Epidemiological characterisation of Streptococcus pneumoniae from India using multilocus sequence typing

Objective: The aim of this study was to utilize the multilocus sequence typing (MLST) technique to characterise Streptococcus pneumoniae among clinical isolates in India. MLST was used to determine clonality, to establish genetic relatedness, to check for correlation between serotypes and sequence types (STs) and its relevance associated with antibiotic resistance. Methods: Forty consecutive invasive S. pneumoniae isolates in children <5 years were characterised. Preliminary identification of serotype and antibiotic susceptible profile was followed with MLST technique to identify the STs of the isolates. STs were then analysed for clonality using an eBURST algorithm and genetic relatedness using Sequence Type Analysis and Recombinational Tests version 2 software. Results: The most common ST was ST63. Among the forty isolates, we identified nine novel STs, six of which had known alleles but in new combinations, three of which had new alleles in their sequence profile. The new STs assigned were 8501–8509. One clonal complex was found among the 40 strains characterised. The most common serotypes in this study were serotype 19F, 14 and 5. Non-susceptibility to penicillin and erythromycin was observed in 2.5% and 30% of the isolates, respectively. Conclusion: This study shows a significant number of novel STs among the 40 isolates characterised (9/40, 22.5%), however, internationally recognised strains were also circulating in India, indicating, there could be greater geographical variation in pneumococcal STs in India. Molecular epidemiology data is essential to understand the population dynamics of S. pneumoniae in India before the introduction of pneumococcal vaccines in NIP in India.     

Clonal structure and 21-year evolution of Streptococcus pneumoniae serotype 1 isolates in northern Spain

This study of 135 serotype 1 pneumococcal isolates (88 invasive and 47 non-invasive), collected between 1987 and 2007, gave eight sequence types (217, 227, 228, 304, 305, 306, 3860 and 3861) that group, using eBurst, into three different lineages and one singleton. The annual incidence of serotype 1 invasive episodes per million inhabitants increased from 1.8 in 1987–1993 to 4.0 in 1994–2000, and to 25.6 in 2001–2007. ST228 was the predominant clone until 1998. ST306 first appeared in 1998 and became the most prevalent sequence type (>80%) after the introduction, in June 2001, of the heptavalent pneumococcal conjugate vaccine.     

Invasive pneumococcal disease caused by ceftriaxone-resistant Streptococcus pneumoniae in Taiwan

Background

Invasive pneumococcal disease (IPD) was associated with mortality, but the risk factors associated with mortality remains controversial.

Distribution and clonal relationship of cell surface virulence genes among Streptococcus pneumoniae isolates in Japan

Streptococcus pneumoniae resides on mucosal surfaces in the nasopharynx, where selection for horizontal transfer of antimicrobial resistance genes and virulence factors may provide a survival advantage. We investigated the distribution of genes for pneumococcal cell surface proteins and their correlations with multilocus sequence typing (MLST), Pneumococcal Molecular Epidemiology Network (PMEN) clones and antimicrobial resistance, to identify pneumococcal virulence factors predicting prevalent clones from 156 pneumococcal isolates recovered from adult patients with community-acquired pneumonia in Japan. Pneumococcal eno, pavA, piuA, cbpA and cbpG were present in all isolates, and hyl and piaA were distributed among the clinical isolates. In contrast, pneumococcal rlrA, pclA, psrP, nanC and pspA family 1-type genes were variably distributed and significantly associated with MLST (Wallace coefficients (W) were over 84%). Serotype was a weaker predictor of sequence type (W, 0.75) than vice versa (W, 0.97). A multiple logistic regression analysis adjusted to the presence of virulence genes, pspA family 1 genes and carriage serotypes revealed that pclA and rlrA correlated with PMEN clones and antimicrobial resistance, and are likely to contribute to the selection of prevalent clones.     

