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Carbapenem-resistant Enterobacteriaceae isolates by carbapenemase production are being reported globally with increasing frequency, leading to limited therapeutic options. We therefore developed a loop-mediated isothermal amplification method with hydroxynaphthol blue dye (LAMP-HNB) for rapid confirmation of bla , bla , bla , bla and bla groups. Sixty-two Enterobacteriaceae and Pseudomonas spp. isolates carrying various carbapenemase genes (28 bla , 9 bla , 2 bla , 1 bla , 1 bla , 1 bla , 1 bla , 4 bla , 1 bla , 1 bla & bla , 7 bla , 3 bla and 3 bla ) and 37 non-carbapenemase-producing Enterobacteriaceae isolates as confirmed by the PCR methods were included. Bacterial DNA was extracted by a simple boiling method. The LAMP-HNB method for each target gene was carried out using a set of six primers under isothermal condition at 65 °C in an ordinary water bath within 60 min and visual measurement of reaction by the change from violet to sky blue. This method had high efficiency (100% sensitivity and specificity) for identifying the bla , bla , bla , bla and bla groups compared with the PCR method. The HNB is easy to prepare, inexpensive and provides reliable results. Therefore, this method could be used as a confirmatory carbapenemase test in routine laboratory or for epidemiological purposes. [ABSTRACT FROM AUTHOR]

Sixty-one extended-spectrum beta-lactamase (ESBL)-producing isolates were collected from Srinagarind Hospital, Thailand. These included 43 Enterobacteriaceae and 18 Pseudomonadaceae. The 43 Enterobacteriaceae were found to produce the following ESBLs: 26 (60.5%) SHV-12, 13 (30.2%) SHV-5, two (4.7%) SHV-2a, one (2.3%) VEB-1 and one (2.3%) unidentified. Twenty-four isolates (55.8%) also carried bla(TEM-1B), as well as bla(SHV) or bla(VEB-1). Plasmid DNA from transconjugants carrying the bla(SHV-12) gene showed various restriction patterns, indicating the distribution of the bla(SHV-12) gene among different antibiotic resistance plasmids. In contrast, bla(SHV-5) in 13 isolates was found on a single plasmid of c. 130 kb. Pulsed-field gel electrophoresis (PFGE) analysis of genomic DNA from these isolates revealed that nine of 11 Klebsiella pneumoniae gave the same pattern, indicating clonal spread of the strain within the hospital, together with the occasional spread of the plasmid to other strains. Among the pseudomonad isolates, 16 Pseudomonas aeruginosa and one Pseudomonas putida had bla(VEB-like) and one P. aeruginosa had bla(SHV-12). Nine of the 16 isolates carrying bla(VEB-like) (56.3%) had identical PFGE patterns, suggesting the dissemination of this gene, also by clonal spread. At least six different bla(VEB-like-)containing integrons were found among the 18 isolates. This is the first report of bacteria producing SHV-12 and SHV-2a in Thailand and the first report of SHV-12 in P. aeruginosa, of VEB-1 in Citrobacter freundii and a VEB-1-like beta-lactamase in P. putida. These findings indicate that ESBL genes in the Far East are part of a gene pool capable of broad horizontal gene transfer, in that these genes can transfer between different families of Gram-negative bacilli. [ABSTRACT FROM AUTHOR]

A technically simple method—the MAST double disc (MDD) test—for the detection of extended-spectrum β-lactamase (ESBL) production by bacteria is described. A wide range of ESBL, non-ESBL and Class 1 β-lactamase-producing isolates was examined. The MDD test, which uses discs containing ceftazidime and a complementary disc containing ceftazidime and clavulanate and a second pair containing cefotaxime and cefotaxime and clavulanate was compared with the standard double disc diffusion test and an Etest method. Both the Etest and the MDD correctly identified 93% of ESBL producers. The MDD is an inexpensive alternative to current methods for the detection of ESBL production. [ABSTRACT FROM PUBLISHER]

Vancomycin is widely used for treatment of infection caused by methicillin-resistant Staphylococcus aureus (MRSA) leading to an increasing appearance of low-level vancomycin-resistant isolates called heterogeneous vancomycin-intermediate S. aureus (hVISA). The mechanism of vancomycin tolerance in hVISA is still unclear. This study aimed to investigate the fatty acid compositions of S. aureus isolates under the stress environment with vancomycin. The different responses of hVISA and vancomycin-susceptible S. aureus (VSSA) may lead to more understanding the mechanism. The bacterial lipid profiles were tested three times from three extractions of each isolate cultured on tryptic soy agar (TSA) and TSA with vancomycin. Of the 30 MRSA isolates studied, 13, 12, and 5 isolates were VSSA, hVISA, and VISA, respectively. The analysis of bacterial lipid profiles showed that under vancomycin stress, there was a reduction of straight chain fatty acids (SCFAs) in VSSA isolates but an increase in branched chain fatty acids (BCFAs). In contrast, the hVISA group exhibited an increase only in the BCFAs but not in SCFAs. Of interest, vancomycin had no effect on either BCFAs or SCFAs of the VISA cells. This study provided information of bacterial adaptation during stress with vancomycin that may be helpful to overcome the resistant bacteria. [ABSTRACT FROM AUTHOR]

