- Email: [email protected]
Dynamics of classical–El Tor switch of Vibrio cholerae strains isolated from 1961–2010
570
Letters to the Editor / International Journal of Antimicrobial Agents 40 (2012) 562–573
been used in vitro for efficacy testing against Arcobacter, but a few other members of the -lactams such as cefoperazone, cefuroxime sodium and cefalothin were tested and failed to produce a desirable action against Arcobacter [5]. In the current study, a low level of resistance in A. butzleri isolates to gentamicin (M.I.C.E., 26.1%; disk diffusion, 19.6%) and ciprofloxacin (M.I.C.E., 21.7%; disk diffusion, 19.6%) was recorded. Resistance to ciprofloxacin shown by A. butzleri and Arcobacter cryaerophilus is primarily due to mutation in the Thr85 residue of GyrA [1]. Tetracycline showed the highest efficacy, with only 6.5% (M.I.C.E.) and 8.7% (disk diffusion) of A. butzleri isolates found to be resistant. Previous studies have also reported tetracycline as the most effective drug and thus have suggested its usage in the treatment of human and animal infections caused by Arcobacter spp. [2]. Multidrug resistance (resistance to three or more antibiotics) is of major concern, limiting the therapeutic choice of antibiotics and dose calculations. A total of 41.3% (M.I.C.E) and 34.7% (disk diffusion) of A. butzleri isolates were found to be multidrug-resistant. Regarding the antibiotic resistance patterns, 19.6% of isolates were resistant to ampicillin, erythromycin and cefotaxime, whereas 8.6% of isolates were resistant to ampicillin, cefotaxime and gentamicin. Genetic elements such as plasmids, transposons, multidrug efflux pumps and integrons have remained involved in the evolution and dissemination of multidrug resistance in bacteria [2]. Disks and automated systems with truncated MIC ranges are adequate for most routine susceptibility testing, but precise values are needed in order to set the dosage for infections caused by drug-resistant pathogens. MICs of antimicrobial agents were determined for A. butzleri and results were expressed as the MIC50 and MIC90 values. The lowest MIC50 and MIC90 values were recorded for tetracycline (0.25/8 g/mL) and the highest were for ampicillin (32/256 g/mL) (Table 1). Comparative results of M.I.C.E. and disk diffusion methods based on correlation (SPSS v.20; SPSS Inc., Chicago, IL) showed a very high relationship for cefotaxime and ciprofloxacin and a high relationship for ampicillin, tetracycline and gentamicin; however, a moderate correlation was observed for erythromycin (Table 1). Differences in results between the two methods could be due to lack of a specific protocol and breakpoints for Arcobacter spp. Arcobacter butzleri was found to be highly sensitive to tetracycline, thus it is recommended as the first choice in the treatment of Arcobacter infections. Either of the antimicrobial sensitivity methods can be used for in vitro susceptibility testing, but application of M.I.C.E. is advantageous in terms of quantitative results, which are useful to select the proper antibiotic and to estimate the dose of drugs against Arcobacter. Funding: The authors are indebted to Sindh Agriculture University, Tandojam (Pakistan) for a PhD grant (2008–2011) to AHS as well as to Universiti Putra Malaysia (RUGS Project No. 01-0111-1125RU). The authors are also indebted to the World Bank for providing financial support to AHS under the Robert S. McNamara Fellowship (2012) programme. Competing interests: None declared. Ethical approval: Not required. References [1] Shah AH, Saleha AA, Zunita Z, Murugaiyah M. Arcobacter—an emerging threat to animals and animal origin food products? Trends Food Sci Technol 2011;22:225–36. [2] Collado L, Figueras MJ. Taxonomy, epidemiology, and clinical relevance of the genus Arcobacter. Clin Microbiol Rev 2011;24:174–92. [3] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; twentieth informational supplement. Document M100-S20. Wayne, PA: CLSI; 2010.
[4] Clinical and Laboratory Standards Institute. Methods for antimicrobial dilution and disc susceptibility testing of infrequently isolated or fastidious bacteria; approved guideline. Document M45-A. Wayne, PA: CLSI; 2006. [5] Atabay HI, Aydin F. Susceptibility of Arcobacter butzleri isolates to 23 antimicrobial agents. Lett Appl Microbiol 2001;33:430–3.
