Correlation of the Native Inaba Strain With the Dominant Isolated Strains Obtained From Outbreaks in 2013 in Iran

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all documents of the Center for Disease Control of Iran shows that it switched to Inaba in 2013, after 2 years.It could have been re-emerged from native reservoir or from strains imported from abroad outbreaks.Based on a previous study, a few Inaba had been isolated from sporadic cases. 7o lecular typing techniques such as random amplified polymorphic DNA (RAPD), ribotyping, and multilocus enzyme electrophoresis (MEE) have been employed to study genetic relatedness. 8,9However, PFGE has been able to play a considerable role in epidemiological investigations because it has a high discriminatory power. 10,11The pulse field gel electrophoresis (PFGE) protocol for cholera has been validated and standardized.The technique has been accepted to discriminate the data of those participating laboratories and compare their correlations and homology.Therefore it can be applied as a strong tool in monitoring and controlling the enteric pathogen. 12FGE is considered as a "golden standard" molecular typing method for food borne pathogens, illustrating high discriminatory power for epidemiology investigations.This method is able to support epidemiological data in describing how a V. cholerae O1 isolate can emerge from abroad or reemerge from native strains.Based on a previous study, 7 pulsotypes were reported from which 3 types were dominant throughout the country and 4 were sporadic, 7 while in previous studies just two types were detected. 5,6

Objectives
The objective of this study was the analysis of Inaba strains isolated from 2013 outbreak to study the similarity of the isolated strains and compare their homology in order to find out the route of infection whether emerged from foreign strains or reemerged from domestic native strains.

Materials and Methods
All patients suspected for cholera were entered to this study.All V. cholerae isolates were diagnosed in the local laboratories of provinces based on standard procedures. 13,14For re-identification for final confirmation, the first 5 diagnosed V. cholerae strains were transferred from each province to the Health Reference Laboratory as a referral laboratory, which is established as a surveillance system by the Ministry of Health and Medical Education. 15,16hese specimens were examined for specific serogroups by O1 polyvalent and Ogawa/Inaba monospecific antisera (BD, Becton Dickinson Co., USA) after identification by standard biochemical and bacteriological tests.

Antimicrobial Susceptibility
Those confirmed V. cholerae 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), trimethoprim-sulfamethaxazone (SXT), and erythromycin (E).The organisms Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 29213), and Pseudomonas aeru-ginosa (ATCC 27853) were used as quality control strains for MIC E-test. 17lse Field Gel Electrophoresis Genotyping of isolates was performed by PFGE using PulseNet standard procedure for V. cholera.12 The whole agarose-embedded genomic DNA from V. cholera was prepared.The conditions used for separation were as follows: an isolated colony was streaked from test cultures to Trypticase soy agar with 5% defibrinated sheep blood (TSA-SB) plates incubated overnight for confluent growth.7,13 Grown colonies within 14-18 hours were used to prepare cell suspension.Bacterial suspension was prepared in a cell suspension buffer (100 mM Tris:100 mM 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 mM Tris: 50 mM EDTA, pH 8.0 + 1% sarcosyl, and 25 μL proteinase K 20 mg/mL) and washed with pre-heated sterile ultrapure water and sterile TE buffer 6 times in a 54-55°C water bath. Eah plug was digested with 40 units of NotI restriction enzyme (Fermentas).DNA molecular weight size marker was prepared by XbaI digestion of Salmonella enterica serotype Braenderup H9812 plugs.PFGE was carried out with CHEF Mapper XA System (Bio-Rad) using program explained by PulseNet.

