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Indian Journal of Medical Microbiology, Vol. 29, No. 2, April-June, 2011, pp. 158-160 Brief Communication Multi locus sequence type comparison of invasive and commensal Haemophilus influenzae isolates from Delhi KK Saikia1, R Bewal1, D Bansal1, A Kapil1, S Sood1, NK Arora2, BK Das1 1 Department of Microbiology, All India Institute of Medical Sciences, New Delhi - 100 029, India Correspondence Address: B K Das Department of Microbiology, All India Institute of Medical Sciences, New Delhi - 100 029 India tezpur.bimal@gmail.com Date of Submission: 25-Oct-2010 Code Number: mb11036 PMID: 21654111 DOI: 10.4103/0255-0857.81800 Abstract Haemophilus influenzae is a major public health concern in the developing world. The most virulent strain is H. influenzae Type b (Hib). Hib also constitutes a major portion of nasopharyngeal commensal flora in otherwise healthy individuals. Through dendogram based on composite gene sequences of seven multi locus sequence type genes, it was observed that invasive and commensal isolates made two completely separate clusters which are indicative of independent evolution of these two groups of H. influenzae in the Indian subcontinent.Keywords: Commensal, Haemophilus influenzae, invasive, multi locus sequence type Introduction Haemophilus influenzae is a major public health concern even in the post H. influenzae Type b (Hib) conjugate vaccine era, responsible each year for more than three million cases of invasive disease and 400,000 deaths worldwide. [1] There are six capsular serotypes of H. influenzae (a-f) as well as nontypable (unencapsulated) strains. [2] The most virulent strain is H. influenzae Type b (Hib) responsible for 95% of bloodstream and meningeal Haemophilus infections in children. The most important virulence factor is the polyribosyl ribitol phosphate (PRP) capsule. [3] On the other hand Hib also constitutes a major portion of nasopharyngeal commensal flora in otherwise healthy individuals. The carriage rate of Hib varies from place to place. In India Hib carriage of children below two years of age is around 7.72%. [4] Before widespread vaccination, Hib carriage rate in the USA, UK varied from 2-6%. [5] The relationship between carriage rates and the risk of disease is not understood. The spread of infection in the presence of low carriage rates has been described and no clear disease has been reported in spite of high carriage rates. [5] In addition, nasopharyngeal organisms appear to lack certain virulence attributes common in organisms isolated from patients with invasive disease. [6] This study was undertaken to analyze if there exist any significant genetic differences between invasive and commensal H. influenzae isolates. Materials and Methods H. influenzae isolates were collected from August 2005 to August 2007. Isolates causing invasive disease were cultured from sterile body fluid like blood, cerebrospinal fluid and pleural fluid and were designated as invasive isolates. [7] Commensal isolates were obtained from the nasopharynx of healthy schoolgoing children aged 5-14 years through nasopharyngeal swab. The study was cleared by the institutional review board (IRB). In total 15 invasive and 13 commensal isolates were characterized through multi locus sequence typing (MLST). Isolates were cultured on Haemophilus test medium, confirmed as H. influenzae using standard methodology and stored as glycerol stock until further use. [8],[9] capB gene of H. influenzae is known as virulence gene as Type b capsular polysaccharide is a major virulence factor and target for serum antibodies. Isolates with Type b serotype from both invasive and commensal isolates were detected through capB polymerase chain reaction (PCR) as described earlier. [2] The results were also confirmed by slide agglutination serotyping. High-fidelity enzyme AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster city, California, USA) was used for all PCR reactions. For MLST, DNA sequencing of seven housekeeping genes (adk, atp G,0 frd B,0 fuc K,0 mdh, pgi, rec A )0 was carried out for each isolate as done previously. [10] Big Dye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, California, USA) was used for cycle sequencing reactions. Sequencing was performed on Applied Biosystems 3130xl platform. Sequence analysis was done using SeqScape v2.6 software (Applied Biosystems). Phylogenetic analysis was performed using MEGA4. [11] Results Serotyping revealed that all 15 invasive isolates in this study were of Type b. Seven out of 13 commensal isolates were of serotype b and 6/13 were non-Type b. MLST results showed presence of two sequence types (STs) previously described and five new STs among the Indian invasive H. influenzae isolates tested. The most prevalent ST among the invasive Hib isolates was ST118 (7/15, 46.67%) also reported from the United States earlier ( www.haemophilus.mlst.net ). The second most common STs in our study were ST617 and ST24, both represented by two isolates (13.33%) each. ST617, a single locus variant (SLV) of ST118 in the mdh