Genotypic characterization of Streptococcus pneumoniae serotype 19F in Malaysia

Streptococcus pneumoniae is an epidemiologically important bacterial pathogen. Recently, we reported the antibiotic susceptibility patterns of a limited collection of pneumococcal isolates in Malaysia with a high prevalence of erythromycin resistant strains. In the present study, 55 of the pneumococcal isolates of serotype 19F were further analysed by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The generated genotypic patterns were then correlated with the antibiograms previously reported. Forty-seven different PFGE profiles (PTs) were obtained, showing that the isolates were genetically diverse. MLST identified 16 sequence types (STs) with ST-236 being predominant (58.2%), followed by ST-81 (10.3%). Among the ST-236 isolates, 22 were erythromycin resistant S. pneumoniae (ERSP) and 15 were trimethoprim/sulfamethoxazole (TMP/SMX) resistant, while among ST-81, four isolates were ERSP and two were TMP/SMX resistant. The high prevalence of erythromycin resistant serotype 19F isolates of ST-236 in this study has also been reported in other North and South East Asian countries.     

肺炎克雷伯菌单基因分子进化分析及多位点序列分型与基因组系统学研究

目的比较7株已完成全基因组测序的肺炎克雷伯菌单基因分子进化分析及多位点序列(MLST)分型与基因组系统学研究的结果,研究其亲缘关系。方法采用Roche 454高通量测序技术对1株泛耐药肺炎克雷伯菌JM45株做全基因组测序(完成图),再与NCBI已完成全基因组测序的6株肺炎克雷伯菌9种看家基因进行单个基因分子进化分析,将该9个基因序列串联作MLST分型,并将7株肺炎克雷伯菌的全基因组序列作聚类分析(为Fast Minimum Evolutin法)。结果 JM45株测得一条完整的基因组(染色体)序列及两条质粒序列;因为不同的基因进化率不同,7株肺炎克雷伯菌9种看家基因单个基因序列的分子进化图并不一致;7株肺炎克雷伯菌MLST分型可见JM45株与HS11286株相同(即为同一型),1084株与NTUH-K2044株相同,但7株肺炎克雷伯菌基因组系统学分析可见,JM45株与HS11286株并不相同(即非为同一型),JM45株与HS11286株距离最近,与342株距离最远,其他各株介于二者之间。结论采用单个基因序列的分子进化分析法及MLST分型用作菌株亲缘性关系分析法并不可靠,本研究为国内首个单个看家基因分子进化分析及MLST分型与基因组系统学研究的比较报道。     

Population genetics of microbial pathogens estimated from multilocus sequence typing (MLST) data.

The inference of population recombination (rho), population mutation (Theta), and adaptive selection is of great interest in microbial population genetics. These parameters can be efficiently estimated using explicit statistical frameworks (evolutionary models) that describe their effect on gene sequences. Within this framework, we estimated rho and Theta using a coalescent approach, and adaptive (or destabilizing) selection under heterogeneous codon-based and amino acid property models in microbial sequences from MLST databases. We analyzed a total of 91 different housekeeping gene regions (loci) corresponding to one fungal and sixteen bacterial pathogens. Our results show that these three population parameters vary extensively across species and loci, but they do not seem to be correlated. For the most part, estimated recombination rates among species agree well with previous studies. Over all taxa, the rho/Theta ratio suggests that each factor contributes similarly to the emergence of variant alleles. Comparisons of Theta estimated under finite- and infinite-site models indicate that recurrent mutation (i.e., multiple mutations at some sites) can increase Theta by up to 39%. Significant evidence of molecular adaptation was detected in 28 loci from 13 pathogens. Three of these loci showed concordant patterns of adaptive selection in two to four different species.     

