Pseudomonas alcaligenes NCIMB 9867 (strain P25X) is known to synthesize two isofunctional gentisate 1,2-dioxygenases (GDO; EC 1.13.11.4) as well as other enzymes involved in the degradation of xylenols and cresols via the gentisate pathway. The hbzE gene encoding what is possibly the strictly inducible gentisate 1,2-dioxygenase II (GDO-II) was cloned, overexpressed and purified as a hexahistidine fusion protein from Escherichia coli. Active recombinant GDO-II had an estimated molecular mass of 150kDa and is likely a tetrameric protein with a subunit mass of approximately 40kDa, similar to the previously characterized gentisate 1,2-dioxygenase I (GDO-I) encoded by xlnE. However, GDO-II was unable to utilize gentisate that is substituted at the carbon-4 position, unlike GDO-I which had broader substrate specificity. GDO-II also possessed different kinetic characteristics when compared to GDO-I. The hbzE-encoded GDO-II shared higher sequence identities (53%) with GDOs from Ralstonia sp. U2 and Polaromonas naphthalenivorans CJ2, compared with only 35% identity with the xlnE-encoded GDO-I. The hbzE gene was found to be part of a cluster of nine genes including the putative regulatory gene designated hbzR, which encodes an LysR-type regulator and is divergently transcribed from the other genes of the hbzHIJKLFED cluster.
Pseudomonas aeruginosa is the third most common pathogen causing nosocomial infections. The objective of this study was to investigate the antimicrobial resistance profiles and genetic diversity of hospital isolates of P. aeruginosa and to investigate the presence of several resistance genes and integrons.
The emergence of multidrug-resistant (MDR) and extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae poses a serious antibiotic management problem as resistance genes are easily transferred from one organism to another. Fifty-one strains of K. pneumoniae isolated from sporadic cases in various hospitals throughout Malaysia were analysed by antimicrobial susceptibility testing, PCR detection of ESBL-encoding genes and DNA fingerprinting. Although 27 of the 51 K. pneumoniae strains were MDR (i.e. resistant to three or more classes of antibiotics), the majority of the strains (98 %) were sensitive to imipenem. PCR detection using ESBL gene-specific primers showed that 46 of the K. pneumoniae strains harboured bla(SHV), 19 harboured bla(CTX-M), 5 harboured bla(OXA-1) and 4 harboured bla(TEM-1). Class 1 integron-encoded intI1 integrase was detected in 21 of the 51 K. pneumoniae strains and amplification of the integron 5'CS region showed the presence of several known antibiotic resistance gene cassettes of various sizes. Results of conjugation and transformation experiments indicated that some of the ESBL-encoding genes (i.e. bla(SHV), bla(CTX-M) and bla(TEM-1)) were transmissible and were likely plasmid-encoded. DNA fingerprinting using PFGE and PCR-based methods indicated that the 51 K. pneumoniae strains were genetically diverse and heterogeneous.
The emergence of Escherichia coli that produce extended spectrum beta-lactamases (ESBLs) and are multidrug resistant (MDR) poses antibiotic management problems. Forty-seven E. coli isolates from various public hospitals in Malaysia were studied. All isolates were sensitive to imipenem whereas 36 were MDR (resistant to 2 or more classes of antibiotics). PCR detection using gene-specific primers showed that 87.5% of the ESBL-producing E. coli harbored the blaTEM gene. Other ESBL-encoding genes detected were blaOXA, blaSHV, and blaCTX-M. Integron-encoded integrases were detected in 55.3% of isolates, with class 1 integron-encoded intI1 integrase being the majority. Amplification and sequence analysis of the 5'CS region of the integrons showed known antibiotic resistance-encoding gene cassettes of various sizes that were inserted within the respective integrons. Conjugation and transformation experiments indicated that some of the antibiotic resistance genes were likely plasmid-encoded and transmissible. All 47 isolates were subtyped by PFGE and PCR-based fingerprinting using random amplified polymorphic DNA (RAPD), repetitive extragenic palindromes (REPs), and enterobacterial repetitive intergenic consensus (ERIC). These isolates were very diverse and heterogeneous. PFGE, ERIC, and REP-PCR methods were more discriminative than RAPD in subtyping the E. coli isolates.
