J Med Microbiol Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Teyssier, C.
Right arrow Articles by Jumas-Bilak, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Teyssier, C.
Right arrow Articles by Jumas-Bilak, E.
Agricola
Right arrow Articles by Teyssier, C.
Right arrow Articles by Jumas-Bilak, E.
J Med Microbiol 54 (2005), 945-953; DOI: 10.1099/jmm.0.46116-0
© 2005 Society for General Microbiology
ISSN 0022-2615

Molecular and phenotypic features for identification of the opportunistic pathogens Ochrobactrum spp.

Corinne Teyssier1, Hélène Marchandin2, Hélène Jean-Pierre2, Isabelle Diego1, Hélène Darbas2, Jean-Luc Jeannot1, Anne Gouby3 and Estelle Jumas-Bilak1

1Laboratoire de bactériologie, faculté de pharmacie, 15, avenue Charles Flahault, 34093 Montpellier Cedex 5, France 2Laboratoire de bactériologie, hôpital Arnaud de Villeneuve, 34295 Montpellier Cedex 5, France 3Laboratoire de bactériologie, hôpital Carremeau, 30060 îmes, France

Correspondence Estelle Jumas-Bilak ebilak{at}univ-montp1.fr

Received April 10, 2005
Accepted July 6, 2005

Among the six species characterized within the genus Ochrobactrum, Ochrobactrum anthropi and Ochrobactrum intermedium are currently reported as opportunistic pathogens in humans. Since the species identification is mainly based on 16S rDNA analysis, the aim of this study was to search for other characteristics useful for Ochrobactrum species discrimination. Ribotyping, morphological and biochemical analyses, and antimicrobial susceptibility testing were performed for a panel of 35 clinical isolates, first identified to the species level using 16S rDNA sequencing. Type and reference strains of five Ochrobactrum species were comparatively analysed. Commercial identification systems such as API 20NE and VITEK 2 were tested for their ability to identify Ochrobactrum anthropi and to detect other members of the genus Ochrobactrum. An improved protocol for the identification of Ochrobactrum spp. by routine medical microbiology practices is proposed: isolation of a non-fastidious non-fermenting oxidase-positive Gram-negative rod resistant to all ß-lactams except imipenem indicates the genus Ochrobactrum, and the API 20NE system confirms the genus identification for most strains, whereas the VITEK 2 system using ID-GNB cards was less powerful. Urease activity, the mucoidy of the colonies, growth at 45 °C on tryptic soy agar, and susceptibility to colistin, tobramycin and netilmicin should be considered as differential characteristics for identification of O. anthropi and O. intermedium to the species level. However, definitive identification depends on genotyping methods.

Abbreviations: AFLP, amplified fragment length polymorphism; DIG, digoxigenin.

The GenBank/EMBL/DDBJ accession numbers for the 16s rDNA sequences reported in this paper are AF526518–AF526526, AY917104–AY917119 and AY918295–AY918296.


   INTRODUCTION
TOP
 INTRODUCTION
METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The genus Ochrobactrum and its type species Ochrobactrum anthropi were created in 1988 for the organisms formerly known as CDC group Vd (Holmes et al., 1988). Since this description, five other species, Ochrobactrum intermedium (Velasco et al., 1998), Ochrobactrum tritici, Ochrobactrum grignonense (Lebuhn et al., 2000), Ochrobactrum gallinifaecis (Kämpfer et al., 2003) and ‘Ochrobactrum lupini’ (Trujillo et al., 2005) have been characterized, mainly on the basis of 16S rDNA sequencing. They are non-fermentative, strictly aerobic, motile, oxidase-positive and indole-negative, Gram-negative rods. On the basis of phenotypic characteristics, the genus Ochrobactrum could be related to the genera Alcaligenes, Achromobacter, or to the members of Pseudomonadaceae. However, molecular taxonomy places Ochrobactrum in the {alpha}-subgroup of proteobacteria, closely related to the genus Brucella (Lebuhn et al., 2000; Velasco et al., 1998). Surprisingly, 16S rDNA-based phylogeny, as well as protein profiling (Velasco et al., 1998) and AFLP analysis (Leal-Klevezas et al., 2005), place O. intermedium strains closer to Brucella spp. than any other members of the genus Ochrobactrum.

O. anthropi has been isolated from various clinical specimens and is recognized as an opportunistic pathogen. Nosocomial infections due to O. anthropi have been increasingly reported during the last decade, particularly bacteraemia and endocarditis in patients with indwelling central venous catheters (Gill et al., 1997; Mahmood et al., 2000; Stiakaki et al., 2002). Other cases of infections and outbreaks have been described in patients in dialysis (see Daxboeck et al., 2002 for a review), after surgery in ophthalmology (Berman et al., 1997; Greven & Nelson, 2001; Inoue et al., 1999), in neurosurgery (Christenson et al., 1997), after transplantation (Ezzedine et al., 1994), or after valve replacement (Romero Gomez et al., 2004). The virulence of O. anthropi is generally considered to be low, but reports suggested a high virulence for some strains involved in pyogenic infections (Brivet et al., 1993; Cieslak et al., 1996; Wheen et al., 2002). O. anthropi was described as one of the most antibiotic-resistant Gram-negative rods (Nadjar et al., 2001; Higgins et al., 2001). Indeed, clinical strains of O. anthropi are multiresistant to common antibiotics, in particular they are usually resistant to all ß-lactams except imipenem. The resistance to ß-lactams is explained by the presence of an AmpC ß-lactamase described as chromosomal, inducible and resistant to inhibition by clavulanic acid (Nadjar et al., 2001). The majority of Ochrobactrum infections in humans have been imputed to the species O. anthropi, except for a liver abscess caused by O. intermedium (Moller et al., 1999).

