Curr Microbiol (2015) 70:408–414 DOI 10.1007/s00284-014-0725-2

Assessing Clonal Correlation of Epidemic Vibrio cholerae Isolates During 2011 in 16 Provinces of Iran Massoud Hajia • Mohamad Rahbar • Marjan Rahnamye Farzami Hossein Masoumi Asl • Alireza Dolatyar • Mohsen Imani • Roghieh Saburian • Moharam Mafi • Bita Bakhshi



Received: 27 April 2014 / Accepted: 30 September 2014 / Published online: 26 November 2014 Ó Springer Science+Business Media New York 2014

Abstract A total of 1,187 Vibrio cholerae isolates were received during 2011 cholera outbreaks from 16 provinces in different geographical location to Iranian reference Health laboratory. A random selection was performed, and 61 isolates were subjected to further investigations. Cholera cases were come up from May with nine cases and reached to its maximum rate at August (57 cases) and continued to October after which a fall occurred in September. All of the isolates were susceptible to three antimicrobial agents including ciprofloxacin, cefixime, and ampicillin. The highest rate of resistance was seen to nalidixic acid (96.7 %) and co-trimoxazole (91.8 %). Clonality of isolates was investigated through genotyping by PFGE method. A total of seven pulsotypes were obtained from 61 isolates under study. The pulsotypes were highly related with only 1–3 bands differences. Three pulsotypes (PT5, PT6, and PT7) constituted 93.4 % of total isolates. One environmentally isolated strain showed distinct pattern from clinical specimens. This strain although M. Hajia  M. Rahbar Department of Molecular Biology, Research Center of Health Reference Laboratories, Ministry of Health and Medical Education, Tehran, Iran M. Rahnamye Farzami  A. Dolatyar  M. Imani  R. Saburian Research Center of Health Reference Laboratories, Ministry of Health and Medical Education, Tehran, Iran H. Masoumi Asl  M. Mafi Center for Communicable Diseases Control, Ministry of Health and Medical Education, Tehran, Iran B. Bakhshi (&) Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Jalal-Ale-Ahmad Ave, 14117-13116 Tehran, Iran e-mail: [email protected]

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had no any evidence in identified cholera infections, highlighted selecting more environmental specimens in any future outbreaks as long as human samples. In conclusion, emergence and dominance of Ogawa serotypes after about 7 years in Iran are alarming due to fear of import of new V. cholerae clones from out of the country. Approximately, one third of patients in 2011 cholera outbreak in Iran were of Afghan or Pakistani nationality which makes the hypothesis of import of Ogawa serotype strains from neighboring countries more documented and signifies the need to monitor and protect the boundaries.

Introduction Epidemic cholera, caused by toxigenic Vibrio cholerae O1 and O139, continues as a global treat and a major health problem in developing countries including Iran [16, 19, 25]. Recently, reports of cholera outbreaks have been increased and to now seven pandemics of cholera have been recorded [17]. V. cholerae O1 was the only agent of cholera pandemics before emergence of V. cholerae serogroup O139 in Southern India in 1992, which is rapidly spread within the Bangladesh, India, and neighbor countries [21, 23, 24]. Seasonal outbreaks of cholera occur in Southern Asia, parts of Africa and Latin America [3]. Variations which may occur in genetic content of V. cholerae during epidemic and inter-epidemic periods may affect the virulence and genotyping properties of epidemic isolates. Mobile genetic elements including phages, pathogenicity islands, and constins may contribute to genetic variation of V. cholerae [5, 12, 20]. According to performed studies phenotypic characters may not be sufficient to investigate diversity of V. cholerae

M. Hajia et al.: Clonal Correlation of Epidemic V. cholerae Isolates

409

Table 1 MIC determination of 7 antimicrobial agents for 61 V. cholerae isolates under study

Performance of Susceptibility Testing

Antimicrobial agents

Sensitive (n=)

(n=)

Resistant (n=)

CIP

61





NA

2



59

CFM

61





AMP

60

1



TE

43

6

12

SXT

5



56

E

11

14

36

All of 61 isolates were tested by MIC Test Strip Method using Liofilchem (CE IVD approved, Italy) against ciprofloxacin (CIP), nalidixic acid (NA), cefixime (CFM), ampicillin (AMP), tetracycline (TE), co-trimoxazole (SXT), and erythromycin (E) (Table 1). The considered MICs as sensitive, intermediate, and resistant levels are shown in Table 2 [10]. Following organisms were used as quality control strains for MIC E-testing; E.coli (ATCC 25922), S. aureus (ATCC 29213), and P. aeruginosa (ATCC 27853).

