Streptococcus pneumoniae is a major causative agent of severe infections including sepsis, pneumonia, meningitis, and otitis media. Of the 90 capsular types, Seroptypes 4 and 14 and serogroups 6, 7, 9, 18, 19 and 23 are associated with pediatric diseases. Here we studied the serogroup specific distribution of pneumococci isolates to predict the efficacy of the 7-valent conjugate vaccine in Malaysian populations.
Streptococcus pneumoniae is a major causative agent of the morbidity and mortality among young children and adults.(1) Since the emergence of penicillin resistant strain in 1967, antibiotic resistance in S.pneumoniae has spread worldwide. The organism has also been reported to be resistant to other antibiotics such as macrolides and quinolones.(2, 3) An important factor that enables the organism to cause diseases is the production of capsule, a polysaccharide structure which is external to the cell wall. This structure provides resistance to phagocytosis and promotes bacterial evasion from the host immune system.(4) Pneumococci can produce at least 90 immunologically distinct capsules that differ in their chemical properties.(5) Of the 90 capsular types, only a few of these are causative agents of invasive diseases and associated with pediatric diseases.(6) Serotypes 4 and 14 and serogroups 6, 7, 9, 18, 19, and 23 are known to be associated with pediatric diseases.(6) However, the association of the serotypes/serogroups with diseases varies with the geographic distribution and the time period.(6)
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Currently, the management of pneumococcal infections has become difficult with the rapid development of antimicrobial resistance in this organism. The majority of antibiotic resistance strains carry serotypes that are not included in the conjugate vaccines that are currently in use and in development.(7) It has been reported that serotypes reported in both pediatric and adult infections are frequently found among both drug susceptible and resistant strains that colonize healthy children.(8) Therefore, the main focus of better management of pneumococcal infection is currently through widespread usage of vaccination. The available vaccine covers 23 serotypes, which represents 90% of the strains responsible for invasive diseases but are found to be not immunogenic in young children. There is also a new pneumococcal conjugate vaccine (PCV) which has been reported to be immunogenic in children < 2 years old. This vaccine has different formulations depending on the manufacturers, which are 7-, 9-, or 11 valent (9) that confer protection against different number of pneumococcus serotypes.
Since pneumococci has the ability to switch serotypes by horizontal transfer recombination and other genetic events, it is important to monitor the frequency of the serotype exchanges in order to predict long term efficacy of new vaccines. Therefore continuous monitoring of antimicrobial resistance and serotype distribution of S.pneumoniae is important in a population. In this work, we study the development of penicillin resistance and serogroup specific epidemiology of pneumococci among isolates from the University Malaya Medical Centre (UMMC).
Materials and Methods
A total of 151 pneumococcal strains were obtained from clinical samples processed at the Microbiology Laboratory of the University of Malaya Medical Centre, Malaysia from March 1999 to February 2007. Control strains of known serotypes (Quellung reaction) representing different serotypes and serogroups were used in the study. The serotypes included 1, 2, 3, 4, 5, 8, 13, 14, 20, 21, 31, 34, 37, 38, 39, 40, 44, 46, 6A, 7 A, 7B, 7C, 7F, 9A, 9N, 9L, 9L, 10A, 10F, 11 A, 11D, 11F, 12A, 12B, 12F, ISA, 15B, 15C, 15F, 16A, 16F, 17F, 33A, 35B, 35F, 35A, 35C, and 47F. The isolates were obtained from invasive and non-invasive sites of both pediatric and adult patients. The source of the isolates included blood, nasopharyngeal secretion, tracheal secretion, sputum, and bronchoalveolar lavage. Samples were grown on 5% horse blood agar and incubated at 37°C in the presence of 5% CO2 for 12 - 15 hours prior to other biochemical and molecular assays.
The strains were identified as S.pneumoniae using conventional microbiological methods including susceptibility to ethylhydrocupreine disc (optochin), catalase test and bile solubility.
The antibiotic susceptibility of the strains was tested on Mueller Hinton Agar (Oxoid) plates containing 5% sheep blood (Oxoid), incubated at 37°C with 5% CO2 using the agar dilution method as described.(10) The penicillin used was obtained from Sigma Aldrich (Sigma Chemical Co., St. Louis, Mo). S.pneumoniae ATCC 49619 was used as control.
Genomic DNA was extracted from the bacterial culture using a previously described method.(11) Bacterial colonies suspended in 15ïl of dH2O containing 50mg/L were incubated at 37°C for 10 minutes. This was followed by addition of 10ïg/ml Proteinase K and 0.lmM Tris HCL pH 7.5 and incubated at 37°C for another 10 minutes. Subsequently, the suspension was boiled for 5 minutes and finally centrifuged at 13000 rpm for 2 minutes. The supernatant obtained was used as the template in the PCR reaction.
