Classification of Aeromonas spp. strains from human ... · Presumptive Aeromonas veronii/sobria complex and Aeromonas hydrophila complex strains isolated from human intestinal clinical

1,* 1 1 2 1Ivo Sedláček, Eva Kroupová, Michaela Sedláčková, Eva Krejčí, Pavel Švec

1 Czech Collection of Microorganisms, Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic

2 Public Health Institute, Ostrava, Czech Republic

* Corresponding author: [email protected]

Acknowledgements This study was supported by project CEB (CZ.1.07/2.3.00/20.0183).

ReferencesAbbott et al., 2003. The genus Aeromonas: Biochemical characteristics, atypical reactions, and phenotypic identification schemes. J. Clin. Microbiol. 41:2348-2357.Aravena-Román et al., 2011. Aeromonas aquariorum as widely distributed in clinical and environmental specimens and can be misidentified as A. hydrophila. J. Clin. Microbiol. 49:306-308.Austin et al., 1996. The genus Aeromonas. John Wiley & Sons. Chichester, England.MacFaddin, 2000. Biochemical Tests for Identification of Medical Bacteria. Lippincott Williams and Wilkins, Philadelphia.Martin-Carnahan and Joseph, 2005. Genus I. Aeromonas, pp 557-578. Bergey's Manual of Systematic Bacteriology, 2nd edition. Volume 2. The Proteobacteria. Part B The Gammaproteobacteria. Springer, New York.Minana-Galbis et al., 2002. Biochemical identification and numerical taxonomy of Aeromonas spp. isolated from environmental and clinical samples in Spain. J. Appl. Microbiol. 93:420-430.Sedláček et al. 2012. Aeromonas hydrophila subsp. dhakensis – a causative agent of gastroenteritis imported into the Czech Republic. Ann. Agr. Envir. Med. 19:409-413.Švec et al. 2001. Evaluation of ribotyping for characterization and identification of Enterococcus haemoperoxidus and Enterococcus moraviensis strains. FEMS Microbiol. Lett. 203, 23-27.

Fig 1. Dendrogram based on cluster analysis of ribotype patterns of A. hydrophila complex strains.

Fig 1. Dendrogram based on cluster analysis of ribotype patterns of A. veronii/sobria complex strains.

Material and methods

Bacterial strains A collection of 76 presumptive A. hydrophila and A. veronii bv. sobria strains was isolated from human clinical material (faeces samples – acute diarrhoea) on Bile salt Irgasan Brilliant Green Agar intended for isolation of aeromonds. Among them, two strains from A. hydrophila complex have been identified previously as A. hydrophila subsp. dhakensis (Sedláček et al. 2012). Remaining 74 isolates were classified by extensive biotyping, ribotyping and cpn60 sequencing to the species level. The reference type strains of aeromonads were obtained from the Czech Collection of Microorganisms ( ). The isolates and reference strains were grown at 37°C for 24 h on blood agar during all http://www.sci.muni.cz/ccmexperiments.

Biotyping Basic phenotypic classification of isolates was performed using set of key conventional tube tests (Abbott et al., 2003; Austin et al., 1996, MacFaddin, 2000; Martin-Carnahan and Joseph, 2005). Further extensive phenotypic characterization and identification were achieved using the commercial identification system Biolog with the GN2 MicroPlates.

Ribotyping Ribotyping of isolated strains and reference cultures was performed using PvuII restriction enzyme and DNA probe complementary to 16S and 23S rRNA (Roche Diagnostics) according to Švec et al. (2001). Obtained ribotype patterns were processed by BioNumerics v. 6.6 software (Applied Maths). The dendrogram was calculated by means of the UPGMA clustering method based on the Dice coefficient.

cpn60 gene sequencing DNA isolation, the PCR conditions and primer sequences were used according to Minana-Galbis et al. (2009) and sequencing was performed by Eurofins MWG Operon (Ebersberg, Germany) classification service.Results

Aeromonas hydrophila complex

Ribotyping clearly distinguished members of A. hydrophila complex and A. veronii/sobria complex, because the ribotypes of A. hydrophila complex strains revealed typical band profiles with absence of bands smaller than 2.000 bp.