Table 2 The level of MICs which was considered as resistant, intermediate, or sensitive SensitiveB

Intermediate

ResistantC

CIP

1

2

4

NA

16



32

CFM

1

2

4

AMP

8

16

32

TE SXT

4 2

8 –

16 4

E

2

3

4

strains because of instability of expressed phenotypes or insufficient discriminatory for strain differentiation [14, 27]. Many molecular methods are increasingly used as a tool for typing toxigenic V. cholerae strains among which Pulsed-Field Gel Electrophoresis (PFGE) is more commonly used by laboratories focusing cholera investigations due its reliability, discriminatory power, and typeability [8]. This method has been also reported as useful method in discriminating the two biotype (Classic and El Tor) of V. cholerae isolates from different origins [13]. The aim of this study was to investigate the epidemiological surveillance of cholera and comparative analysis of clonal relationships among isolated strains in recent cholera outbreak in Iran (2011).

Determination of Genetic Relatedness of V. cholerae Isolates Genotyping of isolates was performed by pulsed-field gel electrophoresis using PulseNet standard procedure for V. cholerae [11]. Briefly, bacterial suspension was prepared in a buffer (100 mmol l-1 Tris, 100 mmol l-1 EDTA, pH 8.0) and adjusted to absorbance values of 0.8–1.0 at a wavelength of 610 nm, after which plugs were prepared with SeaKem Gold agarose (Lonza, Rockland, ME, USA) and proteinase K. Bacterial plugs were lysed (50 mmol l-1 Tris, 50 mmol l-1 EDTA, 1 % sarcosine, and 0.5 mg of proteinase K, pH 8.0)and washed after which digested with 40 units of NotI restriction enzyme (Fermentase). DNA molecular weight size marker was prepared by XbaI digestion of Salmonella enterica serotype BraenderupH9812 plugs. CHEF Mapper XA System (BioRad) was applied for electrophoresis using program explained by PulseNet. PFGE banding patterns were analyzed by BioNumerics software version 6.1 (Applied Maths, Kortrijk, Belgium) and a dendrogram was produced using Dice coefficient and the unweighted pair-group method with arithmetic mean algorithm (UPGMA) and a position tolerance of 1.3 %.

Results Materials and Methods

V. cholerae Isolates and Cholera Patients

V. cholerae Isolates and Data Collection

A total of 1,187 V. cholerae isolates were obtained during 2011 cholera outbreaks from the whole country, which affected 16 provinces in different geographical location in Iran. All of the isolates from provinces with lower cholera cases were selected and included in the study, while in provinces with more cholera cases (Qom, Baluchistan and Tehran provinces) a random selection was performed and finally 61 isolates were selected for further investigations. Cholera cases were come up from May with 9 cases and

The V. cholerae isolates were received from 16 provinces of Iran during May to October, 2011. Identification of the isolates was confirmed in our laboratory using standard biochemical and bacteriological tests. All isolates were examined for specific serogroups by O1 polyvalent and Ogawa/Inaba monospecific antisera (BD, Becton–Dickinson Co.USA) [9, 18, 22].