Image Analysis
The PFGE fingerprint pattern was analyzed using the computer software package BioNumerics 6.6 (Applied Maths, Belgium).After background subtraction and gel normalization, the fingerprint patterns were subjected to typing on the basis of banding similarity and dissimilarity using Dice similarity coefficient and clustering based on the unweighted-pair group method using average linkages (UPGMA), as recommended by the software manufacturer, and results were graphically represented as dendrograms.
According to the issued instructions released by Center for Disease Control and Prevention, just 118 specimens were sent for confirmation and 104 out of them were confirmed as cholera cases, including 3 Ogawa and 101 Inaba strains.These three Ogawa strains were not considered in this comparison as they did not play an important role in the current outbreak.
Antibiotic susceptibility test revealed 100% resistance of Inaba serotypes to NA, TE, and SXT; while all of them were sensitive to CIP, CFM, and AMP.Susceptibility test showed that only 23% that were isolated from both Afghan travelers and Iranian citizens, were sensitive to erythromycin; although all strains showed intermediate pattern from the second month of outbreak.Isolated specimen in 2011 (30-90) had different patterns.It was sensitive to all above antibiotics except to SXT (Table 1).
The genomic DNA of 31 selected strains plus one belonging to the outbreak in 2011 were digested by NotI restriction enzyme, creating 17 to 20 fragments, the sizes of which ranged from 20.5 to 668.9 kb.Cluster analysis by dendrogram of the gel images separated the V. cholerae biotype strains into some major clusters, although generally all analyzed strains in 2013 showed 92% homology.These strains were located in 8 clusters.Strains isolated in 2011 had a homology less than 80% and were located in a totally distinct cluster from all strains isolated in 2013.
PFGE analysis revealed no correlation between the stains resistant and sensitive to erythromycin (Figure 1), although susceptible strains were seen at first 2 weeks of commencing outbreaks (Table 2).

Discussion
Mafi et al studied the cholera outbreaks in Iran since 2010 to 2014. 18They gathered all the data and concluded that the rate of Ogawa strains was reduced from 100% in 2010 to 1.17% in 2013 and rate of Inaba strains was increased to 98.83% in 2013 instead.However, similar studies indicated the trend of isolates toward Inaba during 2005-2010 outbreaks. 5mong total cases, 83.65% of all the cases involved with cholera were reported from Baluchestan and Kerman provinces.The Afghan travelers had the major contribution in this cholera outbreak (81.71%).Therefore, it is expected the infection to be transmitted from abroad by the travelers and spread to other provinces from these two provinces like previous outbreaks 18,19 ; however, this is needed to be confirmed by a molecular typing method.
Isolated strains were analyzed by the PFGE method using software package BioNumerics for the outbreak in 2011. 7Results of PFGE analysis in this study revealed the cholera isolates were in different clusters.Ogawa serotype was a main type of the outbreak of 2011.This serotype was distributed through the country with 6 different patterns, while a few Inaba strains were only isolated with differentiated patterns from Ogawa serotype. 7In the present study, the diversity of cholera strains in 2013 outbreak was studied and the results were compared with the homology of detected Inaba strains in 2011.We concluded the previous native Inaba strain could not have a role in 2013 outbreak.
PFGE results showed no correlation between pattern of our pulsotypes and susceptibility testing results.This result was also supported by different researches. 20,21The  V. cholerae strains isolated in this study had nearly similar susceptibility pattern, except to erythromycin.The specimens isolated in 2011 that were located in a separate cluster had different susceptibility patterns, although the Inaba isolated strains with different patterns of susceptibility to erythromycin had no distinct PFGE patterns.However, it seems this issue needs to be more investigated in other researches.Therefore, we suggest that the susceptibility testing be performed for all confirmed isolates during the outbreaks, not just for the first identified strains.It means, we need to revise our previously released sampling procedure. 22,23n recent years, new pathogenic variants of V. cholerae have emerged and spread throughout many Asian and African countries. 2,24On the other hand, the emergence of multidrug resistant V. cholera isolates has been reported frequently that is a major problem in developing coun-tries, particularly in Iran which is surrounded by several neighboring countries engaging with cholera problem. 25he number of male cholera patients, their nationality, and the mean age all obviously confirmed the emergence of cholera from abroad.Regarding above mentioned issue, performing an active surveillance program at least during summer for all passengers from Afghanistan and Pakistan is recommended.
Although the main aim of establishment of PulseNet in eastern Mediterranean region is to help the early detection, investigation on bacterial causes of outbreaks could not be traced in the member countries, which causes a gap.It is clear that using recommended standardized procedures for PFGE would help to compare obtained data from different countries.

Conclusion
This study proved that isolated Inaba strains were emerged from neighboring countries, with distinct clonality pattern in PFGE compared to the native strains.The analysis results may support epidemiological data in describing how a V. cholerae may distribute through the country.This study also underlined the contribution of new variant of cholera El Tor that had some dissimilarity with the previously isolated strains in outbreak of 2011.The study was supported by Health Reference Laboratory and Center for Disease Control and Prevention of Ministry of Health and medical Education.We hereby thank all staff in local centers of provinces of Iran whose sent specimens helped us to perform this study.

Table 2 .
Susceptibility Results of Isolated Strains to Erythromycin