A.H. Shah ∗ Department of Animal Products Technology, Sindh Agriculture University, Tandojam 70060, Pakistan A.A. Saleha ∗ Z. Zunita M. Murugaiyah A.B. Aliyu Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia ∗ Corresponding
authors. Tel.: +60 389 463 458; fax: +60 389 471 972. E-mail addresses: vetdr [email protected] (A.H. Shah), [email protected] (A.A. Saleha) 24 March 2012 doi:10.1016/j.ijantimicag.2012.08.001
Dynamics of classical–El Tor switch of Vibrio cholerae strains isolated from 1961–2010 Sir, Vibrio cholerae O1 and O139 are the serogroups mainly responsible for the disease cholera. Vibrio cholerae isolates belonging to the O1 serogroup are further classified into two biotypes, namely classical and El Tor [1]. The year 1992 is a milestone in the history of cholera as it was the year when V. cholerae O139 emerged in Chennai, India [2] and subsequently in other parts of the globe. This study promoted us to monitor how far this variation in the ctxB gene has been incorporated into the representative V. cholerae O1 isolates from different parts of the world from 1961–2010. Among the two cholera toxin genes (ctxA and ctxB), ctxB has received much more attention owing to the presence of genetic variability within it. Recently, three variants of V. cholerae O1 biotype El Tor were reported, namely Matlab variants (mix of both classical and El Tor traits), Mozambique variants (typical El Tor genome, with a tandem repeat of classical CTX prophage) and altered El Tor variants (typical El Tor biotype with El Tor CTX prophage but producing classical cholera toxin) [3]. Integrons (not autonomously mobile) are an important mechanism to capture, integrate and express resistance gene cassettes in their variable region in many bacteria. Among the three classes of integrons, class 1 integrons are most prevalent among clinical isolates and consist of two conserved segments [the 5 conserved segment (5 -CS) and the 3 conserved segment (3 -CS)] and an internal variable region that contains gene cassettes encoding antibiotic resistance determinants [4]. The 5 -CS encodes the type I integrase gene (intI1) and the recombination site attI1; the 3 -CS encodes the antiseptic resistance gene qacE1 and the sulfonamide resistance gene sul1. To monitor the effect of genetic variation on phenotypic traits, 256 clinical isolates collected worldwide spanning a period of 50 years (1961–2010) were analysed by mismatch amplification mutation assay PCR (MAMA-PCR) (ctxB), simplex PCR (class 1 integron, ctxA and wbe) and a phage typing technique. MAMA-PCR results clustered all the isolates into three groups according to the presence of classical and El Tor ctxB genes: CT-B I, isolates with both classical and El Tor ctxB; CT-B II, isolates with only El Tor ctxB; and CT-B III, isolates with only classical ctxB. Phage typing and
Letters to the Editor / International Journal of Antimicrobial Agents 40 (2012) 562–573
571
Fig. 1. Graphical distribution of Vibrio cholerae O1 biotype El Tor isolates based on heterogeneity of the ctxB gene determined by mismatch amplification mutation assay PCR (MAMA-PCR), by year.