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410

M. Hajia et al.: Clonal Correlation of Epidemic V. cholerae Isolates

Table 3 Characterization of V. cholerae isolates under study Province

Nationality of patients

Pulsotype group 7

Tehran

5

6

MIC of isolates 4

3

2

1

CIP

NA

CFM

AMP

TE

SXT

E

Iranian

j

0.75

256C

0.064

6

4

32C

1.5

Iranian

j

0.5

256C

0.125

4

3

32C

1.5

0.094

2

0.094

4

1.5

0.25

1.5

j

Iranian Iranian

j

0.75

256C

0.064

3

32

32C

4

Iranian

j

0.75

256C

0.094

4

3

32C

2

Iranian

j

0.50

256C

0.125

6

4

32C

1.5

Iranian Iranian

j j

0.50 0.50

256C 256C

0.064 0.064

3 3

32 24

32C 32C

3 4

Afghan

j

0.75

256C

0.125

6

4

32C

1.5

Iranian

j

1

256C

0.125

4

3

32C

2

j

4

Kashan

Pakistani

0.75

256C

0.064

3

16

32C

Baluchestan

Pakistani

j

0.50

256C

0.47

2

16

32C

4

Pakistani

j

0.50

256C

0.094

2

16

32C

2

Markazi-Arak

Golestan

Pakistani

j

0.50

256C

0.064

1.5

24

32C

3

Pakistani

j

0.75

256C

0.094

2

2

0.47

3

Pakistani

j

1

256C

0.094

6

32

32C

3

Pakistani

j

0.75

256C

0.064

3

32

32C

3

Pakistani

j

0.75

256C

0.064

3

24

32C

3

Afghan

j

0.50

256C

12

3

16

32C

4

Afghan

j

0.75

256C

0.064

3

24

32C

4

Afghan

j

0.50

256C

0.064

4

24

32C

4

Iranian Iranian

j j

0.75 0.75

256C 256C

0.047 0.064

4 12

16 24

32C 32C

3 3

Iranian

j

0.75

256C

0.064

3

24

32C

6

Iranian

j

0.50

256C

0.094

6

4

32C

1.5

Afghan

j

0.50

256C

0.094

4

3

32C

1.5

j

Iranian Iranian Iranian Afghan

Zanjan

123

0.064

4

32

32C

8

0.094

6

3

32C

2

j

0.75

256C

0.064

3

32

32C

6

0.50

256C

0.094

3

16

32C

3

j

0.75

256C

0.064

2

32

32C

3 2

Iranian

j

0.75

256C

0.094

4

3

32C

Iranian

j

0.75

256C

0.094

3

24

32C

2

0.75

256C

0.094

6

1

32C

2

j

Iranian

Ghilan

256C 256C

j

Iranian Qom

0.75 0.75

j

Iranian

j

0.50

256C

0.094

3

3

32C

1.5

Iranian

j

0.75

256C

0.094

4

2

32C

2

Iranian

j

Nonclinical Iranian Iranian

0.75

256C

0.064

4

3

32C

1.5

j

0.004 0.50

1.5 256C

0.064 0.094

3 6

3 3

0.50 32C

4 1.5

j

0.75

256C

0.094

6

3

32C

1.5

j

Iranian

j

0.75

256C

0.094

4

1.5

32C

1

Iranian

j

0.75

256C

0.094

3

3

32C

2

Iranian

j

0.75

256C

0.094

3

24

32C

1.5

Iranian

j

0.50

256C

0.094

3

24

32C

1.5

Iranian

j

0.75

256C

0.064

3

24

32C

3

Iranian

j

0.50

256C

0.064

3

24

32C

3

Iranian

j

0.50

256C

0.094

3

24

32C

3

M. Hajia et al.: Clonal Correlation of Epidemic V. cholerae Isolates

411

Table 3 continued Province

Nationality of patients

Pulsotype group 7

Ghazvin

5

Semnan Khoozesstan

4

3

2

1

CIP

NA

CFM

AMP

TE

SXT

E

Iranian

j

1

256C

0.125

3

2

32C

2

Iranian

j

0.75

256C

0.125

4

4

32C

1.5

j

0.50

256C

0.064

6

4

32C

1.5

0.75

256C

0.064

4

2

32C

2

Iranian Alborz

6

MIC of isolates

j

Iranian Iranian

j

0.50

256C

0.094

2

128

32C

2

Iranian

j

1

256C

0.094

2

64

32C

2

Iranian

j

0.75

256C

0.064

3

16

32C

3

Iranian

j

Iraniana

j j

0.50

256C

0.064

4

16

32C

1.5

0.064

4

0.094

1.5

2

0.50

1.5

0.094

4

0.064

2

3

0.25

2

0.75

256C

0.064

2

16

32C

1.5

Boushehr

Iranian

Hormozgan

Iranian

Fars

Afghan

j

0.50

256C

0.064

2

16

32C

1.5

Afghan

j

0.75

256C

0.125

4

4

32C

1.5

Iranian

j

0.75

256C

0.094

3

24

32C

2

Jiroft a

j

Inaba

Table 4 Frequency and distribution of V. cholerae isolates in relation to their pulsotypes and patients nationality

