Potential targets by pentacyclic triterpenoids from Callicarpa farinosa against methicillin-resistant and sensitive Staphylococcus aureus.

The evolution of antibiotic resistance in Staphylococcus aureus showed that there is no long-lasting remedy against this pathogen. The limited number of antibacterial classes and the common occurrence of cross-resistance within and between classes reinforce the urgent need to discover new compounds targeting novel cellular functions not yet targeted by currently used drugs. One of the experimental approaches used to discover novel antibacterials and their in vitro targets is natural product screening. Three known pentacyclic triterpenoids were isolated for the first time from the bark of Callicarpa farinosa Roxb. (Verbenaceae) and identified as α-amyrin [3ß-hydroxy-urs-12-en-3-ol], betulinic acid [3ß-hydroxy-20(29)-lupaene-28-oic acid], and betulinaldehyde [3ß-hydroxy-20(29)-lupen-28-al]. These compounds exhibited antimicrobial activities against reference and clinical strains of methicillin-resistant (MRSA) and methicillin-sensitive S. aureus (MSSA), with minimum inhibitory concentration (MIC) ranging from 2 to 512 µg/mL. From the genome-wide transcriptomic analysis to elucidate the antimicrobial effects of these compounds, multiple novel cellular targets in cell division, two-component system, ABC transporters, fatty acid biosynthesis, peptidoglycan biosynthesis, aminoacyl-tRNA synthetases, ribosomes and ß-lactam resistance pathways are affected, resulting in destabilization of the bacterial cell membrane, halt in protein synthesis, and inhibition of cell growth that eventually lead to cell death. The novel targets in these essential pathways could be further explored in the development of therapeutic compounds for the treatment of S. aureus infections and help mitigate resistance development due to target alterations.

Identification, by gene expression profiling analysis, of novel gene targets in Staphylococcus aureus treated with betulinaldehyde.

Staphylococcus aureus has become a serious concern in hospitals and community due to rapid adaptation to existing antimicrobial agents. Betulinaldehyde [3ß-hydroxy-20(29)-lupen-28-al (BE)] belongs to pentacyclic triterpenoids that are based on a 30-carbon skeleton comprising four six-membered rings and one five-membered ring. In a preliminary study, BE exhibited antimicrobial activity against reference strains of methicillin-resistant and methicillin-sensitive S. aureus. However, the response mechanism of S. aureus to this compound is not known. In this study, the global gene expression patterns of both the reference strains in response to sub-inhibitory concentrations of BE were analyzed using DNA microarray to identify gene targets, particularly essential targets in novel pathways, i.e. not targeted by currently used antibiotics, or novel targets in existing pathways. The transcriptome analysis revealed repression of genes in the aminoacyl-tRNA synthetase and ribosome pathways in both the reference strains. Other pathways such as cell division, two-component systems, ABC transporters, fatty acid biosynthesis and peptidoglycan biosynthesis were affected only in the reference strain of methicillin-resistant S. aureus. The findings suggest that BE regulates multiple desirable targets which could be further explored in the development of therapeutic agents for the treatment of S. aureus infections.

Transcriptional profiles of the response of methicillin-resistant Staphylococcus aureus to pentacyclic triterpenoids.

Staphylococcus aureus is an important human pathogen in both hospital and the community that has demonstrated resistance to all currently available antibiotics over the last two decades. Multidrug-resistant isolates of methicillin-resistant S. aureus (MRSA) exhibiting decreased susceptibilities to glycopeptides has also emerged, representing a crucial challenge for antimicrobial therapy and infection control. The availability of complete whole-genome nucleotide sequence data of various strains of S. aureus presents an opportunity to explore novel compounds and their targets to address the challenges presented by antimicrobial drug resistance in this organism. Study compounds α-amyrin [3ß-hydroxy-urs-12-en-3-ol (AM)], betulinic acid [3ß-hydroxy-20(29)-lupaene-28-oic acid (BA)] and betulinaldehyde [3ß-hydroxy-20(29)-lupen-28-al (BE)] belong to pentacyclic triterpenoids and were reported to exhibit antimicrobial activities against bacteria and fungi, including S. aureus. The MIC values of these compounds against a reference strain of methicillin-resistant S. aureus (MRSA) (ATCC 43300) ranged from 64 µg/ml to 512 µg/ml. However, the response mechanisms of S. aureus to these compounds are still poorly understood. The transcription profile of reference strain of MRSA treated with sub-inhibitory concentrations of the three compounds was determined using Affymetrix GeneChips. The findings showed that these compounds regulate multiple desirable targets in cell division, two-component system, ABC transporters, fatty acid biosynthesis, peptidoglycan biosynthesis, aminoacyl-tRNA synthetase, ribosome and ß-lactam resistance pathways which could be further explored in the development of therapeutic agents for the treatment of S. aureus infections.