Type II (proteic) toxin-antitoxin systems (TAS) are ubiquitous among bacteria. In the chromosome of the pathogenic bacterium Streptococcus pneumoniae, there are at least eight putative TAS, one of them being the yefM-yoeB(Spn) operon studied here. Through footprinting analyses, we showed that purified YefM(Spn) antitoxin and the YefM-YoeB(Spn) TA protein complex bind to a palindrome sequence encompassing the -35 region of the main promoter (P(yefM2)) of the operon. Thus, the locus appeared to be negatively autoregulated with respect to P(yefM2), since YefM(Spn) behaved as a weak repressor with YoeB(Spn) as a corepressor. Interestingly, a BOX element, composed of a single copy (each) of the boxA and boxC subelements, was found upstream of promoter P(yefM2). BOX sequences are pneumococcal, perhaps mobile, genetic elements that have been associated with bacterial processes such as phase variation, virulence regulation, and genetic competence. In the yefM-yoeB(Spn) locus, the boxAC element provided an additional weak promoter, P(yefM1), upstream of P(yefM2) which was not regulated by the TA proteins. In addition, transcriptional fusions with a lacZ reporter gene showed that P(yefM1) was constitutive albeit weaker than P(yefM2). Intriguingly, the coupling of the boxAC element to P(yefM1) and yefM(Spn) in cis (but not in trans) led to transcriptional activation, indicating that the regulation of the yefM-yoeB(Spn) locus differs somewhat from that of other TA loci and may involve as yet unidentified elements. Conservation of the boxAC sequences in all available sequenced genomes of S. pneumoniae which contained the yefM-yoeB(Spn) locus suggested that its presence may provide a selective advantage to the bacterium.
Maslinic acid, a natural pentacyclic triterpene has been shown to inhibit growth and induce apoptosis in some tumour cell lines. We studied the molecular response of Raji cells towards maslinic acid treatment. A proteomics approach was employed to identify the target proteins. Seventeen differentially expressed proteins including those involved in DNA replication, microtubule filament assembly, nucleo-cytoplasmic trafficking, cell signaling, energy metabolism and cytoskeletal organization were identified by MALDI TOF-TOF MS. The down-regulation of stathmin, Ran GTPase activating protein-1 (RanBP1), and microtubule associated protein RP/EB family member 1 (EB1) were confirmed by Western blotting. The study of the effect of maslinic acid on Raji cell cycle regulation showed that it induced a G1 cell cycle arrest. The differential proteomic changes in maslinic acid-treated Raji cells demonstrated that it also inhibited expression of dUTPase and stathmin which are known to induce early S and G2 cell cycle arrests. The mechanism of maslinic acid-induced cell cycle arrest may be mediated by inhibiting cyclin D1 expression and enhancing the levels of cell cycle-dependent kinase (CDK) inhibitor p21 protein. Maslinic acid suppressed nuclear factor-kappa B (NF-κB) activity which is known to stimulate expression of anti-apoptotic and cell cycle regulatory gene products. These results suggest that maslinic acid affects multiple signaling molecules and inhibits fundamental pathways regulating cell growth and survival in Raji cells.
A cholera outbreak in Terengganu, Malaysia, in November 2009 was caused by 2 El Tor Vibrio cholerae variants resistant to typical antimicrobial drugs. Evidence of replacement of treatable V. cholerae infection in the region with antimicrobial-resistant strains calls for increased surveillance and prevention measures.
The emergence of Escherichia coli resistant to extended-spectrum cephalosporins (ESCs) is of concern as ESC is often used to treat infections by Gram-negative bacteria. One-hundred and ten E. coli strains isolated in 2009-2010 from children warded in a Malaysian tertiary hospital were analyzed for their antibiograms, carriage of extended-spectrum beta-lactamase (ESBL) and AmpC genes, possible inclusion of the beta-lactamase genes on an integron platform, and their genetic relatedness. All E. coli strains were sensitive to carbapenems. About 46% of strains were multidrug resistant (MDR; i.e., resistant to ≥3 antibiotic classes) and almost half (45%) were nonsusceptible to ESCs. Among the MDR strains, high resistance rates were observed for ampicillin (98%), tetracycline (75%), and trimethoprim/sulfamethoxazole (73%). Out of 110 strains, bla(TEM-1) (49.1%), bla(CTX-M) (11.8%), and bla(CMY-2) (6.4%) were detected. Twenty-one strains were ESBL producers. CTX-M-15 was the predominant CTX-M variant found and this is the first report of a CTX-M-27-producing E. coli strain from Malaysia. Majority (3.1%) of the strains harbored class 1 integron-encoded integrases with a predominance of aadA and dfr genes within the integron variable region. No gene cassette encoding ESBL genes was found and integrons were not significantly associated with ESBL or non-ESBL producers. Possible clonal expansion was observed for few CTX-M-15-positive strains but the O25-ST131 E. coli clone known to harbor CTX-M-15 was not detected while CMY-2-positive strains were genetically diverse.