The aim of this study was to determine genotypic and phenotypic features allowing discrimination between the Ochrobactrum species. We carried out comparative analyses of 35 clinical isolates to type or reference strains of O. anthropi, O. intermedium, O. tritici, O. grignonense and O. gallinifaecis. ‘O. lupini' was not included in the study due to its very recent description (Trujillo et al., 2005). Comparative analysis with clinically relevant {alpha}-proteobacterial genera, such as Brucella, Agrobacterium, Sinorhizobium and Inquilinus is also presented. The isolates were first identified by 16S rDNA sequencing and analysed by ribotyping. Then, we studied cell and colony morphology, growth conditions, biochemical traits on commercial identification systems, and antibiotic susceptibility, in order to define characteristics that could be used for species identification in a routine medical microbiology protocol.


   METHODS
TOP
 INTRODUCTION
 METHODS
RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Bacterial strains.

Thirty-five clinical isolates were obtained during a 5 year period from patients hospitalized in the academic hospitals of Montpellier, îmes and Clermont-Ferrand (France). The type strains of O. anthropi (ATCC 49188T) and O. intermedium (LMG 3301T, deposited as O. anthropi but now transferred to O. intermedium as the type strain; Velasco et al., 1998), were obtained from the American Type Culture Collection (ATCC) and the Collection Fraçaise des Bactéries Phytopathogènes, respectively. The reference strains of O. tritici (DSM 13340T and DSM 13341), O. grignonense (DSM 13338T and DSM 13339) and O. gallinifaecis (DSM 15295T) were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). Agrobacterium tumefaciens C58 and Sinorhizobium meliloti 1021 were gifts from X. Nesmes and M. Fernandez (Laboratoire d'Écologie Microbienne du Sol, Université Claude Bernard Lyon I, Villeurbanne, France) (Jumas-Bilak et al., 1998). Inquilinus limosus LMG 20952T was obtained from Laboratorium voor Microbiologie, Universiteit Gent, Belgium (LMG).

16S rRNA gene analysis.

Bacteria were grown on tryptic soy agar (TSA) for 24 h at 37 °C. One colony was suspended in 50 µl sterile water, and the DNA was liberated by a boiling-freezing method (Teyssier et al., 2003). The 16S rRNA gene was selectively amplified from this crude lysate by PCR using universal primers 27f and 1492r, as previously described (Teyssier et al., 2003). PCR products of about 1400 bp were sequenced directly on an Applied Biosystems Automatic Sequencer (Genome Express). Partial 16S rDNA sequences were compared with sequences deposited in databases using the standard nucleotide-nucleotide Basic Local Alignment Search Tool (BLAST) program (http://www.ncbi.nlm.nih.gov/blast/). The genetic distances between strains were calculated using the DNADIST program (option similarity table) in the PHYLIP package (www.pasteur.fr) after sequence alignment by the DIALIGN software (www.expasy.org).

Ribotyping.

Intact genomic DNAs were prepared in agarose plugs for enzymic digestion as previously described (Teyssier et al., 2003). DNAs were digested with 40 U HindIII or EcoRI (New England Biolabs), and then electrophoresed for 3 h at 80 V in a 0.8 % agarose gel in 0.5x TBE. Gels were transferred onto nylon membrane by vacuum blotting (vacuum blotter; Bio-Rad) in 20x SSC. The 16S rDNA digoxigenin (DIG)-labelled probe was obtained by PCR using the 27f/1492r pair of primers with a dNTPs mixture containing 0.1 mM DIG-dUTP (Roche), and with the DNA of O. intermedium LMG 3301T as a template. The hybridization of the probe was detected by the CSPD chemiluminescent system (Roche).

Phenotypic analysis.

Cultures were grown at 30, 37 and 45 °C on TSA, blood Columbia agar (bioMérieux), cetrimide agar, Drigalski medium and MacConkey medium (Difco BRL) for 24 h. Cell morphology was observed by photonic microscopy after Gram staining. Identification systems API 20E, API 20NE and VITEK 2, ID-GNB card version WSVT2-R03.01 (bioMérieux), were used according to the supplier's recommendations, and particular care was taken to obtain the recommended turbidity for the bacterial suspensions. Urease activity was detected after bacterial growth on urea-indole medium (bioMérieux). The strain's susceptibility to antibiotics was determined by the disk-diffusion assay on Mueller–Hinton agar, according to the guidelines of the Comité de l'Antibiogramme de la Société Fraçaise de Microbiologie (Members of the SFM Antibiogram Committee, 2003). The antibiotic disks (Bio-Rad) used were as follows: amoxycillin (25 µg), amoxycillin/clavulanic acid (20 µg/10 µg), ticarcillin (75 µg), ticarcillin/clavulanic acid (75 µg/10 µg), piperacillin (75 µg), piperacillin/tazobactam (75 µg/10 µg), imipenem (10 µg), cefalotine (30 µg), cefotaxime (30 µg), ceftazidime (30 µg), cefpirome (30 µg), cefepime (30 µg), latamoxef (30 µg), aztreonam (30 µg), gentamicin (15 µg ), tobramycin (10 µg), netilmicin (30 µg), nalidixic acid (30 µg), pefloxacin (5 µg), ofloxacin (5 µg), ciprofloxacin (5 µg), rifampicin (30 µg), colistin (50 µg), chloramphenicol (30 µg) and trimethoprim/sulfamethoxazole (1.25 µg/23.75 µg).