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The primers used in this study were extracted from previously published sequences and the PCR protocol was as previously described.(12) Briefly, the primers were grouped into seven multiplex reactions as shown in (Figure 1) and each reaction was designed to include four primer pairs targeting four different serotypes and another primer pair targeting the common region of the cps operon as an internal positive control. The optimal PCR condition for a 25ï1 reaction included 1X PCR buffer (Fermentas, Lithuania), 2.5mM MgCl2, 0.2 mM dNTP mix, 2U Taq Polymerase (Fermentas) and primer pairs at varying concentrations as shown in Table I.(12) The PCR cycling parameters were as follows: An initial denaturation step at 94°C for 4 minutes, 30 cycles of amplification performed as follows: denaturation at 94°C for 45s, annealing temperature at 54°C for 45s and extension temperature at 65°C for 2 minutes and 30s and finally completed with an extension at 72°C for 2 minutes. The amplification reaction was performed in an Eppendorf Gradient Mastercycler. The PCR product was electrophoresed on a 2% TAB agarose gel for 1 hour at 70V and the bands were analyzed using a UV transilluminator.
Minimal inhibitory concentrations
The stains were categorized based on their susceptibility to penicillin, termed as Penicillin Sensitive S.pneumoniae (PSSP), Penicillin Intermediate S.pneumoniae (PISP), and Penicillin Resistant S.pneumoniae (PRSP). Of the 151 isolates, there were 21.2% PRSP, 29.1% PISP, and 49.7% PSSP (Table II).
Serotypes that were detected among the Malaysian isolates were 1, 3, 10A, 11A/11D, 12F/12A, 14, 15A, 15B/15C, 16F, 18C/18B/18A/18F, 19A, 19F, 23F, 35B, 35F/47F, 6A/6B, 7C/7B/40, 7F/7A, 9V/9A, and 34 (Table III). The most predominant serotype out of all groups was 19F. Serotypes 19F and 23F were observed to be common among the PRSP strains, with 20/32 strains serotyped as 19F while 6/32 strains were serotyped as 23F. Figure 2 shows a representative complete seven multiplex reactions performed on 10 PRSP strains. Only one PRSP strain belongs to each serotype 6A/6B and 9V/9A. Serotype 19F was also predominant among the PISP strains. Other serotypes detected among the PISP strains were 1, 14, 11A/11D, 19A, 23F, 35B, 35F/47F, and 6A/6B. However, the distributions of these serotypes were not common. The PSSP strains were detected to have other serotypes:- 3, 10A, 12F/12A, 15A, 15B/15C, 16F, 18C/18B/18A/18F, 7C/7B/40, and 7F/7A. The distribution of serotype 19F in PSSP was less abundant as compared to PISP and PRSP groups.
The emergence of antibiotic resistance in S.pneumoniae has increased the urge for researchers to find new therapeutic agents. Although the prevalence of penicillin resistant pneumococci observed in this study only represents reports of pneumococcal incidence from one centre in Malaysia, the rate of resistance is significantly high with a prevalence rate of 21.2%. Therefore, this study was carried out to investigate the distribution of serotype specific strains in the centre to promote surveillance of pneumococcal infection in Malaysia. Previous study has reported serogroups 6, 9, 14 and 23 to be associated with penicillin resistance (9). These serogroups have also been associated with carriage. However, our findings showed that serotype 19F and 23F to be predominant in the penicillin non-susceptible strains, especially the PRSP strains. This is particularly interesting as how these serotypes are related to penicillin-resistance and the alterations associated that lead to the changes in response against penicillin stress. Many other minor and non-prevelance serotypes have also been detected among all three groups. This may be due to variations in the geographical area, disease and age groups of the patients. However, the high prevalence of penicillin resistance in this study suggests faster spread of resistant clones among pneumococci strains in the region. Therefore, the study supports the need for a long term surveillance program and also vaccination as the measures for prevention and control of pneumococcal infections.
In conclusion, we observe serotype 19F and 23F to be prevalent among the penicillin resistant strains, which has been included in the 7-valent conjugate vaccine. Therefore, the efficacy of this vaccine should be adequate in our population. However, it is not possible to predict serotypes that might become predominant in the future.
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This work was supported by grant provided by the Ministry of Science and Technology, Malaysia: 36-02-03-6027. No conflict of interest.