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Genotypic characterization of Malaysian human isolates of Streptococcus pneumoniae from carriage and clinical sources

Shakrin NN, Masri SN, Taib NM, Nordin SA, Jamal F, Desa MN

Comp Immunol Microbiol Infect Dis, 2014 Dec;37(5-6):347-54.
PMID: 25467035 DOI: 10.1016/j.cimid.2014.10.005

This study characterized carriage and clinical pneumococcal isolates for serotypes, penicillin susceptibility, virulence genes and restriction fragment length polymorphism (RFLP) pattern of penicillin binding protein (PBP) genes. DNA fingerprint of isolates was generated by BOX-PCR. Majority of serotypes were 23F followed by 19F, 19A and 6A. Twenty-four percent of isolates were penicillin non-susceptible (PNSP). All of the targeted virulence genes were detected in all isolates with the exception of pili; 20.6% (n=22) for PI-1 and 14.0% (n=15) for PI-2. Of the 13 isolates which carried both PI-1 and PI-2, 10 were of clinical origin. Digested pbp-DNA produced three PBP-RFLP profiles for pbp1a (A1 to A3), six profiles for pbp2b (B1 to B6) and seven for pbp2x (X1 to X7) mostly in PNSPs. Based on BOX-PCR analysis, the majority of isolates were genetically diverse with a small number of potentially related isolates carrying pili genes. No obvious genotypic association was observed pertaining to carriage and clinical origin of isolates.

Matched MeSH terms: Penicillin-Binding Proteins/genetics*; Penicillin-Binding Proteins/metabolism
Fulltext Quintuplex PCR to detect antibiotic resistance genes in Streptococcus pneumoniae

Navindra Kumari Palanisamy, Parasakthi Navaratnam, Shamala Devi Sekaran

Journal of Clinical and Health Sciences, 2016;1:22-28.

Introduction: Streptococcus pneumoniae is an important bacterial pathogen, causing respiratory infection. Penicillin resistance in S. pneumoniae is associated with alterations in the penicillin binding proteins, while resistance to macrolides is conferred either by the modification of the ribosomal target site or efflux mechanism. This study aimed to characterize S. pneumoniae and its antibiotic resistance genes using 2 sets of multiplex PCRs. Methods: A quintuplex and triplex PCR was used to characterize the pbp1A, ermB, gyrA, ply, and the mefE genes. Fifty-eight penicillin sensitive strains (PSSP), 36 penicillin intermediate strains (PISP) and 26 penicillin resistance strains (PRSP) were used. Results: Alteration in pbp1A was only observed in PISP and PRSP strains, while PCR amplification of the ermB or mefE was observed only in strains with reduced susceptibility to erythromycin. The assay was found to be sensitive as simulated blood cultures showed the lowest level of detection to be 10cfu. Conclusions: As predicted, the assay was able to differentiate penicillin susceptible from the non-susceptible strains based on the detection of the pbp1A gene, which correlated with the MIC value of the strains.

Matched MeSH terms: Penicillin-Binding Proteins
Fulltext Quintuplex PCR to detect antibiotic resistance genes in Streptococcus pneumoniae

Navindra Kumari Palanisamy, Parasakthi Navaratnam, Shamala Devi Sekaran

Journal of Clinical and Health Sciences, 2016;1(2):22-28.
MyJurnal

Introduction: Streptococcus pneumoniae is an important bacterial pathogen, causing respiratory infection. Penicillin resistance in S. pneumoniae is associated with alterations in the penicillin binding proteins, while resistance to macrolides is conferred either by the modification of the ribosomal target site or efflux mechanism. This study aimed to characterize S. pneumoniae and its antibiotic resistance genes using 2 sets of multiplex PCRs. Methods: A quintuplex and triplex PCR was used to characterize the pbp1A, ermB, gyrA, ply, and the mefE genes. Fifty-eight penicillin sensitive strains (PSSP), 36 penicillin intermediate strains (PISP) and 26 penicillin resistance strains (PRSP) were used. Results: Alteration in pbp1A was only observed in PISP and PRSP strains, while PCR amplification of the ermB or mefE was observed only in strains with reduced susceptibility to erythromycin. The assay was found to be sensitive as simulated blood cultures showed the lowest level of detection to be 10cfu. Conclusions: As predicted, the assay was able to differentiate penicillin susceptible from the non-susceptible strains based on the detection of the pbp1A gene, which correlated with the MIC value of the strains.

