacks L.V.
      • Labriola A.M.
      • Gill V.J.
      • Gordin F.M.
      Use of ciprofloxacin for successful eradication of bacteremia due to Campylobacter cinaedi in a human immunodeficiency virus-infected person.
      ]. Antimicrobial susceptibility testing for H. cinaedi isolates has been carried out using the agar dilution method [
      • Tee W.
      • Street A.C.
      • Spelman D.
      • Munckhof W.
      • Mijch A.
      Helicobacter cinaedi bacteraemia: varied clinical manifestations in three homosexual males.
      ,
      • Orlicek S.L.
      • Welch D.F.
      • Kuhls T.L.
      Septicemia and meningitis caused by Helicobacter cinaedi in a neonate.
      ,
      • Kiehlbauch J.A.
      • Brenner D.J.
      • Cameron D.N.
      • Steigerwalt A.G.
      • Makowski J.M.
      • Baker C.N.
      • et al.
      Genotypic and phenotypic characterization of Helicobacter cinaedi and Helicobacter fennelliae strains isolated from humans and animals.
      ], which is too cumbersome to carry out routinely in hospital laboratories. The E-test is an alternative method used to measure antimicrobial susceptibility [
      • Holst H.
      • Andresen K.
      • Blom J.
      • Højlyng N.
      • Kemp M.
      • Krogfelt K.A.
      • et al.
      A case of Helicobacter cinaedi bacteraemia in a previously healthy person with cellulitis.
      ,
      • Cederbrant G.
      • Kahlmeter G.
      • Ljungh A.
      The E test for antimicrobial susceptibility testing of Helicobacter pylori.
      ]; however, because H. cinaedi has a migratory growth pattern, the E-test may be inaccurate due to unclear edges around the growth inhibition zone.
      Comparative analysis of the growth ability of H. cinaedi isolates in some broth media revealed that modified Levinthal broth is suitable for supporting the growth of H. cinaedi strains in 96-well format microplates [
      • Tomida J.
      • Oumi A.
      • Okamoto T.
      • Morita Y.
      • Okayama A.
      • Misawa N.
      • et al.
      Comparative evaluation of agar dilution and broth microdilution methods for antibiotic susceptibility testing of Helicobacter cinaedi.
      ]. Minimum inhibitory concentration (MIC) values obtained from the broth microdilution method using the modified Levinthal broth are almost same as those obtained from the traditional agar dilution method. From these data, Tomida et al. [
      • Tomida J.
      • Oumi A.
      • Okamoto T.
      • Morita Y.
      • Okayama A.
      • Misawa N.
      • et al.
      Comparative evaluation of agar dilution and broth microdilution methods for antibiotic susceptibility testing of Helicobacter cinaedi.
      ] concluded that a broth microdilution method for antimicrobial susceptibility testing of H. cinaedi, which can be performed easily, is a reliable method for determining the MIC.
      H. cinaedi strains generally show low MIC values for carbapenems, aminoglycosides, and tetracycline (MIC90 ≦1 μg/ml for imipenem, gentamicin, and tetracycline). Penicillins and cephalosporins show moderate MIC values (MIC90 = 16 μg/ml for ampicillin, and carbenicillin, MIC90 = 8 μg/ml for amoxicillin, cefepime, and ceftriaxone). In contrast, H. cinaedi, which has well known resistance to macrolides [
      • Kiehlbauch J.A.
      • Brenner D.J.
      • Cameron D.N.
      • Steigerwalt A.G.
      • Makowski J.M.
      • Baker C.N.
      • et al.
      Genotypic and phenotypic characterization of Helicobacter cinaedi and Helicobacter fennelliae strains isolated from humans and animals.
      ,
      • Kuijper E.J.
      • Stevens S.
      • Imamura T.
      • de Wever B.
      • Claas E.C.J.
      Genotypic identification of erythromycin-resistant Campylobacter isolates as Helicobacter species and analysis of resistance mechanism.
      ], has particularly high MIC values (MIC90 > 64 μg/ml for erythromycin). Although there are some reports from before the current decade describing susceptibility to quinolones [
      • Tee W.
      • Street A.C.
      • Spelman D.
      • Munckhof W.
      • Mijch A.
      Helicobacter cinaedi bacteraemia: varied clinical manifestations in three homosexual males.
      ,
      • Burman W.J.
      • Cohn D.L.
      • Reves R.R.
      • Wilson M.L.
      Multifocal cellulitis and monoarticular arthritis as manifestations of Helicobacter cinaedi bacteremia.
      ,
      • Lasry S.
      • Simon J.
      • Marais A.
      • Pouchot J.
      • Vinceneux P.
      • Boussougant Y.
      Helicobacter cinaedi septic arthritis and bacteremia in an immunocompetent patient.
      ,
      • Kiehlbauch J.A.
      • Brenner D.J.
      • Cameron D.N.
      • Steigerwalt A.G.
      • Makowski J.M.
      • Baker C.N.
      • et al.
      Genotypic and phenotypic characterization of Helicobacter cinaedi and Helicobacter fennelliae strains isolated from humans and animals.
      ,
      • Kiehlbauch J.A.
      • Tauxe R.V.
      • Baker C.N.
      • Wachsmuth I.K.
      Helicobacter cinaedi – associated bacteremia and cellulitis in immunocompromised patients.
      ], more recently in Japan and elsewhere H. cinaedi isolates have shown high resistance to quinolones (MIC90 = 64 μg/ml for ciprofloxacin and levofloxacin) due to point mutation(s) of DNA gyrase genes. Almost the same MIC values are reported by other researchers [
      • Rimbara E.
      • Mori S.
      • Kim H.
      • Matsui M.
      • Suzuki S.
      • Takahashi S.
      • et al.
      Helicobacter cinaedi and Helicobacter fennelliae transmission in a hospital from 2008 to 2012.
      ]. MIC values of recent isolates from several hospitals in Japan are summarized in Table 3.
      Table 3Pattern of primary antibiotics of H. cinaedi isolates.
      Range of MIC (μg/ml)MIC50 (μg/ml)MIC90 (μg/ml)n
      Penicillins
      Ampicilllin0.5–3281679
      Amoxicillin1–168845
      (0.5–32)(4)(8)(46)
      Carbenicilin0.5–1681638
      Cephalosporins
      Cefepime0.5–324845
      Ceftriaxone2–8488
      Carbapenems
      Meropenem also showed low MIC values (≦0.12 μg/ml) in our small scale examination (n = 4).
