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Characterization of Vibrio cholerae from 1986 to 2012 in Yunnan Province, southwest China bordering Myanmar
Infection, Genetics and Evolution 21 (2014) 1–7
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Characterization of Vibrio cholerae from 1986 to 2012 in Yunnan Province, southwest China bordering Myanmar Wenpeng Gu a, Jianwen Yin a, Jianbin Yang a, Chaoqun Li a, Yujuan Chen a, Jie Yin a, Wen Xu a, Shiwen Zhao a, Junrong Liang b, Huaiqi Jing b, Xiaoqing Fu a,⇑ a
Department of Acute Infectious Diseases Control and Prevention, Yunnan Provincial Centre for Disease Control and Prevention, 650022 Kunming, China National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory for Infectious Disease Prevention and Control, 102206 Beijing, China b
a r t i c l e
i n f o
Article history: Received 18 July 2013 Received in revised form 18 October 2013 Accepted 18 October 2013 Available online 28 October 2013 Keywords: Vibrio cholerae El Tor variants Pulse-field gel electrophoresis Imported strains
a b s t r a c t Vibrio cholerae is an important infectious pathogen causing serious human diarrhea. We analyzed 568 V. cholerae strains isolated from 1986 to 2012 in Yunnan province, southwest China bordering Myanmar. Polymerase chain reactions for detecting virulence genes, antibiotic susceptibility tests and pulse-field gel electrophoresis (PFGE) were performed. The results showed all the strains were El Tor biotype from 1986. The ctxB subunit sequence analysis for all strains have shown that cholera between 1986 and 1995 was associated with mixed infections with El Tor and El Tor variants, while infections after 1996 were all caused by El Tor variant strains. All of the strains were sensitive to aminoglycosides and quinolone antibiotics while resistant to b-lactamase and carbapenem antibiotics increased gradually. 568 V. cholerae were divided into 218 PFGE-NotI patterns, and the isolates before 2001 and after 2011 were separated into two groups according to PFGE results. The strains isolated before 2001 were mainly referred to native cholera in Yunnan, and after 2011 were primarily referred to as imported strains from Myanmar, which showed the variation of V. cholerae in this area. The molecular characteristics of V. cholerae indicated regularity in bacterial variation and evolution in Yunnan province. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Vibrio cholerae is an important entero-infectious pathogen, causing serious human diarrhea and having severe effects on human society and economic development (Kaper et al., 1995). At present, seven cholera pandemics have occurred in the world; it is generally considered the seventh pandemic began in 1961, was caused by the El Tor biotype, and is still ongoing (Faruque et al., 1998). Asia is the major epidemic focus of cholera in the world, especially Southeast Asia (Kaper et al., 1995). Several studies show the V. cholerae El Tor variants (harboring the classical cholera toxin) gradually became the predominant strains all over the world, e.g. in Asia, Africa, and America (Alam et al., 2010; Ceccarelli et al., 2011; Mohamed et al., 2012; Nair et al., 2006; Safa et al., 2006).
Yunnan located in southwest China, bordering Myanmar, Vietnam and Laos, has an extended frontier, and being one of the minority habitations in China. From our continuous surveillance for V. cholerae, several cholera outbreaks occurred in Yunnan since 1986. Because of its special location, it was very important to study the molecular characteristics of V. cholerae in this area. Pulse-field gel electrophoresis (PFGE) is considered as the ‘‘golden standard’’ for pathogen molecular typing, and shows high discriminatory power for epidemiology investigations (Cameron et al., 1994). Our research was based on the PFGE method including virulence gene identification, and susceptibility tests; and was performed to analyze the characteristics of V. cholerae in Yunnan Province to investigate the molecular features of the isolates in this area.
2. Materials and methods Abbreviations: V. cholerae, Vibrio cholerae; PFGE, pulse-field gel electrophoresis; RT-PCR, real-time polymerase chain reaction. ⇑ Corresponding author. Address: Department of Acute Infectious Diseases Control and Prevention, Yunnan Provincial Centre for Disease Control and Prevention, Dongsi Street 158, 650022 Kunming, China. Tel./fax: +86 0871 63632033. E-mail address: [email protected] (X. Fu). 1567-1348/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2013.10.015
2.1. Bacterial strains 568 V. cholerae strains were isolated and stored in our laboratory from 1986 to 2012; all the strains were O1 serogroup except three O139 isolates. 436 isolates were Ogawa serotype, and 129
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Fig. 1. The location of Yunnan province and its neighboring nations or provinces. The cholera happened in different years and counties of Yunnan in the history were shown with red star. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