Matched MeSH terms: Penicillin-Binding Proteins
Detection of pbp2b and ermB genes in clinical isolates of Streptococcus pneumoniae

Kumari N, Navaratnam P, Sekaran SD

J Infect Dev Ctries, 2008 Jun 01;2(3):193-9.
PMID: 19738350

BACKGROUND: Streptococcus pneumoniae is a major human pathogen. The emergence of penicillin resistant strains since the 1970s has been life threatening and the evolution of the bacteria have enabled itself to develop resistance to many other antibiotics such as the macrolides and the fluoroquinolones. This study aims to characterize S. pneumoniae isolates for the presence of penicillin and macrolide resistance genes.
METHODOLOGY: One hundred and twenty clinical isolates of S. pneumoniae were obtained from patients of University Malaya Medical Centre (UMMC). The strains were screened using a multiplex real-time PCR method for the presence of alterations in the genes encoding the penicillin binding proteins: pbp2b, macrolide resistance determinant ermB and the pneumolysin gene, ply. Dual-labelled Taqman probes were used in the real-time detection method comprising three different genes labeled with individual fluorophores at different wavelengths. One hundred and twenty isolates from bacterial cultures and isolates directly from blood cultures samples were analyzed using this assay.
RESULTS: A multiplex PCR comprising the antibiotic resistance genes, ermB and and pneumolysin gene (ply), a S. pneumoniae species specific gene, was developed to characterize strains of S. pneumoniae. Out of the 120 pneumococcal isolates, 58 strains were categorized as Penicillin Sensitive Streptococcus pneumoniae (PSSP), 36 as Penicillin Intermediate Streptococcus pneumoniae (PISP) and 26 as Penicillin Resistant Streptococcus pneumoniae (PRSP). All the 58 PSSP strains harboured the pbp2b gene while the 36 PISP and 26 PRSP strains did not harbour this gene, thus suggesting reduced susceptibility to penicillin. Resistance to erythromycin was observed in 47 of the pneumococcal strains while 15 and 58 were intermediate and sensitive to this drug respectively. Susceptibility testing to other beta-lactams (CTX and CRO) also showed reduced susceptibility among the strains within the PISP and PRSP groups but most PSSP strains were sensitive to other antibiotics.
CONCLUSION: The characterization of pneumococcal isolates for penicillin and erythromycin resistance genes could be useful to predict the susceptibility of these isolates to other antibiotics, especially beta-lactams drugs. We have developed an assay with a shorter turnaround time to determine the species and resistance profile of Streptococcus pneumoniae with respect to penicillin and macrolides using the Real Time PCR format with fluorescent labeled Taqman probes, hence facilitating earlier and more definitive antimicrobial therapy which may lead to better patient management.

Matched MeSH terms: Penicillin-Binding Proteins/genetics*; Penicillin-Binding Proteins/isolation & purification
Fulltext Inhibitory effect of Duabanga grandiflora on MRSA biofilm formation via prevention of cell-surface attachment and PBP2a production

Santiago C, Lim KH, Loh HS, Ting KN

Molecules, 2015 Mar 10;20(3):4473-82.
PMID: 25764489 DOI: 10.3390/molecules20034473

Formation of biofilms is a major factor for nosocomial infections associated with methicillin-resistance Staphylococcus aureus (MRSA). This study was carried out to determine the ability of a fraction, F-10, derived from the plant Duabanga grandiflora to inhibit MRSA biofilm formation. Inhibition of biofilm production and microtiter attachment assays were employed to study the anti-biofilm activity of F-10, while latex agglutination test was performed to study the influence of F-10 on penicillin-binding protein 2a (PBP2a) level in MRSA biofilm. PBP2a is a protein that confers resistance to beta-lactam antibiotics. The results showed that, F-10 at minimum inhibitory concentration (MIC, 0.75 mg/mL) inhibited biofilm production by 66.10%; inhibited cell-surface attachment by more than 95%; and a reduced PBP2a level in the MRSA biofilm was observed. Although ampicilin was more effective in inhibiting biofilm production (MIC of 0.05 mg/mL, 84.49%) compared to F-10, the antibiotic was less effective in preventing cell-surface attachment. A higher level of PBP2a was detected in ampicillin-treated MRSA showing the development of further resistance in these colonies. This study has shown that F-10 possesses anti-biofilm activity, which can be attributed to its ability to reduce cell-surface attachment and attenuate the level of PBP2a that we postulated to play a crucial role in mediating biofilm formation.