Genetic Diversity and Allelic Variation in South Indian Isolates of Group A Streptococci Causing Invasive Disease

Background: Reported literature on invasive group A streptococcal isolates in India is very scanty. This study was undertaken to determine the molecular heterogeneity of such isolates as seen in a tertiary care center. Materials and Methods: Thirty two blood culture isolates and 18 from other sterile body fluids were characterized by emm gene sequencing and multilocus sequence typing. Results: Forty two emm types were identified including 25 from 32 blood isolates and 17 from 18 other body fluid isolates. Types 110, 74, 63, 85, 102, 105, 124 and st854.1 were common to both groups and accounted for 40% of the isolates. Two types namely, stKNB6 and stKNB9 were newly identified types. MLST identified forty eight sequence types (MLST - ST) of which 31 were from 32 blood isolates and 17 from 18 body fluid isolates; thirty three of them were hitherto unrecognized at the time of identification. Two blood isolates of emm 85 had the same MLST - ST 484 while three blood isolates of emm 110 had three different STs namely, ST 493, 494 and 497. Two types, ST 493 and ST497 had single locus variation while ST 497 had a double locus variation. Conclusions: Our study shows that subtle allelic variations in the house keeping genes results in the development of new strains in a given emm type and contribute significantly to the existing high diversity of strains circulating in the community.     

Molecular Biology of Group A Streptococcus and its Implications in Vaccine Strategies

Infections due to Streptococcus pyogenes and their complications are a problem of major concern in many countries, including India. Primary prophylaxis with benzathine penicillin is the key to control and prevent sequelae such as acute rheumatic fever and rheumatic heart disease (RF/RHD) or post-streptococcal glomerulonephritis (PSGN). Non-compliance to prophylaxis due to fear of injection and anaphylaxis is major issues in RF/RHD control in India and leads to continued high prevalence of infection and post-streptococcal sequelae. Differing reports on the efficacy of two weekly, three weekly or monthly injections raise questions on the actual dosages to be administered. Availability of more effective antibiotics with better dosages has replaced the use of penicillin; hence, companies are reluctant to manufacture penicillin preparations in India. It is in this context that a concept of a Group A streptococci vaccine is looked at and whether or not a globally designed vaccine will be useful in the Indian context. Modern molecular techniques and genomic analysis of S. pyogenes have identified many molecules as vaccine candidates among which the M-protein has attracted the most attention. High diversity of M (emm) types in endemic regions raises questions about the efficacy of such a vaccine. A recent 30-valent M-protein-based vaccine that elicits antibodies to homologous as well as non-vaccine M types looks promising. This review will discuss the genomics of S. pyogenes, the various candidate vaccine molecules and highlight their efficacy in the Indian context where control of post-streptococcal sequelae remains a challenge.     

Molecular Capsular Typing and Multi Locus Sequence Typing of Invasive,Non-invasive and Commensal Streptococcus pneumoniae Isolates from North India

Purpose: The purpose of this study is to determine the antimicrobial susceptibility pattern, serotype distribution and sequence type (ST) of Streptococcus pneumoniae isolates from invasive and non-invasive infection and correlate it with isolates from commensal nasopharyngeal flora to ascertain their role in infection. Materials and Methods: S. pneumoniae isolates from blood, cerebrospinal fluid, pleural fluid and respiratory secretions (sputum, bronchoalveolar lavage and nasopharyngeal swab/throat swab) were analysed to determine ST, serotype and antimicrobial susceptibility pattern. Serotyping was performed by multiplex polymerase chain reactions as well as by quellung reaction. Antimicrobial susceptibility testing was determined using Kirby Bauer’s disc diffusion method as per the Clinical Laboratory Standards Institute guidelines. Minimum inhibitory concentration was determined using E-test for penicillin. Multilocus sequence typing (MLST) was done to understand genetic relatedness and evolutionary relationship among strains. Results: A total of 125 S. pneumoniae isolates were collected, including 25 from invasive pneumococcal disease, 25 from non-invasive and 75 from nasopharyngeal swab of healthy children (Commensal). Resistance to penicillin, erythromycin, and co-trimoxazole was observed in 14.4%, 12% and 81.6% of the isolates, respectively, by KirbyBauer’s disc diffusion method. Serotype 14 was found to be the most prevalent in invasive and non-invasive isolates, while serotype 6 was the most common in commensal isolates. New STs were found among invasive (ST13826, ST13827), non-invasive (ST13823, ST13824, and ST13961) and commensal (ST13825) isolates. Conclusion: MLST sequence analysis shows that invasive isolates were found to be clustered with non-invasive and commensal isolates. Analysis of MLST suggests the possibility of genetic relatedness and exchange of genetic material between invasive, non-invasive and commensal isolates.     