   RESULTS AND DISCUSSION
TOP
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
ACKNOWLEDGEMENTS
 REFERENCES
 
Genotyping

Sequence accession numbers and 16S rDNA-based identifications are given in Tables 1 and 2 for Ochrobactrum spp., and for type or reference strains of the genera Brucella, Agrobacterium, Sinorhizobium and Inquilinus. Sequencing of 16S rDNA clearly differentiated members of the genus Ochrobactrum from related genera since the sequence similarity was always below 95.5 %, except for Brucella spp. and Ochrobactrum spp., which displayed a sequence similarity of over 97 % for most of the strains. The clinical isolates of Ochrobactrum spp. were identified to the species level by 16S rRNA gene sequencing. All 16S rDNA sequences obtained matched with sequences deposited either for O. anthropi or O. intermedium after BLAST analysis. No isolates were affiliated to the species O. tritici, O. grignonense or O. gallinifaecis. The genetic distance calculated on a 534 bp alignment showed that the 20 strains affiliated to O. anthropi displayed strictly identical 16S rDNA sequences. Sequences of isolates affiliated to O. intermedium (n = 15) were more polymorphic, with identity ranging from 99.4 to 100 %. These results suggested a higher genetic divergence in the species O. intermedium than that observed in O. anthropi, and contrasted with AFLP results recently reported on a very small number of O. intermedium strains (Leal-Klevezas et al., 2005). Sequence similarity between O. anthropi and O. intermedium ranged from 97.9 to 98.7 % according to the strains. Despite the fact that there is no generally accepted cut-off value for the bacterial species delineation, a 97 % similarity level in 16S rDNA has been proposed (Stackebrandt & Goebel, 1994). According to this value, O. anthropi and O. intermedium were not separated as well as O. intermedium and Brucella melitensis. As a consequence, the efficiency of 16S rDNA sequencing for the identification of Ochrobactrum to the species level could be questioned. In order to confirm the splitting of our collection of isolates into two clusters corresponding to O. anthropi and O. intermedium, we tested ribotyping as an alternative genotyping method. Although ribotyping has been frequently used to investigate the intra-species level, it is known that in the genus Brucella, which is phylogenetically related to Ochrobactrum, ribotyping patterns are species-specific and not strain-specific (Verger et al., 2000). Ribotyping has also been shown to be efficient for the identification of species belonging to the genus Agrobacterium (Clermont et al., 2001). We tested ribotyping as a genotyping method to discriminate between Ochrobactrum species, and between Ochrobactrum and related genera. The clinical isolates showed two groups of HindIII ribotypes named A and B (Fig. 1), clearly differentiated on the basis of their hybridization patterns. Ribogroup A grouped the O. intermedium type strain and all the strains affiliated to O. intermedium by 16S rDNA sequencing, whereas ribotype B grouped the O. anthropi type strain and all O. anthropi clinical isolates (Fig. 1). Three other ribogroups named C, D and E corresponded to the type and reference strains of O. grignonense, O. tritici and O. gallinifaecis, respectively (Fig. 1). I. limosus and A. tumefaciens displayed poor HindIII ribotype patterns (F and G) with only one hybridizing band of about 20 and 4.8 kb, respectively, whereas we did not obtain any exploitable pattern for Sinorhizobium meliloti. The Brucella pattern (H, a schematic representation) determined from the complete genome sequence (DelVecchio et al., 2002) clearly differed from those of all Ochrobactrum strains and other genera. The partition of the clinical strains into two ribogroups, named A' and B', was also observed after EcoRI restriction (Fig. 2). The two types of EcoRI pattern corresponded to the patterns of O. anthropi and O. intermedium type strains. Therefore, the distribution of the strains according to their ribotype was consistent with 16S rDNA-based identification (Tables 1 and 2). The other species of the genus Ochrobactrum and the related genera displayed different and specific EcoRI patterns, except for Agrobacterium and Sinorhizobium (Fig. 2).


View this table:
[in this window]
[in a new window]
  		Table 1. Genotypic and phenotypic identification of O. anthropi strains, including the type strain, based on 16S rDNA sequencing, ribotyping, and API 20NE and VITEK 2 system analyses NFGNB, non-fermenting Gram-negative bacilli; EI, excellent identification; VGI, very good identification; GI, good identification; AI, acceptable identification; LD, low discrimination.
 

View this table:
[in this window]
[in a new window]
  		Table 2. Genotypic and phenotypic identification of strains of O. intermedium, other Ochrobactrum species and related genera, including reference and type strains based on 16S rDNA sequencing, ribotyping, and API 20NE and VITEK 2 system analyses NFGNB, non-fermenting Gram-negative bacilli; EI, excellent identification; VGI, very good identification; GI, good identification; AI, acceptable identification; LD, low discrimination.
 


