New aminoglycoside acetyltransferase gene, aac(3)-Id, in a class 1 integron from a multiresistant strain of Vibrio fluvialis isolated from an infant aged 6 months

Ashraf M. Ahmed1, Tomoko Nakagawa2, Eiji Arakawa3, Thandavarayan Ramamurthy4, Sumio Shinoda2 and Tadashi Shimamoto1,*

1 Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima; 2 Graduate School of Natural Science and Technology, Okayama University, Okayama; 3 Department of Bacteriology, National Institute of Infectious Diseases, Tokyo, Japan; 4 National Institute of Cholera and Enteric Diseases, Calcutta, India

Received 23 December 2003; returned 2 February 2004; revised 2 March 2004; accepted 3 March 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: To characterize the molecular basis of antibiotic resistance in a multidrug-resistant clinical isolate of Vibrio fluvialis H-08942.

Patient and methods: V. fluvialis H-08942 was isolated from a hospitalized infant aged 6 months suffering from cholera-like diarrhoea in India in 2002. The broth microdilution method was used to determine the MICs of a range of antibiotics for this strain. PCR, DNA sequencing, Southern hybridization, cloning and expression were used to characterize the molecular basis of antibiotic resistances.

Results: V. fluvialis H-08942 showed resistance to chloramphenicol, streptomycin, spectinomycin, co-trimoxazole, ampicillin, furazolidone, nalidixic acid and gentamicin. A class 1 integron that contains a novel aminoglycoside acetyltransferase gene, aac(3)-Id, and aminoglycoside adenyltransferase gene, aadA7, was characterized. The aac(3)-Id gene product was found to share 50%, 45% and 44% identity to AAC(3)-Ic, AAC(3)-Ia, and AAC(3)-Ib, respectively. Both aac(3)-Id and aadA7 genes were cloned and expressed in Escherichia coli. Phylogenetic analysis suggested that the aac(3)-Id represents a fourth evolutionary lineage in the aminoglycoside acetyltransferase genes. Southern hybridization showed that this integron is located in the chromosome.

Conclusions: In this study we identified a new type of aminoglycoside acetyltransferase gene, aac(3)-Id. In addition, this is the first report of identification of antibiotic resistance genes and a class 1 integron in V. fluvialis.

Keywords: gentamicin resistance, aminoglycoside adenyltransferase, aadA7, cholera-like diarrhoea


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Vibrio fluvialis is a food-borne pathogenic bacterium, and has been implicated in outbreaks and sporadic cases of diarrhoea.1 It was first identified in 1975 in Bahrain in a patient with diarrhoea as group F vibrio,2 and then renamed as V. fluvialis.3 The largest outbreak of V. fluvialis infection was reported in Bangladesh, between October 1976 and November 1977.4 It involved more than 500 patients, half of whom were young children. In the USA, V. fluvialis accounted for 10% of clinical cases in a survey of vibrio infections along the Gulf Coast,5 and has also been associated with enterocolitis in infants.6 Recently, it was reported to be associated with acute diarrhoea in Indonesia.7 Moreover, V. fluvialis has an infectious importance, as the clinical symptoms of gastroenteritis caused by V. fluvialis and Vibrio cholerae are very similar, and recently an enterotoxigenic El Tor-like haemolysin, which represents one of the virulence factors of V. cholerae, was characterized in V. fluvialis.8

Integrons are DNA elements that capture genes, especially antibiotic resistance genes, by a site-specific recombination system.9 Class 1 integrons are widespread among multiresistant Gram-negative bacteria.10 Some of the most captured antibiotic resistance genes in this class are those that confer resistance to aminoglycosides. The aminoglycosides are highly potent, broad-spectrum antibiotics with many desirable properties for the treatment of life-threatening infections.11 Several mechanisms have been proposed for bacterial resistance to aminoglycoside antibiotics. They include decreased antibiotic uptake and accumulation, modification of the ribosomal target, efflux of antibiotic, and enzymatic modification of aminoglycosides.12 One of the most important modifying aminoglycosides enzymes is N-acetyltransferase (AAC), which uses acetyl-coenzyme A as a donor and affects amino functions of aminoglycosides on positions 2', 6', 1 and 3. Therefore, there are four classes of aminoglycoside acetyltransferases: AAC(2'), AAC(6'), AAC(1) and AAC(3).12 AAC(3)-I enzymes produce resistance to gentamicin, sisomicin and fortimicin.12 Until now, at least three types of AAC(3) genes have been identified: aac(3)-Ia, aac(3)-Ib and aac(3)-Ic, which were identified in 1989,13 199514 and 2003,15 respectively.