Ribotyping showed high heterogeneity of ribotype patterns and also proved occurrence of a rare pathogen A. hydrophila subsp. dhakensis in diarrheic stool samples. Several isolates revealed very close or almost identical ribotype profiles suggesting clonal distribution of isolates.

Biochemical identification of presumptive A. hydrophila complex (species and subspecies level) was based on results of oxidase, catalase, Voges-Proskauer reaction, esculin, gelatine, DNA and Tween 80 hydrolysis, gas from glucose, D-L lactate utilization, acid production from L-arabinose, salicin, celobiose, lactose, sucrose, rhamnose and sorbitol, lysine decarboxylase, and indole production.

Biolog identification system with the GN2 MicroPlates identified 33 strains as A. hydrophila (HG 1); one isolate P999 was classified as Aeromonas sp., one isolate P959 as A. encheleia and one P1026 as A. bestiarum (HG 2). In our opinion, the Biolog system is more convenient for aeromonads identification than other commercial kits intended for fermenting rods (e.g. API 20NE, ENTEROtest 24).

The taxonomic position of selected A. hydrophila strains was determined by cpn60 sequencing (analysed strains are marked with asterisks in Fig 1.).

Obtained results did not prove presence of Aeromonas aquariorum. According to Aravena-Román et al. (2011) this species is frequently occurring in clinical specimens but is often misidentified as A. hydrophila.

Aeromonas veronii/sobria complex

In total, 28 strains revealing multiple bands ranging from 1000 to 2000 bp were assigned by ribotyping into A. veronii/sobria complex. Ribotyping clearly distinguished this group from type strains of A. hydrophila complex. Only one couple of isolates from A. veronii/sobria complex (P1031 and P1036) had identical ribotype profiles showing clonal occurrence.

In general, ribotyping with PvuII restriction enzyme showed high heterogeneity of ribotype patterns among analysed strains and also proved occurrence of rare pathogens Aeromonas allosaccharophila (P1051) and Aeromonas jandaei (P960) in diarrheic stool samples. Ribotyping clustered strains P1091, P1004, P948 and P1159 very closely to A. veronii bv. veronii

TCCM 4359 ; with an exception of P948 the three remaining isolates were confirmed as A. veronii bv. veronii based on positive ornithine decarboxylase results.

Biochemical identification of the presumptive A. veronii/sobria complex strains was based on results of oxidase, catalase, Voges-Proskauer reaction, esculin, gelatine, DNA and Tween 80 hydrolysis, gas from glucose, susceptibility to cephalothin, acid production from L-arabinose, salicin, celobiose, lactose, sucrose and mannose, lysine and ornithine decarboxylase, and indole production.

GN2 MicroPlate identified only 15 strains as A. veronii bv. sobria (HG 8); 11 isolates were classified as Aeromonas sp., one isolate P1044 as A. encheleia and one P1004 as A. sobria (HG 7). Biolog system showed acceptable identification level for A. veronii/sobria complex.

The taxonomic position of selected A. veronii bv. sobria strains was determined by cpn60 gene sequencing (analysed strains are marked with asterisks in Fig 2.).

There is a problem with identification and differentiation of A. veronii bv. sobria and A. jandaei because only one feature (cephalothin susceptibility) is able to reliably distinguish these taxa.

