Enterobacteriaceae isolated from clinical sources were examined for antibiotic resistance and the ability to transfer resistance to Escherichia coli. Twenty-nine out of 80 strains tested transferred part or all oftheir resistance genes. The strains carrying R plasmids included the genera Escherichia, Klebsiella, Salmonella, Enterobacter, Proteus, Providencia and Citrobacter. These results indicate that R plasmids possibly play a major role in the emergence of antibiotic resistance among clinical isolates of Enterobacteriaceae.
Forty samples of Malaysian cooked foods were examined for the presence of antibiotic-resistant coliforms and R plasmids. Twenty seven (68%) of the foods had antibiotic-resistant coliforms and 5 (13%) had R plasmids. Nineteen samples (48%) had total bacterial counts over 10(6) per gm and in 5 samples, no coliforms were detected. Our findings show that cooked food may be one possible way by which R plasmids are spread. The control of the spread of R plasmids is discussed.
Extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases (AmpCs)-producing Enterobacteriaceae have been increasingly reported and imposing significant threat to public. Livestock production industry might be the important source for clinically important ESBL-producing Enterobacteriaceae. This study aims to investigate the resistance profile, phenotypic ESBL production, beta-lactamase genes, virulence factors, and plasmid replicon types among 59 Enterobacteriaceae strains isolated from poultry faecal samples in Malaysia's commercial poultry farm. There were 38.7% and 32.3% of Escherichia coli resistant to cefotaxime and cefoxitin, respectively, while Klebsiellaspp. demonstrated resistance rate of 52.6% to both mentioned antimicrobials. Majority of the E. coli isolates carried blaTEM and blaCMY-2 group. blaSHV was the most prevalent gene detected in Klebsiellaspp., followed by blaDHA and blaTEM. Resistance to extended spectrum cephalosporin in our isolates was primarily mediated by plasmid mediated AmpC beta-lactamase such as CMY-2 group and DHA enzyme. The CTX-M genes were found in two ESBL-producing E. coli. IncF, IncI1, and IncN plasmids were most frequently detected in E. coli and Klebsiellaspp. The virulence factor, including EAST1 and pAA were identified at low frequency. This study highlights the poultry as a reservoir of resistance and virulence determinants and prevalence of plasmids in Enterobacteriaceae might drive their dissemination.
Kosakonia radicincitans (formerly known as Enterobacter radicincitans), an endophytic bacterium was isolated from the symptomatic tissues of bacterial wilt diseased banana (Musa spp.) plant in Malaysia. The total genome size of K. radicincitans UMEnt01/12 is 5 783 769 bp with 5463 coding sequences (CDS), 75 tRNAs, and 9 rRNAs. The annotated draft genome of the K. radicincitans UMEnt01/12 strain might shed light on its role as a bacterial wilt-associated bacterium.
Bacterial heart rot of pineapple reported in Hawaii in 2003 and reoccurring in 2006 was caused by an undetermined species of Dickeya. Classification of the bacterial strains isolated from infected pineapple to one of the recognized Dickeya species and their phylogenetic relationships with Dickeya were determined by a multilocus sequence analysis (MLSA), based on the partial gene sequences of dnaA, dnaJ, dnaX, gyrB and recN. Individual and concatenated gene phylogenies revealed that the strains form a clade with reference Dickeya sp. isolated from pineapple in Malaysia and are closely related to D. zeae; however, previous DNA-DNA reassociation values suggest that these strains do not meet the genomic threshold for consideration in D. zeae, and require further taxonomic analysis. An analysis of the markers used in this MLSA determined that recN was the best overall marker for resolution of species within Dickeya. Differential intraspecies resolution was observed with the other markers, suggesting that marker selection is important for defining relationships within a clade. Phylogenies produced with gene sequences from the sequenced genomes of strains D. dadantii Ech586, D. dadantii Ech703 and D. zeae Ech1591 did not place the sequenced strains with members of other well-characterized members of their respective species. The average nucleotide identity (ANI) and tetranucleotide frequencies determined for the sequenced strains corroborated the results of the MLSA that D. dadantii Ech586 and D. dadantii Ech703 should be reclassified as Dickeya zeae Ech586 and Dickeya paradisiaca Ech703, respectively, whereas D. zeae Ech1591 should be reclassified as Dickeya chrysanthemi Ech1591.