were Inaba. 429 strains were isolated from patients, 77 from water (contaminated water or monitoring water), and the other 62 were isolated from the environment (soil or surface of objects) (Table 1). 2.2. Resuscitation and confirmation of V. cholerae strains All the isolates were stored at 80 °C in glycerin broth. The bacteria were inoculated on Brain Heart Infusion agar (Oxoid, UK) and incubated at 37 °C for 24 h. Slide agglutination test (National Institute for the Control of Pharmaceutical and Biological Products, China) was performed to confirm the serogroup of the strains, and reconfirmed by PCR test for rfb genes of O1 and O139 serotypes. The phenotypic tests: hemolysis test, susceptibility to polymyxin B and Voges–Proskauer test were analyzed for biotype investigation. The biotype methods were all performed at 37 °C for 24 h, and the API20E (Biomerieux, France) was used for Voges–Proskauer reaction. 2.3. Antibiotic susceptibility The antimicrobial susceptibility of all the V. cholerae strains was determined using the disc diffusion method on Mueller–Hinton
agar (MH). The antibiotics (Oxoid, UK) used were: ampicillin, amoxicillin/clavulanic acid, piperacillin, cefazolin, ceftazidime, ceftriaxone, cefepime, aztreonam, ertapenem, imipenem, meropenem, amikacin, gentamicin, ciprofloxacin, levofloxacin, tetracycline, nitrofurantoin, and trimethoprim/sulfamethoxazole. ATCC strain 25922 (Escherichia coli) served as the quality control. The results were interpreted based on the Clinical and Laboratory Standards Institute (CLSI version 2011). 2.4. PCR detection of virulence genes and ctxB sequencing Genomic DNA was extracted from each isolate using a DNA extraction kit (Tiangen, Beijing) according to the manufacturers’ instructions. The presences of rfb genes for O1 and O139 serotypes were determined. The primers used were: rfbO1 forward (50 -GGAAT AACTCAAGGCGATGAAGTG-30 ) and reverse (50 -TAGAGACTCACCT TCGATTTCAGC-30 ); rfbO139 forward (50 - CGATGGCGTGTTCATTAG AAGG-30 ), and reverse (50 - TCCCTTTCCACCTCGGTATTTC-30 ). SYBR premix Taq (TaKaRa, Japan) was used, and the amplification conditions were 95 °C for 30 s followed by 40 cycles: 95 °C for 5 s and 60 °C for 20 s. The virulence genes for ctxAB, ompU, ace, zot, toxR, rtxC, rstR (Classical/El Tor) and tcpA (Classical/El Tor) were
Table 1 The distribution and information of strains and PCR results for virulence genes in this study. Years
Isolated counties
Serotype
Source
PCR results
Ogawa
Inaba
O139
Patient
Water
Environment
rstR
tcpA
Rugose numbers
Changes of resistant antibiotics
AM(76),AMC(72),PIP(32),CZ(10),CAZ(14),FEP(63),ATM(55),IPM,CRO(6) AM(3),AMC(2),PIP(2),CAZ(1),ATM(2),IPM,MEM(2) AM,AMC(2),PIP(3),CZ(3),FEP(2),IPM
Gengma Yongde Ruili
– – –
86 4 5
– – –
74 3 3
11 1 1
1 – 1
ET(76)/ET,CL(10) ET,CL ET,CL
ET ET ET
18 1 3
1989
Gengma Ruili
4 16
3 2
– –
5 12
1 2
1 4
ET ET(14)/ET,CL(4)
ET ET
5 2
AM(6),AMC(3),PIP(2),CAZ,FEP,ATM,IPM,CRO(3),MEM(2) AM,AMC(5),PIP(3),CZ(7),CAZ,FEP,ATM,IPM
1991
Gengma Ruili Yuanmou
11 10 –
2 1 2
– – –
13 11 2
– – –
– – –
ET(8)/ET,CL(5) ET(4)/ET,CL(7) ET(1)/ET,CL(1)
ET ET ET
3 1 0
AM(9),AMC,PIP(6),CZ(5),CAZ,FEP,ATM,IPM,CRO(5) AM,AMC,CAZ,FEP,ATM,IPM,MEM(7) AM,AMC,PIP(1),CZ,FEP,ATM,IPM
Gengma Ruili Yuanmou Jinghong Cangyuan Dali Longchuan Mangshi Longling Yongshan
23 20 8 40 5 21 5 23 7 11
2 2 1 1 – 2 1 – – 3
– – – – – – – – – –
22 21 7 31 5 18 5 12 5 10
3 – – 9 – 5 1 11 2 4
– 1 2 1 – – – – – –
ET(2)/ET,CL(23) ET(2)/ET,CL(20) ET,CL ET,CL ET,CL ET,CL ET,CL ET,CL ET,CL ET,CL
ET ET ET ET ET ET ET ET ET ET
10 6 2 1 3 5 0 2 2 5
1997
Yuanmou Wuding
2 –
5 4
7 4
– –
– –
ET,CL ET,CL
ET ET
1 0
AM,AMC,PIP,FEP,ATM,IPM,CRO(3),MEM(3)TC,NIT,SXT AM,AMC,PIP,CZ(2),CAZ(2),FEP,ATM,IPM,TC,NIT,SXT
1998
Ruili Yuanmou Mangshi Yanshan Guangnan
21 22 21 15 25
– 1 – – –
3 – – – –
19 19 17 13 22
– – – – –
5 4 4 2 3
ET(3)/ET,CL(21) ET,CL ET,CL ET,CL ET,CL
ET ET ET ET ET
1 2 0 0 0
AM,AMC,PIP(7),CZ(7),CAZ(9),FEP(9),ATM,IPM,MEM(5),TC,NIT,SXT AM,AMC,PIP,CZ(6),CAZ(6),FEP(4),ATM,IPM,CRO(7),TC,NIT,SXT AM,AMC,PIP(11),CZ(9),CAZ(7),ATM,IPM,MEM(7),TC,NIT,SXT AM,AMC,PIP,CZ,FEP,ATM,IPM,TC,NIT,SXT AM,AMC,PIP,ATM,IPM,CRO(2),TC,NIT,SXT
1999
Dali Gejiu Yuanyang Kunming
8 13 23 1
– – – –
– – – –
6 5 12 1
2 8 11 –
– – – –
ET,CL ET,CL ET,CL ET,CL
ET ET ET ET
1 4 4 0
AM,AMC,CZ,CAZ(3),FEP(1),ATM,IPM,TC,NIT,SXT AM,AMC,PIP,CZ,ATM,IPM,MEM(5),TC,NIT,SXT AM,AMC,PIP,CZ,CAZ(8),FEP(8),ATM,IPM,CRO(8),ETP(3),TC,NIT,SXT AM,AMC,CZ,FEP,ATM,IPM,TC,NIT,SXT
2001
Yuanmou Mangshi
6 65
– 2
–
5 34
1 –
– 33
ET,CL ET,CL
ET ET
2 12
2011
Ruili Mangshi Ruili
2 7 1
– – –
– – –
2 3 1
– 4 –
– – –
ET,CL ET,CL ET,CL
ET ET ET
0 0 0
1994 1995
1996
2012
AM,AMC,PIP(11),CZ(7),CAZ(9),FEP(11),ATM(11),IPM,CRO(8),TC,NIT,SXT AM,AMC,PIP(7),CZ(4),CAZ(9),FEP(7),ATM(9),IPM,MEM(5),TC,NIT,SXT AM,AMC,CZ(2),FEP(3),IPM,TC,NIT,SXT AM,AMC,PIP(17),CAZ(11),FEP(11),ATM(17),IPM,CRO(5),MEM(6),TC,NIT,SXT AM,AMC,PIP,IPM,TC,NIT,SXT AM,AMC,PIP(8),CZ(8),CAZ(7),FEP(8),ATM(8),IPM,TC,NIT,SXT AM,AMC,PIP,IPM,TC,NIT,SXT AM,AMC,PIP(5),CZ(11),FEP(9),ATM(9),IPM,TC,NIT,SXT AM,AMC,PIP,ATM,IPM,TC,NIT,SXT AM,AMC,PIP,CZ(8),CAZ(8),FEP,ATM,IPM,CRO(4),MEM(7),TC,NIT,SXT
AM,AMC,PIP,CZ,CAZ,FEP,ATM,IPM,TC,NIT,SXT AM,AMC,PIP,CZ,CAZ,FEP,ATM,IPM,CRO(10),MEM(8),TC,NIT,SXT AM,AMC,PIP,CZ,CAZ,FEP,ATM,IPM,TC,NIT,SXT AM,AMC,PIP,CZ,CAZ,FEP,ATM,IPM,TC,NIT,SXT AM,AMC,PIP,CZ,CAZ,FEP,ATM,IPM,TC,NIT,SXT
W. Gu et al. / Infection, Genetics and Evolution 21 (2014) 1–7
1986
Note: ET, El Tor biotype; CL, Classical biotype. The numbers in the bracket represented the strains possessed the ET or ET, CL alleles in different counties and years, no bracket represented all the isolates had the same allele. AM, ampicillin; AMC, amoxicillin/clavulanic acid; PIP, piperacillin; CZ, cefazolin; CAZ, ceftazidime; FEP, cefepime; ATM, aztreonam; IPM, imipenem; CRO, ceftriaxone; ETP, ertapenem; MEM, meropenem; TC, tetracycline; NIT, nitrofurantoin; SXT, trimethoprim/sulfamethoxazole. The numbers in the bracket represented the resistant strain numbers for each antibiotic; otherwise, no bracket represented all the isolates were resistant.
