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 ~  Abstract
 ~ Introduction
 ~ Subjects and Methods
 ~ Results
 ~ Discussion
 ~ Acknowledgments
 ~  References
 ~  Article Figures
 ~  Article Tables

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  Table of Contents  
BRIEF COMMUNICATION
Year : 2012  |  Volume : 30  |  Issue : 4  |  Page : 467-469
 

Markers of pathogenicity islands in strains of Aeromonas species of clinical and environmental origin


1 Medical Bacteriology Laboratory, Department of Microbiology, National School of Biological Sciences, Institute National Polytechnic (IPN); Department of Microbiology, Pediatrics Hospital, National Medical Center Siglo XXI, IMSS, Mexico, DF
2 Medical Bacteriology Laboratory, Department of Microbiology, National School of Biological Sciences, Institute National Polytechnic (IPN), Mexico, DF

Date of Submission08-Mar-2012
Date of Acceptance26-May-2012
Date of Web Publication24-Nov-2012

Correspondence Address:
G Castro-Escarpulli
Medical Bacteriology Laboratory, Department of Microbiology, National School of Biological Sciences, Institute National Polytechnic (IPN), Mexico, DF

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Source of Support: Research and graduate office grants SIP20100252 and SIP20110191, Conflict of Interest: None


DOI: 10.4103/0255-0857.103772

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 ~ Abstract 

The aim of this study was to investigate the presence of markers of pathogenicity islands that may be informative to detect the virulent PAI carriers of clinical and environmental strains of Aeromonas spp. isolated in Mexico. virB2, virB9 and virB11 genes were found in Aeromonas strains isolated from environmental and clinical sources while cagE and tfc16 genes were only in strains of environmental origin. Having performed the wide screening presented in this study, we now have a set of strains to map and confirm the presence of a pathogenicity island in Aeromonas strains isolated in Mexico.


Keywords: Aeromonas, pathogenicity island, cagE, cagA, TIVSS, T4SS


How to cite this article:
Ruiz-Ruiz J M, Aguilera-Arreola M G, Castro-Escarpulli G. Markers of pathogenicity islands in strains of Aeromonas species of clinical and environmental origin. Indian J Med Microbiol 2012;30:467-9

How to cite this URL:
Ruiz-Ruiz J M, Aguilera-Arreola M G, Castro-Escarpulli G. Markers of pathogenicity islands in strains of Aeromonas species of clinical and environmental origin. Indian J Med Microbiol [serial online] 2012 [cited 2019 Sep 22];30:467-9. Available from: http://www.ijmm.org/text.asp?2012/30/4/467/103772



 ~ Introduction Top


Aeromonas is a genus of gram-negative rod-shaped facultative anaerobic bacteria. These bacteria are widely distributed throughout nature and are considered important fish pathogens. In addition, Aeromonas spp. are also pathogens of other cold and warm-blooded animals. [1] For several years, the significance of Aeromonas spp. as human diarrhea-causing agents was controversial; several studies demonstrated that the pathogenic mechanism is multifactorial because many virulence factors are involved. [2] Previously, Datta et al. [3] and Alvarado-Hernández et al. [4] described the presence of the cagA gene, a marker of the cag pathogenicity island (cag-PAI) of Helicobacter pylori in Aeromonas spp. and demonstrated its horizontal transfer among genera. [3],[4] Currently, the cagE gene is considered the functional marker of cag-PAI because it shows ATPase activity that is typical of Type IV Secretion System (T4SS) and is homologous to such genes as tfc16 from Haemophilus influenzae, which shows the same activity. [5],[6]


 ~ Subjects and Methods Top


This study included 63 clinical isolates from Hidalgo, Mexico; 41 strains of environmental origin from Veracruz beach, and 14 reference strains of Aeromonas spp. Primers for amplification of the virB2, virB9, and virB11 genes of the T4SS were designed based on the sequence of plasmid pFBAOT6 from A. culicicola (GenBank Accession Number: DQ890522.1). A bioinformatics analysis was performed using DNAMAN v5 and BioEdit v7.0.9 software to confirm the absence of interactions between all primers and to determine other physicochemical values to standardize a triplex PCR (t-PCR) technique. E. coli J53 containing the pFBAOT6 plasmid was used as a positive control and E. coli K12 was used as a negative control. The amplicons from the plasmid and an A. bestiarum strain, AhA4, isolated from the environment were purified and sequenced with an ABI-PRISMTM 3100 (Applied Biosystems, CA, USA) following the standard methodology outlined by the manufacturer. The consensus sequence was analysed with the Basic Local Alignment Search Tool (BLAST) provided by the National Center of Biotechnology Information (NCBI). The presence of T4SS-like ATPases homologous to tfc16 (H. influenzae) and cagE (H. pylori) was determined by single-PCR. [6],[7] The obtained amplicons were sequenced and analyzed using the same strategy described above. H. influenzae ATCC 33930 and H. pylori ATCC 43504 were used as positive controls and E. coli K12 was used as a negative control. [Table 1] lists all the primers used in this study.
Table 1: Oligonucleotide sequences of primers and PCR product sizes