Synergistic antimicrobial activity between pentacyclic triterpenoids and antibiotics against Staphylococcus aureus strains.

BACKGROUND: There has been considerable effort to discover plant-derived antibacterials against methicillin-resistant strains of Staphylococcus aureus (MRSA) which have developed resistance to most existing antibiotics, including the last line of defence, vancomycin. Pentacyclic triterpenoid, a biologically diverse plant-derived natural product, has been reported to show anti-staphylococcal activities. The objective of this study is to evaluate the interaction between three pentacyclic triterpenoid and standard antibiotics (methicillin and vancomycin) against reference strains of Staphylococcus aureus. METHODS AND RESULTS: The activity of the standard antibiotics and compounds on reference methicillin-sensitive and resistant strains of S. aureus were determined using the macrodilution broth method. The minimum inhibitory concentration (MIC) of the compounds was compared with that of the standard antibiotics. The interaction between any two antimicrobial agents was estimated by calculating the fractional inhibitory concentration (FIC index) of the combination. The various combinations of antibiotics and compounds reduced the MIC to a range of 0.05 to 50%. CONCLUSION: Pentacyclic triterpenoids have shown anti-staphylococcal activities and although individually weaker than common antibiotics produced from bacteria and fungi, synergistically these compounds may use different mechanism of action or pathways to exert their antimicrobial effects, as implicated in the lowered MICs. Therefore, the use of current antibiotics could be maintained in their combination with plant-derived antibacterial agents as a therapeutic option in the treatment of S. aureus infections.

High Helicobacter pylori resistance to metronidazole but zero or low resistance to clarithromycin, levofloxacin, and other antibiotics in Malaysia.

OBJECTIVE: Bacterial resistance to antibiotics is the single most important determinant of treatment success. The objective of this study was to determine the prevalence of Helicobacter pylori resistance to clarithromycin, amoxicillin, metronidazole, tetracycline, levofloxacin, rifabutin, and furazolidone in our local bacterial strains. METHODS: Samples from consecutive ninety patients were obtained for culture and sensitivity testing. Resistance to individual antibiotics were tested using the E-test and MIC(90) read from the strips. Resistance to rifampicin and nitrofurantoin were used as a surrogate for rifabutin and furazolidine. RESULTS: There was a high prevalence of resistance to metronidazole 68/90 (75.5%). No male (34/45 (75.5%) versus female (35/45 (77.7%) difference in frequency of metronidazole resistance was noted (p = 1.000). There was zero resistance (0) to clarithromycin, levofloxacin, amoxicillin, and nitrofurantoin/furazolidone. Resistance to rifampicin/rifabutin was for breakpoints of 1 and 4 µg/mL of 14.4 and 2.2% respectively. CONCLUSIONS: Although there was high bacterial resistance to metronidazole, the absence of resistance particularly to the key antibiotics used in H. pylori eradication therapy: clarithromycin and levofloxacin is reassuring to note. Continued monitoring of antibiotic resistance should be carried out.

Pneumococcal replicative state in relation to its adherence capacity to A549-cell line: A preliminary in vitro analysis

This study was to compare the replication capacity of pneumococcal isolates (serotypes 1, 7F, 19F and 23F) with their adherence pattern to monolayer cells (A549). For standardization purposes, all isolates showed a normal growth curve in both bacteriological (THB + 0.5% yeast extract with and without 2% FBS) and cell culture media (RPMI + 2% FBS). In the former media, a shorter lag phase was observed for isolate serotypes 1 and 7F in presence of serum while in the later; growth yield was lower for all isolates with stationary phase approaching OD600 of 0.01 as compared to 1.0 in bacteriological media. In the replicative analysis at different growth phases of the isolates in cell culture media, growth capacity at 3 h post-incubation was frequently twice as that at 1 h, and that at early-log phase was frequently higher than that at mid-log phase at both post-incubation times. Adherence was frequently the least at early-log phase although the isolates were in the most active state of replication to increase the number of pneumococcal cells to adhere. At mid- and late-log phases, pneumococcal adherence was frequently higher although the replication was reduced. This study marks the potential correlation between pneumococcal growth fitness and adherence capacity whereby the later may not be superior during the early growth phase.