3
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Fig. 2. The ctxB subunit sequencing results for all the isolates in Yunnan. Three isolates in 1995 and four in 1998 could not amplify in the study.
amplified using Taq premix (TaKaRa, Japan), the primers and amplification procedures were as described previously (Chow et al., 2001; O’Shea et al., 2004; Singh et al., 2002, 2001). All of the strains were sequenced for ctxB gene subunit to further identify the characters of the isolates, Taq premix (TaKaRa, Japan) was used as described above, and amplification processes were performed as previously described (Goel et al., 2010). The amplification products were sent for bidirectional sequencing (TaKaRa, Japan), and the results were analyzed using DNAStar and MEGA4 software. The ctxB sequences of N16961 of El tor V. cholerae (Genebank: NC-002505) and O395 Classical strain (Genebank: NC-012582) were used as the standards for comparison. 2.5. PFGE PFGE was performed based on the PulseNet protocol for V. cholerae and procedures described previously (Cameron et al., 1994). The enzyme digestion for each plug was NotI 40U at 37 °C for 4 h and SfiI 50U at 50 °C for 4 h. The CHEF-Mapper (Bio-Rad) was used for electrophoresis, and the pulse time ranged from 1 to 20 s for 13 h, and 20 to 25 s for 6 h. The gel was stained using Gelred (Biofilm) and visualized using the gel imaging system (Bio-Rad, Gel DocXR). PFGE patterns were analyzed with BioNumerics version 5.10, and a dendrogram was produced using the Dice coefficient and un-weighted pair group method with arithmetic mean algorithm (UPGMA). A pairwise distance matrix and a minimum spanning tree were also created. 3. Results 3.1. Phenotypic characteristics of the strains 568 V. cholerae were isolated from 1986 to 2012, in Gengma, Yongde, Ruili, Yuanmou and Jinghong, etc. The outbreaks occurred at intervals of one or two years between 1986 and 2001; then the frequency of cholera diminished after 2001. There were two imported cases from Myanmar in 2011 and one in 2012, both of the strains were isolated in Ruili. In Fig. 1 each county in which cholera was observed in marked with a red star and the year of its occurrence, and the information of the isolates involved in our study were shown in Table 1. All the strains were O1 serogroup except three that were O139 serotype, and the RT-PCR results for rfb genes of O1 and O139 were
consistent with the phenotypic assays. Serotype Inaba was the predominant type before 1995; the Ogawa serotype became the major type after 1995. A portion of the isolates expressed hemolysis and were sensitive to polymyxin B, while others were not; no regularity was found. However, the Voges–Proskauer test for all the strains was positive. In the process of our study, we found a portion of the isolates assumed the rugose survival form when inoculated on nonselective agars (Table 1). 3.2. Susceptibility test All the isolates were sensitive to gentamicin, ciprofloxacin, and levofloxacin, regardless of the year isolated, county, or source. Most of the V. cholerae were sensitive to ceftriaxone, ertapenem, and meropenem, while completely drug resistant isolates were seldom found. Some of the isolates were sensitive to ampicillin, amoxicillin/clavulanic acid, piperacillin, cefazolin, ceftazidime, cefepime, aztreonam, and imipenem and the proportion of resistant strains to these antibiotics was increased gradually. The strains isolated from 1986 to 1994 were sensitive to tetracycline, nitrofurantoin, and trimethoprim/sulfamethoxazole; after 1995, all of the isolates were resistant to these antibiotics (Table 1). 3.3. PCR test for virulence genes and ctxB sequencing The ctxAB, ompU, ace, zot, toxR, and rtxC for all of the isolates were positive; tcpAEl Tor was positive for all of the isolates as well, while tcpAClassical was negative. For the rstR, most of the strains carried rstREl Tor and rstRClassical, however, some of the strains possessed only rstREl Tor (Table 1). The results showed mixed infection with El Tor type (harboring ctxBEl Tor) and El Tor variant strains (harboring ctxBClassical) before 1995; after 1996 all of the isolates harbored the ctxBClassical except three O139 V. cholerae that possessed ctxBEl Tor. The percentage of El Tor type strains decreased, while the El Tor variants gradually increased over time, and all the strains became El Tor variants after 1996 for O1 serogroup (Fig. 2). 3.4. PFGE The 568 V. cholerae were divided into 218 PFGE-NotI patterns and formed two major groups in the dendrogram (Fig. 3). The PFGE-NotI patterns for strains isolated from 1986 to 2001 formed
W. Gu et al. / Infection, Genetics and Evolution 21 (2014) 1–7
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Fig. 3. PFGE-NotI dendrogram for representative V. cholerae in this study.
group A, and the patterns for strains isolated in 2011 and 2012 and the O139 serotypes from 1998 formed group B. The same molecular typing characteristic was also found using PFGE-SfiI. The same cluster result was shown with minimum spanning tree based on PFGE results (Fig. 4). Two major cluster groups were also identified,
group A in the pink area contained isolates collected before 2001, and group B in the green area contained O1 serogroup strains from 2011 and 2012 and all O139 strains in 1998. PFGE-SfiI was performed for strains possessed the same PFGENotI patterns. Some of the strains isolated from different counties
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Fig. 4. The minimum spanning tree of V. cholerae in Yunnan based on PFGE results. Group A in the pink area: isolates before 2001; group B in the green area: isolates after 2011 and all the O139 isolates. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. The distribution of V. cholerae showing identical PFGE-NotI and SfiI patterns. The pattern with the square frame was the imported strain in Ruili.
and years showed the same PFGE-NotI and SfiI patterns, e.g. Gengma in 1995, Yanshan in 1998, Yuanyang in 1999, and Mangshi in 2001; compared to Yuanmou in 1994, Dali and Gengma in 1995, Yongshan in 1996, Mangshi in 1998, and Gejiu in 1999; Mangshi in 1998 and 2001, Ruili in 1989 and Yuanyang in 1999 (Fig. 5A–C). The bacteria isolated from different counties but the same years also had the same PFGE-NotI and SfiI patterns, e.g. Dali, Jinghong, Longchuan and Ruili in 1995; and Yanshan, Guangnan and Ruili in 1998; Gejiu and Yuanyang in 1999 (Fig. 5D–F). Furthermore, the identical result was reconfirmed that strains isolated from patients and water or environment had the close relations, in Fig. 5. A–G, the same PFGE patterns were found from different source, in different counties and years, showed the identical similarities.