Molecular epidemiology of penicillin-non-susceptible Streptococcus pneumoniae isolates from children with invasive pneumococcal disease in Germany

A population-based nationwide surveillance of antibiotic resistance associated with invasive pneumococcal disease (IPD) in children and adolescents (aged<16 years) was performed in Germany between 1997 and 2004. In total, 1517 isolates were collected, of which 5.1% and 1.1% were intermediately- or fully-resistant, respectively, to penicillin G. During the 8-year study period, an increase in resistance to both penicillin G and erythromycin A was observed, and the frequency of isolates exhibiting reduced susceptibility to penicillin G or erythromycin A increased from 1.4% and 11.1%, respectively, in 1997, to 8.7% and 29.0%, respectively, in 2004. Among the penicillin non-susceptible pneumococcal isolates, serotypes 14 (24.5% of isolates), 23F (16.0%) and 6B (16.0%) were found most frequently. Multilocus sequence typing of 58 (62%) penicillin G non-susceptible isolates revealed that sequence type (ST) 156 (Spain9V-3 clone) and its single-locus variant ST 557 were widespread in Germany. Moreover, 17 new penicillin G non-susceptible STs were defined for the first time. The study illustrated the genetic heterogeneity of antibiotic-resistant pneumococcal isolates in Germany.     

Molecular epidemiology of OXA-48-producing Klebsiella pneumoniae in France

We characterized 53 OXA-48-producing Klebsiella pneumoniae (OXA-48-Kp) isolated between 2011 and 2013 in 21 French hospitals. All the isolates were genotyped using MLST and PFGE and the population structure of the species was determined by a nucleotide-based analysis of the entire K. pneumoniae MLST database. Most of the OXA-48-Kp isolates also produced CTX-M-15 and remained susceptible to imipenem and meropenem. The isolates were distributed into 20 STs, of which five were dominant (ST15, ST101, ST147, ST395 and ST405). All the OXA-48-Kp clustered in the major clade of K. pneumoniae KpI.     

Pneumococcal Conjugate Vaccine Rollout in India: Expectations and Challenges

India is one among the four Asian countries with the greatest number of deaths due to pneumococcal infection among children under 5 years. pneumococcal conjugate vaccine (PCV) has been introduced in a phased manner in five major Indian states. Ambiguity remains in choosing the appropriate type of PCV and optimum schedule with maximum effectiveness specific for each country. Here, we discuss the evidences with respect to serotype coverage, immunogenicity, reactogenicity and dosage schedule for introduction of PCV13 in India. In addition, the expected PCV impact and the challenges are detailed. PCV13 is expected to provide >75% serotype coverage for invasive pneumococcal disease (IPD) serotypes in Indian children combined with the replacement by nonvaccine serotypes which is unpredictable due to lack of complete data. Nasopharyngeal (NP) surveillance is easy, feasible and can replace IPD surveillance in resource-poor settings. Continuous IPD as well as NP surveillance in all the regions are necessary to assess the impact of PCV in India.     