肺炎克雷伯菌脉冲场凝胶电泳及多位点序列分型技术的联合应用

目的 联合使用脉冲场凝胶电泳(PFGE)及多位点序列分型(MLST)对肺炎克雷伯菌进行分型研究.方法 参照美国疾病预防控制中心及亚太地区PulseNet提供的PFGE相关标准化操作程序,优化相关电泳条件,对某儿童医院于2005-2006年分离到的59株菌分型分析,对其中高度相似性菌株进行MLST分型分析.结果 59株菌经PFGE分型分析,可分成47个PFGE型,19个PFGE群,具有高度相似性的菌株经MLST分型,其型别分别为ST-340、ST-342、ST-343、ST-344、ST-345,其中ST-342、ST-343、ST-344、ST-345为MLST数据库中中国新发现的ST型.结论 具有PFGE高度相似性的肺炎克雷伯菌可以通过MLST进一步确认或区分.     

肺炎克雷伯菌脉冲场凝胶电泳及多位点序列分型技术的联合应用

Objective To type Klebsiella pneumonia through methods including pulse-field gel electrophoresis (PFGE) in combination with multilocus sequence typing. Methods Four selected different Eps, referring to the Standard Operating Procedure of PulseNet China, were used. The single colony of Klebsiella pneumonia was quantified after enriched culture. Embedding organisms in agarose and genome DNA were lysed with Proteinase K and then digested by restriction endonuclease Xba Ⅰ , to produce agarose gel. Fingerprint was obtained through PFGE and bands were marked with their molecular weights and then analyzed by BioNumerics software. Using MLST to analyze the strains that were highly similar, by PFGE typing Results By comparing the four results from each Eps, fk3 (switch time from 6s to 36s,total run time is 18.5 hours) seemed to be better than the others.59 strains of Klebsiella pneumonia were divided into 47 PFGE types and 19 PFGE clusters. The highly similar strains could be typed into ST-340、ST-342、ST-343、ST-344、ST-345 by MLST. Among them, ST-342、 ST-343、 ST-344、 ST-345 types were all new MLST types that were reported in China.Conclusion Highly similar Klebsiella pneumonias typed by PFGE could also be typed by MLST.     

肺炎克雷伯菌脉冲场凝胶电泳及多位点序列分型技术的联合应用

Objective To type Klebsiella pneumonia through methods including pulse-field gel electrophoresis (PFGE) in combination with multilocus sequence typing. Methods Four selected different Eps, referring to the Standard Operating Procedure of PulseNet China, were used. The single colony of Klebsiella pneumonia was quantified after enriched culture. Embedding organisms in agarose and genome DNA were lysed with Proteinase K and then digested by restriction endonuclease Xba Ⅰ , to produce agarose gel. Fingerprint was obtained through PFGE and bands were marked with their molecular weights and then analyzed by BioNumerics software. Using MLST to analyze the strains that were highly similar, by PFGE typing Results By comparing the four results from each Eps, fk3 (switch time from 6s to 36s,total run time is 18.5 hours) seemed to be better than the others.59 strains of Klebsiella pneumonia were divided into 47 PFGE types and 19 PFGE clusters. The highly similar strains could be typed into ST-340、ST-342、ST-343、ST-344、ST-345 by MLST. Among them, ST-342、 ST-343、 ST-344、 ST-345 types were all new MLST types that were reported in China.Conclusion Highly similar Klebsiella pneumonias typed by PFGE could also be typed by MLST.     

Clonal diversity of Streptococcus pyogenes within some M-types revealed by multilocus enzyme electrophoresis.

Twenty-two reference isolates and 30 local isolates of group A Streptococci were classified into 36 electrophoretic types (ET) on the basis of allozyme variation at 27 enzyme loci. Local isolates were characterized by a high frequency of M-non typable strains. M-type and ET were more closely associated in local isolates from an endemically-infected population; nevertheless, amongst the local isolates there were also strains of the same ET type with different M-types. A possible explanation is that genetic exchange between strains may introduce different M-types into strains of defined ET when these are exposed to strong selection in the presence of heavy loads of infection. In contrast to the reported clustering of strains associated with toxic shock-like syndrome into two closely related ET clones, we found no relationship of ET phenotype to acute poststreptococcal glomerulonephritis or rheumatic fever.     