4. Discussion In our study, the results showed all the strains possessed the rtxC and tcpAEl Tor genes, and the tcpAClassical was not found in all
the isolates; therefore, 568 V. cholerae isolated from 1986 to 2012 in Yunnan were all El Tor biotype. Most of the strains had the rstREl Tor and rstRClassical genes, while some had only one of them as shown in other studies (Alam et al., 2010). The distribution of the rstREl Tor was mainly referred to Gengma and Ruili from 1986 to 1995, and after 1996 all the isolates had rstREl Tor and rstRClassical, regardless of which county and year it was collected. In general, V. cholerae in Yunnan were totally sensitive to aminoglycosides and quinolone antibiotics. While, in the earlier years of surveillance, some of the strains were sensitive to b-lactamase and carbapenem antibiotics, and the proportion of resistant strains to these antibiotics increased in recent years. For tetracycline and trimethoprim/sulfamethoxazole, all the isolates were sensitive before 1994, and after 1995 all the strains were resistant. These changes in the resistance pattern in Yunnan reflected the antibiotics used in this area a period, the antibiotics resistance has become a serious problem although completely drug resistant isolates were seldom found. Goel et al. (2010) showed the strains were sensitive to b-lactamase, quinolone and tetracycline antibiotics in India, 2004; Tran et al. (2012) found that the V. cholerae in Vietnam from
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2007 to 2010 were sensitive to b-lactamase and quinolone antibiotics, resistant to tetracycline and trimethoprim/sulfamethoxazole; Sjolund-Karlsson et al. (2011) demonstrated the Haitian V. cholerae strains were sensitive to tetracycline, but were resistant to quinolones, aminoglycosides, and trimethoprim/sulfamethoxazole in 2010. There was no similarity for bacterial antibiotic susceptibility based on these studies. In recent studies, the El Tor variants (harboring classical cholera enterotoxin) have become widely distributed all over the world, especially in Asia. V. cholerae El Tor variants became the predominate strains and were isolated in India in 2004 (Goel et al., 2010), Bangladesh since 2001 (Nair et al., 2006), Vietnam in 2007 to 2008 (Nguyen et al., 2009), Thailand from 2007 to 2010 (Okada et al., 2012), Malaysia in 2009 (Ang et al., 2010), Mexico since 1991 (Alam et al., 2010), and Angola in 2006 (Ceccarelli et al., 2011). Use of ctxB sequencing, showed the El Tor variants appeared early in 1986 in Yunnan, southwest China, and substituted for the typical El Tor biotypes after 1996. The strains after 1996 were all El Tor variants, except O139 isolates that had the typical El Tor cholera enterotoxin in 1998. This possibly indicates the close evolutionary relations within the El Tor biotypes. The variation of the El Tor biotype changes reflected in our study possibly represented the features of V. cholerae in Yunnan province, and the El Tor variants had existed early in 1986 in southwest of China. In our study, the strains isolated from patients and environment or water shown the high similarity (Figs. 3 and 5) based on PFGE two enzyme digestion results, indicated the close transmission relations of these bacteria. The variation of V. cholerae in Yunnan was divided into two groups based on PFGE results (Figs. 3 and 4), and suggested the different source of the pathogen transmitted in this area. The strains before 2001 formed group A, and the isolates after 2011 and three O139 strains in 1998 formed group B. Furthermore, the cholera isolates in Yunnan showed the same PFGE patterns with the imported strain from Myanmar in 2011 (Fig. 5G), indicated the close transmitted relationship between two nations at the frontier. Therefore, it was very important to enhance the surveillance in the bordering areas in Yunnan. 5. Conclusions The 568 V. cholerae isolated from 1986 to 2012 in Yunnan Province, southwest China were El Tor biotype, and the typical El Tor biotype was later replaced by the El Tor variant strains. The PFGE patterns of the isolates also changed and divided into two major groups as shown during the surveillance years, and indicated regularity in bacterial variation and evolution. Acknowledgments This work was supported by National Sci-Tech key project (2012ZX10004-201, 2012ZX10004-212 and 2013ZX10004-203002). We thank Dr. Jim Nelson for critical reading of our manuscript.
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References Alam, M., Nusrin, S., Islam, A., Bhuiyan, N.A., Rahim, N., Delgado, G., Morales, R., Mendez, J.L., Navarro, A., Gil, A.I., Watanabe, H., Morita, M., Nair, G.B., Cravioto, A., 2010. Cholera between 1991 and 1997 in Mexico was associated with infection by classical, El Tor, and El Tor variants of Vibrio cholerae. J. Clin. Microbiol. 48, 3666–3674. Ang, G.Y., Yu, C.Y., Balqis, K., Elina, H.T., Azura, H., Hani, M.H., Yean, C.Y., 2010. Molecular evidence of cholera outbreak caused by a toxigenic Vibrio cholerae O1 El tor variant strain in Kelantan, Malaysia. J. Clin. Microbiol. 48, 3963–3969. Cameron, D.N., Khambaty, F.M., Wachsmuth, I.K., Tauxe, R.V., Barrett, T.J., 1994. Molecular characterization of Vibrio cholerae O1 strains by pulsed-field gel electrophoresis. J. Clin. Microbiol. 32, 1685–1690. Ceccarelli, D., Spagnoletti, M., Bacciu, D., Cappuccinelli, P., Colombo, M.M., 2011. New V. cholerae atypical El Tor variant emerged during the, epidemic outbreak in Angola. BMC Microbiol. 11, 130. Chow, K.H., Ng, T.K., Yuen, K.Y., Yam, W.C., 2001. Detection of RTX toxin gene in Vibrio cholerae by PCR. J. Clin. Microbiol. 39, 2594–2597. Faruque, S.M., Albert, M.J., Mekalanos, J.J., 1998. Epidemiology, genetics, and ecology of toxigenic Vibrio cholerae. Microbiol. Mol. Biol. Rev. 62, 1301–1314. Goel, A.K., Jain, M., Kumar, P., Jiang, S.C., 2010. Molecular characterization of Vibrio cholerae outbreak strains with altered El Tor biotype from southern India. World J. Microbiol. Biotechnol. 26, 281–287. Kaper, J.B., Morris Jr., J.G., Levine, M.M., 1995. Cholera. Clin. Microbiol. Rev. 8, 48–86. Mohamed, A.A., Oundo, J., Kariuki, S.M., Boga, H.I., Sharif, S.K., Akhwale, W., Omolo, J., Amwayi, A.S., Mutonga, D., Kareko, D., Njeru, M., Li, S., Breiman, R.F., Stine, O.C., 2012. Molecular epidemiology of geographically dispersed Vibrio cholerae, Kenya, January 2009–May 2010. Emerg. Infect. Dis. 18, 925–931. Nair, G.B., Qadri, F., Holmgren, J., Svennerholm, A.M., Safa, A., Bhuiyan, N.A., Ahmad, Q.S., Faruque, S.M., Faruque, A.S., Takeda, Y., Sack, D.A., 2006. Cholera due to altered El Tor strains of Vibrio cholerae O1 in Bangladesh. J. Clin. Microbiol. 