Acinetobacter baumannii is a major cause of nosocomial infection worldwide. We report the draft genome sequence of A. baumannii AC12, a multidrug-resistant nosocomial strain with additional resistance to carbapenems and polymyxin. The genome data will provide insights into the genetic basis of antimicrobial resistance and its adaptive mechanism.
Escherichia coli is an important etiologic agent of lower respiratory tract infections (LRTI). Multidrug-resistant E. coli EC302/04 was isolated from a tracheal aspirate, and its genome sequence is expected to provide insights into antimicrobial resistance as well as adaptive and virulence mechanisms of E. coli involved in LRTI.
Staphylococcus aureus is a persistent human pathogen responsible for a variety of infections ranging from soft-tissue infections to bacteremia. The objective of this study was to determine genetic relatedness between methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains. We isolated 35 MRSA and 21 MSSA strains from sporadic cases at the main tertiary hospital in Terengganu, Malaysia, screening them for the presence of virulence genes. Their genetic relatedness was determined by accessory gene regulator (agr) types, PCR-restriction fragment length polymorphism (RFLP) of the coa gene, pulsed-field gel electrophoresis (PFGE), S. aureus protein A (spa), and multilocus-sequence typing (MLST). We found that 57% of MRSA and 43% of MSSA strains harbored enterotoxin genes. The majority (87.5%) of the strains were agr type I. PCR-RFLP and PFGE genotyping of the coa gene revealed that MRSA strains were genetically related, whereas MSSA strains had higher heterogeneity. The combined genotype, MLST-spa type ST239-t037, was shared among MRSA and MSSA strains, indicating that MRSA strains could have evolved from MSSA strains. Two combined MLST-spa types were present in MRSA strains, whereas 7 different MLST-spa types were detected in MSSA strains, including 2 combined types (ST779-t878 and ST1179-t267) that have not been reported in Malaysia. In conclusion, enterotoxin genes were more prevalent in MRSA than in MSSA strains in the Terengganu hospital. The MSSA strains were genetically more diverse than the MRSA strains.
Postweaning diarrhea caused by pathogenic Escherichia coli, in particular verotoxigenic E. coli (VTEC), has caused significant economic losses in the pig farming industry worldwide. However, there is limited information on VTEC in Malaysia. The objective of this study was to characterize pathogenic E. coli isolated from post-weaning piglets and growers with respect to their antibiograms, carriage of extended-spectrum beta-lactamases, pathotypes, production of hemolysins and fimbrial adhesins, serotypes, and genotypes.
This study aimed to investigate the survival of Vibrio cholerae O1 in 3 types of preparation for cooked rice, Oryza sativa L., (plain rice, rice with coconut milk, and rice with ginger); coffee, Coffea canephora, (plain coffee, coffee with sugar, and coffee with sweetened condensed milk); and tea, Camellia sinensis, (plain tea, tea with sugar, and tea with sweetened condensed milk) held at room temperature (27°C). The survival of V. cholerae O1 was determined by spread plate method on TCBS agar. Initial cultures of 8.00 log CFU/mL were inoculated into each food sample. After 6 h incubation, significant growth was only detected in rice with coconut milk (9.67 log CFU/mL; P < 0.05). However, all 3 types of rice preparation showed significant growth of V. cholerae after 24 h (P < 0.05). For coffee and tea preparations, V. cholerae survived up to 6 h in tea with condensed milk (4.72 log CFU/mL) but not in similar preparation of coffee. This study showed evidence for the survivability of V. cholerae in rice, coffee, and tea. Thus, holding these food and beverages for an extended period of time at room temperature should be avoided.