Clonal dissemination of multilocus sequence type 11 Klebsiella pneumoniae carbapenemase – producing K. pneumoniae in a Chinese teaching hospital

Klebsiella pneumoniae carbapenemase (KPC)‐producing K. pneumoniae has disseminated rapidly in China. We aimed to analyze the molecular epidemiology of four KPC‐producing K. pneumoniae strains isolated from a suspected clonal outbreak during a 3‐month period and to track the dissemination of KPC‐producing K. pneumonia retrospectively. We created antimicrobial susceptibility profiles using an automated broth microdilution system and broth microdilution methods. We screened carbapenemase and KPC phenotypes using the modified Hodge test and meropenem–boronic acid (BA) disk test, respectively. We identified β‐lactamase genes with PCR and sequencing. We investigated clonal relatedness for epidemiological comparison using pulsed‐field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). All isolates expressed multidrug resistance and yielded positive results for the modified Hodge and meropenem‐BA disk tests. The isolates all carried bla_KPC‐2, and coproduced CTX‐M–type extended‐spectrum β‐lactamase. PFGE and MLST showed that the isolates were clonally related. The PFGE patterns of these isolates had ≥90% similarity. We found a single clone, sequence type (ST) 11, and its typical dissemination mode resembled clonal spread. The dissemination of KPC‐producing K. pneumoniae is clonally related and there is probable local transmission of a successful ST11 clone.     

Prevalence of Campylobacter Enteritis in Children under 5 Years Hospitalised for Diarrhoea in Two Cities of Northeast India

Background: Campylobacter enteritis is the major cause of bacterial gastroenteritis worldwide. In recent years, there has been a rise in global incidence of campylobacteriosis. There are no available data on prevalence of Campylobacter diarrhoea from Northeast India. Materials and Methods: The study investigated archival stool samples collected between 2014 and 2016 from two hospitals of Northeast India. A total of 407 archival stool samples from cases of diarrhoea under 5 years of age were screened for Campylobacter spp. using commercial probe-based real-time polymerase chain reaction assay. Results: Campylobacter spp. was detected in overall 10.1% (41/407; 95% confidence interval: 7.4%–13.3%) in children under 5 years hospitalised for diarrhoea. The prevalence was significantly higher from Dibrugarh, Assam, compared to Dimapur, i.e., 13.4% (27/201) versus 6.8% (14/206), respectively (P = 0.02). Campylobacter detection was highest in the month of June and July compared to December and January (20%–18.8% vs. 8.9%–6.2%, respectively). Further, Campylobacter infection was higher in the age group below 24 months (11.7%) compared to above 24 months (7.0%). Campylobacter jejuni was detected in 80.5% of the positive cases. Conclusion: The present study reveals that Campylobacter infection is endemic in the studied regions of Northeast India and microbiological laboratories of the region should actively pursue the isolation or detection of Campylobacter spp. in cases of diarrhoea in routine stool cultures.     

Management of antibiotic-resistant Streptococcus pneumoniae infections and the use of pneumococcal conjugate vaccines

The epidemiology of Streptococcus pneumoniae , a major cause of meningitis, pneumonia, bacteraemia and acute otitis media in both children and adults, has been altered by the availability of the seven-valent pneumococcal conjugate vaccine (PCV7) and selection pressure from broad-spectrum antibiotics. In Spain, the rates of antimicrobial consumption and resistance are high; of all S. pneumoniae isolates collected between 2001 and 2003, 9.2% were penicillin-resistant and 26.4% were penicillin-intermediate. These rates were even higher in children aged ≤4 years; 52.3% of isolates were penicillin-resistant or penicillin-intermediate. Serogroup 14 comprises nearly 9% of pneumococcal strains and exhibits the highest resistance to penicillin (69%). Since the introduction of PCV7 in Madrid, the isolates collected from hospitalized children aged <15 years from 21 hospitals revealed that only 8% of cases involving S. pneumoniae isolates were due to PCV7 serotypes, and no PCV7 vaccination failures were identified. These isolates demonstrated low rates of penicillin–non-susceptible and erythromycin–non-susceptible strains. Among multiresistant strains, serotype 19A was identified as most important. Although the recent WHO position paper on childhood immunizations speaks to the growing resistance of pneumococci, antibiotic resistance in Spain is noted to be decreasing and must be evaluated in conjunction with global studies.     