To date, nothing is known about the genetic bases of antibiotic resistance in V. fluvialis. In an attempt to determine the genetic mechanism of this resistance, V. fluvialis H-08942 was tested for the presence of a class 1 integron.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Source and characterization of V. fluvialis H-08942

V. fluvialis H-08942 used in this study was isolated from the stool specimen of an infant aged 6 months suffering from cholera-like watery diarrhoea admitted to the Infectious Diseases Hospital, Calcutta, India, in 2002. Collection, transportation and bacteriological examination of the stool sample, and identification of V. fluvialis H-08942, were carried out as described previously for other vibrio isolates.16 Strain identification of V. fluvialis H-08942 was confirmed with the Biolog GN2 microplate (Biolog, Inc., Hayward, CA, USA) according to the manufacturer’s instructions.

Antibiotic susceptibility testing

At present, there are no interpretive standards for susceptibility testing for Vibrio species other than V. cholerae. Therefore, the MICs of the antibiotics for V. fluvialis H-08942 were determined by the broth microdilution method as previously described for V. cholerae.17 The MICs were tested using the following antibiotics supplied by different commercial companies: ampicillin, chloramphenicol, ciprofloxacin, furazolidone, gentamicin, kanamycin, nalidixic acid, norfloxacin, spectinomycin, streptomycin, tetracycline and co-trimoxazole (trimethoprim/sulfamethoxazole). MIC breakpoints were evaluated according to NCCLS guidelines.18

Bacterial DNA preparation, PCR and DNA sequencing

Preparation of bacterial DNA template and also PCR conditions for detection of a class 1 integron were carried out as described previously.19 Class 1 integron primers (5'-CS, 5'-GGCATCCAAGCAGCAAG-3' and 3'-CS, 5'-AAGCAGACTTGACCTGA-3'),20 which amplify the region between the 5'-CS and the 3'-CS, were used. The PCR fragment was purified using a QIAquick Gel Extraction Kit (Qiagen K. K.), and then sequenced in an ABI automatic DNA sequencer (Model 373; Perkin-Elmer) using class 1 integron primers. According to the preliminary DNA sequencing results, two other primers (IntVf-F, 5'-ACTCAACGTCATCGGGCG-3' and IntVf-B, 5'-AGCTACAGTCACCAGCAAA-3') were designed. These primers are located within the PCR fragment and were used for complete sequencing of both strands of the whole integron segment.

Plasmid isolation, preparation of probe and Southern blot hybridization

Plasmids were isolated from V. fluvialis H-08942 by the alkaline lysis method as described by Sambrook & Russell.21 After agarose gel electrophoresis, DNA fragments in the gel were transferred onto Hybond-N+ nylon membranes (Amersham Biosciences) according to the manufacturer’s instructions. The purified PCR fragment of the class 1 integron was labelled with alkaline phosphatase using AlkPhos Direct Labeling System (Amersham Biosciences), and then used as a DNA probe. All steps of hybridization were carried out according to the manufacturer’s protocol. The hybridization was performed at 55°C for 12 h.

Transformation using V. fluvialis H-08942 plasmid preparation

V. fluvialis H-08942 plasmid preparation was used to transform Escherichia coli TG1 competent cells. After transformation, E. coli TG1 transformants were cultured on LB agar plates containing streptomycin (10 mg/L) or gentamicin (10 mg/L). The plates were then incubated for 24 h at 37°C.

Cloning and expression of aminoglycoside determinants in E. coli

The purified PCR fragment of the class 1 integron of V. fluvialis H-08942 was cloned into the EcoRV site of pBluescript II SK(–). The new recombinant plasmid was named pSVF1. In addition, to confirm specifically the contribution of aac(3)-Id to the resistance phenotype, another recombinant plasmid, pSVF2, was constructed. pSVF2 contains only the cloned aac(3)-Id after removal of aadA7 from pSVF1 using SalI digestion and re-ligation. Both recombinant plasmids were used to transform E. coli TG1 for the expression of the cloned genes. MICs of selected aminoglycoside antibiotics, including sisomicin, were tested for both types of E. coli. On the other hand, V. fluvialis H-08942 and E. coli TG1 host carrying pBluescript II SK(–) were also included for comparison.

Computer analysis of the sequenced data

The similarity search was carried out using the BLAST program,22 while multiple amino acid sequence alignment of aminoglycoside acetyltransferases was carried out using the CLUSTAL W program.23 The phylogenetic tree was constructed based on the amino acid sequences of aminoglycoside acetyltransferases by using GENETYX-MAC software (GENETYX, Inc., Tokyo, Japan).