antimicrobial susceptibility results demonstrated that all of the isolates were sensitive to El Tor group V phage, resistant to classical group IV phage and resistant to polymyxin B, which concluded the fact that all V. cholerae isolates incorporated in this study belonged to O1 biotype El Tor. The graphical presentation of MAMA-PCR results in Fig. 1 clustered V. cholerae isolates from Chennai, India, during 1980–2010 into four periods: Period I, 1980–1984; Period II, 1985–1991; Period III, 1992; and Period IV, 1993–2010. These periods reflected predominance of the CT-B I variant, a mosaic combination both of CT-B I and II variants, appearance of CT-B III along with the CT-B II variant, and uninterrupted prevalence of the CT-B III variant, respectively. Among these 256 isolates 100 representative isolates were selected to confirm the presence and absence of class 1 integron by PCR amplification of the 3 -CS. Of the 100 V. cholerae isolates, 60 were found to be positive for 3 -CS; no 3 -CS amplification was found within the 40 V. cholerae isolates from 1961–1969. Phage typing of each variant (CT-B I, II and III) showed a similar typing pattern dominated by type 27 and followed by types 24 and 26. It was clearly found from these data that the influence of the classical biotype still remained incessantly in the isolates from Chennai up to 1984. Interestingly, this influence of the classical biotype became sporadic with the appearance of typical El Tor variants (CT-B II) from the year 1961 and finally disappeared in 1991. This study clarifies that V. cholerae O139 has changed the cholera scenario following its emergence in 1992 in Chennai. Since the onset of O139 (in 1992), we found altered El Tor variants and from the year 1993 it was still continuing. These findings are in accordance with previous reports [5] which postulated that the classical cholera toxin gene has reappeared but its carrier has been El Tor, and this study showed that its first appearance was in 1966. MAMA-PCR and class 1 integron and phage typing results concluded that the genotypic variation, such as polymorphic traits in ctxB genes, did not influence the antibiotic susceptibility and phenotypic behaviour of V. cholerae O1 biotype El Tor strains. The pathogenic effect of El Tor vibrios harbouring the classical ctxB gene is still an unsolved mystery but it is clear that a cryptic change in the cholera scenario has taken place and the consequences are still being evaluated from the viewpoint of clinical appearance. This subtle genetic change in V. cholerae could result in some alterations in clinical manifestations of cholera. In future, this observation will be helpful to understand the epidemiology of cholera in a more precise way. Funding: All the research works of this manuscript have been performed in the National Institute of Cholera and Enteric Diseases (Kolkata, India) with the support of institutional funding. Competing interests: None declared. Ethical approval: Not required.
References [1] Safa A, Sultana J, Dac Cam P, Mwansa JC, Kong RY. Vibrio cholerae O1 hybrid El Tor strains, Asia and Africa. Emerg Infect Dis 2008;14:987–8. [2] Ramamurthy T, Garg S, Sharma R, Bhattacharya SK, Nair GB, Shimada T, et al. Emergence of novel strain of Vibrio cholerae with epidemic potential in southern and eastern India. Lancet 1993;341:703–4. [3] Morita M, Ohnishi M, Arakawa E, Bhuiyan NA, Nusrin S, Alam M, et al. Development and validation of a mismatch amplification mutation PCR assay to monitor the dissemination of an emerging variant of Vibrio cholerae O1 biotype El Tor. Microbiol Immunol 2008;52:314–17. [4] Hall RM, Stokes HW. Integrons: novel DNA elements which capture genes by site-specific recombination. Genetica 1993;90:115–32. [5] Ansaruzzaman M, Bhuiyan NA, Nair GB, Sack DA, Lucas M, Deen JL, et al. Cholera in Mozambique, variant of Vibrio cholerae. Emerg Infect Dis 2004;10: 2057–9.
Mayukh Das Texas A&M University, College Station, TX, USA Abhishek Jaiswal Subharthi Pal National Institute of Cholera and Enteric Diseases, Kolkata, India Tushar Suvra Bhowmick Texas A&M University, College Station, TX, USA Amit Ghosh National Institute of Cholera and Enteric Diseases, Kolkata, India A.K. Goel Defence Research and Development Establishment, Gwalior, India B.L. Sarkar ∗ National Institute of Cholera and Enteric Diseases, Kolkata, India ∗ Corresponding
author. Present address: Vibrio Phage Reference Laboratory, Division of Bacteriology, National Institute of Cholera and Enteric Diseases, P-33, C. I. T. Road, Scheme XM, Beliaghata, Kolkata 700 010, India. Tel.: +91 33 2370 1176; fax: +91 33 2370 5066. E-mail address: bl [email protected] (B.L. Sarkar) 3 August 2012 doi:10.1016/j.ijantimicag.2012.08.005