      Imipenem0.06–40.120.529
      (0.031–0.25)(0.063)(0.125)(46)
      Aminoglycosides
      Gentamicin0.12–20.5179
      (0.25–1)(0.5)(0.5)(46)
      Tetracycline
      Tetracycline0.06–40.25179
      Minocycline
      MIC value of minocycline in our small scale examination also showed ≦0.12 μg/ml (n = 4).
      (0.016–0.25)(0.063)(0.125)(46)
      Macrolides
      Erythromycin16–>64>64>648
      Clarithromycin(2–>128)(64)(>128)(46)
      Quinolones
      Ciprofloxacin0.06–64166479
      (16–128)(64)(128)(46)
      Levofloxacin0.06–>6446475
      Moxifloxacin0.06–320.5875
      Metronidazole
      Metronidazole0.06–>6426411
      The data were basically collected by the broth microdilition method
      • Tomida J.
      • Oumi A.
      • Okamoto T.
      • Morita Y.
      • Okayama A.
      • Misawa N.
      • et al.
      Comparative evaluation of agar dilution and broth microdilution methods for antibiotic susceptibility testing of Helicobacter cinaedi.
      .
      MIC values reported by Rimbra et al.
      • Rimbara E.
      • Mori S.
      • Kim H.
      • Matsui M.
      • Suzuki S.
      • Takahashi S.
      • et al.
      Helicobacter cinaedi and Helicobacter fennelliae transmission in a hospital from 2008 to 2012.
      using agar dilution method are described in the parentheses.
      a Meropenem also showed low MIC values (≦0.12 μg/ml) in our small scale examination (n = 4).
      b MIC value of minocycline in our small scale examination also showed ≦0.12 μg/ml (n = 4).
      It is well known that efflux pumps contribute to antimicrobial resistance in many cases. The resistance nodulation cell division (RND) type multidrug efflux transporters are the clinically relevant chromosomally encoded fundamental antimicrobial resistance mechanisms in Gram-negative bacteria [
      • Poole K.
      Efflux-mediated multi resistance in gram-negative bacteria.
      ]. We identified two genes (locus-tags HCN_0595 and HCN_1563) in the chromosome of H. cinaedi PAGU 611 encoding the hydrophobe/amphiphile efflux-1 sub-family of the RND family [
      • Goto T.
      • Ogura Y.
      • Hirakawa H.
      • Tomida J.
      • Morita Y.
      • Akaike T.
      • et al.
      Complete genome sequence of Helicobacter cinaedi strain PAGU611, isolated in a case of human bacteremia.
      ,
      • Morita Y.
      • Tomida J.
      • Kawamura Y.
      Multidrug efflux systems in Helicobacter cinaedi.
      ]. The genes were also conserved in other H. cinaedi genomic strains of CCUG 18818 and ATCC BAA-847 [
      • Miyoshi-Akiyama T.
      • Takeshita N.
      • Ohmagari N.
      • Kirikae T.
      Complete genome sequence of Helicobacter cinaedi type strain ATCC BAA-847.
      ]. HCN_0595 orthologs are found in various species among the genus Helicobacter (e.g. H. pylori 26695 and H. hepaticus ATCC 51449), while HCN_1563 orthologs are found only in enterohepatic Helicobacter species (e.g. H. hepaticus ATCC 51449) examined. A phylogenetic tree was constructed using COBALT software (Fig. 6) [
      • Papadopoulos J.S.
      • Agarwala R.
      COBALT: constraint-based alignment tool for multiple protein sequences.
      ]. HCN_1563 pump is between CmeB of C. jejuni and BepE pump of Brucella suis, both of which are major antimicrobial resistance contributors, while HCN_0595 pump is between HefC of H. pylori and CmeF of C. jejuni, both of which are likely to be small or secondary contributors. The CmeABC pump contributes to the acquired resistance of C. jejuni to macrolides and fluoroquinolones [
      • Cagliero C.
      • Mouline C.
      • Payot S.
      • Cloeckaert A.
      Involvement of the CmeABC efflux pump in the macrolide resistance of Campylobacter coli.
      ,
      • Yan M.
      • Sahin O.
      • Lin J.
      • Zhang Q.
      Role of the CmeABC efflux pump in the emergence of fluoroquinolone-resistant Campylobacter under selection pressure.
      ]; HCN_1563 pump of H. cinaedi may be associated with resistance to these drugs. Another eight putative drug transporter genes (one belonging to the major facilitator family, one to the ATP-binding cassette family, two to the multidrug and toxin extrusion family, and four to the small multidrug resistance family) are found in the genome of H. cinaedi PAGU 611. The orthologs are also encoded in H. cinaedi ATCC BAA-847 and H. hepaticus ATCC 51449 [
      • Morita Y.
      • Tomida J.
      • Kawamura Y.
      Multidrug efflux systems in Helicobacter cinaedi.
      ]. It is not clear how the gene products operate and how they contribute to antimicrobial resistance, and further investigation is needed.
      Figure thumbnail gr6
      Fig. 6Two RND multidrug efflux pumps in H. cinaedi PAGU 611 are similar to those of CmeB of C. jejuni and HefC of H. pylori, respectively. The tree constructed using the COBALT program
      [
      • Papadopoulos J.S.
      • Agarwala R.
      COBALT: constraint-based alignment tool for multiple protein sequences.
      ]
      is shown. The accession number of each pump is in the text.

      6. Chemotherapy and recurrence

      Various antibiotic agents alone or in combination have been successfully used for treating infections caused by H. cinaedi [
      • Tee W.
      • Street A.C.
      • Spelman D.
      • Munckhof W.
      • Mijch A.
      Helicobacter cinaedi bacteraemia: varied clinical manifestations in three homosexual males.
      ,
      • Burman W.J.
      • Cohn D.L.
      • Reves R.R.
      • Wilson M.L.
      Multifocal cellulitis and monoarticular arthritis as manifestations of Helicobacter cinaedi bacteremia.
      ,
      • Kitamura T.
      • Kawamura Y.
      • Ohkusu K.
      • Masaki T.
      • Iwashita H.
      • Sawa T.
      • et al.
      Helicobacter cinaedi cellulitis and bacteremia in immunocompetent hosts after orthopedic surgery.
      ,
      • Minauchi K.
      • Takahashi S.
      • Sakai T.
      • Kondo M.
      • Shibayama K.
      • Arakawa Y.
      • et al.
      The nosocomial transmission of Helicobacter cinaedi infections in immunocompromised patients.
      ,
      • Kiehlbauch J.A.
      • Tauxe R.V.
      • Baker C.N.
      • Wachsmuth I.K.
      Helicobacter cinaedi – associated bacteremia and cellulitis in immunocompromised patients.