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Antimicrobial susceptibility

Multidrug resistance phenotypes were observed for V. fluvialis H-08942. This strain showed resistance to streptomycin (MIC > 64 mg/L), gentamicin (64 mg/L), spectinomycin (>64 mg/L), chloramphenicol (16 mg/L), co-trimoxazole (>64 mg/L), furazolidone (8 mg/L), ampicillin (32 mg/L) and nalidixic acid (32 mg/L). On the other hand, it was susceptible to tetracycline (0.25 mg/L), kanamycin (4 mg/L), neomycin (2 mg/L), norfloxacin (2 mg/L) and ciprofloxacin (0.25 mg/L). Among the Vibrio species, resistance to streptomycin and furazolidone were previously observed for the first time in V. cholerae O139 isolated after 1992,24 and then these phenotypes associated with resistance to chloramphenicol were detected in many recent Asian clinical V. cholerae O1 El Tor and O139 isolates,25 and recently in some clinical isolates of non-O1, non-O139.26 The spread of these resistances in V. cholerae is mainly due to a new type of conjugative transposon called the SXT element or constin.24 Recently, we have detected a variant type of the SXT element in V. fluvialis H-08942 (Ahmed, A. M., Shimada, S. & Shimamoto, T., submitted for publication).

Class 1 integron

To date, there have been no reports on the antibiotic resistance genes in V. fluvialis or even on the mechanisms of antibiotic resistance in this important pathogen. The PCR result showed the presence of a class 1 integron in V. fluvialis H-08942 with an amplicon size of 1.5 kb (data not shown). DNA sequencing results of the purified PCR fragment showed the presence of two antibiotic resistance gene cassettes inside the class 1 integron (Figure 1). The first one is a novel aminoglycoside acetyltransferase gene cassette that is related to aac(3)-I class, and confers resistance to aminoglycosides, mainly gentamicin.12 The second is related to aminoglycoside adenyltransferase gene, aadA7, and confers resistance to streptomycin and spectinomycin. The aadA7 gene of V. fluvialis H-08942 is an open reading frame that encodes a protein of 265 amino acid residues. This protein showed 99% (264/265) identity with the novel streptomycin resistance gene, aadA7, which has been identified recently in E. coli.27 Therefore, aadA7 of V. fluvialis H-08942 is considered the second report for this gene.



View larger version (5K):
[in this window]
[in a new window]
 
Figure 1. Organization of the class 1 integron of V. fluvialis. Black filled boxes represent 5'- and 3'-conserved sequences, hatched arrows are the antibiotic gene cassettes, aminoglycoside acetyltransferase [aac(3)-Id] and aminoglycoside adenyltransferase (aadA7) genes, and the hatched circles represent their 59-base elements (59-be).

 
Class 1 integron is chromosomal

Southern hybridization was used to determine the location of the class 1 integron in V. fluvialis H-08942. Even though several plasmids of different sizes were detected in V. fluvialis H-08942, a positive hybridization signal was detectable only in the chromosomal band (Figure 2). A transformation experiment was also carried out using a V. fluvialis H-08942 plasmid preparation and E. coli TG1 competent cells to confirm the Southern hybridization result. The transformation results supported the chromosomal location of the class 1 integron as no transformant cells were obtained on LB medium containing streptomycin or gentamicin. This is similar to the cases of class 1 integrons of aac(3)-Ib and aac(3)-Ic genes where they were identified in the chromosome of Pseudomonas aeruginosa.14,15



View larger version (35K):
[in this window]
[in a new window]
 
Figure 2. Agarose gel electrophoresis and Southern hybridization for the chromosomally encoded class 1 integron in V. fluvialis. Lanes M1 and M2 are {lambda} DNA digested with HindIII and Salmonella typhimurium LT2 plasmid (90 kb) used as size markers, respectively, while lane V is the plasmids of V. fluvialis including part of its chromosome. The hybridization signal is in the chromosome (right panel).

 
Cloning and expression of aac(3)-Id and aadA7 of V. fluvialis H-08942 in E. coli

We have constructed two recombinant plasmids: one contains the whole integron, pSVF1, while the other contains only the aac(3)-Id gene, pSVF2. The MIC results of aminoglycoside antibiotics (Table 1) for the transformants showed that the E. coli TG1 transformant carrying pSVF1 displayed a notable resistance to gentamicin, sisomicin, streptomycin and spectinomycin. However, the E. coli TG1 transformant carrying pSVF2 showed a notable resistance to gentamicin and sisomicin only. Both types of transformants were susceptible to kanamycin and neomycin. For comparison, both V. fluvialis H-08942 and E. coli TG1 host carrying pBluescript II SK(–) were included. These results clearly confirmed that the aac(3)-Id gene is a new type of AAC(3) gene that confers resistance to gentamicin and sisomicin, and aadA7 could contribute to the streptomycin and spectinomycin resistance in V. fluvialis H-08942.