View larger version (31K):
[in this window]
[in a new window]
  		Fig. 1. Hybridization patterns of DIG-labelled 16S rRNA gene probe with Southern blotted HindIII restriction fragments from type strains and selected isolates of Ochrobactrum spp. and related genera. A, B, C, D and E correspond to the five Ochrobactrum ribogroups. F, G and H correspond to I. limosus LMG 20952T, A. tumefaciens C58 and B. melitensis 16M ribogroups, respectively. H is a schematic representation deduced from the complete genome sequence. Molecular mass marker: bacteriophage {lambda} digested by HindIII (data not shown).

 


View larger version (24K):
[in this window]
[in a new window]
  		Fig. 2. Hybridization patterns of DIG-labelled 16S rRNA gene probe with Southern blotted EcoRI restriction fragments from type strains and selected isolates of Ochrobactrum spp. and related genera A', B', C', D' and E' correspond to the five Ochrobactrum ribogroups. F', G' and H' correspond to I. limosus LMG 20952T, A. tumefaciens C58 and Sinorhizobium meliloti 1021, and B. melitensis 16M ribogroups, respectively. H' is a schematic representation deduced from the complete genome sequence. Molecular mass marker: bacteriophage {lambda} digested by HindIII (data not shown).

 

Phenotypic traits of Ochrobactrum spp.

All the strains were Gram-negative short rods, straight or slightly curved with one end flame shaped. Wet-mount microscopy examination showed that the cells were highly motile. After 24 h incubation, colony aspect at 30 and 37 °C on TSA, Drigalski medium and MacConkey medium clearly differed among species. O. anthropi and O. grignonense developed circular, smooth, shiny colonies, whereas the colonies of O. intermedium and O. tritici were mucoid, opaque and quickly became confluent. The strains of O. intermedium were the sole strains able to grow at 45 °C on TSA. No strain grew on cetrimide agar. Haemolysis was never observed on blood Columbia agar. The brown pigment classically described for the genus Ochrobactrum was hardly visible on TSA and blood Columbia agar, even when a colony was harvested on a swab.

Using the API 20E and API 20NE systems, all the strains were negative for indole, H2S and acetoin production, carbohydrate fermentation, utilization of citrate, and assimilation of adipate and phenylacetate. Moreover, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, ß- galactosidase and gelatinase activities were not detected. All the strains were positive for the assimilation of glucose, arabinose, mannose, N-acetylglucosamine, maltose and malate. Other characteristics varied among strains of a same species except for urease activity. Positive urease activity, detected either by urea-indole medium (bioMérieux) or on API strips was shown for all O. anthropi except strain ADV 45, and for O. tritici and O. gallinifaecis strains. In contrast, none of the O. intermedium and O. grignonense strains displayed urease activity, whatever the method used to detect it (Table 3). However, an inoculum heavier than that recommended by manufacturers led to positive reactions for most of these strains. The VITEK 2 ID-GNB card gave urease results consistent with the urea-indole medium and the API systems, except for the results for three strains of O. intermedium (strains ADV1, ADV14 and ADV36), which showed urease activity only when analysed on the VITEK 2 apparatus. All these discrepancies suggested that O. intermedium possessed a faint urease activity as previously underlined by Lebuhn et al. (2000). This could explain the discrepancy observed between the urease activity results reported for O. intermedium in previous studies (Lebuhn et al., 2000; Moller et al., 1999; Velasco et al., 1998). Urease activity should no longer be considered as a criterion for genus identification, but rather as a biochemical characteristic useful for the discrimination of the two Ochrobactrum species of medical interest, O. anthropi and O. intermedium.


View this table:
[in this window]
[in a new window]
  		Table 3. Differential phenotypic features of Ochrobactrum species The number of strains with the indicated characteristic is given in parentheses. ND, not determined; TSA, tryptic soy agar; R, resistant; S, susceptible.
 

Only a few of the 41 characteristics tested by the ID-GNB card of the VITEK 2 system were positive for Ochrobactrum spp. Pyrrolidonyl arylamidase (PyrA), L-proline arylamidase (ProA) and alkaline phosphatase (PHOS) had been previously described as very useful tests for the identification of non-fermenting Gram-negative rods (Laffineur et al., 2002). The authors found that 100 % of the Ochrobactrum spp. strains were positive for PyrA and ProA, and negative for PHOS. Our results were in accordance for PyrA (42+/42) and PHOS (42–/42), but ProA was not positive for all strains (28+/42). The results for {gamma}-glutamyl transferase (17+/42), adonitol (17+/42), D-maltose (12+/42), palatinose (11+/42), sucrose (9+/42) and L-lysine arylamidase (5+/42) were variable between strains, without correlation with species identification. The glu-gly-arg arylamidase (GGAA) was positive for O. grignonense strains only, but additional strains of this species need to be tested to determine if it is a species-specific characteristic.