Resistance to extended-spectrum beta-lactams by the emergence of SHV-12 and the loss of OmpK35 in Klebsiella pneumoniae and Escherichia coli in Malaysia.

BACKGROUND AND PURPOSE: In addition to beta-lactamase production, loss of porins confers resistance to extended-spectrum beta-lactams in Klebsiella pneumoniae and Escherichia coli infection. This study describes the detection of SHV-12 extended-spectrum beta-lactamase (ESBL) subtype and the loss of OmpK35 porin in 4 strains of K. pneumoniae and E. coli. METHODS: Isoelectric focusing was performed to detect beta-lactamases in 4 strains of K. pneumoniae and E. coli. The presence of the SHV gene in the 4 isolates was characterized by polymerase chain reaction, DNA sequencing, and DNA hybridization. Loss of porin in these strains was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis. RESULTS: The strains of K. pneumoniae and E. coli were confirmed to be ESBL producers and were resistant to cefoxitin, with minimal inhibitory concentration values of 512 microg/mL. All 4 strains had beta-lactamases with an isoelectric value of 8.2. The SHV gene from these strains was characterized to be the SHV-12 subtype and was plasmid-borne. The deduced amino acid sequence showed that the SHV-12 beta-lactamase was a derivative of the more common ESBL, SHV-5 subtype. All the strains showed absence of the OmpK35 porin. CONCLUSION: Resistance of the strains towards extended-spectrum beta-lactams was a result of a dual-mechanism - the production of SHV-12 enzymes and loss of the OmpK35 porin.

Detection and characterization of class 1 integrons among carbapenem-resistant isolates of Acinetobacter spp. in Malaysia.

BACKGROUND AND PURPOSE: To detect and characterize class 1 integrons among carbapenem-resistant strains of Acinetobacter spp. at University Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia. METHODS: Thirty nine carbapenem-resistant Acinetobacter strains were obtained from UMMC from August 2003 to March 2004 and analyzed for the presence of bla(IMP) genes and class 1 integrons. RESULTS: Class 1 integrons were detected in 31 of 39 strains. Two Acinetobacter calcoaceticus strains harbored an integron-borne bla(IMP-4) metallo-beta-lactamase, 1 of which was located on a 36-kb plasmid. Two different amplified products were found in the 31 isolates with 3 restriction pattern profiles 1, 2, and 3. Correlation was observed between carriage of class 1 integrons and genomic relatedness among these isolates, indicating that particular mechanisms of carbapenem resistance could have been acquired by genotypically distinct clinical isolates of Acinetobacter spp. CONCLUSION: Although class 1 integrons are widely disseminated among clinical isolates of Acinetobacter spp. they do not play a major role in the spread of carbapenem resistance.

Expression analysis of adherence-associated genes in pneumococcal clinical isolates during adherence to human respiratory epithelial cells (in vitro) by real-time PCR.

Pneumococcal virulence determinants have been extensively studied but molecular evidence on virulence gene expression pattern is still lacking. We undertook this study to analyze the regulation pattern of adherence-associated genes; psaA, pspC, cbpG, including ply of serotypes 1, 7F, 19F and 23F clinical isolates during the bacterial adherence to human lung epithelial cells (in vitro), by real-time PCR. We were able to harvest the bacterial RNA (0.5-1 microg microL(-1)) from the infected host cell and analysis showed a consistent upregulation of psaA. Differential expressions were observed for pspC, cbpG and ply genes but the former was mostly upregulated whereas the later two frequently showed either no significant change or a downregulation. Partial nucleotide sequences of psaA, cbpG and ply were highly homologous among the isolates as well as against GenBank sequences (99%) whereas those for pspC were similar (98%) to allelic variants pspC-3 and pspC-5.

Detection of pbp2b and ermB genes in clinical isolates of Streptococcus pneumoniae.