Matched MeSH terms: Penicillin-Binding Proteins/antagonists & inhibitors
Fulltext Reversal of ampicillin resistance in MRSA via inhibition of penicillin-binding protein 2a by Acalypha wilkesiana

Santiago C, Pang EL, Lim KH, Loh HS, Ting KN

Biomed Res Int, 2014;2014:965348.
PMID: 25101303 DOI: 10.1155/2014/965348

The inhibitory activity of a semipure fraction from the plant, Acalypha wilkesiana assigned as 9EA-FC-B, alone and in combination with ampicillin, was studied against methicillin-resistant Staphylococcus aureus (MRSA). In addition, effects of the combination treatment on PBP2a expression were investigated. Microdilution assay was used to determine the minimal inhibitory concentrations (MIC). Synergistic effects of 9EA-FC-B with ampicillin were determined using the fractional inhibitory concentration (FIC) index and kinetic growth curve assay. Western blot experiments were carried out to study the PBP2a expression in treated MRSA cultures. The results showed a synergistic effect between ampicillin and 9EA-FC-B treatment with the lowest FIC index of 0.19 (synergism ≤ 0.5). The presence of 9EA-FC-B reduced the MIC of ampicillin from 50 to 1.56 μg mL(-1). When ampicillin and 9EA-FC-B were combined at subinhibitory level, the kinetic growth curves were suppressed. The antibacterial effect of 9EA-FC-B and ampicillin was shown to be synergistic. The synergism is due the ability of 9EA-FC-B to suppress the activity of PBP2a, thus restoring the susceptibility of MRSA to ampicillin. Corilagin was postulated to be the constituent responsible for the synergistic activity showed by 9EA-FC-B.

Matched MeSH terms: Penicillin-Binding Proteins/biosynthesis
Fulltext A persistent antimicrobial resistance pattern and limited methicillin-resistance-associated genotype in a short-term Staphylococcus aureus carriage isolated from a student population

Mat Azis N, Pung HP, Abdul Rachman AR, Amin Nordin S, Sarchio SNE, Suhaili Z, et al.

J Infect Public Health, 2017 Mar-Apr;10(2):156-164.
PMID: 27033676 DOI: 10.1016/j.jiph.2016.02.013

The aim of the present study was to assess and compare the antimicrobial susceptibility pattern against a panel of antibiotics and molecular and methicillin resistance-associated genotypes of 120 carriage S. aureus isolates previously isolated from a student population at two isolation events within a one-month interval. The antibiotic susceptibility of isolates was determined using the Kirby-Bauer disc-diffusion method (cefoxitin by Etest). The MRSA was screened using polymerase chain reaction for the presence of the mecA gene. The mecA-positive isolates were subjected to staphylococcal cassette chromosome (SCC) mec typing, multilocus sequence typing (MLST) and eBURST analysis. All isolates were characterized for the presence of the Panton-Valentine leukocidin (PVL) gene, an enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) pattern and the spa type. For the two occasions where S. aureus was isolated, the highest frequency of resistance was observed for penicillin (70% and 65%, respectively), with a lower rate against erythromycin and tetracycline (<12%). All isolates were susceptible to ciprofloxacin and gentamycin. As for methicillin resistance, eight isolates had minimum inhibitory concentrations (MIC) of resistant categories, but 10 isolates (8.33%) were positive for the mecA gene. The mecA-positive isolates belonged to SCCmec types I (n=9) and V (n=1). MLST was resolved for only three MRSAs, ST508 (n=1), ST88 (n=1) and ST96 (n=1). The results of the eBURST analysis showed that the MRSA isolates analyzed in the present study were potentially related to MRSA identified in other countries. Approximately half of the persistent S. aureus carriers harbored S. aureus of a similar spa type in the respective individuals during both isolation events. A persistent antimicrobial pattern and limited distinct MRSAs were observed over the short study period. The latter frequently exhibited SCCmec type I, commonly associated with hospital-acquired (HA) characteristics, but further delineation is needed to justify the origins of these bacteria.