      ]. Symptoms caused by this microorganism, such as fever or cellulitis, resolve after 2 or 3 days of drug therapy. In some cases the symptoms disappear spontaneously and the patients becomes asymptomatic, although the microorganisms are still present in the patients and will recur [
      • Uçkay I.
      • Garbino J.
      • Dietrich P.Y.
      • Ninet B.
      • Rohner P.
      • Jacomo V.
      Recurrent bacteremia with Helicobacter cinaedi: case report and review of the literature.
      ]. The prognosis is generally good, but it should be recognized that about 30–60% of patients have recurrent symptoms [
      • Kitamura T.
      • Kawamura Y.
      • Ohkusu K.
      • Masaki T.
      • Iwashita H.
      • Sawa T.
      • et al.
      Helicobacter cinaedi cellulitis and bacteremia in immunocompetent hosts after orthopedic surgery.
      ,
      • Murakami H.
      • Goto M.
      • Ono E.
      • Sawabe E.
      • Iwata M.
      • Okuzumi K.
      • et al.
      Isolation of Helicobacter cinaedi from blood of an immunocompromised patient in Japan.
      ,
      • Kiehlbauch J.A.
      • Tauxe R.V.
      • Baker C.N.
      • Wachsmuth I.K.
      Helicobacter cinaedi – associated bacteremia and cellulitis in immunocompromised patients.
      ,
      • Sullivan A.K.
      • Nelson M.R.
      • Walsh J.
      • Gazzard B.G.
      Recurrent Helicobacter cinaedi cellulitis and bacteraemia in a patient with HIV infection.
      ,
      • Shimizu S.
      • Inokuma D.
      • Watanabe M.
      • Sakai T.
      • Yamamoto S.
      • Tsuchiya K.
      • et al.
      Cutaneous manifestations of Helicobacter cinaedi infection.
      ]. The CDC recommends long-term therapy of about 2–6 weeks, rather than short-term therapy of up to 10 days [
      • Kiehlbauch J.A.
      • Tauxe R.V.
      • Baker C.N.
      • Wachsmuth I.K.
      Helicobacter cinaedi – associated bacteremia and cellulitis in immunocompromised patients.
      ]. Many other reports also describe long-term chemotherapy for the prevention of recurrent symptoms [
      • Lasry S.
      • Simon J.
      • Marais A.
      • Pouchot J.
      • Vinceneux P.
      • Boussougant Y.
      Helicobacter cinaedi septic arthritis and bacteremia in an immunocompetent patient.
      ,
      • Kiehlbauch J.A.
      • Tauxe R.V.
      • Baker C.N.
      • Wachsmuth I.K.
      Helicobacter cinaedi – associated bacteremia and cellulitis in immunocompromised patients.
      ,
      • Uçkay I.
      • Garbino J.
      • Dietrich P.Y.
      • Ninet B.
      • Rohner P.
      • Jacomo V.
      Recurrent bacteremia with Helicobacter cinaedi: case report and review of the literature.
      ].
      No recommended guidelines are available for the treatment of a diagnosed infection of H. cinaedi. As an alternative guide to chemotherapy, it may be noted that in testing for H. cinaedi in recurrent-bacteremia cases, the approach used is an interpretation of the susceptibility to clarithromycin based on the CLSI (Clinical and Laboratory Standards Institute; http://clsi.org/) guidelines for H. pylori and on published reports for metronidazole and amoxicillin [
      • Kim J.J.
      • Reddy R.
      • Lee M.
      • Kim J.G.
      • El-Zaatari F.A.
      • Osato M.S.
      • et al.
      Analysis of metronidazole, clarithromycin and tetracycline resistance of Helicobacter pylori isolates from Korea.
      ,
      • Lopez-Brea M.
      • Martinez M.J.
      • Domingo D.
      • Alarcon T.A.
      9 year study of clarithromycin and metronidazole resistance in Helicobacter pylori from Spanish children.
      ]. For other antibiotics, interpretation is based on CLSI guidelines for gram-negative bacilli [
      • Uçkay I.
      • Garbino J.
      • Dietrich P.Y.
      • Ninet B.
      • Rohner P.
      • Jacomo V.
      Recurrent bacteremia with Helicobacter cinaedi: case report and review of the literature.
      ]. Although such a challenging report exists, authorized guidelines for the treatment of H. cinaedi infections, including the clinical breakpoints of antimicrobial agents, have not been established.

      7. Epidemiological analysis

      The infection route of H. cinaedi has not been clarified. However, H. cinaedi has been found in a wide range of animals, from domestic pets to wild animals, including cats, dogs, hamsters, rats, foxes, and rhesus monkeys [
      • Solnick J.V.
      • Schauer D.B.
      Emergence of diverse Helicobacter species in the pathogenesis of gastric and enterohepatic diseases.
      ,
      • On S.L.W.
      • Lee A.
      • O’rourke J.L.
      • Dewhirst F.E.
      • Paster B.J.
      • Fox J.G.
      • et al.
      Genus Helicobacter.
      ,
      • Fox J.G.
      • Handt L.
      • Sheppard B.J.
      • Xu S.
      • Dewhirst F.E.
      • Motzel S.
      • et al.
      Isolation of Helicobacter cinaedi from the colon, liver, and mesenteric lymph node of a rhesus monkey with chronic colitis and hepatitis.
      ,
      • Stills Jr., H.F.
      • Hook Jr., R.R.
      • Kinden D.A.
      Isolation of a Campylobacter-like organism from healthy Syrian hamsters (Mesocricetus auratus).
      ].
      Many reports have raised a suspicion of zoonotic transmission from animals to humans [
      • De Groote D.
      • Ducatelle R.
      • Haesebrouck F.
      Helicobacters of possible zoonotic origin: a review.
      ,
      • Fox J.G.
      The non-H. pylori helicobacters: their expanding role in gastrointestinal and systemic diseases.
      ]. Orlicek et al. [
      • Bartonickova L.
      • Sterzenbach T.
      • Nell S.
      • Kops F.
      • Schulze J.
      • Venzke A.
      • et al.
      Hcp and VgrG1 are secreted components of the Helicobacter hepaticus type VI secretion system and VgrG1 increases the bacterial colitogenic potential.
      ] reported that H. cinaedi was responsible for bacteremia and meningitis in a newborn whose mother cared for a pet hamster during her pregnancy. Lasry et al. [
      • Lasry S.
      • Simon J.
      • Marais A.
      • Pouchot J.
      • Vinceneux P.
      • Boussougant Y.
      Helicobacter cinaedi septic arthritis and bacteremia in an immunocompetent patient.