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.

Rapid detection of ESBL-producing Klebsiella pneumoniae in blood cultures by fluorescent in-situ hybridization.

Multi-resistant Enterobacteriaceae pose a serious threat of hospital acquired infections and their rapid identification is important for better clinical outcome. This study describes the rapid identification of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae of the sulphydryl variable-type by fluorescent in-situ hybridization. The method which rapidly identifies the target genes within 1 h could be a potentially rapid bacterial diagnostic tool.

Evaluation of the monoclonal stool antigen test for Helicobacter pylori in an Asian population with dyspepsia.

OBJECTIVE: Although well established in the West, stool antigen tests (SAT) are not widely used in Asia. Data on the accuracy of this test in Asia is sparse and, to date, there have been no studies looking at the more refined monoclonal SAT. The aim of this study is to validate the diagnostic accuracy of a stool antigen test, Hp STAR, for the detection of Helicobacter pylori. METHODS: Consecutive new patients with dyspepsia, referred to the endoscopy unit were recruited. At endoscopy, biopsies were taken for rapid urease tests and histology. Stool specimens were collected before any therapy was given and were tested for H. pylori using the monoclonal SAT HpSTAR test. Samples were collected from 90 patients. The stool antigen results were compared with the results of rapid urease tests and histopathology, which were used together as the gold standard test in the diagnosis of H. pylori. RESULTS: Of the 90 patients tested, 45 were H. pylori positive and 45 were H. pylori negative. There was one false positive and one false negative test. The sensitivity, specificity, positive predictive value and negative predictive value for the Hp STAR were identical: 97.8% with a 95% confidence interval of 88.2-99.9%. The diagnostic accuracy was 97.8% with a 95% confidence interval of 92.2-99.7%. CONCLUSION: The monoclonal stool antigen test, HpSTAR is a highly accurate test for the diagnosis of H. pylori.

Etiology of blood culture isolates among patients in a multidisciplinary teaching hospital in Kuala Lumpur.

BACKGROUND AND PURPOSE: Bloodstream infections are an important cause of morbidity and mortality among hospitalized patients and the surveillance of etiological agents in these infections is important for their prevention and treatment. Data on common organisms isolated from blood cultures from Malaysia are limited, and our aim was to identify the common bloodstream isolates in hospitalized patients at the University of Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia. METHODS: A retrospective analysis was conducted over a 1-year period from January to December 2004 by reviewing laboratory reports of patients from the UMMC. The clinical significance of the isolates was not analyzed. RESULTS: Coagulase-negative staphylococci were the most common organisms isolated, accounting for 33.0% of the total blood culture isolates, followed by Staphylococcus aureus (10.4%) and Escherichia coli (9.7%). The incidence of methicillin-resistant S. aureus, and extended-spectrum beta-lactamase-producing E. coli and Klebsiella spp. bacteremia was low (2.3% and 1.8% of total isolates, respectively). Non-albicans Candida were the most common fungal isolates. CONCLUSIONS: The high number of coagulase-negative staphylococci should motivate clinicians and microbiologists to re-examine blood culture techniques in our institution. We recommend that further studies be carried out to establish the true significance of this organism among blood culture isolates.

Evaluation of the routine use of the anaerobic bottle when using the BACTEC blood culture system.

The established practice of sending blood cultures in an aerobic-anaerobic pair of bottles has been questioned in recent years, and this study was conducted to evaluate the routine use of an anaerobic bottle in the BACTEC blood culture set at the University of Malaya Medical Centre, Kuala Lumpur, from January to December 2004. A total of 11,663 paired blood culture sets were received, of which 3326 were from pediatric patients and 8337 were from adult patients. The overall positive isolation rate was 15%; the positive isolation rate on excluding the anaerobic bottles was 13%. Overall, there were significantly more organisms isolated from the aerobic bottle (p<0.05); however, the best yield was obtained on using the paired aerobic-anaerobic bottles. Among the positive blood culture sets, organisms were isolated from the anaerobic bottle alone in 15.2% of the pediatric sets and in 18.1% of the adult sets. Organisms that grew more frequently in the anaerobic bottle were anaerobes and some facultative anaerobes; however, the difference was not statistically significant except for anaerobes in the adult sets. We recommend that when culturing blood, an aerobic-anaerobic pair of bottles be used rather than an aerobic-aerobic pair, to optimize the recovery of a wider spectrum of organisms, including obligatory anaerobes.