Spread of the blaIMP-13 gene in French Pseudomonas aeruginosa through sequence types ST621, ST308 and ST111 Sir, IMP-13 is a class B metallo--lactamase (MBL) that was first described in a carbapenem-resistant clinical isolate of Pseudomonas aeruginosa in Italy in 2001 [1]. Since then, IMP-13-producing P. aeruginosa have been implicated in significant outbreaks in Italy
Letters to the Editor / International Journal of Antimicrobial Agents 40 (2012) 562–573
been used in vitro for efficacy testing against Arcobacter, but a few other members of the -lactams such as cefoperazone, cefuroxime sodium and cefalothin were tested and failed to produce a desirable action against Arcobacter [5]. In the current study, a low level of resistance in A. butzleri isolates to gentamicin (M.I.C.E., 26.1%; disk diffusion, 19.6%) and ciprofloxacin (M.I.C.E., 21.7%; disk diffusion, 19.6%) was recorded. Resistance to ciprofloxacin shown by A. butzleri and Arcobacter cryaerophilus is primarily due to mutation in the Thr85 residue of GyrA [1]. Tetracycline showed the highest efficacy, with only 6.5% (M.I.C.E.) and 8.7% (disk diffusion) of A. butzleri isolates found to be resistant. Previous studies have also reported tetracycline as the most effective drug and thus have suggested its usage in the treatment of human and animal infections caused by Arcobacter spp. [2]. Multidrug resistance (resistance to three or more antibiotics) is of major concern, limiting the therapeutic choice of antibiotics and dose calculations. A total of 41.3% (M.I.C.E) and 34.7% (disk diffusion) of A. butzleri isolates were found to be multidrug-resistant. Regarding the antibiotic resistance patterns, 19.6% of isolates were resistant to ampicillin, erythromycin and cefotaxime, whereas 8.6% of isolates were resistant to ampicillin, cefotaxime and gentamicin. Genetic elements such as plasmids, transposons, multidrug efflux pumps and integrons have remained involved in the evolution and dissemination of multidrug resistance in bacteria [2]. Disks and automated systems with truncated MIC ranges are adequate for most routine susceptibility testing, but precise values are needed in order to set the dosage for infections caused by drug-resistant pathogens. MICs of antimicrobial agents were determined for A. butzleri and results were expressed as the MIC50 and MIC90 values. The lowest MIC50 and MIC90 values were recorded for tetracycline (0.25/8 g/mL) and the highest were for ampicillin (32/256 g/mL) (Table 1). Comparative results of M.I.C.E. and disk diffusion methods based on correlation (SPSS v.20; SPSS Inc., Chicago, IL) showed a very high relationship for cefotaxime and ciprofloxacin and a high relationship for ampicillin, tetracycline and gentamicin; however, a moderate correlation was observed for erythromycin (Table 1). Differences in results between the two methods could be due to lack of a specific protocol and breakpoints for Arcobacter spp. Arcobacter butzleri was found to be highly sensitive to tetracycline, thus it is recommended as the first choice in the treatment of Arcobacter infections. Either of the antimicrobial sensitivity methods can be used for in vitro susceptibility testing, but application of M.I.C.E. is advantageous in terms of quantitative results, which are useful to select the proper antibiotic and to estimate the dose of drugs against Arcobacter. Funding: The authors are indebted to Sindh Agriculture University, Tandojam (Pakistan) for a PhD grant (2008–2011) to AHS as well as to Universiti Putra Malaysia (RUGS Project No. 01-0111-1125RU). The authors are also indebted to the World Bank for providing financial support to AHS under the Robert S. McNamara Fellowship (2012) programme. Competing interests: None declared. Ethical approval: Not required. References [1] Shah AH, Saleha AA, Zunita Z, Murugaiyah M. Arcobacter—an emerging threat to animals and animal origin food products? Trends Food Sci Technol 2011;22:225–36. [2] Collado L, Figueras MJ. Taxonomy, epidemiology, and clinical relevance of the genus Arcobacter. Clin Microbiol Rev 2011;24:174–92. [3] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; twentieth informational supplement. Document M100-S20. Wayne, PA: CLSI; 2010.
[4] Clinical and Laboratory Standards Institute. Methods for antimicrobial dilution and disc susceptibility testing of infrequently isolated or fastidious bacteria; approved guideline. Document M45-A. Wayne, PA: CLSI; 2006. [5] Atabay HI, Aydin F. Susceptibility of Arcobacter butzleri isolates to 23 antimicrobial agents. Lett Appl Microbiol 2001;33:430–3.