locus, was a novel ST and ST24, a SLV of ST118 in the recA locus, was observed in the USA earlier. Other STs present amongst the invasive isolates in our study, ST616, -570, -571, and -572, were novel STs. Amongst the 13 nasopharyngeal isolates tested through MLST, nine distinct STs were discovered each of which were novel. The most common ST among the commensal isolates was ST626 represented by three serotype b isolates (23.07%). The second most prevalent commensal ST was found to be ST628 and ST622 represented by two non-Type b isolates each (15.39%). Other STs discovered from commensal isolates in our study were ST618, -629 (non-Type b), -619, -620, -625, and -627 (Type b). Mean age of invasive isolates was 748.27±276.80 (Mean±SD) days and that of commensal isolates was 763.15±232.02 days respectively. There was no significant age difference between the invasive and commensal isolates (P=0.880) as revealed by unpaired t-test (SPSS Version 11.5). By sequence comparison of invasive and commensal isolates through dendogram based on composite gene sequences of seven conserved MLST genes, it was observed that invasive and commensal isolates made two completely separate clusters [Figure - 1]. The dendogram presented three distinct clusters of Type b invasive isolates, Type b commensal isolates and non-Type b commensal isolates. EU103610, EU103611, FJ010204, FJ010205, FJ010206 and FJ867898 were genebank accession numbers obtained from this study. Discussion Traditionally, Type b H. influenzae isolates are found to be associated with 95% cases of invasive diseases. In the recent years, the nontypeable H. influenzae (NTHi) has emerged as an important cause of acute respiratory tract infection replacing Hib due to successful implementation of conjugate vaccine. Invasive Hib predominantly causes invasive disease in young infants (< two years old). Presence of predisposing factors, e.g. absence of maternal antibodies, inability to deal with complex carbohydrate antigen and concurrent viral infection may play a role in invasive disease due to Hib. Type b H. influenzae does not cause disease in older children presumably due to acquisition of herd immunity. Carriage of Hib among healthy individuals had been observed frequently. The reported carriage varies among different geographical regions. [12],[13] In a study carried out in healthy schoolgoing children in North India overall Hib carriage was found to be 13.17% and that of non-Type b was found to be 28.54%. [14] After the introduction of conjugate vaccine, the Hib carriage rate has reduced in the industrialized countries to almost zero in vaccinated children from pre-vaccination levels of 5-10%. [15] However, NTHi carriage rate among children has been reported to vary between 25 and 81%. [16] Presence of healthy Hib carrier in the community may help in inducing herd immunity by transmitting the bug to younger individuals (natural vaccination). Alternatively, presence of Hib carrier may also endanger susceptible individuals by acting as a source of invasive infection. It is unclear whether commensal Hib carry genetic attributes similar to that of invasive Hib. Typing methods that are based on antibodies against capsular polysaccharide fail to differentiate between invasive and commensal Hib. In our study of MLST involving Hib of invasive and commensal origin, it was observed that invasive Hib did not demonstrate much variation in their genome. In contrast, genetic diversity among commensal Hib was much more common implying existence of two distinct evolutionary lineages among invasive and commensal Hib. The commensal carriers and invasive isolates were age-matched in this study. The significance of the existence of two genetically distinct clusters of Hib is, at the moment, not clearly understood. However, it is pertinent that the pathogenic potential of the commensal Hib needs to be ascertained. In addition, protection against invasive Hib disease is primarily attributed to the induction of antibodies against Type b capsular polysaccharide. Invasive H. influenzae with conserved genetic attributes pose no threat to a successful program as these isolates belong to a close cluster. Commensal Hib with wider genetic diversity may carry polymorphism within antigens that induces protection. If true, existence of polymorphism within the protective antigens may warrant wider representation of protective antigens from genetically distinct clusters. Therefore it will be necessary to determine, in future studies, the pathogenic potential of the evolutionarily distinct commensal Hib and examine these isolates for presence of polymorphism within the protective antigens. This will ensure that a future vaccine against H. infleunzae will induce a robust immune response. Hence, exploration involving larger studies that looks at molecular evolution of invasive and commensal H. influenzae isolates is of paramount importance. Acknowledgement This study was supported by a grant from Department of Science and Technology, Government of India. References
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