Lumyai Wonglakorn

Emergence of NDM-1- and IMP-14a-producing Enterobacteriaceae in Thailand

OBJECTIVES To detect carbapenemases in clinical isolates of Enterobacteriaceae collected from patients in a university hospital in Thailand between October 2010 and August 2011. METHODS A total of 4818 Enterobacteriaceae isolates were screened for the presence of carbapenemases by ertapenem and imipenem disc diffusion tests. All positive screening isolates were subjected to modified Hodge test, phenylboronic acid- and EDTA-carbapenem combined disc tests and two multiplex PCRs of bla(IMP), bla(VIM), bla(SPM), bla(SIM) and bla(GIM), and of bla(KPC), bla(NDM) and bla(OXA-48). Carbapenemase-producing isolates were typed by PFGE and then characterized by antimicrobial susceptibility tests. Conjugation was performed using a broth culture mating method. RESULTS Two isolates each of Escherichia coli, Klebsiella pneumoniae and Citrobacter freundii produced NDM-1, whereas two other isolates of K. pneumoniae produced IMP-14a. DNA fingerprints revealed that the metallo-β-lactamase (MBL)-producing isolates were of different strains except for clonal strains of C. freundii. In vitro transfer of carbapenem resistance was successful for the eight MBL-producing isolates. All MBL producers were susceptible to colistin and tigecycline. The six NDM-producing isolates were recovered from the urine of three patients, who had no history of travel outside Thailand. Interestingly, one patient had chronic urinary tract infections caused by a K. pneumoniae strain and two strains of E. coli producing NDM-1. CONCLUSIONS Surveillance of carbapenemases, particularly NDM-1, in Enterobacteriaceae is urgently needed to control and prevent the spread of these resistance determinants in our country.

SCCmec IX in meticillin-resistant Staphylococcus aureus and meticillin-resistant coagulase-negative staphylococci from pigs and workers at pig farms in Khon Kaen, Thailand

Livestock-associated meticillin-resistant Staphylococcus aureus, clonal complex (CC) 398, has been reported in Europe, whereas CC9 MRSA has mostly been found in Asia. Therefore, we aimed to detect MRSA on pig farms in north-eastern Thailand. A total of 257 nasal swabs (159 samples from pigs and 98 from pig-farm workers) were collected from three pig farms in north-eastern Thailand from 2010 to 2011. MRSA isolates were confirmed for femA and mecA genes by PCR. The MICs of eight antimicrobials, namely vancomycin (VA), cefazolin (CZ), ofloxacin (OF), tetracycline (TET), erythromycin (ER), oxacillin (OX), cefoxitin (FOX) and gentamicin (GN), were tested by agar dilution method. The virulence genes for Panton-Valentine leukocidin toxin (lukSF-PV), toxic shock syndrome toxin-1 (tst) and α-haemolysin (hla) were detected by PCR. Strain typing was performed by staphylococcal cassette chromosome (SCC) mec, agr, spa and multilocus sequence typing. Four MRSA were isolated: three from workers and one from a pig. All the MRSA isolates were resistant to OX, GN, ER, TET and CZ, and they all carried hla only. Two MRSA from humans carried SCCmec II-sequence type (ST)764-agrII, whereas the two remaining MRSA (one each from a human and a pig) contained SCCmec IX-ST9-agrII. Interestingly, meticillin-resistant coagulase-negative Staphylococcus isolates carrying SCCmec IX were also obtained from five workers and three pigs. This study suggests that the SCCmec IX element is distributed among the Staphylococcus found in pigs and pig-farm workers, and pigs may be a reservoir for MRSA in the community.

Molecular Characterization of Carbapenemase-Nonproducing Clinical Isolates of Escherichia coli (from a Thai University Hospital) with Reduced Carbapenem Susceptibility

Twelve nonreplicate carbapenemase-negative ertapenem (ETP)-nonsusceptible (CNENS) Escherichia coli isolates obtained at a Thai university hospital between 2010 and 2014 were characterized and compared with 2 carbapenemase-producing E. coli isolates from the same hospital. Eight unique pulsed-field gel electrophoresis patterns were obtained. All the isolates produced CTX-M-15 β-lactamase and 2 either coexpressed CMY-2 cephalosporinase or showed increased efflux pump activity. Amino acid sequence analysis revealed that an OmpF defect (in 7 isolates) due to mutations generating truncated proteins or an IS1 insertion was more prevalent than a defect in OmpC was (no truncated proteins detected). Seven out of 10 isolates possessing OmpC variants with any OmpF defect were weakly ETP-resistant (minimum inhibitory concentrations [MICs] of 1-4 μg/mL) and imipenem (IPM)- and meropenem (MEM)-susceptible (MICs 0.125-0.5 μg/mL). Two isolates with ompC PCR-negative results and an OmpF defect showed higher carbapenem MICs (8-32, 1-8, and 1-4 μg/mL for ETP, IPM, and MEM, respectively) with the highest MICs associated with the additional efflux pump activity. Both carbapenemase producers possessing CTX-M-15 and a porin background identical to that in the CNENS isolates showed ETP, IPM, and MEM MICs of 128-256, 8, and 2-32 μg/mL, respectively. These findings suggest that a porin defect combined with CTX-M-15 production is the major mechanism of low carbapenem susceptibility among our CNENS isolates, which have potential to become strongly carbapenem-resistant because of additional carbapenemase or efflux pump activities.