Pulsotypes

Provinces

PT7

PT6

PT5

PT4

PT3

PT2



3-a

6 (5 a, 1 b)

1IR





10 (9 IR, 1 AF)

Tehran



3- a



PT1









3 (a)

Qazvin

2-a







2 (a)

Zanjan











3-a

2-a



1-a

1-a











1 (a)

Semnan

1-a 2-b

– –



– –

1-a –

– –

2 (a) 2 (b)

Khuzestan Fars



8-a

1-IR





1-d

10 (9 a, 1 d)

qom



2-a

2 (1-a,1-b)







4 (3 a-1 b)

Markazi-Arak





1-c







1 (c)

Kashan

1-b











1 (a)

KermanJIRoft

2-c

9 (8-c,1-a)



11 (7 c, 3 b,1a)

Baluchestan

1aR











1 (a)

Boushehr

1 (a)

Hormozgan



3-a

3 (2-a,1-b)







10

28

19

1

1

1

1- IaR a: Iranian, b: Afghan, c: Pakistani, d: non clinical

Number of tested isolates

reached to its maximum rate at August (57 cases) and continued to October after which a fall occurred in September. All of the isolates belonged to Ogawa serotype except for one isolate from Khuzestan province. One isolate which was collected from contaminated vegetables was also included in the study. The majority of

1

3 (a)

Ghilan

3 (a)

Alborz

6 (5 a, 1 b)

Golestan

44aR, 8 b, 8 c, 1 d

Total

cholera patients were of Iranian (73.3 %), Afghan (13.3 %) and Pakistani (13.3 %) nationality (Tables 3, 4). Determination of MIC of Antimicrobial Agent All of the isolates were susceptible to 3 antimicrobial agents including ciprofloxacin, cefixime, and ampicillin.

123

100

95

90

85

Fig. 1 PFGE banding patterns were analyzed with BioNumerics software version 6.1

M. Hajia et al.: Clonal Correlation of Epidemic V. cholerae Isolates

80

412

PT5 PT7 PT6 PT3 PT4 PT1 PT2

The highest rate of resistance was seen to nalidixic acid (96.7 %) and co-trimoxazole (91.8 %).The second most resistance was seen to erythromycin (59 %) (Table 3). Clonal Relationship of V. cholerae Isolates Clonality of isolates was investigated through genotyping by PFGE method. A total of seven pulsotypes were obtained from 61 isolates under study (Fig. 1). The pulsotypes were highly related with only 1–3 bands differences. Three pulsotypes (PT5, PT6, and PT7) constituted 93.4 % of total isolates. Each of pulsotypes PT1–PT4 was seen in only one isolate from different provinces. Pulsotype PT2 was related to an environmental isolate from Qom province. A summary of different pulsotypes in relation to the number of isolates with the same pulsotype and their province of isolation is shown in Table 4.

Discussion Previous studies have shown the dominance of V. cholerae Ogawa serotype in 2004(11, 16) outbreaks in Iran, while similar studies indicated the trend of isolates toward Inaba during 2005–2010 outbreaks [2]. Emergence and dominance of Ogawa serotypes after about 7 years in Iran are alarming due to fear of import of new V. cholerae clones from out of the country. Furthermore, genotyping results of the newly emerged Ogawa serotypes showed no similar or identical pulsotypes with those previously reported for Ogawa serotype strains in this country (5). These foreigner strains which may persist and spread in the incoming years could be easily added to the native reservoirs and reemerge as new cholera genotypes. It has been shown that Inaba V. cholerae isolates may arise from prevailing Ogawa strains due to a single point mutation within the coding sequence of wbeT gene of O-antigen biosynthesis cluster which could make the