Comparison of API 20NE strip and VITEK 2 ID-GNB card for Ochrobactrum identification

It should be noted that O. anthropi is the sole species of the genus Ochrobactrum included in the databases of the API and VITEK 2 systems. Using the API 20NE system, nearly all the strains were identified as O. anthropi at 24 h, with the mention ‘very good identificatio’ or ‘excellent identificatio’ (Tables 1 and 2). The two O. grignonense strains were unidentified, or misidentified as Pseudomonas fluorescens with low discrimination. Moreover, the O. gallinifaecis type strain remained unidentified (Table 2). The reading of the strips after 48 h gave the same identification, but associated with the mentions ‘good identificatio’ or ‘acceptable identification'. Thus, despite the supplier's recommendations, the more confident identification of the genus was obtained after 24 h incubation of the strips. The absence of urease in O. intermedium strains did not alter the confidence level of identification, since urease was presented as an inconstant characteristic for O. anthropi (84 %) on the API 20NE technical sheet. It is likely that the original calibration of the system used O. anthropi strain samples that were actually a mix of O. anthropi and O. intermedium strains. Thus, the API 20NE system gave satisfactory identification of the genus Ochrobactrum, except for O. grignonense and O. gallinifaecis strains.

The identification reports given by the VITEK 2 system after analysis of the ID-GNB card are shown in Tables 1 and 2. In 8 cases out of 42, the identification of O. anthropi was assessed with the following confidence levels: ‘excellent identificatio’ (n = 2), ‘very good identificatio’ (n = 3), ‘good identificatio’ (n = 1), and ‘acceptable identificatio’ (n = 2). The identification of O. anthropi was proposed with a low discrimination for 16 strains; the identification of Pseudomonas aeruginosa is proposed as an alternative choice for nine of these. To sum up, the VITEK 2 gave or suggested the identification of O. anthropi for 25 strains among the 42 tested. This identification system appeared to be less powerful than the API 20NE system for the identification of the genus Ochrobactrum. Particularly, the VITEK 2 ID-GNB card gave the identification of Pseudomonas aeruginosa with the mention ‘very good identificatio’ for 3 strains and did not achieve identification for 11 strains. The three misidentified isolates (ADV3, ADV9, ADV21) were readily identified, at least to the genus level, by using the API 20NE system (Table 2). The VITEK 2 proposed the name O. anthropi for 16 out of 21 O. anthropi strains, but for only 6 out of 16 O. intermedium strains. The two O. tritici strains were identified as O. anthropi, but O. grignonense and O. gallinifaecis strains were not identified. The results suggested that the VITEK 2 system placed urease as a major characteristic for identification. As a consequence, the species without detectable urease activity were not fully identified as members of the genus Ochrobactrum.

Antibiotic susceptibility testing

All the O. anthropi clinical isolates and the type strains were highly resistant to all ß-lactams except imipenem (no growth inhibition was observed for most of the ß-lactams). This resistance profile is consistent with the expression of the AmpC ß-lactamase characterized in O. anthropi (Higgins et al., 2001; Nadjar et al., 2001). Identical susceptibility patterns were obtained for the strains of O. intermedium and O. tritici, suggesting that a ß-lactamase of the same class, or the same enzyme, could be expressed in these two species. The two O. grignonense strains were also resistant to all ß-lactams except imipenem, but in contrast to the strains of O. anthropi, O. intermedium and O. tritici, growth inhibition was observed around amoxycillin (12 mm diameter), cefotaxime (20 mm diameter), cefpirome (15 mm diameter), cefepime (19 mm diameter) and latamoxef (20 mm diameter) disks. Moreover, the partial restoration of the amoxycillin and ticarcillin activities by clavulanic acid was not in agreement with the presence of a class 1 AmpC ß-lactamase. These observations suggested that the mechanism of resistance of O. grignonense differed from that of the three other species, but this needs further investigation. In contrast with all the other members of the genus, the type strain of O. gallinifaecis was susceptible to all ß-lactams except aztreonam.

We observed a general susceptibility of the strains to gentamicin, rifampicin and fluoroquinolones. Susceptibility to netilmicin and tobramycin varied according to the species. All the O. anthropi strains were susceptible to netilmicin and tobramycin, whereas all the O. intermedium strains were resistant to these aminoglycosides (Table 3). Moreover, we confirmed in our collection of 35 clinical isolates the susceptibility of O. anthropi to colistin, and the resistance of O. intermedium to this antibiotic, as reported by Velasco et al. (1998) (Table 3). Susceptibility to trimethoprim/ sulfamethoxazole was observed for all strains of O. anthropi, O. intermedium, O. tritici and O. gallinifaecis, whereas the two O. grignonense strains were resistant to this association. Moreover, the O. gallinifaecis type strain was the sole strain found to be susceptible to chloramphenicol.

Improving the identification of Ochrobactrum species in medical microbiology

An efficient procedure for identifying Ochrobactrum to the species level is necessary to evaluate the role of each species in human infections, as well as for epidemiological investigations. The results we obtained for isolates and type strains of O. anthropi and O. intermedium allowed us to propose a routine protocol for identifying these two Ochrobactrum species, which were currently the only ones involved in human pathology. Firstly, the isolation of a non-fastidious non-fermenting Gram-negative rod, which is oxidase-positive and resistant to all ß-lactams except for imipenem should indicate both the genera Ochrobactrum and Inquilinus (Coenye et al., 2002). However, Inquilinus spp. displays a remarkably huge inhibition diameter (40–60 mm) around an imipenem disk (Chiron et al., 2005). In addition, I. limosus can be differentiated from Ochrobactrum spp. by its growth on Drigalski agar but not on MacConkey agar plates. Although Brucella spp. and Ochrobactrum spp. are genetically very closely related, they show clear differences in their phenotypes, i.e. Brucella spp. are fastidious and slow-growing Gram-negative cocco-bacilli.