Matched MeSH terms: Penicillin-Binding Proteins/genetics
Fulltext Inhibition of penicillin-binding protein 2a (PBP2a) in methicillin resistant Staphylococcus aureus (MRSA) by combination of ampicillin and a bioactive fraction from Duabanga grandiflora

Santiago C, Pang EL, Lim KH, Loh HS, Ting KN

BMC Complement Altern Med, 2015;15:178.
PMID: 26060128 DOI: 10.1186/s12906-015-0699-z

The inhibition of penicillin-binding protein 2a (PBP2a) is a promising solution in overcoming resistance of methicillin resistance Staphylococcus aureus (MRSA). A potential approach in achieving this is by combining natural product with currently available antibiotics to restore the activity as well as to amplify the therapeutic ability of the drugs. We studied inhibition effects of a bioactive fraction, F-10 (isolated from the leaves of Duabanga grandiflora) alone and in combination with a beta-lactam drug, ampicillin on MRSA growth and expression of PBP2a. Additionally, phytochemical analysis was conducted on F-10 to identify the classes of phytochemicals present.

Matched MeSH terms: Penicillin-Binding Proteins/antagonists & inhibitors*
Fulltext Prevention of cell-surface attachment and reduction of penicillin-binding protein 2a (PBP2a) level in methicillin-resistant Staphylococcus aureus biofilms by Acalypha wilkesiana

Santiago C, Lim KH, Loh HS, Ting KN

BMC Complement Altern Med, 2015;15:79.
PMID: 25880167 DOI: 10.1186/s12906-015-0615-6

Formation of biofilm is known to enhance the virulence of methicillin-resistance Staphylococcus aureus (MRSA), which is associated with persistent infections in hospital settings. The biofilm layer essentially forms a protective barrier encapsulating the bacterial colony and thus reduces the effectiveness of chemotherapeutics. We have isolated 9EA-FC-B bioactive fraction from Acalypha wilkesiana Müll. Arg. that reverses ampicillin resistant in MRSA through inhibition of the antibiotic resistant protein, penicillin-binding protein 2a (PBP2a). In this study, we aimed to investigate the effects of 9EA-FC-B on MRSA biofilm forming capacity.

Matched MeSH terms: Penicillin-Binding Proteins/metabolism
Phenotypic and Genotypic Characterization of Neisseria gonorrhoeae Isolates from New Zealand with Reduced Susceptibility to Ceftriaxone

Jamaludin N, Gedye K, Collins-Emerson J, Benschop J, Nulsen M

Microb Drug Resist, 2019 Sep;25(7):1003-1011.
PMID: 31021281 DOI: 10.1089/mdr.2018.0111

Aim:
To characterize mutations in penA, mtrR, ponA, and porBIB, considered target genes for antimicrobial resistance, in Neisseria gonorrhoeae isolates with elevated minimum inhibitory concentrations (MICs) of ceftriaxone cultured from patients in New Zealand.
Results:
Out of 28 isolates supplied by the Institute of Environmental Science and Research Limited (ESR), Porirua, New Zealand, 14 were found to show reduced susceptibility to ceftriaxone (MIC of 0.06 mg/L) according to criteria used by the ESR and the Australian Gonococcal Surveillance Programme (AGSP) when tested in our laboratory. Rates of resistance to ciprofloxacin, azithromycin, penicillin, and tetracycline were 100% (28/28), 7% (2/28), 36% (10/28), and 25% (7/28), respectively. Ten different penA (Penicillin binding protein 2 [PBP2]) sequences were observed. The most common mosaic penA M-1 resembled mosaic penA XXXIV, which has been associated with ceftriaxone treatment failures in other countries. Four semimosaic PBP2 sequences were observed and may be novel PBP sequences, while four out of five nonmosaic PBP2 sequences were similar to PBP2 sequences reported in Australia. Twenty-one isolates harbored mutations in all 4 genes (penA, mtrR, porBIB, and ponA), and 13 of these exhibited reduced susceptibility to ceftriaxone.
Conclusion:
Mutations in penA, mtrR, porBIB, and ponA observed in this study may have contributed to reduced susceptibility to ceftriaxone among New Zealand gonococcal isolates. Over half (16/22) of mosaic penA sequences from the gonococcal isolates resembled penA XXXIV.