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associated protein product incapable of accomplishing transferase activity [26]. Regarding this point that, the rate of shift back mutation to the ancestor intact sequence is dramatically low which makes it almost impossible; therefore, the emergence of Ogawa serotype strains in Iran in 2011 is probably due to import of Ogawa serotype strains from out of the country. Considering the nationality of patients affected in recent outbreaks in Iran (26.6 % Afghan or Pakistani) especially in Baluchistan province which has a long-line common boundaries with both Afghanistan and Pakistan countries, makes the hypothesis of import of Ogawa serotype strains from neighboring countries more documented [15]. Genotyping analysis by PFGE method revealed seven related genotypes among the total isolates which are indicative of a high level of homogeneity among the isolates. Approximately, 93.4 % of isolates commonly showed three genotypes, with all of the isolates obtained from patients with Afghan or Pakistani nationality have fallen in this group. This may clearly reflect the import of foreign genotypes from beyond the boundaries to inside the country, probably through travelers, which has been spread and disseminated to 16 provinces. Previous studies in Iran have shown identical V. cholerae PFGE genotypes with those reported from India, Nepal, and Pakistan, and it was suggested that travelers may have played a key role in the import of foreign genotypes to the country [5]. Four single genotypes were obtained from patients with Iranian nationality in four different provinces. These genotypes are more distinct from the first three common genotypes. This may be due to (i) genetic variations, especially point mutations, which may occur within the genome of imported strains and result to single new genotypes with 1–3 bands differences, or (ii) the presence of different native genotypes within the country with lower epidemic potential which may be related to their different CTX phage content and arrangement [2, 6, 26].

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Comparative analysis of genotypes obtained in 2011 indicates significant differences with those previously reported from Iran, providing additional reason for foreigner strains [5]. The probable emergence and spread of foreigner V. cholerae strains move us toward establishing and developing an extra-national database of epidemic V. cholerae isolates occurring in neighboring countries and Middle East. One environmental specimen was received and examined for isolation of V. cholerae that showed distinct pattern from clinical isolates even with those obtained from the same province (Qom). This isolated strain although had no evidence in identified cholera infections, highlighted selecting more environmental specimens in any future outbreaks as long as human specimens, because previous reports underlines that they are more related to food born transmission rout rather than water born. The presence of V. cholerae strains with toxigenic and pathogenic potential in the surface waters was previously reported from the same province (2006) which signifies the need to monitor the environmental reservoirs for V. cholerae maintenance and spread [4]. In our study, resistance to co-trimoxazole was 91.8 % among the isolates similar to previously reports. Adabi and colleagues have shown a high rate of resistance to cotrimoxazole among V. cholerae isolates due to the presence of SXT constin within the genome of Iranian isolates [1]. This constin, which is a commonly occurring genetic element of V. cholerae, is responsible for conferring resistance to many antimicrobial agents including cotrimoxazole, chloramphenicol, and streptomycin. The high rate of resistance to co-trimoxazole recalls the crucial role of SXT constin in conferring resistance to this antibacterial drug. Although rehydration is main choice for treatment of cholera infection, prescribing antibiotics has an important role in shortening the course of trend of disease. Unfortunately, emerging resistance for some essential antibiotics such as tetracycline has caused difficulty in treatment. Tetracycline resistance was a common phenotype among the 2011 isolates (54.1 %). This phenomenon was previously reported to be less than 20 % in Iranian isolates [1]. Considering the fact that tetracycline is still prescribed by Iranian physicians for cholera treatment, makes such rate of resistance more sensible, and imposes additional challenges for cholera management and control in this country. Tetracycline resistance in many other countries also has shown a significant rise. Bhattacharya and colleagues have shown an increase in tetracycline resistance rate among V. cholerae strains isolated in Kolkata, India, from 2004 through 2009 [7].

In conclusion, emergence and spread of Ogawa serotype strains after about 7 years in Iran is considered to be due to import of new V. cholerae isolates from out of the country. Tetracycline resistance is increasing during recent years in Iran and other countries encountering cholera outbreaks. Approximately, one third of patients in 2011 cholera outbreak in Iran were of Afghan or Pakistani nationality which makes the hypothesis of import of Ogawa serotype strains from neighboring countries more liable and signifies the need to monitor and protect the boundaries. Acknowledgments The study was supported by Iranian ministry of health and medical education and a grant from research council of Tarbiat Modares University.

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Assessing clonal correlation of epidemic Vibrio cholerae isolates during 2011 in 16 provinces of Iran.

A total of 1,187 Vibrio cholerae isolates were received during 2011 cholera outbreaks from 16 provinces in different geographical location to Iranian ...
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