Secondly, commercial identification systems can confirm the genus identification. Members of the genera Agrobacterium and Sinorhizobium were both identified by the API 20NE system as Agrobacterium radiobacter with a high level of confidence (Table 2). The use of the API 20NE system gave, for most of the strains tested, the identification of O. anthropi. Since O. intermedium is not included in the commercial identification systems’ databases, the identification of O. anthropi ought to be considered as genus identification. The VITEK 2 system appeared to be less efficient than the API 20NE system at identifying the genus Ochrobactrum. However, the newly available VITEK 2 detection system (VITEK 2 Advanced Colorimetry) needs to be evaluated. For routine identification to the species level, the urease test should be taken into account as a good indication (Table 3). Then, the aspect of the colonies, their growth at 45 °C on TSA and their susceptibility to colistin, tobramycin and netilmicin should be considered as additional characteristics allowing the species identification (Table 3).

We cannot propose a robust methodology for the identification of O. tritici, O. grignonense and O. gallinifaecis due to the low number of isolates characterized to date for these species. However, the preliminary results suggest that O. tritici strains are readily identified as members of the genus Ochrobactrum by the use of an API 20NE strip. The two strains are highly related to O. anthropi, with a positive urease test and susceptibility to colistin, netilmicin and tobramycin, but they differ from this species by their highly mucoid colonies on TSA. The criteria for differentiating O. grignonense and O. gallinifaecis from other members of the genus are given in Table 3. However, their genus affiliation is hard to assign since identification systems fail to class them as members of the genus Ochrobactrum, and their ß-lactam resistance patterns are atypical. Therefore, O. grignonense and O. gallinifaecis can only be readily identified by molecular means.

Conclusion

To our knowledge, we have presented here the largest collection of Ochrobactrum spp. clinical isolates identified to the species level by genotyping methods. The literature included only one case of human infection caused by O. intermedium (Moller et al., 1999), suggesting that amongst Ochrobactrum species O. anthropi has the predominant role in human disease. However, most of the infections involving O. anthropi were reported before the description of the other Ochrobactrum species, and/or by the use of non-discriminating methods. Thus, the role of each species in human infections needs to be revisited. Our collection showed a nearly equivalent distribution of clinical isolates between O. anthropi and O. intermedium. This indicates that the role of O. intermedium in human infections is probably underestimated due to the lack of a convenient identification system and should be further evaluated.

The phenotypic study of the collection allowed us to determine some characteristics useful for species identification using routine medical microbiology. However, further large-scale studies including more isolates are clearly required to confirm the efficiency of our approach for the identification of Ochrobactrum spp. More generally, this study could be considered as an improvement of the identification of Gram-negative non-fermenting rods by conventional methods. Indeed, their identification is often difficult, and the commercial systems are not always reliable, especially for some genera and species (Laffineur et al., 2002; VanPelt et al., 1999). Moreover, recent taxonomic studies resulted in the description of an increasing number of new taxa involved in nosocomial infections, and requiring additional tests for identification. This was particularly true for the genus Ochrobactrum.


   ACKNOWLEDGEMENTS
TOP
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
REFERENCES
 
We gratefully acknowledge Catherine Chanal for providing bacterial strains. We also wish to thank Agès Masnou, for excellent technical assistance, Ghislaine Dusart and Régine Liparoti-Baylac for their contributions. This work was supported by the association ADEREMPHA, Montpellier, France.


   REFERENCES
TOP
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 

  • Berman, A. J., Del Priore, L. V. & Fischer, C. K. (1997). Endogenous Ochrobactrum anthropi endophthalmitis. Am J Ophthalmol 123, 560–562.[Medline]

  • Brivet, F., Guibert, M., Kiredjian, M. & Dormont, J. (1993). Necrotizing fasciitis, bacteremia, and multiorgan failure caused by Ochrobactrum anthropi. Clin Infect Dis 17, 516–518.[Medline]

  • Chiron, R., Marchandin, H., Counil, F. & 8 other authors (2005). Clinical and microbiological features of Inquilinus sp.isolates from five patients with cystic fibrosis. J Clin Microbiol 43, 3938–3943.[Abstract/Free Full Text]

  • Christenson, J. C., Pavia, A. T., Seskin, K., Brockmeyer, D., Korgenski, E. K., Jenkins, E., Pierce, J. & Daly, J. A. (1997). Meningitis due to Ochrobactrum anthropi: an emerging nosocomial pathogen.A report of 3 cases. Pediatr Neurosurg 27, 218–221.[Medline]

  • Cieslak, T. J., Drabick, C. J. & Robb, M. L. (1996). Pyogenic infections due to Ochrobactrum anthropi. Clin Infect Dis 22, 845–847.[Medline]

  • Clermont, D., Harmant, C. & Bizet, C. (2001). Identification of strains of Alcaligenes and Agrobacterium by a polyphasic approach. J Clin Microbiol 39, 3104–3109.[Abstract/Free Full Text]

  • Coenye, T., Goris, J., Spilker, T., Vandamme, P. & LiPuma, J. J. (2002). Characterization of unusual bacteria isolated from respiratory secretions of cystic fibrosis patients and description of Inquilinus limosus gen.nov., sp. nov. J Clin Microbiol 40, 2062–2069.[Abstract/Free Full Text]