Contribution of serotype-specific IgG concentration, IgG subclasses and relative antibody avidity to opsonophagocytic activity against Streptococcus pneumoniae

The contribution of serotype-specific IgG concentration, subclasses, and avidity to opsonophagocytic activity (OPA) against Streptococcus pneumoniae (Pnc) was evaluated in sera of adults and infants immunized with different pneumococcal vaccines. Antibody concentrations and avidities were measured by enzyme immunoassay (EIA) and OPAs by killing assay of Pnc. The most important factor contributing positively to OPA was the specific IgG level. In infants, a tendency to negative correlation was found between the concentration needed for killing of bacteria and avidity, suggesting that less antibodies of high rather than low avidity were required for killing. No such correlation was seen in adults. However, in adults the avidity was high already before vaccination and the variation was narrow. Thus, avidity was probably not a limiting factor influencing OPA. The effect of IgG2/IgG1 ratio on OPA was mostly negative but insignificant.     

Multiclonal epidemic of Klebsiella pneumoniae isolates producing DHA-1 in a Spanish hospital

Between June 2007 and January 2008, 26 Klebsiella pneumoniae isolates carrying bla_DHA-1 on an IncL/M plasmid were obtained from clinical samples at Granollers Hospital, Barcelona, Spain. Three of the isolates also carried a bla_CTX-M-15 gene. The 26 isolates showed 11 pulsed-field gel electrophoresis (PFGE) patterns. Multilocus sequence typing showed that PFGE patterns A, B and C belonged to sequence type (ST)17, D to ST13, E to ST427, F and G to ST416, H to ST37, I to ST440, J to ST326, and K to ST428. Results demonstrated the effectiveness of the infection control programme in place at the centre. This study reports the first characterization of STs for bla_DHA-1-producing K. pneumoniae isolates.     

High prevalence of multidrug-resistant Pneumococcal molecular epidemiology network clones among Streptococcus pneumoniae isolates from adult patients with community-acquired pneumonia in Japan

A total of 141 Streptococcus pneumoniae isolates from patients with community-acquired pneumonia were collected from May 2003 through October 2004. The strains were tested for antimicrobial agent susceptibility, serotype and genotype by multilocus sequence typing (MLST) and the presence of the pilus rlrA islet. MLST analysis identified 49 sequence types (STs), of which 19 were novel. eBURST analysis using the MLST database (3773 STs) grouped the isolates into 27 clonal complexes and three singletons. A total of 92 (65.2%) isolates were related to ten of the 43 international Pneumococcal Molecular Epidemiology Network (PMEN) clones; major clones found were multidrug-resistant Netherlands³-31 [clonal complex (CC) 180], Taiwan^19F-14 (CC271), Taiwan^23F-15 (CC242), and Colombia^23F-26 (CC138) (the latter new to Asia). We adopted univariate and multiple logistic regression models to identify factors associated with PMEN CCs. Multivariate analysis showed that multidrug resistance (OR 6.3; 95% CI 2.0–22.9), carriage serogroups (OR 7.2; 95% CI 2.5–23.7), prevalence of rlrA (OR 12.6; 95% CI 3.6–59.7) and central nervous system-related disorders (OR 7.7; 95% CI 1.8–48.4) were independently associated with PMEN CCs. Our data indicate that multidrug-resistant PMEN clones are highly prevalent, contributing to the high frequency of resistance to antimicrobial agents in Japan, and suggest that certain predisposing factors in patients contribute to the high frequency of these clones.     