      ] reported H. cinaedi-related septic arthritis and bacteremia in an immunocompetent patient who worked occasionally as a shepherd and had contact with cows and farm animals. Indeed, H. cinaedi is reported to be a member of the normal intestinal flora in hamsters [
      • Gebhart C.J.
      • Fennell C.L.
      • Murtaugh M.P.
      • Stamm W.E.
      Campylobacter cinaedi is normal intestinal flora in hamsters.
      ]. Another report [
      • Recordati C.
      • Gualdi V.
      • Craven M.
      • Sala L.
      • Luini M.
      • Lanzoni A.
      • et al.
      Spatial distribution of Helicobacter spp. in the gastrointestinal tract of dogs.
      ] describes certain enterohepatic Helicobacter species, including H. cinaedi, are located mainly in sites in the lower intestinal tract such as the cecum and colon in dogs, rather than in the upper parts such as the as duodenum, jejunum, or ileum. It is likely that contact infection has occurred from animal to human. However, there are no reports of the simultaneous isolation of H. cinaedi in human patients and close contact animals. It is noteworthy that H. cinaedi isolates from human, dog, and hamster formed distinct ribotype pattern groups according to their host source [
      • Kiehlbauch J.A.
      • Brenner D.J.
      • Cameron D.N.
      • Steigerwalt A.G.
      • Makowski J.M.
      • Baker C.N.
      • et al.
      Genotypic and phenotypic characterization of Helicobacter cinaedi and Helicobacter fennelliae strains isolated from humans and animals.
      ]. This kind of discrepancy between human and animal isolates has been recently recognized by using the matrix-assisted laser desorption/ionization time of flight mass spectrometry system [
      • Taniguchi T.
      • Sekiya A.
      • Higa M.
      • Saeki Y.
      • Umeki K.
      • Okayama A.
      • et al.
      Rapid identification and subtyping of Helicobacter cinaedi strains by intact-cell mass spectrometry profiling with the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry.
      ].
      Epidemiological analysis methods such as plasmid profiles, pulse-field gel electrophoresis, randomly amplified polymorphic DNA, and multilocus sequence typing have been proposed for H. cinaedi isolates [
      • Kitamura T.
      • Kawamura Y.
      • Ohkusu K.
      • Masaki T.
      • Iwashita H.
      • Sawa T.
      • et al.
      Helicobacter cinaedi cellulitis and bacteremia in immunocompetent hosts after orthopedic surgery.
      ,
      • Rimbara E.
      • Mori S.
      • Matsui M.
      • Suzuki S.
      • Wachino J.
      • Kawamura Y.
      • et al.
      Molecular epidemiologic analysis and antimicrobial resistance of Helicobacter cinaedi isolated from seven hospitals in Japan.
      ,
      • Kiehlbauch J.A.
      • Brenner D.J.
      • Cameron D.N.
      • Steigerwalt A.G.
      • Makowski J.M.
      • Baker C.N.
      • et al.
      Genotypic and phenotypic characterization of Helicobacter cinaedi and Helicobacter fennelliae strains isolated from humans and animals.
      ].
      We have developed a nested PCR system, as mentioned above [
      • Oyama K.
      • Khan S.
      • Okamoto T.
      • Fujii S.
      • Ono K.
      • Matsunaga T.
      • et al.
      Identification of and screening for human Helicobacter cinaedi infections and carriers via nested PCR.
      ], to directly catch the bacterial DNA (antigen detecting system) in the clinical specimens, and have established an immunological diagnosis method (antibody detecting test) with high specificity to detect the exposure history of H. cinaedi [
      • Iwashita H.
      • Fujii S.
      • Kawamura Y.
      • Okamoto T.
      • Sawa T.
      • Masaki T.
      • et al.
      Identification of the major antigenic protein of Helicobacter cinaedi and its immunogenicity in humans with H. cinaedi infections.
      ]. Using these methods, we have analyzed many healthy subjects working in a hospital (doctors, nurses, staff members, etc.) and found some healthy individuals infected with H. cinaedi [
      • Oyama K.
      • Khan S.
      • Okamoto T.
      • Fujii S.
      • Ono K.
      • Matsunaga T.
      • et al.
      Identification of and screening for human Helicobacter cinaedi infections and carriers via nested PCR.
      ]. This finding suggests asymptomatic carriers exist, and may be related to nosocomial infections.
      Further investigations are needed to clarify the complete infection route and the nosocomial transmission route of H. cinaedi infection.

      8. Conclusion

      It appears that, because H. cinaedi is thought not to cause acute severe disease, little importance has been placed on this organism. However, we now know that it likely causes nosocomial infections, is difficult to eradicate, and has a high incidence of recurrence. Furthermore, an association with chronic illnesses such as arrhythmia and arteriosclerosis has been pointed out in recent years.
      Therefore, there is a need to rapidly establish guidelines for the use of antimicrobial agents, susceptibility testing, and the treatment regimen in diagnosed H. cinaedi infection cases. In addition, it is important to elucidate the infection route. To our knowledge, no medical center or clinic that has detected recurrent H. cinaedi infection has successfully eradicated it. Taking into account the variety of environmental or animal vector routes, both the route and the mechanism of infection by this microorganism should be clarified. Furthermore, we need to carefully monitor and understand the trends in H. cinaedi infections.

      Conflict of interest

      Authors declare no conflict of interest.

      Acknowledgments

      We thank the following persons for their helpful discussions and cooperation in medical, genetic, or biochemical analysis; Takatsugu Goto, Gifu University; Hideki Hirakawa, Kazusa DNA Research Institute; Tetsuro Matsunaga, Tohoku University Graduate School of Medicine; Masaru Baba, Toranomon Hospital.
      We are grateful to the following individuals for providing the H. cinaedi isolates used in this study: Shunji Takahashi, Sapporo City General Hospital; Masashi Narita, Ohta-nishinouchi Hospital; Ayako Oumi, Social Insurance Chuo General Hospital; Ken Kikuchi, Juntendo University; Yoshihito Otsuka, Kameda Medical Center; Haruki Sawamura and Hiroshige Mikamo, Aichi Medical University; Yoko Kawakami, National Hospital Organization Kyushu Cancer Center; Toshio Kitamura, Shuichi Higashi, Keita Yamakawa, and Itsuo Honda, Kumamoto Orthopedic Hospital.

      References

        • Fennell C.L.
        • Totten P.A.
        • Quinn T.C.
        • Patton D.L.
        • Holmes K.K.
        • Stamm W.E.
        Characterization of Campylobacter-like organisms isolated from homosexual men.
        J Infect Dis. 1984; 149: 58-66
        • Totten P.A.
        • Fennell C.L.
        • Tenover F.C.
        • Wezenberg J.M.