Twenty-five strains of enterobacteria, isolated from man in Peninsular Malaysia and consisting of seven Enterobacter spp., five Escherichia coli, five Salmonella spp., four Klebsiella spp., two Shigella spp., one Proteus sp. and and one Providencia sp., were tested for antibiotic resistance and conjugative R plasmids. They were all sensitive to nalidixic acid and resistant to at least three antibiotics. The number of resistances ranged from 3 to 11 antibiotics, including cefoperazone and sisomicin (two) newly released antibiotics), in addition to common drugs of current use. Of the 25 isolates, 19 (76%) conjugally transferred, at varied frequencies, at least two resistance determinants. Results from equilibrium density gradient centrifugation, agarose gel electrophoresis and transformation experiments provided proof that the transferable resistances were plasmid-mediated. Restriction endonuclease cleavage patterns showed that the plasmids from Proteus strain K005 and Providencia strain K001 may be identical.
The increased frequency of antibiotic resistance is known to be associated with the dissemination of integrons in the Enterobacteriaceae. This study determined the prevalence and type of integrons amongst 160 extended-spectrum beta-lactamase producing enterobacterial isolates kept in our culture collection. Integrons were detected in 98(61.3%) isolates, including 28(62.2%) Escherichia coli, 34(64.2%) Klebsiella spp., 27(61.4%), Enterobacter spp. and 9(50.0%) Citrobacter spp. investigated in this study. Restriction analysis of the integron gene fragments revealed that class I integron was the principal integron detected in 92(57.5%) of our isolates. Class II integron was detected in 6(3.8%) of our isolates, while no class III integron was detected in this study. The high rates of integron prevalence particularly of the class I integron in the E. coli and Klebsiella spp. concur with previous studies in other geographical regions. The higher (≥50%) integron prevalence of Citrobacter and Enterobacter isolates comparing to previous studies suggests the potential of these isolates as sources for dissemination of resistance determinants. The finding in this study serves as a basis for further study on the antibiotic resistance mechanisms of enterobacterial species in this teaching hospital.
Dickeya solani is an emerging pathogen that causes soft rot and blackleg diseases in several crops including Solanum tuberosum, but little is known about its genomic diversity and evolution.
Since multidrug resistant bacteria are frequently reported from Southeast Asia, our study focused on the occurrence of ESBL-producing Enterobacteriaceae in fresh imported herbs from Thailand, Vietnam and Malaysia. Samples were collected from fresh culinary herbs imported from Southeast Asia in which ESBL-suspected isolates were obtained by selective culturing. Analysis included identification by MALDI-TOF mass spectrometry, susceptibility testing, XbaI-PFGE, microarray, PCR and sequencing of specific ESBL genes, PCR based replicon typing (PBRT) of plasmids and Southern blot hybridization. In addition, the quinolone resistance genotype was characterized by screening for plasmid mediated quinolone resistance (PMQR) genes and mutations in the quinolone resistance determining region (QRDR) of gyrA and parC. The study encompassed fifty samples of ten batches of culinary herbs (5 samples per batch) comprising nine different herb variants. The herbs originated from Thailand (Water morning glory, Acacia and Betel leaf), Vietnam (Parsley, Asian pennywort, Houttuynia leaf and Mint) and Malaysia (Holy basil and Parsley). By selective culturing 21 cefotaxime resistant Enterobacteriaceae were retrieved. Array analysis revealed 18 isolates with ESBL genes and one isolate with solely non-ESBL beta-lactamase genes. Mutations in the ampC promoter region were determined in two isolates with PCR and sequencing. The isolates were identified as Klebsiella pneumoniae (n=9), Escherichia coli (n=6), Enterobacter cloacae complex (n=5) and Enterobacter spp. (n=1). All isolates tested were multidrug resistant. Variants of CTX-M enzymes were predominantly found followed by SHV enzymes. PMQR genes (including aac(6')-1b-cr, qnrB and qnrS) were also frequently detected. In almost all cases ESBL and quinolone resistance genes were located on the same plasmid. Imported fresh culinary herbs from Southeast Asia are a potential source for contamination of food with multidrug resistant bacteria. Because these herbs are consumed without appropriate heating, transfer to human bacteria cannot be excluded.
A minisequencing method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) was developed for rapid identification of single nucleotide polymorphisms at bla(TEM) gene codons 104, 164 and 238 associated with extended-spectrum activity on TEM-type beta-lactamases. The method was validated by testing the Escherichia coli and Klebsiella pneumoniae strains possessing the known bla(TEM) gene sequences.