Clonal spread of SCCmec type IV methicillin-resistant Staphylococcus aureus between community and hospital

The staphylococcal chromosome cassette (SCC)mec types of 382 hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) isolates in Taiwan were analysed over a 7-year period (1999-2005). There was an abrupt increase in SCCmec type IV in HA-MRSA during 2005. The molecular epidemiology of a subset (n = 69) of HA-MRSA isolates with SCCmec types III, IV or V was characterised and compared with that of community-acquired MRSA (CA-MRSA) (n = 26, collected during 2005). Pulsed-field gel electrophoresis revealed three major pulsotypes (A, B and C) and 15 minor clones. Pulsotypes B and C, which contained isolates carrying SCCmec types IV and V, respectively, included both CA-MRSA and HA-MRSA isolates. Among 24 toxin genes analysed, five genes had significant differential distribution between CA-MRSA and SCCmec type III HA-MRSA. Furthermore, among SCCmec type IV isolates, the seb gene was detected more commonly in HA-MRSA. Analysis of representative members of the three major pulsotypes by multilocus sequence typing revealed two sequence types (STs), namely ST239 (SCCmecIII) and ST59 (SCCmecIV or SCCmecV). This suggests that ST59:SCCmecIV, which is usually community-acquired, has become an important nosocomial pathogen in the hospital studied.     

广州地区老年人肺炎链球菌临床分离株的青霉素耐药研究与分子分型

目的调查广州地区老年患者肺炎链球菌分离株对青霉素的敏感性，并分析其亲缘关系。方法K—B纸片法对33株分离自老年住院病人的肺炎链球菌进行青霉素药敏试验；应用PCR技术检测青霉素结合蛋白基因pbp1a，pbp2x，pbp2b；用盒式PCR（BOX—PCR）分析菌株间亲缘关系。用多位点测序分型技术（multilocus sequence typing，MLST）检测青霉素耐药菌株的分子分型。结果青霉素的耐药率为3．03％（1／33）：用PCR方法鉴定PSSP的准确率为68．75％；BOX-PCR可将这33株肺炎链球菌分为21型。MIST分型显示，青霉素耐药菌株属ST271型。结论广州地区老年患者肺炎链球菌对青霉素耐药率较低．用PCR方法检测PSSP有一定的可行性。BOX—PCR显示了较高的分辨率，能快速可靠地检测菌株间的亲缘关系。广州地区流行的耐药克隆与Taiwan^19F-14株同源。     

Invasive pneumococcal disease among children in a health district of Barcelona: early impact of pneumococcal conjugate vaccine

This study evaluated the impact of heptavalent pneumococcal conjugate vaccine (HPCV) on invasive pneumococcal disease (IPD) in children aged < or = 5 years in Barcelona, Spain. The incidence of IPD, vaccine uptake and prevalence of nasopharyngeal colonisation were analysed in two different periods: 1999-2001 (pre-licence period), and 2002-2004 (post-licence period). In total, 121 cases of IPD were identified. The overall incidence of IPD decreased from 96.9 cases/100,000 to 90.6 cases/100,000 (OR 0.93, 95% CI 0.69-1.26, p 0.71) between the two periods. The proportion of cases caused by non-vaccine-related serotypes (NVS) increased from 21% to 43.7% (OR 2.9, 95% CI 1.2-7, p 0.01). IPD was diagnosed in seven vaccinated children, six of whom were infected by NVS. There was a trend of diminishing prevalence of resistance to penicillin and macrolides in 2002-2004. The incidence of empyema increased from 1.7 to 8.5/100,000 (OR 4.5, 95% CI 0.91-18, p 0.06). The rate of vaccination ranged from 4.8% to 34%. It was concluded that the rates of IPD in this area did not decrease following the introduction of HPCV. The low uptake of vaccine and the greater proportion of colonisation/infection by NVS probably explain these findings. A trend of increasing empyema was also apparent. A decrease in the prevalence of penicillin and macrolide resistance paralleled the progressive uptake of vaccine.