Matched MeSH terms: Penicillin-Binding Proteins/genetics
Fulltext Molecular detection and genotypic characterisation of Streptococcus pneumoniae isolated from children in Malaysia

Goh SL, Kee BP, Abdul Jabar K, Chua KH, Nathan AM, Bruyne J, et al.

Pathog Glob Health, 2020 02;114(1):46-54.
PMID: 32003298 DOI: 10.1080/20477724.2020.1719325

Streptococcus pneumoniae (S. pneumoniae) is one of the main causative agents of pneumococcal diseases. To date, more than 90 distinct serotypes have been identified. Implementation of vaccines has caused a drastic reduction in vaccine-serotype pneumococcal diseases but increase in cases due to non-vaccine serotype has been observed in Malaysia. However, further investigation on different serotype incidence in Malaysia is needed and the rate of pneumococcal vaccination for new-born babies in Malaysia remains low. The recent emergence of drug-resistant S. pneumoniae (DRSP) has also been a global concern, especially penicillin resistance. This study determined the serotypes of S. pneumoniae strains (n = 95) isolated from nasopharyngeal specimens from children admitted to UMMC from 2013 to 2015. In accordance with previous studies, PCR result showed 40% of NT isolates were successfully typed as 3 less common serotypes, namely 9N/L, 17A, and 23B. The repetitive-element PCR (REP-PCR) result revealed genetic variations among the strains whereby five major clusters were observed at the similarity of 80% by clustering analysis based on fingerprint data. Penicillin-binding proteins (pbps) of selected isolates were studied by PCR and sequencing. Three strains with ≤19-mm diameter zone for Oxacillin Disc Diffusion (ODD) test previously were recorded to have mutation on all pbp1a, pbp2b, and pbp2x with MIC of 4 µg/ml, which were penicillin-intermediate resistance according to the CLSI breakpoints.

Matched MeSH terms: Penicillin-Binding Proteins/genetics
Fulltext In vitro transfer of methicillin resistance determinants mecA from methicillin resistant Staphylococcus aureus (MRSA) to methicillin susceptible Staphylococcus aureus (MSSA)

Bitrus AA, Zunita Z, Bejo SK, Othman S, Nadzir NA

BMC Microbiol, 2017 04 04;17(1):83.
PMID: 28376716 DOI: 10.1186/s12866-017-0994-6

BACKGROUND: Staphylococcus aureus more than any other human pathogen is a better model for the study of the adaptive evolution of bacterial resistance to antibiotics, as it has demonstrated a remarkable ability in its response to new antibiotics. This study was designed to investigate the in vitro transfer of mecA gene from methicillin resistant S. aureus to methicillin susceptible S. aureus.
RESULT: The recipient transconjugants were resistant to erythromycin, cefpodoxime and were mecA positive. PCR amplification of mecA after mix culture plating on Luria Bertani agar containing 100 μg/mL showed that 75% of the donor and 58.3% of the recipient transconjugants were mecA positive. Additionally, 61.5% of both the donor cells and recipient transconjugants were mecA positive, while 46.2% and 41.75% of both donor and recipient transconjugants were mecA positive on LB agar containing 50 μg/mL and 30 μg/mL respectively.
CONCLUSION: In this study, the direction of transfer of phenotypic resistance as well as mecA was observed to have occurred from the donor to the recipient strains. This study affirmed the importance of horizontal transfer events in the dissemination of antibiotics resistance among different strains of MRSA.