Development of a multilocus sequence typing tool for high-resolution subtyping and genetic structure characterization of Cryptosporidium ubiquitum

Cryptosporidium ubiquitum is an emerging zoonotic pathogen in humans. Recently, a subtyping tool targeting the 60-kDa glycoprotein (gp60) gene was developed for C. ubiquitum, and identified six subtype families (XIIa-XIIf). In this study, we selected five genetic loci known to be polymorphic in C. hominis and C. parvum for the development of a multilocus subtyping tool for C. ubiquitum, including CP47 (cgd6_1590), MSC6-5 (cgd6_4290), cgd6_60, cgd2_3690, and cgd4_370. PCR primers for these targets were designed based on whole genome sequence data from C. ubiquitum. DNA sequence analyses of 24 C. ubiquitum specimens showed the presence of 18, 1, 5, 4, and 5 subtypes at the CP47, MSC6-5, cgd6_60, cgd2_3690, and cgd4_370 loci, respectively. Altogether, 18 multilocus sequence typing (MLST) subtypes were detected among the 19 specimens successfully sequenced at all polymorphic loci. Phylogenetic analyses of the MLST data indicated that the rodent subtype families of XIIe and XIIf were highly divergent from others, and the ruminant XIIa subtype family formed a monophyletic group genetically distant from other rodent subtype families XIIb, XIIc, and XIId. The latter showed no consistent grouping of specimens and formed one large cluster in phylogenetic analysis of concatenated multilocus sequences. This was supported by results of STRUCTURE and F_ST analyses, which further suggested that XIIa originated from one common ancestor whereas XIIb, XIIc, and XIId contained mixed ancestral types, reflecting a close relatedness of the three subtype families and the likely occurrence of genetic recombination among them. Thus, an MLST tool was developed for high-resolution subtyping of C. ubiquitum and results of preliminary characterizations of specimens from humans and animals supported the conclusion on the existence of ruminant and rodent-adapted C. ubiquitum groups.     

The molecular epidemiology of serial Candida tropicalis isolates from ICU patients as revealed by multilocus sequence typing and pulsed-field gel electrophoresis

The genetic profiles of 50 Candida tropicalis isolates serially collected from 14 patients during a prospective surveillance study in adult intensive care units (ICUs) were characterized by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) of NaeI restriction fragments. A total of 21 diploid sequence types (DSTs) and 43 genotypes were differentiated by MLST and PFGE, respectively. Significant correlations were found between PFGE genotypes and DST types (P < 0.05). Dendrograms generated by either MLST or PFGE-NaeI showed that most isolates from the same patient co-clustered with high similarity regardless of the anatomical source of isolation. Maintenance, microvariation or replacement of C. tropicalis isolates could be observed within the individual patients by further analysis of variations in MLST sequence data. Antifungal susceptibility testing revealed that 17 (34%) of 50 isolates presented high MICs to flucytosine (MIC ≥ 8 μg/mL). Sixteen (94%) of these isolates belonged to DST 164, and these were collected from four patients with different PFGE genotypes. Isolates sharing the same DST may represent a common clone that underwent extensive mutation over time to cope with drug selection pressure, different hosts or different geographic environments.     

Prevalence and genetic structures of Streptococcus pneumoniae serotype 6D, South Korea

To determine prevalence and genetic structures of new serotype 6D strains of pneumococci, we examined isolates from diverse clinical specimens in South Korea during 1991-2008. Fourteen serotype 6D strains accounted for 10.4% of serogroup 6 pneumococci from blood, sputum, nasopharynx, and throat samples. Serotype 6D strains consisted of 3 sequence types.     

