Response to letter: Glycerophospholipid-cholesterol acyltransferase gene (gcat) sequence is present in other species of Aeromonas and not specific to only Aeromonas hydrophila

Abstract

We thank to Dr. Ogueri Nwaiwu (2019) for his great interest and thoughtful review of our article. In his letter, he mentioned the multiplex PCR method that we used the glycerophospholipid-cholesterol acyltransferase gene (gcat) was not enable to make a specific and valid identification of Aeromona hydrophila. Many virulence genes, such as heat-labile cytotonic enterotoxin (alt), serine protease (ser), heat-stable cytotonic enterotoxin (ast), and lipase (lip), were reported to have specifically associated with A. hydrophila; however, these genes also could be detected among A. veronii, A. caviae, and A. salmonicida (Khor et al., 2015Khor W.C. Puah S.M. Tan J.A. Puthucheary S.D. Chua K.H. Phenotypic and genetic diversity of Aeromonas species isolated from fresh water lakes in Malaysia.PLoS One. 2015; 10e0145933Google Scholar, Onuk et al., 2013Onuk E.E. Findik A. Turk N. et al.Molecular identification and determination of some virulence genes of Aeromonas spp. in fish and water from Turkish coastal regions.Rev Méd Vét. 2013; 164: 200-206Google Scholar, Puthucheary et al., 2012Puthucheary S.D. Puah S.M. Chua K.H. Molecular characterization of clinical isolates of Aeromonas species from Malaysia.PLoS One. 2012; 7e30205Crossref PubMed Scopus (66) Google Scholar). Although the gcat gene was found to be present in some Aeromonas strains and had a high identity match of A. hydrophila, A. dhakensis, A.caviae, A. salmonicida, and A. encheleia (Ogueri, 2019Ogueri N. Glycerophospholipid-cholesterol acyltransferase gene (gcat) is present in other species of Aeromonas and not specific to only Aeromonas hydrophila.Int J Infect Dis. 2019; (pii: S1201-9712(19)30142-0)Google Scholar), some literature reported that the gcat gene was mainly found in A. hydrophila and A. salmonicida (Balakrishna et al., 2010Balakrishna K. Murali H.S. Batra H.V. Detection of toxigenic strains of Aeromonas species in food by a multiplex PCR assay.Indian J Microbiol. 2010; 50: 139-144Google Scholar, Onuk et al., 2013Onuk E.E. Findik A. Turk N. et al.Molecular identification and determination of some virulence genes of Aeromonas spp. in fish and water from Turkish coastal regions.Rev Méd Vét. 2013; 164: 200-206Google Scholar). In fact, monomicrobial necrotizing fasciitis (NF) caused by A. hydrophila revealed a high mortality rate of 50% in our institution (Tsai et al., 2012Tsai Y.H. Huang K.C. Shen S.H. et al.Microbiology and surgical indicators of necrotizing fasciitis in a tertiary hospital of southwest Taiwan.Int J Infect Dis. 2012; 16: e159-165Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar and Tsai et al., 2015Tsai Y.H. Shen S.H. Yang T.Y. Chen P.H. Huang K.C. Lee Mel S. Monomicrobial necrotizing fasciitis caused by Aeromonas hydrophila and Klebsiella pneumoniae.Med Princ Pract. 2015; 24: 416-423Crossref PubMed Scopus (24) Google Scholar), but we did find A. salmonicida in the human infections. These 5 primers were initially evaluated by the uniplex PCR for testing the tissue samples from the patients with positive wound culture by microbiological laboratory before multiplex PCR (Figure 1). Due to the high sensitivity and specificity of gcat gene in uniplex PCR, we selected the gcat gene to target A. hydrophila in multiplex PCR (Mendes-Marques et al., 2013Mendes-Marques C.L. Hofer E. Leal N.C. Development of duplex-PCR for identification of Aeromonas species.Rev Soc Bras Med Trop. 2013; 46: 355-357Google Scholar). Furthermore, Dr. Ogueri mentioned that the genes amplified in the multiplex reaction were not labelled with a labelled ladder in our figures. I add Figure 2 to clarify that we used 100 bp DNA ladder for labelling. This figure also explains the initial results of multiplex PCR by comparing the reference pathogen and tested sample from the NF patient. Again, we would like to thank Dr. Ogueri for his comments on our study, and we can clarify the contents of this study. Indeed, to find a realistic, highly accurate and more specific method for quickly diagnosing lethal pathogens in NF patients is not easy. This study demonstrated that our multiplex PCR method for detection of these lethal microorganisms in tissue samples may be a useful diagnostic tool that revealed good results in microbiology laboratory. However, more research must be done to provide further results in clinical practice.