HUITAI rapid urease test: a new ultra-rapid biopsy urease test for the diagnosis of Helicobacter pylori infection.

BACKGROUND: The gastric biopsy urease test is an accurate and robust diagnostic test for Helicobacter pylori infection. Large endoscopy units use their own homemade unbuffered ultra-rapid urease test for diagnosis of H. pylori infection but several commercial rapid urease tests are available. OBJECTIVE: To compare the accuracy and reaction time of a new biopsy urease test, HUITAI rapid urease test, in the diagnosis of H. pylori infection. METHODS: Consecutive patients presenting with dyspepsia to the endoscopy unit, University of Malaya Medical Center were recruited for the study. Patients who were previously treated for H. pylori infection or who had received antibiotics, proton pump inhibitors or bismuth compounds in the preceding 4 weeks were excluded. The H. pylori diagnosis was made based on the homemade rapid urease test, histology and culture of gastric biopsies. Biopsies from the antrum and corpus of the stomach were taken for this purpose. In addition, two antral and corpus biopsies were taken for the HUITAI rapid urease test. A positive diagnosis of H. pylori infection was made when the culture was positive or if both histology and the rapid urease test were positive. A negative diagnosis was made when all tests were negative. The positive reaction time of the HUITAI rapid urease test was carefully timed up to 60 min. RESULTS: Two hundred and six patients were recruited in the study. One hundred and twelve were diagnosed as having an H. pylori infection while the other 94 patients were regarded as negative. There were no spoiled tests and no indeterminate results. The sensitivity of the HUITAI rapid urease test was 98.2% (95% confidence interval (CI): 93.7%, 99.8%), specificity, 99.0% (95% CI: 94.2%, 100%), positive predictive value, 99.0% (95% CI: 95.1%, 100%), negative predictive value, 97.9% (95% CI: 92.6%, 99.7%). The overall diagnostic accuracy for the HUITAI rapid urease test was 98.5% (95% CI: 96.6, 99.9). The median positive reaction time was 1.0 min (25-75% inter-quartile range [IQR]: 1.0-3.0 min). CONCLUSIONS: The HUITAI rapid urease test is highly accurate for the diagnosis of an H. pylori infection and showed a very rapid positive reaction time.

Detection of mecA and ermA genes and simultaneous identification of Staphylococcus aureus using triplex real-time PCR from Malaysian S. aureus strain collections.

A triplex real-time polymerase chain reaction (PCR) assay was used for the simultaneous detection of mecA (methicillin resistance), ermA (erythromycin resistance) and femA (Staphylococcus aureus identification) genes in a single assay. Among 93 clinical S. aureus hospital isolates, there were 48 methicillin-resistant S. aureus (MRSA) and 45 methicillin-sensitive S. aureus (MSSA) isolates. Screening the isolates using the triplex real-time PCR assay, the mecA, ermA and femA genes were detected in all MRSA isolates. The triplex real-time PCR assay was completed within 3h and is a useful genotypic method for detecting the resistance determinants as well as for the identification of S. aureus isolates. These findings will assist the clinical laboratory in identifying these resistance genes and S. aureus rapidly, thus benefiting patient therapy. This study represents a valuable source of information for researchers to study the local antibiotic resistance pattern, which can increase our knowledge of the antibiotic resistance profile, using real-time PCR technology.

Detection of methicillin- and aminoglycoside-resistant genes and simultaneous identification of S. aureus using triplex real-time PCR Taqman assay.

In this study we describe a triplex real-time PCR assay that enables the identification of S. aureus and detection of two important antibiotic resistant genes simultaneously using real-time PCR technology in a single assay. In this triplex real-time PCR assay, the mecA (methicillin resistant), femA (species specific S. aureus) and aacA-aphD (aminoglycoside resistant) genes were detected in a single test using dual-labeled Taqman probes. The assay gives simultaneous information for the identification of S. aureus and detection of methicillin and aminoglycoside resistance in staphylococcal isolates. 152 clinical isolates were subjected to this triplex real-time PCR assay. The results of the triplex real-time PCR assay correlated with the results of the phenotypic antibiotic susceptibility testing. The results obtained from triplex real-time PCR assay shows that the primer and probe sets were specific for the identification of S. aureus and were able to detect methicillin- and aminoglycoside-resistant genes. The entire assay can be performed within 3 h which is a very rapid method that can give simultaneous information for the identification of S. aureus and antibiotic resistance pattern of a staphylococcal isolate. The application of this rapid method in microbiology laboratories would be a valuable tool for the rapid identification of the S. aureus isolates and determination of their antibiotic resistance pattern with regards to methicillin and aminoglycosides.