Carriage prevalence,serotype distribution,antimicrobial resistance,pspA typing and pilus islets of Streptococcus pneumoniae isolated from adults living in a Brazilian urban slum

IntroductionFor Brazilian adults, pneumococcal vaccines have been usually taken only by those who are at higher risk for development of pneumococcal diseases. Since populations from lower socioeconomic status are at high risk of acquiring pneumococcal infections, we investigated the carriage prevalence, colonization risk factors, capsular and surface protein types, and antimicrobial resistance among pneumococcal isolates recovered from adults living in a Brazilian urban slum.MethodsBetween September-December 2016, we conducted a cross-sectional study among individuals aged ≥ 18 years who attended a public primary clinic in Niterói/RJ, Brazil. Pneumococci were isolated by culture on sheep blood agar plates with and without gentamicin. Antimicrobial susceptibility was determined for all isolates. We used PCR to determine capsular types, PspA families (Fam) and pilus islets (PI).ResultsOf 385 adults, 32 (8.3 %) were pneumococcal carriers. Three carriers had two different pneumococci, totaling 35 isolates. After multivariate analysis, smoking, previous hospitalization, alcohol consumption and co-habitation with children aged < 6 years increased the odds of pneumococcal carriage, but antibiotic use in the previous 2 weeks was found to be a protective factor. Fourteen different serogroups/serotypes were detected and the prevalent ones were 9 N/L, 10A, 15B/C and 35F/47F (n = 3; 8.6 % each). Non-typeable (NT) isolates made up 31.4 %. All isolates were susceptible to chloramphenicol, levofloxacin and vancomycin. We found eight (22.9 %) penicillin non-susceptible pneumococci (PNSP) with minimum inhibitory concentrations (MICs) of 0.38–1.5 μg/mL. The two (5.7 %) erythromycin-resistant isolates had MIC > 256 μg/mL, cMLS_B phenotype and the erm(B) gene. Twelve (34.3 %) and 17 (48.6 %) isolates had PspA Fam1 and Fam2, respectively. Three (8.6 %) isolates had genes for pilitwo PI-1 and one PI-2.ConclusionWe detected a low frequency of pneumococcal carriage among the adult population, but a high diversity of serotypes. Frequencies of PNSP and NT isolates resistant to antimicrobial agents are concerning.     

Antimicrobial susceptibility and serotype distribution of Streptococcus pneumoniae isolated from patients with community-acquired pneumonia and molecular analysis of multidrug-resistant serotype 19F and 23F strains in Japan

A nationwide study was undertaken to determine the susceptibility to penicillin and serotypes of Streptococcus pneumoniae in Japan. S. pneumoniae was isolated from 114 adult patients with community-acquired pneumonia over 22 months at 20 hospitals and medical centres in different regions in Japan. All but five isolates were from sputum. Forty-eight isolates (42.1%) were susceptible, 40 (35.1%) showed intermediate resistance (MIC, 0.12-1.0 microg/ml) and 26 (22.8%) were resistant (MIC, >or=2.0 microg/ml) to penicillin G. All isolates were susceptible to ceftriaxone (breakpoint 1 microg/ml), imipenem (4 microg/ml) and vancomycin (4 microg/ml). Most were resistant to erythromycin, clarithromycin and azithromycin; only two were resistant to levofloxacin. Differences were found in the distribution of serotypes among isolates showing susceptibility to penicillin (predominant types 3, 6B, and 19F), intermediate resistance (6B, 14, 19F, and 23F) and full resistance (19F and 23F). PFGE typing showed that 14 of the 25 strains of serotype 19F had a single DNA profile, pattern A, a pattern closely similar to that of the Taiwan multidrug-resistant 19F clone. Twelve pattern A strains were not susceptible to penicillin but carried the macrolide resistance gene mef(A). The DNA profiles of the 15 strains of 23F were also heterogeneous but six were highly similar (pattern b) yet distinct from the Spanish multidrug-resistant 23F clone although possibly related to the Taiwan multidrug-resistant 23F clone. The pattern b strains were not susceptible to penicillin and also harboured either mef(A) or erm(B). Our results indicate that multidrug-resistant pneumococci are spreading rapidly in Japan. Efforts to prevent the spread of the pandemic multidrug-resistant serotypes should be intensified.     