        • Perine P.L.
        • Stamm W.E.
        • et al.
        Campylobacter cinaedi (sp. nov.) and Campylobacter fennelliae (sp. nov.): two new Campylobacter species associated with enteric disease in homosexual men.
        J Infect Dis. 1985; 151: 131-139
        • Goodwin C.S.
        • Armstrong J.A.
        • Chilvers T.
        • Peters M.
        • Collins M.D.
        • Sly L.
        • et al.
        Transfer of Campylobacter pylori and Campylobacter mustelae to Helicobacter gen nov as Helicobacter pylon comb nov and Helicobacter mustelae comb nov, respectively.
        Int J Syst Bacteriol. 1989; 39: 397-405
        • Vandamme P.
        • Falsen E.
        • Rossau R.
        • Hoste B.
        • Segers P.
        • Tytgat R.
        • et al.
        Revision of Campylobacter, Helicobacter, and Wolinella taxonomy: emendation of generic descriptions and proposal of Arcobacter gen. nov.
        Int J Syst Bacteriol. 1991; 41: 88-103
        • Solnick J.V.
        • Schauer D.B.
        Emergence of diverse Helicobacter species in the pathogenesis of gastric and enterohepatic diseases.
        Clin Microbiol Rev. 2001; 14: 59-97
        • Bryner J.H.
        • Littleton J.
        • Gates C.
        • Kirkbride C.A.
        • Richie A.E.
        Flexispira rappini gen. nov., sp. nov., a gram-negative rod from mammalian fetus and feces.
        in: XIV International Congress of Microbiology, Manchester, UK. 1986
        • Bryner J.H.
        Flexispira rappini, gen. nov., sp. nov. A motile, urease-producing rod similar to Campylobacter pyloridis.
        in: Kaijer B. Falsen E. Proceedings of the fourth international workshop on Campylobacter infections, Goteborg, Sweden. University Goteborg, Sweden1987: P440-P442
        • Tee W.
        • Leder K.
        • Karroum E.
        • Dyall-Smith M.
        “Flexispira rappini” bacteremia in a child with pneumonia.
        J Clin Microbiol. 1998; 36: 1679-1682
        • Sorlin P.
        • Vandamme P.
        • Nortier J.
        • Hoste B.
        • Rossi C.
        • Pavlof S.
        • et al.
        Recurrent “Flexispira rappini” bacteremia in an adult patient undergoing hemodialysis: case report.
        J Clin Microbiol. 1999; 37: 1319-1323
        • Tee W.
        • Jenney A.
        • McPhee A.
        • Mijch A.
        • Dyall-Smith M.
        “Helicobacter rappini” isolates from 2 homosexual men.
        Clin Infect Dis. 2001; 33: e8-11
        • Dewhirst F.E.
        • Fox J.G.
        • Mendes E.N.
        • Paster B.J.
        • Gates C.E.
        • Kirkbride C.A.
        • et al.
        'Flexispira rappini' strains represent at least 10 Helicobacter taxa.
        Int J Syst Evol Microbiol. 2000; 50: 1781-1787
        • Hänninen M.L.
        • Utriainen M.
        • Happonen I.
        • Dewhirst F.E.
        Helicobacter sp. flexispira 16S rDNA taxa 1, 4 and 5 and finnish porcine Helicobacter isolates are members of the species Helicobacter trogontum (taxon 6).
        Int J Syst Evol Microbiol. 2003; 53: 425-433
        • Hänninen M.L.
        • Kärenlampi R.I.
        • Koort J.M.
        • Mikkonen T.
        • Björkroth K.J.
        Extension of the species Helicobacter bilis to include the reference strains of Helicobacter sp. flexispira taxa 2, 3 and 8 and finnish canine and feline flexispira strains.
        Int J Syst Evol Microbiol. 2005; 55: 891-898
        • Trivett-Moore N.L.
        • Rawlinson W.D.
        • Yuen M.
        • Gilbert G.L.
        Helicobacter westmeadii sp. nov., a new species isolated from blood cultures of two AIDS patients.
        J Clin Microbiol. 1997; 35: 1144-1150
        • On S.L.W.
        International Committee on Systematics of Prokaryotes: subcommittee on the taxonomy of Campylobacter and related bacteria.
        Int J Syst Evol Microbiol. 2002; 52: 2339-2341
        • Gaudreau C.
        • Lachance N.
        Infection with Helicobacter cinaedi resistant to ciprofloxacin in men with AIDS.
        Clin Microbiol Newslett. 1995; 17: 150-152
        • Mammen Jr., M.P.
        • Aronson N.E.
        • Edenfield W.J.
        • Endy T.P.
        Recurrent Helicobacter cinaedi bacteremia in a patient infected with human immunodeficiency virus: case report.
        Clin Infect Dis. 1995; 21: 1055
        • Tee W.
        • Street A.C.
        • Spelman D.
        • Munckhof W.
        • Mijch A.
        Helicobacter cinaedi bacteraemia: varied clinical manifestations in three homosexual males.
        Scand J Infect Dis. 1996; 28: 199-203
        • Simons E.
        • Spacek L.A.
        • Lederman H.M.
        • Winkelstein J.A.
        Helicobacter cinaedi bacteremia presenting as macules in an afebrile patient with X-linked agammaglobulinemia.
        Infection. 2004; 32: 367-368
        • Pasternak J.
        • Bolivar R.
        • Hopfer R.L.
        • Fainstein V.
        • Mills K.
        • Rios A.
        • et al.
        Bacteremia caused by Campylobacter-like organisms in two male homosexuals.
        Ann Intern Med. 1984; 101: 339-341
        • Burman W.J.
        • Cohn D.L.
        • Reves R.R.
        • Wilson M.L.
        Multifocal cellulitis and monoarticular arthritis as manifestations of Helicobacter cinaedi bacteremia.
        Clin Infect Dis. 1995; 20: 564-570
        • Lasry S.
        • Simon J.
        • Marais A.
        • Pouchot J.
        • Vinceneux P.
        • Boussougant Y.
        Helicobacter cinaedi septic arthritis and bacteremia in an immunocompetent patient.
        Clin Infect Dis. 2000; 31: 201-202
        • Van Genderen P.J.
        • Goessens W.H.
        • Petit P.L.
        Helicobacter cinaedi-associated bacteraemia and erysipelas in an immunocompetent host: a diagnostic challenge.
        Scand J Infect Dis. 2005; 37: 382-385
        • Kitamura T.
        • Kawamura Y.
        • Ohkusu K.
        • Masaki T.
        • Iwashita H.
        • Sawa T.
        • et al.
