Chanwit Tribuddharat

Nosocomial Outbreak of Carbapenem-Resistant Pseudomonas aeruginosa with a New blaIMP Allele, blaIMP-7

ABSTRACT Pseudomonas aeruginosa isolates from an outbreak in Canada were highly resistant to carbapenems and ceftazidime but not piperacillin. They produced a novel integron-associated metallo-β-lactamase, designated IMP-7, with 91% identity to IMP-1. blaIMP-7 was not detected with standard blaIMP-specific primers, owing to mismatches in the forward primer.

Burkholderia pseudomallei Class A β-Lactamase Mutations That Confer Selective Resistance against Ceftazidime or Clavulanic Acid Inhibition

ABSTRACT Burkholderia pseudomallei, the causative agent of melioidosis, is inherently resistant to a variety of antibiotics including aminoglycosides, macrolides, polymyxins, and β-lactam antibiotics. Despite resistance to many β-lactams, ceftazidime and β-lactamase inhibitor-β-lactam combinations are commonly used for treatment of melioidosis. Here, we examine the enzyme kinetics of β-lactamase isolated from mutants resistant to ceftazidime and clavulanic acid inhibition and describe specific mutations within conserved motifs of the β-lactamase enzyme which account for these resistance patterns. Sequence analysis of regions flanking the B. pseudomallei penA gene revealed a putative regulator gene located downstream of penA. We have cloned and sequenced the penA gene from B. mallei and found it to be identical to penA from B. pseudomallei.

Molecular analyses of TEM genes and their corresponding penicillinase-producing Neisseria gonorrhoeae isolates in Bangkok, Thailand

ABSTRACT Neisseria gonorrhoeae is a major public health problem globally, especially because the bacterium has developed resistance to most antimicrobials introduced for first-line treatment of gonorrhea. In the present study, 96 N. gonorrhoeae isolates with high-level resistance to penicillin from 121 clinical isolates in Thailand were examined to investigate changes related to their plasmid-mediated penicillin resistance and their molecular epidemiological relationships. A β-lactamase (TEM) gene variant, blaTEM-135, that may be a precursor in the transitional stage of a traditional blaTEM-1 gene into an extended-spectrum β-lactamase (ESBL), possibly causing high resistance to all extended-spectrum cephalosporins in N. gonorrhoeae, was identified. Clonal analysis using multilocus sequence typing (MLST) and N. gonorrhoeae multiantigen sequence typing (NG-MAST) revealed the existence of a sexual network among patients from Japan and Thailand. Molecular analysis of the blaTEM-135 gene showed that the emergence of this allele might not be a rare genetic event and that the allele has evolved in different plasmid backgrounds, which results possibly indicate that it is selected due to antimicrobial pressure. The presence of the blaTEM-135 allele in the penicillinase-producing N. gonorrhoeae population may call for monitoring for the possible emergence of ESBL-producing N. gonorrhoeae in the future. This study identified a blaTEM variant (blaTEM-135) that is a possible intermediate precursor for an ESBL, which warrants international awareness.

Serotype coverage of pneumococcal conjugate vaccine and drug susceptibility of Streptococcus pneumoniae isolated from invasive or non-invasive diseases in central Thailand, 2006-2009.

The serotype of 172 S. pneumoniae isolates obtained from normally sterile sites from January 2006 to February 2009 in Thai patients was evaluated. The most common serotypes were 6B, 23F, 14, 19F, and 19A in patients <5 year-old, and 6B, 19A, 23F, 4, 9V in patients >65-year old. Seven-valent pneumococcal conjugated vaccine (PCV-7) covered 70.3%, 43.6%, and 43.5% of patients <5, 5-64 and > or = 65 years of age, respectively, while PCV-13 covered 81.2%, 59.7%, and 60.9%, respectively. PCV-9, PCV-10, PCV-11 had very similar coverage as PCV-7. The antibiotic susceptibility rates of the isolates from sterile sites were 88.7-95.7% for penicillin, 90.6-98.4% for cefotaxime, 92.2-100% for ofloxacin and 100% for ciprofloxacin. PCV-7 covered 83% and 100%, respectively, of penicillin and cefotaxime non-susceptible isolates in patients <5-year old.

Application of immunoproteomics to leptospirosis: towards clinical diagnostics and vaccine discovery

Each of the currently available methods for serodiagnosis of leptospirosis, including the microscopic agglutination test (MAT), has its own drawback(s) when used in clinical practice. A new diagnostic test is therefore required for an earlier and more accurate diagnosis of leptospirosis. We applied immunoproteomics to define potential immunogens from five serovars of Leptospira reference strains. A leptospiral whole cell lysate from each serovar was used as the antigen to react with IgG and IgM in the sera from four patients with a positive MAT. Sera from four non‐leptospirosis patients with a negative MAT were pooled and used as the negative control. 2‐D Western blot analysis showed that the degree of immunoreactivity corresponded with the MAT titers. No immunoreactive spots were detected when the pooled control sera were used. A total of 24 protein spots immunoreacted with IgM and/or IgG from patients with leptospirosis. These immunoreactive proteins were identified by MALDI‐TOF MS and were classified into five groups, including flagellar proteins, chaperones/heat shock proteins, transport proteins, metabolic enzymes, and hypothetical proteins. More immunoreactive spots were detected with anti‐human IgG in the sera of all patients and with all the serovars of leptospires used. Some of the identified proteins immunoreacted only with IgG, whereas the others were detectable with both IgM and IgG. Among the immunoreactive proteins identified, FlaB proteins (flagellin and flagellar core protein) have been shown to have a potential role in clinical diagnostics and vaccine development. These data underscore the significant impact of immunoproteomics in clinical applications.