Bacterial toxin-antitoxin (TAs) loci usually consist of two genes organized as an operon, where their products are bound together and inert under normal conditions. However, under stressful circumstances the antitoxin, which is more labile, will be degraded more rapidly, thereby unleashing its cognate toxin to act on the cell. This, in turn, causes cell stasis or cell death, depending on the type of TAs and/or time of toxin exposure. Previously based on in silico analyses, we proposed that Streptococcus pneumoniae, a pathogenic Gram-positive bacterium, may harbor between 4 and 10 putative TA loci depending on the strains. Here we have chosen the pneumococcal strain Hungary(19A)-6 which contains all possible 10 TA loci. In addition to the three well-characterized operons, namely relBE2, yefM-yoeB, and pezAT, we show here the functionality of a fourth operon that encodes the pneumococcal equivalent of the phd-doc TA. Transcriptional fusions with gene encoding Green Fluorescent Protein showed that the promoter was slightly repressed by the Phd antitoxin, and exhibited almost background values when both Phd-Doc were expressed together. These findings demonstrate that phd-doc shows the negative self-regulatory features typical for an authentic TA. Further, we also show that the previously proposed TAs XreA-Ant and Bro-XreB, although they exhibit a genetic organization resembling those of typical TAs, did not appear to confer a functional behavior corresponding to bona fide TAs. In addition, we have also discovered new interesting bioinformatics results for the known pneumococcal TAs RelBE2 and PezAT. A global analysis of the four identified toxins-antitoxins in the pneumococcal genomes (PezAT, RelBE2, YefM-YoeB, and Phd-Doc) showed that RelBE2 and Phd-Doc are the most conserved ones. Further, there was good correlation among TA types, clonal complexes and sequence types in the 48 pneumococcal strains analyzed.
Nosocomial infection caused by Acinetobacter baumannii is of great concern due to its increasing resistance to most antimicrobials. In this study, 54 nonrepeat isolates of A. baumannii from the main tertiary hospital in Terengganu, Malaysia, were analyzed for their antibiograms and genotypes. Out of the 54 isolates, 39 (72.2%) were multidrug resistant (MDR) and resistant to carbapenems whereas 14 (25.9%) were categorized as extensive drug resistant (XDR) with additional resistance to polymyxin B, the drug of "last resort." Pulsed-field gel electrophoresis analyses showed that the polymyxin-resistant isolates were genetically diverse while the carbapenem-resistant isolates were clonally related. The 14 XDR isolates were further investigated for mutations in genes known to mediate polymyxin resistance, namely, pmrCAB, and the lipopolysaccharide biosynthesis genes, lpxA, lpxC, lpxD, and lpsB. All 14 isolates had a P102H mutation in pmrA with no mutation detected in pmrC and pmrB. No mutation was detected in lpxA but each polymyxin-resistant isolate had 2-4 amino acid substitutions in lpxD and 1-2 substitutions in lpxC. Eight resistant isolates also displayed a unique H181Y mutation in lpsB. The extent of polymyxin resistance is of concern and the novel mutations discovered here warrant further investigations.
Here, we report the draft genome sequence of a methicillin-resistant Staphylococcus aureus (MRSA) strain, KT/Y21, isolated from a blood sample of a pediatric patient. This strain belongs to sequence type 772 (ST772), harbors the staphylococcal cassette chromosome mec element (SCCmec) type V, and is positive for the Panton-Valentine leukocidin (PVL) pathogenic determinant.
Acinetobacter baumannii has emerged as an important nosocomial pathogen owing to its increasing resistance to most, if not all, antibiotics in clinical use. We recently reported the occurrence of extensively drug-resistant (XDR) A. baumannii isolates in a Malaysian tertiary hospital. The genome of one of these XDR isolates (A. baumannii AC12) was completely sequenced and comparative genome analyses were performed to elucidate the genetic basis of its antimicrobial resistance. The A. baumannii AC12 genome consists of a 3.8 Mbp circular chromosome and an 8731 bp cryptic plasmid, pAC12. It belongs to the ST195 lineage and is most closely related to A. baumannii BJAB0715 as well as other strains of the international clone III (IC-III) group. Two antibiotic resistance islands (RIs), designated AC12-RI1 and AC12-RI2, were found in the AC12 chromosome along with a 7 kb Tn1548::armA island conferring resistance to aminoglycosides and macrolides. The 22.8 kb AC12-RI1 interrupts the comM gene and harbours the carbapenem resistance gene blaOXA-23 flanked by ISAba1 within a Tn2006-like structure. AC12-RI1 also harbours resistance determinants for aminoglycosides, tetracyclines and sulphonamides. The 10.3 kb IS26-flanked AC12-RI2 is a derivative of AbGRI2-1, containing aphA1b and blaTEM genes (conferring aminoglycoside and β-lactam resistance, respectively). The presence of numerous genes mediating resistance to various antibiotics in novel RI structures as well as other genes encoding drug transporters and efflux pumps in A. baumannii AC12 most likely contributed to its XDR characteristics.