44, 4211–4213. Nguyen, B.M., Lee, J.H., Cuong, N.T., Choi, S.Y., Hien, N.T., Anh, D.D., Lee, H.R., Ansaruzzaman, M., Endtz, H.P., Chun, J., Lopez, A.L., Czerkinsky, C., Clemens, J.D., Kim, D.W., 2009. Cholera outbreaks caused by an altered Vibrio cholerae O1 El Tor biotype strain producing classical cholera toxin B in Vietnam in 2007 to 2008. J. Clin. Microbiol. 47, 1568–1571. O’Shea, Y.A., Reen, F.J., Quirke, A.M., Boyd, E.F., 2004. Evolutionary genetic analysis of the emergence of epidemic Vibrio cholerae isolates on the basis of comparative nucleotide sequence analysis and multilocus virulence gene profiles. J. Clin. Microbiol. 42, 4657–4671. Okada, K., Roobthaisong, A., Nakagawa, I., Hamada, S., Chantaroj, S., 2012. Genotypic and PFGE/MLVA analyses of Vibrio cholerae O1: geographical spread and temporal changes during the 2007–2010 cholera outbreaks in Thailand. PLoS One 7, e30863. Safa, A., Bhuyian, N.A., Nusrin, S., Ansaruzzaman, M., Alam, M., Hamabata, T., Takeda, Y., Sack, D.A., Nair, G.B., 2006. Genetic characteristics of Matlab variants of Vibrio cholerae O1 that are hybrids between classical and El Tor biotypes. J. Med. Microbiol. 55, 1563–1569. Singh, D.V., Isac, S.R., Colwell, R.R., 2002. Development of a hexaplex PCR assay for rapid detection of virulence and regulatory genes in Vibrio cholerae and Vibrio mimicus. J. Clin. Microbiol. 40, 4321–4324. Singh, D.V., Matte, M.H., Matte, G.R., Jiang, S., Sabeena, F., Shukla, B.N., Sanyal, S.C., Huq, A., Colwell, R.R., 2001. Molecular analysis of Vibrio cholerae O1, O139, nonO1, and non-O139 strains: clonal relationships between clinical and environmental isolates. Appl. Environ. Microbiol. 67, 910–921. Sjolund-Karlsson, M., Reimer, A., Folster, J.P., Walker, M., Dahourou, G.A., Batra, D.G., Martin, I., Joyce, K., Parsons, M.B., Boncy, J., Whichard, J.M., Gilmour, M.W., 2011. Drug-resistance mechanisms in Vibrio cholerae O1 outbreak strain, Haiti, 2010. Emerg. Infect. Dis. 17, 2151–2154. Tran, H.D., Alam, M., Trung, N.V., Kinh, N.V., Nguyen, H.H., Pham, V.C., Ansaruzzaman, M., Rashed, S.M., Bhuiyan, N.A., Dao, T.T., Endtz, H.P., Wertheim, H.F., 2012. Multi-drug resistant Vibrio cholerae O1 variant El Tor isolated in northern Vietnam between 2007 and 2010. J. Med. Microbiol. 61, 431–437.
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Characterization of Vibrio cholerae from 1986 to 2012 in Yunnan Province, southwest China bordering Myanmar Wenpeng Gu a, Jianwen Yin a, Jianbin Yang a, Chaoqun Li a, Yujuan Chen a, Jie Yin a, Wen Xu a, Shiwen Zhao a, Junrong Liang b, Huaiqi Jing b, Xiaoqing Fu a,⇑ a
Department of Acute Infectious Diseases Control and Prevention, Yunnan Provincial Centre for Disease Control and Prevention, 650022 Kunming, China National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory for Infectious Disease Prevention and Control, 102206 Beijing, China b
a r t i c l e
i n f o
Article history: Received 18 July 2013 Received in revised form 18 October 2013 Accepted 18 October 2013 Available online 28 October 2013 Keywords: Vibrio cholerae El Tor variants Pulse-field gel electrophoresis Imported strains
a b s t r a c t Vibrio cholerae is an important infectious pathogen causing serious human diarrhea. We analyzed 568 V. cholerae strains isolated from 1986 to 2012 in Yunnan province, southwest China bordering Myanmar. Polymerase chain reactions for detecting virulence genes, antibiotic susceptibility tests and pulse-field gel electrophoresis (PFGE) were performed. The results showed all the strains were El Tor biotype from 1986. The ctxB subunit sequence analysis for all strains have shown that cholera between 1986 and 1995 was associated with mixed infections with El Tor and El Tor variants, while infections after 1996 were all caused by El Tor variant strains. All of the strains were sensitive to aminoglycosides and quinolone antibiotics while resistant to b-lactamase and carbapenem antibiotics increased gradually. 568 V. cholerae were divided into 218 PFGE-NotI patterns, and the isolates before 2001 and after 2011 were separated into two groups according to PFGE results. The strains isolated before 2001 were mainly referred to native cholera in Yunnan, and after 2011 were primarily referred to as imported strains from Myanmar, which showed the variation of V. cholerae in this area. The molecular characteristics of V. cholerae indicated regularity in bacterial variation and evolution in Yunnan province. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Vibrio cholerae is an important entero-infectious pathogen, causing serious human diarrhea and having severe effects on human society and economic development (Kaper et al., 1995). At present, seven cholera pandemics have occurred in the world; it is generally considered the seventh pandemic began in 1961, was caused by the El Tor biotype, and is still ongoing (Faruque et al., 1998). Asia is the major epidemic focus of cholera in the world, especially Southeast Asia (Kaper et al., 1995). Several studies show the V. cholerae El Tor variants (harboring the classical cholera toxin) gradually became the predominant strains all over the world, e.g. in Asia, Africa, and America (Alam et al., 2010; Ceccarelli et al., 2011; Mohamed et al., 2012; Nair et al., 2006; Safa et al., 2006).
Yunnan located in southwest China, bordering Myanmar, Vietnam and Laos, has an extended frontier, and being one of the minority habitations in China. From our continuous surveillance for V. cholerae, several cholera outbreaks occurred in Yunnan since 1986. Because of its special location, it was very important to study the molecular characteristics of V. cholerae in this area. Pulse-field gel electrophoresis (PFGE) is considered as the ‘‘golden standard’’ for pathogen molecular typing, and shows high discriminatory power for epidemiology investigations (Cameron et al., 1994). Our research was based on the PFGE method including virulence gene identification, and susceptibility tests; and was performed to analyze the characteristics of V. cholerae in Yunnan Province to investigate the molecular features of the isolates in this area.