Simultaneous overexpression of multidrug efflux pumps in Pseudomonas aeruginosa non-cystic fibrosis clinical isolates

The purpose of this study was to examine expression and regulation of 6 multidrug efflux systems, including MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY, MexJK, and MexVW, in 13 non-cystic fibrosis (CF) clinical isolates of Pseudomonas aeruginosa. These isolates displayed a high level of resistance to many clinically important antibiotics. Some isolates simultaneously overexpressed up to 4 different Mex systems, as determined by quantitative real-time reverse transcription PCR. None of the isolates overexpressed MexCD-OprJ, and only 1 isolate overproduced MexJK. All the isolates overexpressed MexXY, while overexpression of MexEF-OprN and MexVW was common. DNA sequencing analysis of regulatory genes showed that no clear correlation could be established among (i) the presence of mutations, (ii) the type of mutations, (iii) the expression level of the Mex systems, and (iv) resistance to antibiotic substrates. The results suggest that the concomitant overexpression of some Mex systems may superimpose their antimicrobial drug efflux capabilities, contributing to the multidrug resistance phenotype in the P. aeruginosa non-CF clinical isolates. The existence of uncharacterized regulators for the Mex systems was signified.

Co-existence of beta-lactamases in clinical isolates of Escherichia coli from Kathmandu, Nepal.

BackgroundThe trend of extended-spectrum beta-lactamases producing Escherichia coli (ESBL-EC) is increasing in Nepal. Limited studies have been reported investigating ESBL types and carbapenemases in E. coli.MethodsA cross sectional study was conducted between June 2012 to January 2013 in Kathmandu Medical College and Teaching Hospital, Nepal. Non-repetitive clinical samples from out-patient department (OPD) and Intensive Care Units (ICU) were processed for bacteriological culture and identification of E. coli. Antibiotic susceptibility test, screening and phenotypic confirmation for ESBLs and carbapenemases and PCR (blaCTX-M, blaSHV and blaTEM-type ESBLs, blaVIM, blaIMP and blaNDM-1-type carbapenemases, and class 1 integron element integrase gene) were performed. Clones were resolved by PCR-Randomly Amplified Polymorphic DNA.ResultsOut of 332 non-repetitive clinical specimens processed for culture and identification 160 (48.2%) were culture positive. Of which, 93 (58.1%) were E. coli. Of these, 24 (25.8%) were phenotypically confirmed as ESBL-EC and 3 (12.50%) of 24 ESBL-EC were carbapenemase producers. blaCTX-M-type ESBL was most common (23, 95.8%) followed by blaTEM (7, 29.2%) and blaSHV (3, 12.5%). blaVIM, blaIMP and blaNDM-1 were present in 3, 2 and 2 ESBL-EC, respectively. Class 1 integron element was present in 18 (75.0%) ESBL-EC. Nine isolates possessed more than one type of beta-lactamases. Interestingly, all carbapenemase producers were isolated form ICU and co-existence of blaCTX-M, blaSHV, blaTEM, blaIMP, blaVIM and blaNDM-1 beta-lactamases was documented in one ESBL-EC (EC104). All most all isolates had different RAPD patterns.ConclusionsFor the first time in Nepal, high prevalence of blaCTX-M-type ESBL and co-existence of ESBLs and carbapenemases has been described. Continuous monitoring and surveillance and proper infection control and prevention practices will limit the further spread of these super-bugs within this hospital and beyond.

Role of gyrB Mutations in Pre-extensively and Extensively Drug-Resistant Tuberculosis in Thai Clinical Isolates

ABSTRACT DNA gyrase mutations are a major cause of quinolone resistance in Mycobacterium tuberculosis. We therefore conducted the first comprehensive study to determine the diversity of gyrase mutations in pre-extensively drug-resistant (pre-XDR) (n = 71) and extensively drug-resistant (XDR) (n = 30) Thai clinical tuberculosis (TB) isolates. All pre-XDR-TB and XDR-TB isolates carried at least one mutation within the quinolone resistance-determining region of GyrA (G88A [1.1%], A90V [17.4%], S91P [1.1%], or D94A/G/H/N/V/Y [72.7%]) or GyrB (D533A [1.1%], N538D [1.1%], or E540D [2.2%]). MIC and DNA gyrase supercoiling inhibition assays were performed to determine the role of gyrase mutations in quinolone resistance. Compared to the MICs against M. tuberculosis H37Rv, the levels of resistance to all quinolones tested in the isolates that carried GyrA-D94G or GyrB-N538D (8- to 32-fold increase) were significantly higher than those in isolates bearing GyrA-D94A or GyrA-A90V (2- to 8-fold increase) (P < 0.01). Intriguingly, GyrB-E540D led to a dramatic resistance to later-generation quinolones, including moxifloxacin, gatifloxacin, and sparfloxacin (8- to 16-fold increases in MICs and 8.3- to 11.2-fold increases in 50% inhibitory concentrations [IC50s]). However, GyrB-E540D caused low-level resistance to early-generation quinolones, including ofloxacin, levofloxacin, and ciprofloxacin (2- to 4-fold increases in MICs and 1.5- to 2.0-fold increases in IC50s). In the present study, DC-159a was the most active antituberculosis agent and was little affected by the gyrase mutations described above. Our findings suggest that although they are rare, gyrB mutations have a notable role in quinolone resistance, which may provide clues to the molecular basis of estimating quinolone resistance levels for drug and dose selection.