Population structure and drug resistance patterns of emerging non-PCV-13 Streptococcus pneumoniae serotypes 22F, 15A,and 8 isolated from adults in Ontario,Canada

The introduction of pneumococcal conjugate vaccines has led to the emergence of non-vaccine serotypes, which contributed to invasive pneumococcal disease in Canada and worldwide. A significant increase in the prevalence of non-13-valent pneumococcal conjugate vaccine (PCV-13)-included serotypes 22F, 15A, and 8 was observed from 2009 to 2013 in Ontario (all p values < 0.01). In this study, whole genome sequencing was conducted on the 25 isolates of serotype 22F, seven of 15A and 10 of 8 to investigate the population structure and antibiotic resistance. All seven serotype 15A isolates were found to be multidrug resistant. From whole genome analysis, we observed recombination events among serotypes 22F, 15A and 8 populations. Serotype 22F (ST433) has emerged into two sub-populations, with 28% (7/25) exhibiting recombination events, and five also acquiring macrolide resistance as a result of recombination. This study enhances the knowledge on the molecular evolution of emerging non-PCV-13 vaccine serotype 22F, including acquisition of resistance genes through recombination events. It underpins the importance of whole genome sequencing in studying Streptococcus pneumoniae population structures and dynamics, and its utility in molecular surveillance.     

Use of multilocus sequence typing for the investigation of colonisation by Candida albicans in intensive care unit patients

A prospective study was performed to determine the prevalence of candidal colonisation on the general intensive care unit at a large teaching hospital. Colonisation with Candida spp. was found to be common, occurring in 79% of patients on the unit. C. albicans was the commonest species, colonising 64% of patients, followed by C. glabrata (18%) and C. parapsilosis (14%). Most of the members of staff tested carried Candida spp. at some point, although carriage appeared to be transient. C. parapsilosis was the most commonly isolated species from staff hands, whereas C. albicans was the most commonly isolated species from the mouth. The molecular epidemiology of C. albicans was investigated using Ca3 typing and multilocus sequence typing (MLST). MLST proved to be a reproducible typing method and a useful tool for the investigation of the molecular epidemiology of C. albicans. The results of the molecular typing provided evidence for the presence of an endemic strain on the unit, which was isolated repeatedly from patients and staff. This finding suggests horizontal transmission of C. albicans on the unit though it may also reflect the relative frequency of C. albicans strain types colonising patients on admission. This study has important implications for the epidemiology of systemic candidal infections.     

Phenotype,genotype, and serotype distribution of macrolide resistant invasive and non-invasive Streptococcus pneumoniae strains,in Sousse,Tunisia

ObjectiveWe determined the macrolide resistance phenotypes and genotypes in Streptococcus pneumoniae isolates in Sousse and assessed the serotype distribution.MethodsWe included S. pneumoniae strains isolated at our laboratory (2010–2013). The antimicrobial susceptibility was tested according to CA-SFM specifications. Serotyping was performed by agglutination of latex particles, to identify a subset of serotypes included in pneumococcal conjugate vaccines. The presence of macrolide resistance genes (ermB, mefA, mel) was detected by PCR.ResultsA total of 52.8% of 140 S. pneumoniae isolates were macrolide-resistant: MLSB (89.2%) and M (10.8%). The MLSB phenotypes were genotypically confirmed by ermB gene presence. 62% had decreased susceptibility to penicillin. The serotypes were: 14, 1, 23F, and 19A. Serotype coverage by PCV7, PCV10 and PCV13 was 44.2%, 73.6%, and 75.6% respectively.Conclusion50% of S. pneumoniae isolates were macrolide resistant. The MLSB phenotype encoded by the ermB gene was the most frequent. Serotype coverage seems inadequate.     