Classification of Aeromonas spp. strains from human clinical material

Introduction

Mesophilic aeromonads are widespread in the environment and some of them are important human and fish pathogens. Polyphasic taxonomic studies of aeromonads performed in recent years resulted in description of several novel taxa and extended our knowledge of the biodiversity of the genus Aeromonas inhabiting humans and different environmental niches. Infections caused by Aeromonas species increased interest of microbiologists in these bacteria. The ability to identify organisms rapidly and reliably to the species level is an important step in the treatment of bacterial infections and for monitoring the spread of microorganisms. The present study is a part of the project dealing with the diversity of bacteria in human clinical material. Presumptive Aeromonas veronii/sobria complex and Aeromonas hydrophila complex strains isolated from human intestinal clinical materials were tentatively identified using conventional and commercial biochemical tests. Subsequently, more extensive phenotype characterization and identification of both aforementioned groups was achieved by using the Biolog identification system. It is known that identification of aeromonads to the species level is sometimes problematic, even if widely accepted commercial identification systems are used. Thus characterization using ribotyping with PvuII restriction enzyme was performed and obtained ribotypes were compared with the reference cultures covering all taxa included in A. veronii/sobria complex and A. hydrophila complex. Selected isolates representing different ribotype patterns were determined by cpn60 sequencing to confirm their species identification.

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Conclusions

Our findings demonstrated a continuous problem of Aeromonas spp. biochemical identification which is unreliable and unable to differentiate phenotypically similar species. Therefore it is necessary to apply molecular or chemotaxonomic methods for confirmation of unusual identification results. Combination of key biochemical tests, GN2 MicroPlate results and ribotyping enabled good species/subspecies/biotype identification of aeromonads; sequencing results (cpn60) confirmed identification to the species level.

Both predominant taxa, A. hydrophila subsp. hydrophila and A. veronii bv. sobria showed high heterogeneity of ribotyping patterns distributed in several clusters.

Ribotyping and cpn60 gene sequencing results also proved occurrence of rare pathogens A. hydrophila subsp. dhakensis, A. jandaei, A.veronii bv. veronii and A. allosaccharophila in diarrheic stool samples.

similarity, %

100

90

80

70

60

50

40

21.2

27

5.1

48

(bp)

834

947

1.3

75

1.5

87

2.0

27

3.5

30

4.2

69

1.9

04

A. veronii bv. sobria

A. veronii bv. veronii

A. allosaccharophila

A. jandaei

A. schubertii

A. bestiarum

A. hydrophila subsp. hydrophila




A. hydrophila subsp. dhakensis

A. hydrophila complex


A. aquariorum

A. popoffii






A. piscicola

CCM 1242TCCM 4359TCCM 4363TCCM 4355TCCM 4356TCCM 4707

P1095

P965*

P1066

P1003

P1096

P968

P1074

P996*TCCM 7146

P1094

P1050

P1002

P1070

P1020

P944

P945

P961

P1011

P1084TCCM 7695

P1000

P999

P1007

P1169

P947

P1054

P1092

P1100

P1173*TCCM 4708

P1163

P1015TCCM 7232

P1029

P951

P954*

P940

P946

P959

P1026*TCCM 7715

Lambda DNA/EcoRI+HindIII marker

similarity, %

100

90

80

70

60

50

40

(bp

)

83

49

47

1.9

04

4.2

69

5.1

48

1.3

75

2.0

27

3.5

30

1.5

87

21

.22

7

A. enteropelogenes

A. popoffii

A. hydrophila subsp. dhakensis


A. caviae










A. sobria

A. jandaei




A. jandaei

A. schubertii

TCCM 7243TCCM 4708TCCM 7146TCCM 7232TCCM 4491

P1091*

P948TCCM 4359

P1004

P1159

P1058

P941

P1012

P1090

P1013

P1161

P1069

CCM 1242

P1072

P995TCCM 2808

P960

P1044

P1157

P1027

P1093

P1082

P1018

P998TCCM 4363

*P1051

P1031

P1036

P997

P949*

P970

P1021TCCM 4355TCCM 4356

Lambda DNA/EcoRI+HindIII marker


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Classification of Aeromonas spp. strains from human ... · Presumptive Aeromonas veronii/sobria complex and Aeromonas hydrophila complex strains isolated from human intestinal clinical