2. Materials and methods Abbreviations: V. cholerae, Vibrio cholerae; PFGE, pulse-field gel electrophoresis; RT-PCR, real-time polymerase chain reaction. ⇑ Corresponding author. Address: Department of Acute Infectious Diseases Control and Prevention, Yunnan Provincial Centre for Disease Control and Prevention, Dongsi Street 158, 650022 Kunming, China. Tel./fax: +86 0871 63632033. E-mail address: [email protected] (X. Fu). 1567-1348/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2013.10.015
2.1. Bacterial strains 568 V. cholerae strains were isolated and stored in our laboratory from 1986 to 2012; all the strains were O1 serogroup except three O139 isolates. 436 isolates were Ogawa serotype, and 129
2
W. Gu et al. / Infection, Genetics and Evolution 21 (2014) 1–7
Fig. 1. The location of Yunnan province and its neighboring nations or provinces. The cholera happened in different years and counties of Yunnan in the history were shown with red star. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
were Inaba. 429 strains were isolated from patients, 77 from water (contaminated water or monitoring water), and the other 62 were isolated from the environment (soil or surface of objects) (Table 1). 2.2. Resuscitation and confirmation of V. cholerae strains All the isolates were stored at 80 °C in glycerin broth. The bacteria were inoculated on Brain Heart Infusion agar (Oxoid, UK) and incubated at 37 °C for 24 h. Slide agglutination test (National Institute for the Control of Pharmaceutical and Biological Products, China) was performed to confirm the serogroup of the strains, and reconfirmed by PCR test for rfb genes of O1 and O139 serotypes. The phenotypic tests: hemolysis test, susceptibility to polymyxin B and Voges–Proskauer test were analyzed for biotype investigation. The biotype methods were all performed at 37 °C for 24 h, and the API20E (Biomerieux, France) was used for Voges–Proskauer reaction. 2.3. Antibiotic susceptibility The antimicrobial susceptibility of all the V. cholerae strains was determined using the disc diffusion method on Mueller–Hinton
agar (MH). The antibiotics (Oxoid, UK) used were: ampicillin, amoxicillin/clavulanic acid, piperacillin, cefazolin, ceftazidime, ceftriaxone, cefepime, aztreonam, ertapenem, imipenem, meropenem, amikacin, gentamicin, ciprofloxacin, levofloxacin, tetracycline, nitrofurantoin, and trimethoprim/sulfamethoxazole. ATCC strain 25922 (Escherichia coli) served as the quality control. The results were interpreted based on the Clinical and Laboratory Standards Institute (CLSI version 2011). 2.4. PCR detection of virulence genes and ctxB sequencing Genomic DNA was extracted from each isolate using a DNA extraction kit (Tiangen, Beijing) according to the manufacturers’ instructions. The presences of rfb genes for O1 and O139 serotypes were determined. The primers used were: rfbO1 forward (50 -GGAAT AACTCAAGGCGATGAAGTG-30 ) and reverse (50 -TAGAGACTCACCT TCGATTTCAGC-30 ); rfbO139 forward (50 - CGATGGCGTGTTCATTAG AAGG-30 ), and reverse (50 - TCCCTTTCCACCTCGGTATTTC-30 ). SYBR premix Taq (TaKaRa, Japan) was used, and the amplification conditions were 95 °C for 30 s followed by 40 cycles: 95 °C for 5 s and 60 °C for 20 s. The virulence genes for ctxAB, ompU, ace, zot, toxR, rtxC, rstR (Classical/El Tor) and tcpA (Classical/El Tor) were
Table 1 The distribution and information of strains and PCR results for virulence genes in this study. Years
Isolated counties
Serotype
Source
PCR results
Ogawa
Inaba
O139
Patient
Water
Environment
rstR
tcpA
Rugose numbers
Changes of resistant antibiotics
AM(76),AMC(72),PIP(32),CZ(10),CAZ(14),FEP(63),ATM(55),IPM,CRO(6) AM(3),AMC(2),PIP(2),CAZ(1),ATM(2),IPM,MEM(2) AM,AMC(2),PIP(3),CZ(3),FEP(2),IPM
Gengma Yongde Ruili
– – –
86 4 5
– – –
74 3 3
11 1 1
1 – 1
ET(76)/ET,CL(10) ET,CL ET,CL
ET ET ET
18 1 3
1989
Gengma Ruili
4 16
3 2
– –
5 12
1 2
1 4
ET ET(14)/ET,CL(4)
ET ET
5 2
AM(6),AMC(3),PIP(2),CAZ,FEP,ATM,IPM,CRO(3),MEM(2) AM,AMC(5),PIP(3),CZ(7),CAZ,FEP,ATM,IPM
1991
Gengma Ruili Yuanmou
11 10 –
2 1 2
– – –
13 11 2
– – –
– – –
ET(8)/ET,CL(5) ET(4)/ET,CL(7) ET(1)/ET,CL(1)
ET ET ET
3 1 0
AM(9),AMC,PIP(6),CZ(5),CAZ,FEP,ATM,IPM,CRO(5) AM,AMC,CAZ,FEP,ATM,IPM,MEM(7) AM,AMC,PIP(1),CZ,FEP,ATM,IPM
Gengma Ruili Yuanmou Jinghong Cangyuan Dali Longchuan Mangshi Longling Yongshan
23 20 8 40 5 21 5 23 7 11
2 2 1 1 – 2 1 – – 3
– – – – – – – – – –
22 21 7 31 5 18 5 12 5 10
3 – – 9 – 5 1 11 2 4
– 1 2 1 – – – – – –
ET(2)/ET,CL(23) ET(2)/ET,CL(20) ET,CL ET,CL ET,CL ET,CL ET,CL ET,CL ET,CL ET,CL
ET ET ET ET ET ET ET ET ET ET
10 6 2 1 3 5 0 2 2 5
1997
Yuanmou Wuding
2 –
5 4
7 4
– –
– –
ET,CL ET,CL
ET ET
1 0
AM,AMC,PIP,FEP,ATM,IPM,CRO(3),MEM(3)TC,NIT,SXT AM,AMC,PIP,CZ(2),CAZ(2),FEP,ATM,IPM,TC,NIT,SXT
1998
Ruili Yuanmou Mangshi Yanshan Guangnan
21 22 21 15 25
– 1 – – –
3 – – – –
19 19 17 13 22
– – – – –
5 4 4 2 3
ET(3)/ET,CL(21) ET,CL ET,CL ET,CL ET,CL
ET ET ET ET ET
1 2 0 0 0
AM,AMC,PIP(7),CZ(7),CAZ(9),FEP(9),ATM,IPM,MEM(5),TC,NIT,SXT AM,AMC,PIP,CZ(6),CAZ(6),FEP(4),ATM,IPM,CRO(7),TC,NIT,SXT AM,AMC,PIP(11),CZ(9),CAZ(7),ATM,IPM,MEM(7),TC,NIT,SXT AM,AMC,PIP,CZ,FEP,ATM,IPM,TC,NIT,SXT AM,AMC,PIP,ATM,IPM,CRO(2),TC,NIT,SXT
1999
Dali Gejiu Yuanyang Kunming
8 13 23 1
– – – –
– – – –
6 5 12 1
2 8 11 –
– – – –
ET,CL ET,CL ET,CL ET,CL
ET ET ET ET
1 4 4 0
AM,AMC,CZ,CAZ(3),FEP(1),ATM,IPM,TC,NIT,SXT AM,AMC,PIP,CZ,ATM,IPM,MEM(5),TC,NIT,SXT AM,AMC,PIP,CZ,CAZ(8),FEP(8),ATM,IPM,CRO(8),ETP(3),TC,NIT,SXT AM,AMC,CZ,FEP,ATM,IPM,TC,NIT,SXT
2001
Yuanmou Mangshi
6 65
– 2
–
5 34
1 –
– 33
ET,CL ET,CL
ET ET
2 12
2011
Ruili Mangshi Ruili
2 7 1
– – –
– – –
2 3 1
– 4 –
– – –
ET,CL ET,CL ET,CL
ET ET ET
0 0 0
1994 1995
1996
2012
AM,AMC,PIP(11),CZ(7),CAZ(9),FEP(11),ATM(11),IPM,CRO(8),TC,NIT,SXT AM,AMC,PIP(7),CZ(4),CAZ(9),FEP(7),ATM(9),IPM,MEM(5),TC,NIT,SXT AM,AMC,CZ(2),FEP(3),IPM,TC,NIT,SXT AM,AMC,PIP(17),CAZ(11),FEP(11),ATM(17),IPM,CRO(5),MEM(6),TC,NIT,SXT AM,AMC,PIP,IPM,TC,NIT,SXT AM,AMC,PIP(8),CZ(8),CAZ(7),FEP(8),ATM(8),IPM,TC,NIT,SXT AM,AMC,PIP,IPM,TC,NIT,SXT AM,AMC,PIP(5),CZ(11),FEP(9),ATM(9),IPM,TC,NIT,SXT AM,AMC,PIP,ATM,IPM,TC,NIT,SXT AM,AMC,PIP,CZ(8),CAZ(8),FEP,ATM,IPM,CRO(4),MEM(7),TC,NIT,SXT