International links between Streptococcus pneumoniae vaccine serotype 4 sequence type (ST) 801 in Northern European shipyard outbreaks of invasive pneumococcal disease

BackgroundPneumococcal disease outbreaks of vaccine preventable serotype 4 sequence type (ST)801 in shipyards have been reported in several countries. We aimed to use genomics to establish any international links between them.MethodsSequence data from ST801-related outbreak isolates from Norway (n = 17), Finland (n = 11) and Northern Ireland (n = 2) were combined with invasive pneumococcal disease surveillance from the respective countries, and ST801-related genomes from an international collection (n = 41 of > 40,000), totalling 106 genomes. Raw data were mapped and recombination excluded before phylogenetic dating.ResultsOutbreak isolates were relatively diverse, with up to 100 SNPs (single nucleotide polymorphisms) and a common ancestor estimated around the year 2000. However, 19 Norwegian and Finnish isolates were nearly indistinguishable (0–2 SNPs) with the common ancestor dated around 2017.ConclusionThe total diversity of ST801 within the outbreaks could not be explained by recent transmission alone, suggesting that harsh environmental and associated living conditions reported in the shipyards may facilitate invasion of colonising pneumococci. However, near identical strains in the Norwegian and Finnish outbreaks does suggest that transmission between international shipyards also contributed to those outbreaks. This indicates the need for improved preventative measures in this working population including pneumococcal vaccination.     

Multilocus sequence typing of Candida tropicalis shows clonal cluster enrichment in azole-resistant isolates from patients in Shanghai,China

To explore the putative correlation between the multilocus sequence types (MLST) and antifungal susceptibility of clinical Candida tropicalis isolates in Mainland China. Eighty-two clinical C. tropicalis isolates were collected from sixty-nine patients at Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China, from July 2012 to February 2015, and antifungal susceptibility tests were performed. Genetic profiles of those 82 isolates (30 azole-resistant and 52 azole-susceptible) were characterised by multilocus sequence typing. Phylogenetic analysis of the data was conducted with the clustering method, using UPGMA (unweighted pair group method with arithmetic averages) and the minimal spanning tree algorithm. MLST clonal clusters were analysed using the eBURST V3 package. Of the six gene fragments identified in multilocus sequence typing, SAPT4 presented the highest typing efficiency, whereas SAPT2 was the least efficient. Of the 44 diploid sequence types (DSTs) differentiated, 32 DSTs and 12 genotypes were identified as new to the C. tropicalis DST database. Twenty (45.45%) of the 44 DSTs were assigned to seven major groups based on eBURST analysis. Of these, Group 6, which contained DST 376, DST 505, DST 506 and DST 507, accounted for 76.7% of the 30 azole-resistant isolates. However, the genetic relationships among the azole-susceptible isolates were relatively decentralised. This MLST analysis of the putative correlation between the MLST types and antifungal susceptibility of clinical C. tropicalis isolates in Mainland China shows that DSTs 376, 505, 506 and 507 are closely related azole-resistant C. tropicalis clones.     

Development of a multilocus sequence typing (MLST) scheme for Mannheimia haemolytica and assessment of the population structure of isolates obtained from cattle and sheep