Matched MeSH terms: Penicillin-Binding Proteins/genetics*
Humoral immune consequences of Staphylococcus aureus ST239-associated bacteremia

Ghasemzadeh-Moghaddam H, van Wamel W, van Belkum A, Hamat RA, Tavakol M, Neela VK

Eur J Clin Microbiol Infect Dis, 2018 Feb;37(2):255-263.
PMID: 29103153 DOI: 10.1007/s10096-017-3124-3

The humoral immune responses against 46 different staphylococcal antigens in 27 bacteremia patients infected by clonally related methicillin-resistant Staphylococcus aureus (MRSA) strains of a single sequence type (ST) 239 were investigated. A group of non-infected patients (n = 31) hospitalized for different reasons served as controls. All strains were confirmed as ST 239 by S. aureus and mecA-specific PCR, spa, and multi-locus sequence typing (MLST). In each bacteremia patient, a unique pattern of S. aureus antigen-specific immune responses after infection was observed. Antibody levels among bacteremia patients were significantly higher than controls for HlgB (P = 0.001), LukD (P = 0.009), LukF (P = 0.0001), SEA (P = 0.0001), SEB (P = 0.011), SEC (P = 0.010), SEQ (P = 0.049), IsaA (P = 0.043), IsdA (P = 0.038), IsdH (P = 0.01), SdrD (P = 0.001), SdrE (P = 0.046), EsxA (P = 0.0001), and SA0104 (P = 0.0001). On the other hand, the antibody levels were significantly higher among controls for SSL3 (P = 0.009), SSL9 (P = 0.002), and SSL10 (P = 0.007) when the IgG level on the day of infection was compared with that measured on the day of admission. Diversity was observed in the immune response against the antigens. However, a set of antigens (IsaA, IsdA, IsdH, SdrD, and HlgB) triggered a similar type of immune response in different individuals. We suggest that these antigens could be considered when developing a multi-component (passive) vaccine. SEA and/or its specific antibodies seem to play a critical role during ST239 MRSA bacteremia and SEA-targeted therapy may be a strategy to be considered.

Matched MeSH terms: Penicillin-Binding Proteins/genetics
Spread of drug-resistant Streptococcus pneumoniae in Asian countries: Asian Network for Surveillance of Resistant Pathogens (ANSORP) Study

Song JH, Lee NY, Ichiyama S, Yoshida R, Hirakata Y, Fu W, et al.

Clin Infect Dis, 1999 Jun;28(6):1206-11.
PMID: 10451154

Antimicrobial susceptibility of 996 isolates of Streptococcus pneumoniae from clinical specimens was investigated in 11 Asian countries from September 1996 to June 1997. Korea had the greatest frequency of nonsusceptible strains to penicillin with 79.7%, followed by Japan (65.3%), Vietnam (60.8%), Thailand (57.9%), Sri Lanka (41.2%), Taiwan (38.7%), Singapore (23.1%), Indonesia (21.0%), China (9.8%), Malaysia (9.0%), and India (3.8%). Serotypes 23F and 19F were the most common. Pulsed-field gel electrophoresis (PFGE) of 154 isolates from Asian countries showed several major PFGE patterns. The serotype 23F Spanish clone shared the same PFGE pattern with strains from Korea, Japan, Singapore, Taiwan, Thailand, and Malaysia. Fingerprinting analysis of pbp1a, pbp2x, and pbp2b genes of 12 strains from six countries also showed identical fingerprints of penicillin-binding protein genes in most strains. These data suggest the possible introduction and spread of international epidemic clones into Asian countries and the increasing problems of pneumococcal drug resistance in Asian countries for the first time.

Matched MeSH terms: Penicillin-Binding Proteins
Penicillin susceptibility and molecular characteristics of clinical isolates of Streptococcus pneumoniae at the University of Malaya Medical Center, Kuala Lumpur, Malaysia

Desa MN, Lin TK, Yasin RM, Parasakthi N

Int J Infect Dis, 2003 Sep;7(3):190-7.
PMID: 14563222

To determine the prevalence of penicillin resistance and molecular characteristics of pneumococcal isolates at the University of Malaya Medical Center.

Matched MeSH terms: Penicillin-Binding Proteins

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