A.H. Shah ∗ Department of Animal Products Technology, Sindh Agriculture University, Tandojam 70060, Pakistan A.A. Saleha ∗ Z. Zunita M. Murugaiyah A.B. Aliyu Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia ∗ Corresponding
authors. Tel.: +60 389 463 458; fax: +60 389 471 972. E-mail addresses: vetdr [email protected] (A.H. Shah), [email protected] (A.A. Saleha) 24 March 2012 doi:10.1016/j.ijantimicag.2012.08.001
Dynamics of classical–El Tor switch of Vibrio cholerae strains isolated from 1961–2010 Sir, Vibrio cholerae O1 and O139 are the serogroups mainly responsible for the disease cholera. Vibrio cholerae isolates belonging to the O1 serogroup are further classified into two biotypes, namely classical and El Tor [1]. The year 1992 is a milestone in the history of cholera as it was the year when V. cholerae O139 emerged in Chennai, India [2] and subsequently in other parts of the globe. This study promoted us to monitor how far this variation in the ctxB gene has been incorporated into the representative V. cholerae O1 isolates from different parts of the world from 1961–2010. Among the two cholera toxin genes (ctxA and ctxB), ctxB has received much more attention owing to the presence of genetic variability within it. Recently, three variants of V. cholerae O1 biotype El Tor were reported, namely Matlab variants (mix of both classical and El Tor traits), Mozambique variants (typical El Tor genome, with a tandem repeat of classical CTX prophage) and altered El Tor variants (typical El Tor biotype with El Tor CTX prophage but producing classical cholera toxin) [3]. Integrons (not autonomously mobile) are an important mechanism to capture, integrate and express resistance gene cassettes in their variable region in many bacteria. Among the three classes of integrons, class 1 integrons are most prevalent among clinical isolates and consist of two conserved segments [the 5 conserved segment (5 -CS) and the 3 conserved segment (3 -CS)] and an internal variable region that contains gene cassettes encoding antibiotic resistance determinants [4]. The 5 -CS encodes the type I integrase gene (intI1) and the recombination site attI1; the 3 -CS encodes the antiseptic resistance gene qacE1 and the sulfonamide resistance gene sul1. To monitor the effect of genetic variation on phenotypic traits, 256 clinical isolates collected worldwide spanning a period of 50 years (1961–2010) were analysed by mismatch amplification mutation assay PCR (MAMA-PCR) (ctxB), simplex PCR (class 1 integron, ctxA and wbe) and a phage typing technique. MAMA-PCR results clustered all the isolates into three groups according to the presence of classical and El Tor ctxB genes: CT-B I, isolates with both classical and El Tor ctxB; CT-B II, isolates with only El Tor ctxB; and CT-B III, isolates with only classical ctxB. Phage typing and
Letters to the Editor / International Journal of Antimicrobial Agents 40 (2012) 562–573
571
Fig. 1. Graphical distribution of Vibrio cholerae O1 biotype El Tor isolates based on heterogeneity of the ctxB gene determined by mismatch amplification mutation assay PCR (MAMA-PCR), by year.