View this table:
[in this window]
[in a new window]
 
Table 1. MICs of aminoglycoside antibiotics used for V. fluvialis H-08942 and E. coli TG1 transformants
 
Comparison of AAC(3)-Id with other related AAC(3)-I proteins

The aac(3)-Id gene encodes the aminoglycoside acetyltransferase protein, AAC(3)-Id, which is 158 amino acids long. A similarity search for AAC(3)-Id in the GenBank database showed 50%, 45% and 44% identity to AAC(3)-Ic, AAC(3)-Ia and AAC(3)-Ib, respectively (Figure 3a). These results somewhat matched the results of Riccio et al.,15 who found that AAC(3)-Ic is 59% and 57% identical to AAC(3)-Ia and AAC(3)-Ib, respectively.



View larger version (39K):
[in this window]
[in a new window]
 
Figure 3. Alignment (a) and phylogenetic tree of the proposed evolutionary relationships (b) of the four aminoglycoside acetyltransferases AAC(3)-Ia,13 AAC(3)-Ib,14AAC(3)-Ic15 and AAC(3)-Id. Multiple conserved motifs marked by asterisks are present among the four types, and the novel AAC(3)-Id enzyme represents a fourth evolutionary lineage in the aminoglycoside acetyltransferases group.

 
Interestingly, the phylogenetic tree based on the amino acid sequences of AAC(3)-I enzymes (Figure 3b) showed that AAC(3)-Ia and AAC(3)-Ib are evolutionarily closely related, while AAC(3)-Ic is closely related to AAC(3)-Id. This phylogenetic analysis also matched the identification dates of AAC(3)-Ia,13 AAC(3)-Ib14 and AAC(3)-Ic,15 suggesting that AAC(3)-Id is a fourth evolutionary lineage in this group.

In conclusion, in this study we identified a new type of aminoglycoside acetyltransferase gene, designated aac(3)-Id. In addition, this document characterizes for the first time some of the genetic mechanisms of antimicrobial resistance in V. fluvialis.

Nucleotide sequence accession number

The nucleotide sequence of the V. fluvialis class 1 integron, including aac(3)-Id and aadA7 genes, has been assigned GenBank accession no. AB114632.


    Acknowledgements
 
This work was supported by a Grant-in-Aid for scientific research to T.S. from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


    Footnotes
 
* Corresponding author. Tel/Fax: +81-824-24-7897; E-mail: tadashis{at}hiroshima-u.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Oliver, J. D. & Kaper, J. B. (2001). Vibrio species. In Food Microbiology: Fundamentals and Frontiers, 2nd edn (Doyle, M. P., Beuchat, L. R. & Montville, T. J., Eds), pp. 263–300. ASM Press, Washington, DC, USA.

2 . Furniss, A. L., Lee, J. V. & Donovan, T. J. (1977). Group F, a new vibrio? Lancet 10, 73–94.

3 . Lee, J. V., Shread, P., Furniss, A. L. et al. (1980). Taxonomy and description of Vibrio fluvialis sp nov (synonym group F vibrios, group EF-6). Journal of Applied Bacteriology 50, 73–94.[ISI]

4 . Huq, M. I., Alam, A. K. M. J., Brenner, D. J. et al. (1980). Isolation of vibrio-like group EF-6, from patients with diarrhea. Journal of Clinical Microbiology 11, 621–4.[ISI][Medline]

5 . Levine, W. C., Griffin, P. M. & the Gulf Coast Vibrio Working Group. (1993). Vibrio infections on the Gulf Coast: the results of a first year of regional surveillance. Journal of Infectious Diseases 167, 479–83.[ISI][Medline]

6 . Bellet, J., Klein, B., Alteri, M. et al. (1989). Vibrio fluvialis, an unusual pediatric enteric pathogen. Pediatric Emergency Care 5, 27–8.[Medline]

7 . Lesmana, M., Subekti, D. S., Tjaniadi, P. et al. (2002). Spectrum of Vibrio species associated with acute diarrhea in North Jakarta, Indonesia. Diagnostic Microbiology and Infectious Disease 43, 91–7.[CrossRef][ISI][Medline]

8 . Kothary, M. H., Lowman, H., McCardell, B. A. et al. (2003). Purification and characterization of enterotoxigenic El Tor-like hemolysin produced by Vibrio fluvialis. Infection and Immunity 71, 3213–20.[Abstract/Free Full Text]