Mannheimia haemolytica is an important veterinary pathogen affecting cattle and sheep. Previous typing methods, including restriction enzyme analysis, pulsed-field gel electrophoresis and multilocus enzyme electrophoresis (MLEE) have indicated a clonal population structure. Multilocus sequence typing (MLST) has now almost replaced MLEE and is a definitive and portable typing method, allowing global data exchange. The purpose of this study was to develop a MLST scheme for M. haemolytica. A collection of isolates from 10 countries, including the type strain and reference strains for all recognized serotypes were included in the study. Partial sequences of the housekeeping genes adk, aroE, deoD, gapDH, gnd, mdh and zwf were used to define the MLST scheme. The 95 isolates demonstrated 34 different sequence types (ST) of which 19 were connected in three clonal complexes (CC). ST1 constituted more than one-third of the isolates and was most frequently demonstrated among isolates from bovine sources. The analysis indicated a common evolutionary origin of 33 isolates from the French alps, collected from domestic and wild animals and demonstrating several related STs. An analysis of 17 isolates from the USA demonstrated the same ST in 14 of the isolates. In conclusion, an unambiguous typing scheme is presented for M. haemolytica and results obtained confirm previous observations of a clonal population of the organism.     

An extended multi-locus molecular typing schema for Streptococcus pneumoniae demonstrates that a limited number of capsular switch events is responsible for serotype heterogeneity of closely related strains from different countries

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. Pneumococcal strains are classified according to their capsular serotype and through a Multi-Locus Sequence Typing schema (MLST) based on the sequencing of seven housekeeping genes. However, strains with a defined allelic profile (Sequence Type, ST) can have different serotypes, suggesting that the micro-evolution of the MLST lineages leads to a considerable degree of phenotypic variability. To better investigate the genetic diversity within these lineages, we set-up and then validated an extended molecular typing schema (96-MLST) based on the sequencing of ninety-six genomic loci. 96-MLST loci were designed within core-genes in a collection of 39 complete genomes of S. pneumoniae. None of the capsular genes was included in the schema. When tested on a collection of 69 isolates, 96-MLST was able to partition strains with the same ST and diverse serotypes into groups that were homogenous for capsular serotype, improving our understanding of the evolution of epidemiologically relevant lineages. Phylogenetic sequence analysis showed that the capsular heterogeneity of three STs that were sampled more extensively could be traced back to a limited number of capsular switch events, indicating that changes of serotype occur occasionally during the short term expansion of clones. Moreover, a geographical structure of ST156 was identified, suggesting that the resolution guaranteed by this method is sufficient for phylogeographic studies. In conclusion, we showed that an extended typing schema was able to characterize the expansion of individual lineages in a complex species such as S. pneumoniae.     

Multilocus sequence typing and genetic structure of Cryptosporidium hominis from children in Kolkata, India.

Endemicity of cryptosporidiosis in India has been documented with little genetic characterization of the parasites. Fifty Cryptosporidium-positive specimens collected between 2001 and 2004 from pediatric patients in Kolkata, India were analyzed for parasite genetic structure using multilocus sequence typing (MLST). Genotype analyses showed the presence of Cryptosporidium hominis, Cryptosporidium meleagridis and Cryptosporidium felis in 49, 2 and 1 patients, respectively (two patients had mixed infections of C. hominis and C. meleagridis). To assess the extent of genetic heterogeneity of C. hominis, minisatellites, microsatellites and polymorphic markers in three different chromosomes were sequenced, including genes encoding the 60kDa glycoprotein (GP60), a 47kDa protein (CP47), a mucin-like protein (Mucin1), a serine repeat antigen (MSC6-7), and a 56kDa trans-membrane protein (CP56) in chromosome 6, the 70kDa heat shock protein (HSP70) in chromosome 2, and a T-rich gene fragment (Chrom3T) in chromosome 3. Population sub-structure of C. hominis based on multilocus gene sequences showed that there were 25 multilocus subtypes defined by combined sequence length and nucleotide polymorphism, which formed four distinct groups in this population. Significant intra- and inter-genic linkage disequilibria were observed with minimum recombination or expansion of limited subtypes, all indicative of a mostly clonal population structure. The results highlight the importance of high resolution MLST in studying Cryptosporidium population sub-structure especially when length polymorphism may be inadequate in identifying unique subtypes. The significance of the diverse MLST within C. hominis in relation to geographical and temporal factors and clinical manifestations of disease warrants further investigations.