antimicrobial susceptibility results demonstrated that all of the isolates were sensitive to El Tor group V phage, resistant to classical group IV phage and resistant to polymyxin B, which concluded the fact that all V. cholerae isolates incorporated in this study belonged to O1 biotype El Tor. The graphical presentation of MAMA-PCR results in Fig. 1 clustered V. cholerae isolates from Chennai, India, during 1980–2010 into four periods: Period I, 1980–1984; Period II, 1985–1991; Period III, 1992; and Period IV, 1993–2010. These periods reflected predominance of the CT-B I variant, a mosaic combination both of CT-B I and II variants, appearance of CT-B III along with the CT-B II variant, and uninterrupted prevalence of the CT-B III variant, respectively. Among these 256 isolates 100 representative isolates were selected to confirm the presence and absence of class 1 integron by PCR amplification of the 3 -CS. Of the 100 V. cholerae isolates, 60 were found to be positive for 3 -CS; no 3 -CS amplification was found within the 40 V. cholerae isolates from 1961–1969. Phage typing of each variant (CT-B I, II and III) showed a similar typing pattern dominated by type 27 and followed by types 24 and 26. It was clearly found from these data that the influence of the classical biotype still remained incessantly in the isolates from Chennai up to 1984. Interestingly, this influence of the classical biotype became sporadic with the appearance of typical El Tor variants (CT-B II) from the year 1961 and finally disappeared in 1991. This study clarifies that V. cholerae O139 has changed the cholera scenario following its emergence in 1992 in Chennai. Since the onset of O139 (in 1992), we found altered El Tor variants and from the year 1993 it was still continuing. These findings are in accordance with previous reports [5] which postulated that the classical cholera toxin gene has reappeared but its carrier has been El Tor, and this study showed that its first appearance was in 1966. MAMA-PCR and class 1 integron and phage typing results concluded that the genotypic variation, such as polymorphic traits in ctxB genes, did not influence the antibiotic susceptibility and phenotypic behaviour of V. cholerae O1 biotype El Tor strains. The pathogenic effect of El Tor vibrios harbouring the classical ctxB gene is still an unsolved mystery but it is clear that a cryptic change in the cholera scenario has taken place and the consequences are still being evaluated from the viewpoint of clinical appearance. This subtle genetic change in V. cholerae could result in some alterations in clinical manifestations of cholera. In future, this observation will be helpful to understand the epidemiology of cholera in a more precise way. Funding: All the research works of this manuscript have been performed in the National Institute of Cholera and Enteric Diseases (Kolkata, India) with the support of institutional funding. Competing interests: None declared. Ethical approval: Not required.
References [1] Safa A, Sultana J, Dac Cam P, Mwansa JC, Kong RY. Vibrio cholerae O1 hybrid El Tor strains, Asia and Africa. Emerg Infect Dis 2008;14:987–8. [2] Ramamurthy T, Garg S, Sharma R, Bhattacharya SK, Nair GB, Shimada T, et al. Emergence of novel strain of Vibrio cholerae with epidemic potential in southern and eastern India. Lancet 1993;341:703–4. [3] Morita M, Ohnishi M, Arakawa E, Bhuiyan NA, Nusrin S, Alam M, et al. Development and validation of a mismatch amplification mutation PCR assay to monitor the dissemination of an emerging variant of Vibrio cholerae O1 biotype El Tor. Microbiol Immunol 2008;52:314–17. [4] Hall RM, Stokes HW. Integrons: novel DNA elements which capture genes by site-specific recombination. Genetica 1993;90:115–32. [5] Ansaruzzaman M, Bhuiyan NA, Nair GB, Sack DA, Lucas M, Deen JL, et al. Cholera in Mozambique, variant of Vibrio cholerae. Emerg Infect Dis 2004;10: 2057–9.
Mayukh Das Texas A&M University, College Station, TX, USA Abhishek Jaiswal Subharthi Pal National Institute of Cholera and Enteric Diseases, Kolkata, India Tushar Suvra Bhowmick Texas A&M University, College Station, TX, USA Amit Ghosh National Institute of Cholera and Enteric Diseases, Kolkata, India A.K. Goel Defence Research and Development Establishment, Gwalior, India B.L. Sarkar ∗ National Institute of Cholera and Enteric Diseases, Kolkata, India ∗ Corresponding
author. Present address: Vibrio Phage Reference Laboratory, Division of Bacteriology, National Institute of Cholera and Enteric Diseases, P-33, C. I. T. Road, Scheme XM, Beliaghata, Kolkata 700 010, India. Tel.: +91 33 2370 1176; fax: +91 33 2370 5066. E-mail address: bl [email protected] (B.L. Sarkar) 3 August 2012 doi:10.1016/j.ijantimicag.2012.08.005
Spread of the blaIMP-13 gene in French Pseudomonas aeruginosa through sequence types ST621, ST308 and ST111 Sir, IMP-13 is a class B metallo--lactamase (MBL) that was first described in a carbapenem-resistant clinical isolate of Pseudomonas aeruginosa in Italy in 2001 [1]. Since then, IMP-13-producing P. aeruginosa have been implicated in significant outbreaks in Italy