Methicillin-resistant Staphylococcus aureus (MRSA) has spread worldwide. It is a major cause of hospital-acquired infections in most hospitals for nearly half century. The present study was conducted to examine the antimicrobial susceptibilities and staphylococcal cassette chromosome mec (SCCmec)-type for MRSA isolates from 237 patients treated at Srinagarind Hospital between September 2002 and August 2003. Antimicrobial susceptibility testing for all isolates was performed using an agar dilution method and SCCmec-types of 81 representatives from 237 isolates were determined using multiplex PCR. The minimum inhibitory concentration (MIC) ranges for the MRSA isolates were as follows: cefazolin 8 to > or =64; erythromycin < or = 0.5 to > or =64; gentamicin < or = 0.5 to > or =64; imipenem < or = 0.5 to >16; ofloxacin < or = 0.5 to > or =64; oxacillin 16 to > or =64; tetracycline 2 to > or =64 and vancomycin < or = 0.5 to 2 microg/ml. All MRSA isolates were susceptible to vancomycin, but only 0.4% to 8.9% was susceptible to the remaining antimicrobial agents. Of the 81 isolates tested, 2 types of SCCmec were found (76 with type III and 2 with type II) and no mecA gene was detected in 3 isolates. Sixty-seven of the 78 isolates carried the mercury-resistant operon. The multilocus sequence type in isolates with type III SCCmec was ST239 and in isolates with type II SCCmec was ST5.

This study presents updates on molecular epidemiology of extended-spectrum beta-lactamases (ESBLs) in clinical isolates of Enterobacteriaceae from Srinagarind Hospital, Khon Kaen University, Thailand. All isolates were screened for the presence of ESBL genes, bla(TEM), bla(SHV), bla(VEB) and bla(CTX-M), using PCR followed by nucleotide sequence determination. The results revealed that beta-lactamase genes among 48 isolates collected between 1998 and 1999 were bla(SHV) (79%), bla(CTX-M-9) (52%), bla(TEM-1) (48%) and bla(VEB) (33%), whereas those found in 52 isolates collected in 2003 were bla(TEM-1) (79%), bla(CTX-M-15) (44%), bla(SHV) (36%), bla(VEB) (36%), bla(CTX -M-14) (11%) and bla(CTX-M-9) (10%). In addition, 45 isolates carried at least two different ESBL genes. Using PCR, part of insertion sequence ISEcpl was found in the upstream regions of bla(CTX-M-14) and bla(CTX-M-15). ERIC-PCR analysis revealed that most ESBL-producing isolates were of different strains. This is the first report of CTX-M-9, CTX-M-14 and CTX-M-15 beta-lactamase genes in Enterobacteriaceae in Thailand.

Heterogeneous, intermediate-vancomycin-resistant Staphylococcus aureus (hVISA) represents a threat of an incurable infection since the first report in 1997. The method used to detect hVISA isolates is a population analysis profile (PAP); however, it is impractical for routine laboratory analysis. We therefore tested a simple, reliable and inexpensive method for the detection of hVISA. Eighteen isolates of hVISA and 22 of vancomycin-sensitive S. aureus (VSSA) were included. The organisms were tested by the disk diffusion method, using 15-microg vancomycin disks on four different media: Mueller-Hinton agar (MHA), MHA plus 2% NaCI (MHAS), Brain Heart Infusion agar (BHA), and BHA plus 2% NaCl (BHAS). In addition, two different inoculum sizes, bacterial suspensions adjusted to 0.5 and 2.0 McFarland, were tested. The inhibition zone was read independently by three medical technologists after incubation at 37 degrees C for 24 and 48 hours. The use of MHAS with an inoculum size of 2.0 McFarland and 48-hour incubation period yielded the highest sensitivity (94.4%), specificity (81.8%), positive predictive value (80.9%), and negative predictive value (94.7%). The disk diffusion test with 15-microg vancomycin disk is simple and may be used as a screening method for the detection of hVISA.