        Helicobacter cinaedi cellulitis and bacteremia in immunocompetent hosts after orthopedic surgery.
        J Clin Microbiol. 2007; 45: 31-38
        • Holst H.
        • Andresen K.
        • Blom J.
        • Højlyng N.
        • Kemp M.
        • Krogfelt K.A.
        • et al.
        A case of Helicobacter cinaedi bacteraemia in a previously healthy person with cellulitis.
        Open Microbiol J. 2008; 2: 29-31
        • Murakami H.
        • Goto M.
        • Ono E.
        • Sawabe E.
        • Iwata M.
        • Okuzumi K.
        • et al.
        Isolation of Helicobacter cinaedi from blood of an immunocompromised patient in Japan.
        J Infect Chemother. 2003; 9: 344-347
        • Matsumoto T.
        • Goto M.
        • Murakami H.
        • Tanaka T.
        • Nishiyama H.
        • Ono E.
        • et al.
        Multicenter study to evaluate bloodstream infection by Helicobacter cinaedi in Japan.
        J Clin Microbiol. 2007; 45: 2853-2857
        • Rimbara E.
        • Mori S.
        • Matsui M.
        • Suzuki S.
        • Wachino J.
        • Kawamura Y.
        • et al.
        Molecular epidemiologic analysis and antimicrobial resistance of Helicobacter cinaedi isolated from seven hospitals in Japan.
        J Clin Microbiol. 2012; 50: 2553-2560
        • Minauchi K.
        • Takahashi S.
        • Sakai T.
        • Kondo M.
        • Shibayama K.
        • Arakawa Y.
        • et al.
        The nosocomial transmission of Helicobacter cinaedi infections in immunocompromised patients.
        Intern Med. 2010; 49: 1733-1739
        • Lawson A.J.
        Helicobacter.
        in: Versalovic J. Carrill K.C. Funke G. Jorgensen J.H. Landry M.L. Warnock D.W. Manual of clinical microbiology. 10th ed. American Society for Microbiology, Washington DC2010: 900-915
        • Sugiyama A.
        • Mori M.
        • Ishiwada N.
        • Himuro K.
        • Kuwabara S.
        First adult case of Helicobacter cinaedi meningitis.
        J Neurol Sci. 2014; 336: 263-264
        • Suematsu Y.
        • Morizumi S.
        • Okamura K.
        • Kawata M.
        A rare case of axillobifemoral bypass graft infection caused by Helicobacter cinaedi.
        J Vasc Surg. 2013; https://doi.org/10.1016/j.jvs.2013.08.048
        • Khan S.
        • Okamoto T.
        • Enomoto K.
        • Sakashita N.
        • Oyama K.
        • Fujii S.
        • et al.
        Potential association of Helicobacter cinaedi with atrial arrhythmias and atherosclerosis.
        Microbiol Immunol. 2012; 56: 145-154
        • Khan S.
        • Rahman H.N.A.
        • Okamoto T.
        • Matsunaga T.
        • Fujiwara Y.
        • Sawa T.
        • et al.
        Promotion of atherosclerosis by Helicobacter cinaedi infection that involves macrophage-driven proinflammatory responses.
        Sci Rep. 2014; https://doi.org/10.1038/srep04680
        • Libby P.
        • Ridker P.M.
        • Hansson G.K.
        Inflammation in atherosclerosis: from pathophysiology to practice.
        J Am Coll Cardiol. 2009; 54: 2129-2138
        • Tabas I.
        Macrophage death and defective inflammation resolution in atherosclerosis.
        Nat Rev Immunol. 2010; 10: 36-46
        • Oyama K.
        • Khan S.
        • Okamoto T.
        • Fujii S.
        • Ono K.
        • Matsunaga T.
        • et al.
        Identification of and screening for human Helicobacter cinaedi infections and carriers via nested PCR.
        J Clin Microbiol. 2012; 50: 3893-3900
        • Moazed T.C.
        • Kuo C.
        • Grayston J.T.
        • Campbell L.A.
        Murine models of Chlamydia pneumoniae infection and atherosclerosis.
        J Infect Dis. 1997; 175: 883-890
        • Li L.
        • Messas E.
        • Batista Jr., E.L.
        • Levine R.A.
        • Amar S.
        Porphyromonas gingivalis infection accelerates the progression of atherosclerosis in a heterozygous apolipoprotein E-deficient murine model.
        Circulation. 2002; 105: 861-867
        • Taylor N.S.
        • Ge Z.
        • Shen Z.
        • Dewhirst F.E.
        • Fox J.G.
        Cytolethal distending toxin: a potential virulence factor for Helicobacter cinaedi.
        J Infect Dis. 2003; 188: 1892-1897
        • Jinadasa R.N.
        • Bloom S.E.
        • Weiss R.S.
        • Duhamel G.E.
        Cytolethal distending toxin: a conserved bacterial genotoxin that blocks cell cycle progression, leading to apoptosis of a broad range of mammalian cell lineages.
        Microbiology. 2011; 157: 1851-1875
        • Charoenlap N.
        • Shen Z.
        • McBee M.E.
        • Muthupalani S.
        • Wogan G.N.
        • Fox J.G.
        • et al.
        Alkyl hydroperoxide reductase is required for Helicobacter cinaedi intestinal colonization and survival under oxidative stress in BALB/c and BALB/c interleukin-10-/- mice.
        Infect Immun. 2012; 80: 921-928
        • Goto T.
        • Ogura Y.
        • Hirakawa H.
        • Tomida J.
        • Morita Y.
        • Akaike T.
        • et al.
        Complete genome sequence of Helicobacter cinaedi strain PAGU611, isolated in a case of human bacteremia.
        J Bacteriol. 2012; 194: 3744-3745
        • Filloux A.
        • Hachani A.
        • Bleves S.
        The bacterial type VI secretion machine: yet another player for protein transport across membranes.
        Microbiology. 2008; 154: 1570-1583
        • Jani A.J.
        • Cotter P.A.
        Type VI secretion: not just for pathogenesis anymore.
        Cell Host Microbe. 2010; 8: 2-6
        • Chow J.
        • Mazmanian S.K.
        A pathobiont of the microbiota balances host colonization and intestinal inflammation.
        Cell Host Microbe. 2010; 7: 265-276
        • Bartonickova L.
        • Sterzenbach T.
        • Nell S.
        • Kops F.
        • Schulze J.
        • Venzke A.
        • et al.
        Hcp and VgrG1 are secreted components of the Helicobacter hepaticus type VI secretion system and VgrG1 increases the bacterial colitogenic potential.
        Cell Microbiol. 2013; 15: 992-1011
        • Miyoshi-Akiyama T.