Simultaneous species identification and detection of methicillin resistance in staphylococci using triplex real-time PCR assay.

For rapid identification of methicillin-resistant Staphylococcus aureus, molecular methods are generally targeting mecA and species-specific genes. Sa442 DNA fragment is a popular species-specific target. However, recently, there have been few reports on S. aureus isolates that are negative for Sa442 fragment; therefore, use of single gene or DNA-fragment-specific polymerase chain reaction (PCR) for identification of microbial isolate may result in misidentification. This study includes CoA gene in parallel with Sa442 marker for identification of S. aureus. This further improves the specificity of the assay by checking for 2 determinants simultaneously for the identification of S. aureus and can prevent misidentification of S. aureus isolates lacking Sa442 DNA fragment. In this study, the newly developed triplex real-time PCR assay was compared with a quadruplex conventional gel-based PCR assay using the same primer sets in both assays. The dual-labeled TaqMan probes (ProOligo, France) for these primers were specifically designed and used in a real-time PCR assay. The clinical isolates (n = 152) were subjected to both PCR assays. The results obtained from both assays proved that the primer and probe sets were 100% sensitive and 100% specific for identification of S. aureus and detection of methicillin resistance. This triplex real-time PCR assay represents a rapid and powerful method for S. aureus identification and detection of methicillin resistance.

Antibiotic resistance patterns in nosocomial gram-negative bacterial infections in units with heavy antibiotic usage.

The pattern of antibiotic resistance amongst gram-negative bacteria (GNB) in paediatric units, which have heavy empirical usage of broad-spectrum antibiotics, was studied prospectively over a 6-month period. A total of 200 consecutive, non-duplicate gram-negative isolates were obtained from 109 patients admitted to intensive care and oncology units in two hospitals. The commonest isolates were Klebsiella spp (36.5 per cent) and Pseudomonas (20.0 per cent). The isolates showed lower susceptibility rates to the third-generation cephalosporins (47-62 per cent) compared with cefepime (91 per cent), imipenem (90 per cent) and ciprofloxacin (99 per cent). Fifty-four (52.8 per cent) Klebsiella and Escherichia coli isolates were determined to be extended-spectrum beta-lactamase (ESBL) producing strains. Antibiotics found to be effective against ESBL-producers were imipenem and ciprofloxacin. The high resistance rate amongst GNB to third-generation cephalosporins is a likely consequence of heavy empirical usage of this group of antibiotics. The carbapenems and quinolones remain useful agents in the management of patients admitted to these units.

Comparison of susceptibility test methods to detect penicillin susceptibility in Streptococcus pneumoniae isolates

The increasing prevalence of penicillin-resistant Streptococuus pneumoniae urges for fast and accurate susceptibility testing methods. This study evaluated the comparability of three commonly used techniques; disk diffusion, E-test and agar dilution, to detect penicillin susceptibility in clinical isolates of S. pneumoniae. Fifty pneumococcal isolates, obtained from patients at the University of Malaya Medical Centre, were selected to include both penicillin-susceptible strains and those that had decreased susceptibility (resistant and intermediate) to penicillin. The minimum inhibitory concentration (MIC) values of penicillin to serve as the reference was determined by the agar dilution method in which, based on the MIC breakpoints recommended by the National Committee for Clinical Laboratory Standards (NCCLS), 27 strains had decreased susceptibility to penicillin with 17 strains resistant and 10 intermediate. Comparing to the agar dilution method, oxacillin disk diffusion test detected all strains with decreased penicillin susceptibility as such while E-test showed a close agreement of susceptibility (92%) of the isolates to penicillin. This confirmed that oxacillin is a good screening test for S. pneumoniae isolates with decreased susceptibility to penicillin while E-test is very reliable for rapid and accurate detection of penicillin susceptibility.