9 . Stokes, H. W. & Hall, R. M. (1989). A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons. Molecular Microbiology 3, 1669–83.[ISI][Medline]

10 . Jones, M. E., Peters, E., Weersink, A. M. et al. (1997). Widespread occurrence of integrons causing multiple antibiotic resistances in bacteria. Lancet 349, 1742–3.[ISI][Medline]

11 . Mingeot-Leclercq, M., Glupczynski, Y. & Tulkens, P. M. (1999). Aminoglycosides: activity and resistance. Antimicrobial Agents and Chemotherapy 43, 727–37.[Free Full Text]

12 . Vakulenko, S. B. & Mobashery, S. (2003). Versatility of aminoglycosides and prospects for their future. Clinical Microbiology Reviews 16, 430–50.[Abstract/Free Full Text]

13 . Wohlleben, W., Arnold, W., Bissonnette, L. et al. (1989). On the evolution of Tn21-like multiresistance transposons: sequence analysis of the gene (aacC1) for gentamicin acetyltransferase-3-I (AAC(3)-I), another member of the Tn21-based expression cassette. Molecular and General Genetics 217, 202–8.[Medline]

14 . Schwocho, L. R., Schaffner, C. P., Miller, G. H. et al. (1995). Cloning and characterization of a 3-N-aminoglycoside acetyltransferase gene, aac(3)-Ib, from Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 39, 1790–6.[Abstract]

15 . Riccio, M. L., Docquier, J. D., Dell’Amico, E. et al. (2003). Novel 3-N-aminoglycoside acetyltransferase gene, aac(3)-Ic, from a Pseudomonas aeruginosa integron. Antimicrobial Agents and Chemotherapy 47, 1746–8.[Abstract/Free Full Text]

16 . Sharma, C., Thungapathra, M., Ghosh, A. et al. (1998). Molecular analysis of non-O1, non-O139 Vibrio cholerae associated with an unusual upsurge in the incidence of cholera-like disease in Calcutta, India. Journal of Clinical Microbiology 36, 756–63.[Abstract/Free Full Text]

17 . Dalsgaard, A., Forslund, A., Sandvang, D. et al. (2001). Vibrio cholerae O1 outbreak isolates in Mozambique and South Africa in 1998 are multiple-drug resistant, contain the SXT element and the aadA2 gene located on class 1 integrons. Journal of Antimicrobial Chemotherapy 48, 827–38.[Abstract/Free Full Text]

18 . National Committee for Clinical Laboratory Standards. (1998). Performance Standards for Antimicrobial Susceptibility Testing: Approved Standard M100-S8. NCCLS, Villanova, PA, USA.

19 . Zhao, S., White, D. G., Ge, B. et al. (2001). Identification and characterization of integron-mediated antibiotic resistance among shiga toxin-producing Escherichia coli isolates. Applied and Environmental Microbiology 67, 1558–64.[Abstract/Free Full Text]

20 . Lévesque, C., Piché, L., Larose, C. et al. (1995). PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrobial Agents and Chemotherapy 39, 185–91.[Abstract]

21 . Sambrook, J. & Russell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.

22 . Altschul, S. F., Madden, T. L., Schäffer, A. A. et al. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389–402.[Abstract/Free Full Text]

23 . Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 4673–80.[Abstract]

24 . Waldor, M. K., Tschäpe, H. & Mekalanos, J. J. (1996). A new type of conjugative transposon encodes resistance to sulfamethoxazole, trimethoprim, and streptomycin in Vibrio cholerae O139. Journal of Bacteriology 178, 4157–65.[Abstract]

25 . Hochhut, B., Lotfi, Y., Mazel, D. et al. (2001). Molecular analysis of the antibiotic resistance gene clusters in the Vibrio cholerae O139 and O1 SXT constins. Antimicrobial Agents and Chemotherapy 45, 2991–3000.[Abstract/Free Full Text]

26 . Thungapathra, M., Amita, Sinha, K. K. et al. (2002). Occurrence of antibiotic resistance gene cassettes aac(6')-Ib, dfrA5, dfrA12, and ereA2 in class 1 integrons in non-O1, non-O139 Vibrio cholerae strains in India. Antimicrobial Agents and Chemotherapy 46, 2948–55.[Abstract/Free Full Text]

27 . Mazel, D., Dychinco, B., Webb, V. A. et al. (2000). Antibiotic resistance in the ECOR collection: integrons and identification of a novel aad gene. Antimicrobial Agents and Chemotherapy 44, 1568–74.[Abstract/Free Full Text]