        • Takeshita N.
        • Ohmagari N.
        • Kirikae T.
        Complete genome sequence of Helicobacter cinaedi type strain ATCC BAA-847.
        J Bacteriol. 2012; 194: 5692
        • Suzuki T.
        • Kawamura C.
        • Yoshimura H.
        • Moriguchi M.
        • Okusu K.
        • Honma M.
        • et al.
        A study of Helicobacter cinaedi detection case reports using BacT/ALERT in 4 clinical facilities.
        J Jpn Soc Clin Microbiol. 2009; 19 ([in Japanese]): 156
        • Orlicek S.L.
        • Welch D.F.
        • Kuhls T.L.
        Septicemia and meningitis caused by Helicobacter cinaedi in a neonate.
        J Clin Microbiol. 1993; 31: 569-571
        • Hosoda T.
        • Hayakawa S.
        • Wakuda M.
        • Higashimoto Y.
        • Furukawa H.
        • Ishikawa T.
        • et al.
        Potential for an early stage detection of Helicobacter cinaedi on automated blood culture systems.
        in: 44th annual meeting of the Japan Society for Clinical Laboratory Automation. Yokohama, Japan. 2012 ([in Japanese])
        • Tomida J.
        • Tsurunaga M.
        • Hosoda T.
        • Hayakawa S.
        • Suematsu H.
        • Sawamura H.
        • et al.
        Evaluation of automated blood culture systems about the detection capability for Helicobacter cinaedi.
        J Jpn Soc Clin Microbiol. 2012; 22 ([in Japanese]): 233
        • Baba M.
        Infectious diseases case conference seminar–from clinical questions to research insights.
        Kansenshogakuzassi. 2012; : 13-15
        • Engberg J.
        • On S.T.W.
        • Harrington C.S.
        • Gerner-Smidt P.
        Prevalence of Campylobacter, Arcobacter, Helicobacter, and Sutterella spp. in human fecal samples as estimated by a reevaluation of isolation methods for Campylobacters.
        J Clin Microbiol. 2000; 38: 286-291
        • Tanaka T.
        • Goto M.
        • Okuzumi K.
        • Yoneyama A.
        • Matsumoto T.
        • Yamaguchi K.
        • et al.
        Isolation and identification of Helicobacter cinaedi-like organisms isolated from blood culture in practical laboratory procedures.
        Kansenshogaku Zasshi. 2007; 81 ([in Japanese]): 700-706
        • Tomida J.
        • Kashida M.
        • Oinishi K.
        • Endo R.
        • Morita Y.
        • Kawamura Y.
        Evaluation of various media for rapid detection of Helicobacter spp.
        J Jpn Soc Clin Microbiol. 2008; 18 ([in Japanese]): 227-235
        • Kiehlbauch J.A.
        • Brenner D.J.
        • Cameron D.N.
        • Steigerwalt A.G.
        • Makowski J.M.
        • Baker C.N.
        • et al.
        Genotypic and phenotypic characterization of Helicobacter cinaedi and Helicobacter fennelliae strains isolated from humans and animals.
        J Clin Microbiol. 1995; 33: 2940-2947
        • On S.L.
        • Holmes B.
        Assessment of enzyme detection test useful in identification of campylobacteria.
        J Clin Microbiol. 1992; 30: 746-749
        • Vandamme P.
        • Clare S.
        • Harrington C.S.
        • Jalava K.
        • On S.L.W.
        Misidentifying Helicobacters: the Helicobacter cinaedi example.
        J Clin Microbiol. 2000; 38: 2261-2266
        • Kuhnert P.
        • Burnens A.
        Misidentifying Helicobacter cinaedi.
        J Clin Microbiol. 2001; 39: 2751-2752
        • Al-Soud W.A.
        • Bennedsen M.
        • On S.L.W.
        • Ouis I.S.
        • Vandamme P.
        • Nilsson H.O.
        • et al.
        Assessment of PCR-DGGE for the identification of diverse Helicobacter species, and application to faecal samples from zoo animals to determine Helicobacter prevalence.
        J Med Microbiol. 2003; 52: 765-771
        • Zakharova N.
        • Paster B.J.
        • Wesley I.
        • Dewhirst F.E.
        • Berg D.E.
        • Severinov K.V.
        Fused and overlapping rpoB and rpoC genes in Helicobacters, Campylobacters, and related bacteria.
        J Bacteriol. 1999; 181: 3857-3859
        • Hannula M.
        • Hänninen M.L.
        Phylogenetic analysis of Helicobacter species based on partial gyrB gene sequences.
        Int J Syst Evol Microbiol. 2007; 57: 444-449
        • Mikkonen T.P.
        • Kärenlampi R.I.
        • Hänninen M.L.
        Phylogenetic analysis of gastric and enterohepatic Helicobacter species based on partial HSP60 gene sequences.
        Int J Syst Evol Microbiol. 2004; 54: 753-758
        • Dewhirst F.E.
        • Shen Z.
        • Scimeca M.S.
        • Stokes L.N.
        • Boumenna T.
        • Chen T.
        • et al.
        Discordant 16S and 23S rRNA gene phylogenies for the genus Helicobacter: implications for phylogenetic inference and systematics.
        J Bacteriol. 2005; 187: 6106-6118
        • Dewhirst F.E.
        • Fox J.G.
        • On S.L.W.
        Recommended minimal standards for describing new species of the genus Helicobacter.
        Int J Syst Evol Microbiol. 2000; 50: 2231-2237
        • Vandamme P.
        • On S.L.W.
        Recommendations of the subcommittee on the taxonomy of Campylobacter and related bacteria.
        Int J Syst Evol Microbiol. 2001; 51: 719-721
        • On S.L.W.
        • Holmes B.
        Effect of inoculum size on the phenotypic characterization of Campylobacter species.
        J Clin Microbiol. 1991; 29: 923-926
        • On S.L.W.
        • Holmes B.
        Reproducibility of tolerance tests that are useful in the identification of campylobacteria.
        J Clin Microbiol. 1991; 29: 1785-1788
        • Sacks L.V.
        • Labriola A.M.
        • Gill V.J.
        • Gordin F.M.
        Use of ciprofloxacin for successful eradication of bacteremia due to Campylobacter cinaedi in a human immunodeficiency virus-infected person.
        Rev Infect Dis. 1991; 13: 1066-1068
        • Cederbrant G.
        • Kahlmeter G.
        • Ljungh A.
        The E test for antimicrobial susceptibility testing of Helicobacter pylori.
        J Antimicrob Chemother. 1993; 31: 65-71
        • Tomida J.
        • Oumi A.
        • Okamoto T.
        • Morita Y.