AM,AMC,PIP,CZ,CAZ,FEP,ATM,IPM,TC,NIT,SXT AM,AMC,PIP,CZ,CAZ,FEP,ATM,IPM,CRO(10),MEM(8),TC,NIT,SXT AM,AMC,PIP,CZ,CAZ,FEP,ATM,IPM,TC,NIT,SXT AM,AMC,PIP,CZ,CAZ,FEP,ATM,IPM,TC,NIT,SXT AM,AMC,PIP,CZ,CAZ,FEP,ATM,IPM,TC,NIT,SXT
W. Gu et al. / Infection, Genetics and Evolution 21 (2014) 1–7
1986
Note: ET, El Tor biotype; CL, Classical biotype. The numbers in the bracket represented the strains possessed the ET or ET, CL alleles in different counties and years, no bracket represented all the isolates had the same allele. AM, ampicillin; AMC, amoxicillin/clavulanic acid; PIP, piperacillin; CZ, cefazolin; CAZ, ceftazidime; FEP, cefepime; ATM, aztreonam; IPM, imipenem; CRO, ceftriaxone; ETP, ertapenem; MEM, meropenem; TC, tetracycline; NIT, nitrofurantoin; SXT, trimethoprim/sulfamethoxazole. The numbers in the bracket represented the resistant strain numbers for each antibiotic; otherwise, no bracket represented all the isolates were resistant.
3
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Fig. 2. The ctxB subunit sequencing results for all the isolates in Yunnan. Three isolates in 1995 and four in 1998 could not amplify in the study.
amplified using Taq premix (TaKaRa, Japan), the primers and amplification procedures were as described previously (Chow et al., 2001; O’Shea et al., 2004; Singh et al., 2002, 2001). All of the strains were sequenced for ctxB gene subunit to further identify the characters of the isolates, Taq premix (TaKaRa, Japan) was used as described above, and amplification processes were performed as previously described (Goel et al., 2010). The amplification products were sent for bidirectional sequencing (TaKaRa, Japan), and the results were analyzed using DNAStar and MEGA4 software. The ctxB sequences of N16961 of El tor V. cholerae (Genebank: NC-002505) and O395 Classical strain (Genebank: NC-012582) were used as the standards for comparison. 2.5. PFGE PFGE was performed based on the PulseNet protocol for V. cholerae and procedures described previously (Cameron et al., 1994). The enzyme digestion for each plug was NotI 40U at 37 °C for 4 h and SfiI 50U at 50 °C for 4 h. The CHEF-Mapper (Bio-Rad) was used for electrophoresis, and the pulse time ranged from 1 to 20 s for 13 h, and 20 to 25 s for 6 h. The gel was stained using Gelred (Biofilm) and visualized using the gel imaging system (Bio-Rad, Gel DocXR). PFGE patterns were analyzed with BioNumerics version 5.10, and a dendrogram was produced using the Dice coefficient and un-weighted pair group method with arithmetic mean algorithm (UPGMA). A pairwise distance matrix and a minimum spanning tree were also created. 3. Results 3.1. Phenotypic characteristics of the strains 568 V. cholerae were isolated from 1986 to 2012, in Gengma, Yongde, Ruili, Yuanmou and Jinghong, etc. The outbreaks occurred at intervals of one or two years between 1986 and 2001; then the frequency of cholera diminished after 2001. There were two imported cases from Myanmar in 2011 and one in 2012, both of the strains were isolated in Ruili. In Fig. 1 each county in which cholera was observed in marked with a red star and the year of its occurrence, and the information of the isolates involved in our study were shown in Table 1. All the strains were O1 serogroup except three that were O139 serotype, and the RT-PCR results for rfb genes of O1 and O139 were
consistent with the phenotypic assays. Serotype Inaba was the predominant type before 1995; the Ogawa serotype became the major type after 1995. A portion of the isolates expressed hemolysis and were sensitive to polymyxin B, while others were not; no regularity was found. However, the Voges–Proskauer test for all the strains was positive. In the process of our study, we found a portion of the isolates assumed the rugose survival form when inoculated on nonselective agars (Table 1). 3.2. Susceptibility test All the isolates were sensitive to gentamicin, ciprofloxacin, and levofloxacin, regardless of the year isolated, county, or source. Most of the V. cholerae were sensitive to ceftriaxone, ertapenem, and meropenem, while completely drug resistant isolates were seldom found. Some of the isolates were sensitive to ampicillin, amoxicillin/clavulanic acid, piperacillin, cefazolin, ceftazidime, cefepime, aztreonam, and imipenem and the proportion of resistant strains to these antibiotics was increased gradually. The strains isolated from 1986 to 1994 were sensitive to tetracycline, nitrofurantoin, and trimethoprim/sulfamethoxazole; after 1995, all of the isolates were resistant to these antibiotics (Table 1). 3.3. PCR test for virulence genes and ctxB sequencing The ctxAB, ompU, ace, zot, toxR, and rtxC for all of the isolates were positive; tcpAEl Tor was positive for all of the isolates as well, while tcpAClassical was negative. For the rstR, most of the strains carried rstREl Tor and rstRClassical, however, some of the strains possessed only rstREl Tor (Table 1). The results showed mixed infection with El Tor type (harboring ctxBEl Tor) and El Tor variant strains (harboring ctxBClassical) before 1995; after 1996 all of the isolates harbored the ctxBClassical except three O139 V. cholerae that possessed ctxBEl Tor. The percentage of El Tor type strains decreased, while the El Tor variants gradually increased over time, and all the strains became El Tor variants after 1996 for O1 serogroup (Fig. 2). 3.4. PFGE The 568 V. cholerae were divided into 218 PFGE-NotI patterns and formed two major groups in the dendrogram (Fig. 3). The PFGE-NotI patterns for strains isolated from 1986 to 2001 formed
W. Gu et al. / Infection, Genetics and Evolution 21 (2014) 1–7
5
Fig. 3. PFGE-NotI dendrogram for representative V. cholerae in this study.