Distribution and expression of the Ade multidrug efflux systems in Acinetobacter baumannii clinical isolates

One hundred Acinetobacter baumannii clinical isolates were examined for inhibitory effect of reserpine and carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the antimicrobial susceptibility and expression of 4 resistant-nodulation-cell division (RND)-type multidrug efflux systems, including AdeABC, AdeDE, AdeIJK, and AdeFGH, using RT-PCR. Ten A. baumannii isolates expressing AdeABC, AdeIJK, or AdeFGH were randomly selected for determination of transcription level and regulatory mutations. While all the isolates were resistant to multiple drugs, the reserpine and CCCP experiment showed that the multidrug resistance phenotype in most A. baumannii isolates was associated with efflux pumps. Most isolates expressed at least one of the RND-type efflux pumps tested (97%). AdeIJK expression was most common (97%), but none of the isolates produced AdeDE. Fifty-two percent of the A. baumannii isolates simultaneously produced up to 3 RND-type efflux systems (i.e., AdeABC, AdeFGH, and AdeIJK). No good correlation between the expression of RND-type efflux pumps and the type of antimicrobial resistance was observed. Overexpression of AdeABC, AdeIJK, and AdeFGH was not always related to the presence of mutations in their corresponding regulatory genes. This study highlights (i) the universal presence of the RND-type efflux pumps with variable levels of expression level among the A. baumannii in this collection and (ii) the complexity of their regulation of expression.

Prevalence and antimicrobial susceptibility of Haemophilus influenzae and Moraxella catarrhalis isolated from patients in Bangkok, Thailand

Haemophilus influenzae serotype b (Hib) is significant because it causes invasive diseases, e.g. meningitis, pneumonia and bacteraemia. Hib vaccine is available in Thailand, however it is not on the Expanded Program of Immunization in Thailand owing to its high cost. Hib vaccine has limited use in Thailand, mostly in private hospitals. There are eight biotypes of H. influenzae identified by biochemical tests [1]. Production of b-lactamase is the most common reason for b-lactam resistance. Two different types of b-lactamase (TEM-1 and ROB-1 types) have been described in H. influenzae, but TEM-1 is predominant (93.7%) globally [2]. The blaTEM-1 gene is usually located on large (40 kb) chromosomally integrated conjugative elements and less commonly on small plasmids (at least three distinct plasmids with sizes from 4304 bp to 5646 bp have been characterised) [3]. In addition to blactamase, another b-lactam resistance mechanism is mutation in the ftsI gene encoding penicillin-binding protein 3 (PBP3), leading to altered PBP that has low affinity for ampicillin. Isolates with this mechanism of resistance are referred to as b-lactamasenegative ampicillin-resistant (BLNAR). Moraxella catarrhalis is also an important pathogen. The aim of this study was to determine the prevalence of different biotypes and serotypes, b-lactamase production, b-lactamase genes, ftsI gene mutation in H. influenzae and the blaBRO gene in M. catarrhalis as well as antimicrobial susceptibility. A total of 638 H. influenzae and 297 M. catarrhalis non-duplicate isolates were collected from different patients at Siriraj Hospital (Bangkok, Thailand) during January 2007 to December 2011. For H. influenzae, 57.7% of patients were male and 42.3% were female (M:F sex ratio 1.36:1). For M. catarrhalis, 54.5% of patients were male and 45.5% were female (M:F sex ratio 1.20:1). The patient age distribution ranged from 3 months to 85 years. Clinical specimens of H. influenzae were from various sites (Table 1), but specimens of M. catarrhalis were from the respiratory tract and eye. Sputum was acceptable for culture if it contained >25 polymorphonuclear cells and <25 epithelial cells per low-power field. All isolates were either from infections or from colonisation because in some cases they were isolated with other bacteria. Biotypes were determined by indole, urease and ornithine decarboxylase tests. Serotypes b and non-b were determined by agglutination using latex particles coated with serotype b antiserum (Bio-Rad, Hercules, CA). b-Lactamase production was determined by nitrocefin test (Becton Dickinson, Cockeysville, MD). Study of the blaTEM and blaROB-1 genes in b-lactamaseproducing H. influenzae, the ftsI gene in BLNAR H. influenzae and