        • Okayama A.
        • Misawa N.
        • et al.
        Comparative evaluation of agar dilution and broth microdilution methods for antibiotic susceptibility testing of Helicobacter cinaedi.
        Microbiol Immunol. 2013; 57: 353-358
        • Kuijper E.J.
        • Stevens S.
        • Imamura T.
        • de Wever B.
        • Claas E.C.J.
        Genotypic identification of erythromycin-resistant Campylobacter isolates as Helicobacter species and analysis of resistance mechanism.
        J Clin Microbiol. 2003; 41: 3732-3736
        • Kiehlbauch J.A.
        • Tauxe R.V.
        • Baker C.N.
        • Wachsmuth I.K.
        Helicobacter cinaedi – associated bacteremia and cellulitis in immunocompromised patients.
        Ann Intern Med. 1994; 121: 90-93
        • Rimbara E.
        • Mori S.
        • Kim H.
        • Matsui M.
        • Suzuki S.
        • Takahashi S.
        • et al.
        Helicobacter cinaedi and Helicobacter fennelliae transmission in a hospital from 2008 to 2012.
        J Clin Microbiol. 2013; 51: 2439-2442
        • Poole K.
        Efflux-mediated multi resistance in gram-negative bacteria.
        Clin Microbiol Infect. 2004; 10: 12-26
        • Morita Y.
        • Tomida J.
        • Kawamura Y.
        Multidrug efflux systems in Helicobacter cinaedi.
        Antibiotics. 2012; 2012: 29-43
        • Papadopoulos J.S.
        • Agarwala R.
        COBALT: constraint-based alignment tool for multiple protein sequences.
        Bioinformatics. 2007; 23: 1073-1079
        • Cagliero C.
        • Mouline C.
        • Payot S.
        • Cloeckaert A.
        Involvement of the CmeABC efflux pump in the macrolide resistance of Campylobacter coli.
        J Antimicrob Chemother. 2005; 56: 948-950
        • Yan M.
        • Sahin O.
        • Lin J.
        • Zhang Q.
        Role of the CmeABC efflux pump in the emergence of fluoroquinolone-resistant Campylobacter under selection pressure.
        J Antimicrob Chemother. 2006; 2006: 1154-1159
        • Uçkay I.
        • Garbino J.
        • Dietrich P.Y.
        • Ninet B.
        • Rohner P.
        • Jacomo V.
        Recurrent bacteremia with Helicobacter cinaedi: case report and review of the literature.
        BMC Infect Dis. 2006; 6: 86https://doi.org/10.1186/1471-2334-6-86
        • Sullivan A.K.
        • Nelson M.R.
        • Walsh J.
        • Gazzard B.G.
        Recurrent Helicobacter cinaedi cellulitis and bacteraemia in a patient with HIV infection.
        Int J STD AIDS. 1997; 8: 59-60
        • Shimizu S.
        • Inokuma D.
        • Watanabe M.
        • Sakai T.
        • Yamamoto S.
        • Tsuchiya K.
        • et al.
        Cutaneous manifestations of Helicobacter cinaedi infection.
        Acta Derm Venereol. 2013; 93: 165-167
        • Kim J.J.
        • Reddy R.
        • Lee M.
        • Kim J.G.
        • El-Zaatari F.A.
        • Osato M.S.
        • et al.
        Analysis of metronidazole, clarithromycin and tetracycline resistance of Helicobacter pylori isolates from Korea.
        J Antimicrob Chemother. 2001; 47: 459-461
        • Lopez-Brea M.
        • Martinez M.J.
        • Domingo D.
        • Alarcon T.A.
        9 year study of clarithromycin and metronidazole resistance in Helicobacter pylori from Spanish children.
        J Antimicrob Chemother. 2001; 48: 295-297
        • On S.L.W.
        • Lee A.
        • O’rourke J.L.
        • Dewhirst F.E.
        • Paster B.J.
        • Fox J.G.
        • et al.
        Genus Helicobacter.
        in: Begey's manual of systematic bacteriology 2nd edition. vol. 2. Springer, NewYork2005: 1169-1189
        • Fox J.G.
        • Handt L.
        • Sheppard B.J.
        • Xu S.
        • Dewhirst F.E.
        • Motzel S.
        • et al.
        Isolation of Helicobacter cinaedi from the colon, liver, and mesenteric lymph node of a rhesus monkey with chronic colitis and hepatitis.
        J Clin Microbiol. 2001; 39: 1580-1585
        • Stills Jr., H.F.
        • Hook Jr., R.R.
        • Kinden D.A.
        Isolation of a Campylobacter-like organism from healthy Syrian hamsters (Mesocricetus auratus).
        J Clin Microbiol. 1989; 27: 2497-2501
        • De Groote D.
        • Ducatelle R.
        • Haesebrouck F.
        Helicobacters of possible zoonotic origin: a review.
        Acta Gastro-enterologica Belg. 2000; 63: 380-387
        • Fox J.G.
        The non-H. pylori helicobacters: their expanding role in gastrointestinal and systemic diseases.
        Gut. 2002; 50: 273-283
        • Gebhart C.J.
        • Fennell C.L.
        • Murtaugh M.P.
        • Stamm W.E.
        Campylobacter cinaedi is normal intestinal flora in hamsters.
        J Clin Microbiol. 1989; 27: 1692-1694
        • Recordati C.
        • Gualdi V.
        • Craven M.
        • Sala L.
        • Luini M.
        • Lanzoni A.
        • et al.
        Spatial distribution of Helicobacter spp. in the gastrointestinal tract of dogs.
        Helicobacter. 2009; 14: 180-191
        • Taniguchi T.
        • Sekiya A.
        • Higa M.
        • Saeki Y.
        • Umeki K.
        • Okayama A.
        • et al.
        Rapid identification and subtyping of Helicobacter cinaedi strains by intact-cell mass spectrometry profiling with the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry.
        J Clin Microbiol. 2014; 52: 95-102
        • Iwashita H.
        • Fujii S.
        • Kawamura Y.
        • Okamoto T.
        • Sawa T.
        • Masaki T.
        • et al.
        Identification of the major antigenic protein of Helicobacter cinaedi and its immunogenicity in humans with H. cinaedi infections.
        Clin Vaccine Immunol. 2008; 15: 513-521
        • De Groote D.
        • van Doorn L.J.
        • Ducatelle R.
        • Verschuuren A.
        • Tilmant K.
        • Quint W.G.
        • et al.
        Phylogenetic characterization of 'Candidatus Helicobacter bovis', a new gastric helicobacter in cattle.
        Int J Syst Bacteriol. 1999; 49: 1707-1715