Phylogenetic and taxonomic characterization was performed for bacterium RB-25T, which was isolated from a soil sample collected in a former municipal landfill site in Puchong, Malaysia. Growth occurred at 20-37 °C at pH 5-8 but not in the presence of 9 % (w/v) NaCl or higher. The principal fatty acids were C16:0, C18:1ω7c and summed feature 3 (C16:1ω7c and/or iso-C15:0 2-OH). Ubiquinone-8 was the only isoprenoid quinone detected. Polar lipid analysis revealed the presence of phospholipid, phosphoaminolipid, phosphatidylethanolamine, phosphatidylglycerol and one unidentified aminolipid. DNA G+C content was 50.9 mol% phylogenetic analysis based on 16S rRNA gene sequence showed that strain RB-25T formed a distinct lineage within the family Enterobacteriaceae of the class Gammaproteobacteria. It exhibited a low level of 16S rRNA gene sequence similarity with its phylogenetic neighbours Pantoea rwandensis LMG 26275T (96.6 %), Rahnella aquatilis CIP 78.65T (96.5 %), Pectobacterium betavasculorum ATCC 43762T (96.4 %), Pantoea rodasii LMG 26273T (96.3 %), Gibbsiella dentisursi NUM 1720T (96.3 %) and Serratia glossinae C1T (96.2 %). Multilocus sequence analyses based on fusA, pyrG, rplB, rpoB and sucA sequences showed a clear distinction of strain RB-25T from the most closely related genera. Isolate RB-25T could also be distinguished from members of these genera by a combination of the DNA G+C content, respiratory quinone system, fatty acid profile, polar lipid composition and other phenotypic features. Strain RB-25T represents a novel species of a new genus, for which the name Chaniamultitudinisentens gen. nov., sp. nov. is proposed. The type strain is RB-25T (=DSM 28811T=LMG 28304T).
This study investigated 147 multidrug-resistant Enterobacteriaceae and Pseudomonas aeruginosa isolates from hospitalized patients in Malaysia. Class 1 integrons were the most dominant class identified (45.6%). Three isolates were shown to contain class 2 integrons (2.0%), whilst one isolate harboured both class 1 and 2 integrons. No class 3 integrons were detected in this study. In addition, the sul1 gene was amplified in 35% of isolates and was significantly associated with the presence of integrase genes in an integron structure. RFLP and DNA sequencing analyses revealed the presence of 19 different cassette arrays among the detected integrons. The most common gene cassettes were those encoding resistance towards aminoglycosides (aad) and trimethoprim (dfr). As far as is known, this study is the first to identify integron-carrying cassette arrays such as aadA2-linF, aacC3-cmlA5 and aacA4-catB8-aadA1 in the Malaysian population. Patients' age was demonstrated as a significant risk factor for the acquisition of integrons (P=0.028). Epidemiological typing using PFGE also demonstrated a clonal relationship among isolates carrying identical gene cassettes in Klebsiella pneumoniae and P. aeruginosa but not in Escherichia coli isolates.
The antibiotic, trimethoprim-sulfamethoxazole (TMP-SMX), is generally used for prophylaxis in HIV individuals to protect them from Pneumocystis jiroveci infection. Long-term use of TMP-SMX develops drug resistance among bacteria in HIV patients. The study was aimed to detect the TMP-SMX resistance genes among gram-negative bacteria from HIV patients. TMP-SMX-resistant isolates were detected by the Kirby-Bauer disc diffusion method. While TMP resistance genes such as dfrA1, dfrA5, dfrA7, and dfrA17 and SMX resistance genes such as sul1 and sul2 were detected by multiplex PCR, class 1 and class 2 integrons were detected by standard monoplex PCR. Of the 151 TMP-SMX-resistant bacterial isolates, 3 were positive for sul1 alone, 48 for sul2 alone, 11 for dfrA7 alone, 21 for sul1 and sul2, 1 for sul1 and dfrA7, 23 for sul2 and dfrA7, 2 for sul2 and dfrA5, 41 for sul1, sul2, and dfrA7, and 1 for sul2, dfrA5, and dfrA7. Of 60 TMP-SMX-resistant isolates positive for integrons, 44 had class 1 and 16 had class 2 integrons. It was found that the prevalence of sul genes (n = 202; p