  • Daxboeck, F., Zitta, S., Assadian, O., Krause, R., Wenisch, C. & Kovarik, J. (2002). Ochrobactrum anthropi bloodstream infection complicating hemodialysis. Am J Kidney Dis 40, E17. E17.[CrossRef][Medline]

  • DelVecchio, V. G., Kapatral, V., Redkar, R. J. & 22 other authors (2002). The genome sequence of the facultative intracellular pathogen Brucella melitensis. Proc Natl Acad Sci U S A 99, 443–448.[Abstract/Free Full Text]

  • Ezzedine, H., Mourad, M., Van Ossel, C. & 7 other authors (1994). An outbreak of Ochrobactrum anthropi bacteraemia in five organ transplant patients. J Hosp Infect 27, 35–42.[CrossRef][Medline]

  • Gill, M. V., Ly, H., Mueenuddin, M., Schoch, P. E. & Cunha, B. A. (1997). Intravenous line infection due to Ochrobactrum anthropi (CDC group Vd) in a normal host. Heart Lung 26, 335–336.[CrossRef][Medline]

  • Greven, C. M. & Nelson, K. C. (2001). Chronic postoperative endophtalmitis secondary to Ochrobactrum anthropi. Retina 21, 279–280.[CrossRef][Medline]

  • Higgins, C. S., Avison, M. B., Jamieson, L., Simm, A. M., Bennett, P. M. & Walsh, T. R. (2001). Characterization, cloning and sequence analysis of the inducible Ochrobactrum anthropi AmpC ß-lactamase. J Antimicrob Chemother 47, 745–754.[Abstract/Free Full Text]

  • Holmes, B., Popoff, M., Kiredjian, M. & Kersters, K. (1988). Ochrobactrum anthropi gen.nov., sp. nov. from human clinical specimens and previously known as group Vd. Int J Syst Bacteriol 38, 408–416.

  • Inoue, K., Numaga, J., Nagata, Y., Sakurai, M., Aso, N. & Fujino, Y. (1999). Ochrobactrum anthropi endophthalmitis after vitreous surgery. Br J Ophthalmol 83, 501. 501.[Free Full Text]

  • Jumas-Bilak, E., Michaux-Charachon, S., Bourg, G., Ramuz, M. & Allardet-Servent, A. (1998). Unconventional genomic organization in the alpha subgroup of the Proteobacteria. J Bacteriol 180, 2749–2755.[Abstract/Free Full Text]

  • Kämpfer, P., Buczolits, S., Albrecht, A., Busse, H. J. & Stackebrandt, E. (2003). Towards a standardized format for the description of a novel species (of an established genus): Ochrobactrum gallinifaecis sp.nov. Int J Syst Evol Microbiol 53, 893–896.[Abstract/Free Full Text]

  • Laffineur, K., Janssens, M., Charlier, J., Avesani, V., Wauters, G. & Delmée, M. (2002). Biochemical and susceptibility tests useful for identification of nonfermenting gram-negative rods. J Clin Microbiol 40, 1085–1087.[Abstract/Free Full Text]

  • Leal-Klevezas, D. S., Martinez-de-la-Vega, O., Ramirez-Barba, E. J., Osterman, B., Martinez-Soriano, J. P. & Simpson, J. (2005). Genotyping of Ochrobactrum spp.by AFLP analysis. J Bacteriol 187, 2537–2539.[Abstract/Free Full Text]

  • Lebuhn, M., Achouak, W., Schloter, M., Berge, O., Meier, H., Barakat, M., Hartmann, A. & Heulin, T. (2000). Taxonomic characterization of Ochrobactrum sp.isolates from soil samples and wheat roots, and description of Ochrobactrum tritici sp. nov. and Ochrobactrum grignonense sp. nov. Int J Syst Evol Microbiol 50, 2207–2223.[Abstract]

  • Mahmood, M. S., Sarwari, A. R., Khan, M. A., Sophie, Z., Khan, E. & Sami, S. (2000). Infective endocarditis and septic embolization with Ochrobactrum anthropi: case report and review of literature. J Infect 40, 287–290.[CrossRef][Medline]

  • Members of the SFM Antibiogram Committee (2003). Comité de l'Antibiogramme de la Société Fraçaise de Microbiologie Report 2003. Int J Antimicrob Agents 21, 364–391.[CrossRef][Medline]

  • Moller, L. V., Arends, J. P., Harmsen, H. J., Talens, A., Terpstra, P. & Slooff, M. J. (1999). Ochrobactrum intermedium infection after liver transplantation. J Clin Microbiol 37, 241–244.[Abstract/Free Full Text]

  • Nadjar, D., Labia, R., Cerceau, C., Bizet, C., Philippon, A. & Arlet, G. (2001). Molecular characterization of chromosomal class C ß-lactamase and its regulatory gene in Ochrobactrum anthropi. Antimicrob Agents Chemother 45, 2324–2330.[Abstract/Free Full Text]

  • Romero Gomez, M. P., Peinado Esteban, A. M., Sobrino Daza, J. A., Saez Nieto, J. A., Alvarez, D. & Pena Garcia, P. (2004). Prosthetic mitral valve endocarditis due to Ochrobactrum anthropi: case report. J Clin Microbiol 42, 3371–3373.[Abstract/Free Full Text]

  • Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.[CrossRef]

  • Stiakaki, E., Galanakis, E., Samonis, G., Christidou, A., Maraka, S., Tselentis, Y. & Kalmanti, M. (2002). Ochrobactrum anthropi bacteremia in pediatric oncology patients. Pediatr Infect Dis J 21, 72–74.[Medline]

  • Teyssier, C., Marchandin, H., Siméon de Buochberg, M., Ramuz, M. & Jumas-Bilak, E. (2003). Atypical 16S rRNA gene copies in Ochrobactrum intermedium strains reveal a large genomic rearrangement by recombination between rrn copies. J Bacteriol 185, 2901–2909.[Abstract/Free Full Text]

  • Trujillo, M. E., Willems, A., Abril, A., Planchuelo, A. M., Rivas, R., Ludena, D., Mateos, P. F., Martinez-Molina, E. & Velazquez, E. (2005). Nodulation of Lupinus albus by strains of Ochrobactrum lupini sp.nov. Appl Environ Microbiol 71, 1318–1327.[Abstract/Free Full Text]

  • VanPelt, C., Verduin, C. M., Goessens, W. H. F., Vos, M. C., Tummler, B., Segonds, C., Reubsaet, F., Verbrugh, H. & VanBelkum, A. (1999). Identification of Burkholderia spp.in the clinical microbiology laboratory: comparison of conventional and molecular methods. J Clin Microbiol 37, 2158–2164.[Abstract/Free Full Text]

  • Velasco, J., Romero, C., Lopez-Goni, I., Leiva, J., Diaz, R. & Moriyon, I. (1998). Evaluation of the relatedness of Brucella spp.and Ochrobactrum anthropi and description of Ochrobactrum intermedium sp. nov., a new species with a closer relationship to Brucella spp. Int J Syst Bacteriol 48, 759–768.[CrossRef][Medline]

  • Verger, J. M., Grayon, M., Cloeckaert, A., Lefevre, M., Ageron, E. & Grimont, F. (2000). Classification of Brucella strains isolated from marine mammals using DNA-DNA hybridization and ribotyping. Res Microbiol 151, 797–799.[Medline]

  • Wheen, L., Taylor, S. & Godfrey, K. (2002). Vertebral osteomyelitis due to Ochrobactrum anthropi. Intern Med J 32, 426–428.[CrossRef][Medline]




    This article has been cited by other articles:


    		Home page
    		 Vet PatholHome page
    C. M. Shilton, G. P. Brown, S. Benedict, and R. Shine
    Spinal Arthropathy Associated with Ochrobactrum anthropi in Free-ranging Cane Toads (Chaunus [Bufo] marinus) in Australia
    Vet. Pathol., January 1, 2008; 45(1): 85 - 94.
    [Abstract] [Full Text] [PDF]

    		Home page
    		 J Med MicrobiolHome page
    H. C. Scholz, M. Pfeffer, A. Witte, H. Neubauer, S. Al Dahouk, U. Wernery, and H. Tomaso
    Specific detection and differentiation of Ochrobactrum anthropi, Ochrobactrum intermedium and Brucella spp. by a multi-primer PCR that targets the recA gene
    J. Med. Microbiol., January 1, 2008; 57(1): 64 - 71.
    [Abstract] [Full Text] [PDF]

    		Home page
    		 J Med MicrobiolHome page
    P. Kampfer, D. M. Citron, E. J. C. Goldstein, and H. C. Scholz
    Difficulty in the identification and differentiation of clinically relevant Ochrobactrum species
    J. Med. Microbiol., November 1, 2007; 56(11): 1571 - 1573.
    [Full Text] [PDF]

    		Home page
    		 Int. J. Syst. Evol. Microbiol.Home page
    C. Teyssier, H. Marchandin, H. Jean-Pierre, A. Masnou, G. Dusart, and E. Jumas-Bilak
    Ochrobactrum pseudintermedium sp. nov., a novel member of the family Brucellaceae, isolated from human clinical samples
    Int J Syst Evol Microbiol, May 1, 2007; 57(5): 1007 - 1013.
    [Abstract] [Full Text] [PDF]

    This Article
    Right arrow Abstract Freely available
    Right arrow Full Text (PDF)
    Right arrow Alert me when this article is cited
    Right arrow Alert me if a correction is posted
    Right arrow Citation Map
    Services
    Right arrow Email this article to a friend
    Right arrow Similar articles in this journal
    Right arrow Similar articles in PubMed
    Right arrow Alert me to new issues of the journal
    Right arrow Download to citation manager
    Right arrow reprints & permissions
    Citing Articles
    Right arrow Citing Articles via HighWire
    Right arrow Citing Articles via CrossRef
    Right arrow Citing Articles via Google Scholar
    Google Scholar
    Right arrow Articles by Teyssier, C.
    Right arrow Articles by Jumas-Bilak, E.
    Right arrow Search for Related Content
    PubMed
    Right arrow PubMed Citation
    Right arrow Articles by Teyssier, C.
    Right arrow Articles by Jumas-Bilak, E.
    Agricola
    Right arrow Articles by Teyssier, C.
    Right arrow Articles by Jumas-Bilak, E.

    HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
    INT J SYST EVOL MICROBIOL J MED MICROBIOL MICROBIOLOGY J GEN VIROL ALL SGM JOURNALS