Acinetobacter baumannii is a Gram-negative nosocomial pathogen of importance due to its uncanny ability to acquire resistance to most antimicrobials. These include carbapenems, which are the drugs of choice for treating A. baumannii infections, and polymyxins, the drugs of last resort. Whole genome sequencing was performed on two clinical carbapenem-resistant A. baumannii AC29 and AC30 strains which had an indistinguishable ApaI pulsotype but different susceptibilities to polymyxin. Both genomes consisted of an approximately 3.8 Mbp circular chromosome each and several plasmids. AC29 (susceptible to polymyxin) and AC30 (resistant to polymyxin) belonged to the ST195 lineage and are phylogenetically clustered under the International Clone II (IC-II) group. An AbaR4-type resistance island (RI) interrupted the comM gene in the chromosomes of both strains and contained the bla OXA-23 carbapenemase gene and determinants for tetracycline and streptomycin resistance. AC29 harbored another copy of bla OXA-23 in a large (~74 kb) conjugative plasmid, pAC29b, but this gene was absent in a similar plasmid (pAC30c) found in AC30. A 7 kb Tn1548::armA RI which encodes determinants for aminoglycoside and macrolide resistance, is chromosomally-located in AC29 but found in a 16 kb plasmid in AC30, pAC30b. Analysis of known determinants for polymyxin resistance in AC30 showed mutations in the pmrA gene encoding the response regulator of the two-component pmrAB signal transduction system as well as in the lpxD, lpxC, and lpsB genes that encode enzymes involved in the biosynthesis of lipopolysaccharide (LPS). Experimental evidence indicated that impairment of LPS along with overexpression of pmrAB may have contributed to the development of polymyxin resistance in AC30. Cloning of a novel variant of the bla AmpC gene from AC29 and AC30, and its subsequent expression in E. coli also indicated its likely function as an extended-spectrum cephalosporinase.
Extraintestinal pathogenic Escherichia coli (ExPEC) that causes extraintestinal infections often harbor plasmids encoding fitness traits such as resistance and virulence determinants that are of clinical importance. We determined the complete nucleotide sequence of plasmid pEC302/04 from a multidrug-resistant E. coli EC302/04 which was isolated from the tracheal aspirate of a patient in Malaysia. In addition, we also performed comparative sequence analyses of 18 related IncFIIA plasmids to determine the phylogenetic relationship and diversity of these plasmids. The 140,232 bp pEC302/04 is a multireplicon plasmid that bears three replication systems (FII, FIA, and FIB) with subtype of F2:A1:B1. The plasmid is self-transmissible with a complete transfer region. pEC302/04 also carries antibiotic resistance genes such as bla TEM-1 and a class I integron containing sul1, cml and aadA resistance genes, conferring multidrug resistance (MDR) to its host, E. coli EC302/04. Besides, two iron acquisition systems (SitABCD and IutA-IucABCD) which are the conserved virulence determinants of ExPEC-colicin V or B and M (ColV/ColBM)-producing plasmids were identified in pEC302/04. Multiple toxin-antitoxin (TA)-based addiction systems (i.e., PemI/PemK, VagC/VagD, CcdA/CcdB, and Hok/Sok) and a plasmid partitioning system, ParAB, and PsiAB, which are important for plasmid maintenance were also found. Comparative plasmid analysis revealed only one conserved gene, the repA1 as the core genome, showing that there is an extensive diversity among the IncFIIA plasmids. The phylogenetic relationship of 18 IncF plasmids based on the core regions revealed that ColV/ColBM-plasmids and non-ColV/ColBM plasmids were separated into two distinct groups. These plasmids, which carry highly diverse genetic contents, are also mosaic in nature. The atypical combination of genetic materials, i.e., the MDR- and ColV/ColBM-plasmid-virulence encoding regions in a single ExPEC plasmid is rare but of clinical importance. Such phenomenon is bothersome when the plasmids are transmissible, facilitating the spread of virulence and resistance plasmids among pathogenic bacteria. Notably, certain TA systems are more commonly found in particular ExPEC plasmid types, indicating the possible relationships between certain TA systems and ExPEC pathogenesis.
Strains of Escherichia coli that are non-typeable by pulsed-field gel electrophoresis (PFGE) due to in-gel degradation can influence their molecular epidemiological data. The DNA degradation phenotype (Dnd(+)) is mediated by the dnd operon that encode enzymes catalyzing the phosphorothioation of DNA, rendering the modified DNA susceptible to oxidative cleavage during a PFGE run. In this study, a PCR assay was developed to detect the presence of the dnd operon in Dnd(+) E. coli strains and to improve their typeability. Investigations into the genetic environments of the dnd operon in various E. coli strains led to the discovery that the dnd operon is harboured in various diverse genomic islands.