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 ~ Results Top


The t-PCR technique of three marker genes, virB2 (114 bp), virB9 (278 bp) and virB11 (200 bp), was standardized using the designed primers. With this technique, we determined that 14 (13 %), 56 (54 %) and 67 (64%) of the 104 tested strains from environmental sources and clinical Aeromonas spp. possess the virB2, virB9 and virB11 genes, respectively [Table 2] [Figure 1]a. A total of 23/41 (56%) environmental strains gave an amplicon of 350 bp for the cagE gene essayed by s-PCR. BLASTn analysis showed a 78% similarity between the cagE gene of H. pylori and the A. bestiarum AhA4 cagE gene [Figure 1]b. For the tfc16 gene, 14/41 (34%) strains of environmental origin gave an amplicon of 277 bp [Figure 1]c. This amplicon was 100% identical to the same gene in the genomic island of H. influenzae. However, the 63 clinical isolates from the state of Hidalgo [Table 2] and the 14 reference strains of Aeromonas spp. (data not shown) did not yield a PCR product for the cagE or tfc16 genes.
Table 2: Distribution of pathogenicity island gene markers in all strains included in this study

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Figure 1: PCR products from clinical and environmental strains of Aeromonas spp. (a) t-PCR for T4SS gene markers (virB2: 114 bp, virB9: 278 bp, and virB11: 200 bp). (b) s-PCR for cagE gene (350 bp). (c) s-PCR for tfc16 gene (277 bp).

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 ~ Discussion Top


Aeromonas spp. have been isolated from soil and food samples but are mainly isolated from aquatic environments. These habitats are places shared by Aeromonas and H. pylori that enable greater connectivity between them; Mazari-Hiriart et al. [8] showed that H. pylori can survive for up to 16 days in distilled, saline waters, and even in water with residual chlorine along with other enteropathogenic bacteria, such as A. hydrophila, A. caviae and A. veronii. Therefore, the horizontal transfer of genes between these genera is a likely event, as previously noted in 2003, by Datta et al. These authors demonstrated the presence of a gene-like H. pylori cagA in environmental isolates of Aeromonas spp. from diverse water samples from Calcutta, India, by PCR and solid-phase hybridization (colony blot). [3] The wide screening present in this study reinforce the results from these authors about cagA gene and add information about the presence the other relevant genes, we now have a set of strains to map and confirm the presence of a pathogenicity island in Aeromonas strains isolated in Mexico.


 ~ Acknowledgments Top


This work was supported by the research and graduate office grants SIP20100252 and SIP20110191. MGAA and GCE received support from COFAA, EDI and SNI. We thank: Dr. Glen Rhodes from the Centre for Ecology & Hydrology, UK; Dr. Miroslava Sánchez from Laboratorio de Salud Pública, Hidalgo, Mexico, for providing isolates; and Dr. Ma José Figueras for providing reference strains.

 
 ~ References Top

1.Figueras MJ, Guarro J, Martínez Murcia A. Clinically relevant Aeromonas species. Clin Infect Dis 2000;3:988-9.  Back to cited text no. 1
    
2.Castro-Escarpulli G, Figueras MJ, Aguilera-Arreola G, Soler L, Fernández-Rendón E, Aparicio GO, et al. Characterisation of Aeromonas spp. isolated from frozen fish intended for human consumption in Mexico. Int J Food Microbiol 2003;84:41-9.  Back to cited text no. 2
    
3.Datta S, Khan A, Nandy RK, Rehman M, Sinha S, Chattopadhyay S, et al. Environmental isolates of Aeromonas spp. harboring the cagA-like gene of Helicobacter pylori. Appl Environ Microbiol 2003;69:4291-5.  Back to cited text no. 3
[PUBMED]    
4.Alvarado-Hernández AM. cagA gene of Helicobacter pylori in Aeromonas spp. strains isolated from environmental samples in Mexico. Abstr. 9th International Symposium on Aeromonas and Plesiomonas, Vila Real (Portugal); 2008. p. 95.  Back to cited text no. 4
    
5.Hsu PI, Hwang IR, Cittelly D, Lai KH, El-Zimaity HM, Gutierrez O, et al. Clinical presentation in relation to diversity within the Helicobacter pylori cag phatogenicity island. Am J Gastroenterol 2002;97:2231-8.  Back to cited text no. 5
[PUBMED]    
6.Ikenoue T, Maeda S, Ogura K, Akanuma M, Mitsuno Y, Imai Y, et al. Determination of Helicobacter pylori virulence by simple gene analysis of the cag pathogenicity island. Clin Diagn Lab Immunol 2001;8:181-6.  Back to cited text no. 6
[PUBMED]    
7.Espinoza-Mellado MR. Efecto vacuolizante de sobrenadantes de Haemophilus infuenzae en líneas celulares HEp-2 y su relación con los genes hap y tfo 16 Master's degree thesis. Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico D.F., 2009.  Back to cited text no. 7
    
8.Mazari-Hiriart M, López-Vidal Y, Castillo-Rojas G, Ponce de León S, Cravioto A. Helicobacter pylori and other enteric bacteria in freshwater environments in Mexico City. Arch Med Res 2001;32:458-67.  Back to cited text no. 8
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2]



 

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