group A, and the patterns for strains isolated in 2011 and 2012 and the O139 serotypes from 1998 formed group B. The same molecular typing characteristic was also found using PFGE-SfiI. The same cluster result was shown with minimum spanning tree based on PFGE results (Fig. 4). Two major cluster groups were also identified,
group A in the pink area contained isolates collected before 2001, and group B in the green area contained O1 serogroup strains from 2011 and 2012 and all O139 strains in 1998. PFGE-SfiI was performed for strains possessed the same PFGENotI patterns. Some of the strains isolated from different counties
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Fig. 4. The minimum spanning tree of V. cholerae in Yunnan based on PFGE results. Group A in the pink area: isolates before 2001; group B in the green area: isolates after 2011 and all the O139 isolates. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. The distribution of V. cholerae showing identical PFGE-NotI and SfiI patterns. The pattern with the square frame was the imported strain in Ruili.
and years showed the same PFGE-NotI and SfiI patterns, e.g. Gengma in 1995, Yanshan in 1998, Yuanyang in 1999, and Mangshi in 2001; compared to Yuanmou in 1994, Dali and Gengma in 1995, Yongshan in 1996, Mangshi in 1998, and Gejiu in 1999; Mangshi in 1998 and 2001, Ruili in 1989 and Yuanyang in 1999 (Fig. 5A–C). The bacteria isolated from different counties but the same years also had the same PFGE-NotI and SfiI patterns, e.g. Dali, Jinghong, Longchuan and Ruili in 1995; and Yanshan, Guangnan and Ruili in 1998; Gejiu and Yuanyang in 1999 (Fig. 5D–F). Furthermore, the identical result was reconfirmed that strains isolated from patients and water or environment had the close relations, in Fig. 5. A–G, the same PFGE patterns were found from different source, in different counties and years, showed the identical similarities.
4. Discussion In our study, the results showed all the strains possessed the rtxC and tcpAEl Tor genes, and the tcpAClassical was not found in all
the isolates; therefore, 568 V. cholerae isolated from 1986 to 2012 in Yunnan were all El Tor biotype. Most of the strains had the rstREl Tor and rstRClassical genes, while some had only one of them as shown in other studies (Alam et al., 2010). The distribution of the rstREl Tor was mainly referred to Gengma and Ruili from 1986 to 1995, and after 1996 all the isolates had rstREl Tor and rstRClassical, regardless of which county and year it was collected. In general, V. cholerae in Yunnan were totally sensitive to aminoglycosides and quinolone antibiotics. While, in the earlier years of surveillance, some of the strains were sensitive to b-lactamase and carbapenem antibiotics, and the proportion of resistant strains to these antibiotics increased in recent years. For tetracycline and trimethoprim/sulfamethoxazole, all the isolates were sensitive before 1994, and after 1995 all the strains were resistant. These changes in the resistance pattern in Yunnan reflected the antibiotics used in this area a period, the antibiotics resistance has become a serious problem although completely drug resistant isolates were seldom found. Goel et al. (2010) showed the strains were sensitive to b-lactamase, quinolone and tetracycline antibiotics in India, 2004; Tran et al. (2012) found that the V. cholerae in Vietnam from
W. Gu et al. / Infection, Genetics and Evolution 21 (2014) 1–7
2007 to 2010 were sensitive to b-lactamase and quinolone antibiotics, resistant to tetracycline and trimethoprim/sulfamethoxazole; Sjolund-Karlsson et al. (2011) demonstrated the Haitian V. cholerae strains were sensitive to tetracycline, but were resistant to quinolones, aminoglycosides, and trimethoprim/sulfamethoxazole in 2010. There was no similarity for bacterial antibiotic susceptibility based on these studies. In recent studies, the El Tor variants (harboring classical cholera enterotoxin) have become widely distributed all over the world, especially in Asia. V. cholerae El Tor variants became the predominate strains and were isolated in India in 2004 (Goel et al., 2010), Bangladesh since 2001 (Nair et al., 2006), Vietnam in 2007 to 2008 (Nguyen et al., 2009), Thailand from 2007 to 2010 (Okada et al., 2012), Malaysia in 2009 (Ang et al., 2010), Mexico since 1991 (Alam et al., 2010), and Angola in 2006 (Ceccarelli et al., 2011). Use of ctxB sequencing, showed the El Tor variants appeared early in 1986 in Yunnan, southwest China, and substituted for the typical El Tor biotypes after 1996. The strains after 1996 were all El Tor variants, except O139 isolates that had the typical El Tor cholera enterotoxin in 1998. This possibly indicates the close evolutionary relations within the El Tor biotypes. The variation of the El Tor biotype changes reflected in our study possibly represented the features of V. cholerae in Yunnan province, and the El Tor variants had existed early in 1986 in southwest of China. In our study, the strains isolated from patients and environment or water shown the high similarity (Figs. 3 and 5) based on PFGE two enzyme digestion results, indicated the close transmission relations of these bacteria. The variation of V. cholerae in Yunnan was divided into two groups based on PFGE results (Figs. 3 and 4), and suggested the different source of the pathogen transmitted in this area. The strains before 2001 formed group A, and the isolates after 2011 and three O139 strains in 1998 formed group B. Furthermore, the cholera isolates in Yunnan showed the same PFGE patterns with the imported strain from Myanmar in 2011 (Fig. 5G), indicated the close transmitted relationship between two nations at the frontier. Therefore, it was very important to enhance the surveillance in the bordering areas in Yunnan. 5. Conclusions The 568 V. cholerae isolated from 1986 to 2012 in Yunnan Province, southwest China were El Tor biotype, and the typical El Tor biotype was later replaced by the El Tor variant strains. The PFGE patterns of the isolates also changed and divided into two major groups as shown during the surveillance years, and indicated regularity in bacterial variation and evolution. Acknowledgments This work was supported by National Sci-Tech key project (2012ZX10004-201, 2012ZX10004-212 and 2013ZX10004-203002). We thank Dr. Jim Nelson for critical reading of our manuscript.
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