Aroonwadee Chanawong

Three Cefotaximases, CTX-M-9, CTX-M-13, and CTX-M-14, among Enterobacteriaceae in the People's Republic of China

ABSTRACT Of 15 extended-spectrum β-lactamase (ESBL)-producing isolates of the family Enterobacteriaceae collected from the First Municipal Peoples Hospital of Guangzhou, in the southern part of the Peoples Republic of China, 9 were found to produce CTX-M ESBLs, 3 produced SHV-12, and 3 produced both CTX-M and SHV-12. Eleven isolates produced either TEM-1B or SHV-11, in addition to an ESBL. Nucleotide sequence analysis of the 12 isolates carrying blaCTX-M genes revealed that they harbored three different blaCTX-M genes, blaCTX-M-9 (5 isolates), blaCTX-M-13 (1 isolate), and blaCTX-M-14 (6 isolates). These genes have 98% nucleotide homology with blaToho-2. The blaCTX-M genes were carried on plasmids that ranged in size from 35 to 150 kb. Plasmid fingerprints and pulsed-field gel electrophoresis showed the dissemination of the blaCTX-M genes through transfer of different antibiotic resistance plasmids to different bacteria, suggesting that these resistance determinants are highly mobile. Insertion sequence ISEcp1, found on the upstream region of these genes, may be involved in the translocation of the blaCTX-M genes. This is the first report of the occurrence of SHV-12 and CTX-M ESBLs in China. The presence of strains with these ESBLs shows both the evolution of blaCTX-M genes and their dissemination among at least three species of the family Enterobacteriaceae, Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae, isolated within a single hospital. The predominance of CTX-M type enzymes seen in this area of China appears to be similar to that seen in South America but is different from those seen in Europe and North America, suggesting different evolutionary routes and selective pressures. A more comprehensive survey of the ESBL types from China is urgently needed.

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.

Discrimination of SHV β-Lactamase Genes by Restriction Site Insertion-PCR

ABSTRACT Restriction site insertion-PCR (RSI-PCR) is a simple, rapid technique for detection of point mutations. This technique exploits primers with one to three base mismatches near the 3′ end to modulate a restriction site. We have developed this technique to identify described mutations of the blaSHV genes for differentiation of SHV variants that cannot be distinguished easily by other techniques. To validate this method, eight standard strains were used, each producing a different SHV β-lactamase: SHV-1, SHV-2, SHV-3, SHV-4, SHV-5, SHV-6, SHV-8, and SHV-18. Mismatch primers were designed to detect mutations affecting amino acids at positions 8 (SspI), 179 (HinfI), 205 (PstI), 238 (Gly→Ala) (BsrI), and 240 (NruI) ofblaSHV genes. All amplimers of theblaSHV genes used in this study yielded the predicted restriction endonuclease digestion products. In addition, this study also makes theoretical identification ofblaSHV-6, blaSHV-8, and 12 novel blaSHV variants using the PCR-restriction fragment length polymorphism (RFLP) technique possible. By using a combination of PCR-RFLP and RSI-PCR techniques, up to 27 SHV variants can now be distinguished rapidly and reliably. These simple techniques are readily applied to epidemiological studies of the SHV β-lactamases and may be extended to the characterisation of other resistance determinants.

CMY-2, CMY-8b, and DHA-1 plasmid-mediated AmpC β-lactamases among clinical isolates of Escherichia coli and Klebsiella pneumoniae from a university hospital, Thailand

Between February 2005 and January 2006 in Srinagarind Hospital, Thailand, 44 from 1730 isolates (2.5%) of Escherichia coli and 8 from 982 isolates (0.8%) of Klebsiella pneumoniae were found to produce plasmid-mediated AmpC β-lactamases (pAmpCs) as detected by a cefoxitin-Hodge test followed by a multiplex polymerase chain reaction (PCR) technique. Fifteen of the 52 pAmpC-producing isolates also produced extended-spectrum β-lactamases. The ampC genes found in both organisms were bla(CMY-2) (46 isolates), bla(CMY-8b) (4 isolates), and bla(DHA-1) (2 isolates). These genes were present on plasmids. Twenty-five of the 46 CMY-2-producing isolates could transfer cefoxitin resistance to E. coli UB1637 by conjugation. More than 90% of the pAmpC-producing isolates were resistant to cefoxitin, but 80% to 90% of them were susceptible or intermediately susceptible to ceftazidime or cefotaxime. Enterobacterial repetitive intergenic consensus PCR analysis revealed that most isolates were of different strains, indicating the ease of transmission of these resistance determinants. This is the first report of CMY-2, CMY-8b, and DHA-1 β-lactamases in Thai isolates.

ST9 MRSA strains carrying a variant of type IX SCCmec identified in the Thai community.

BackgroundInfections caused by methicillin-resistant Staphylococcus aureus (MRSA) in Thailand occur most frequently in healthcare facilities. However, reports of community-associated MRSA are limited.MethodsWe characterized 14 MRSA isolates from outpatients (O-1 to O-14) by phenotypic and genotypic methods and compared them with 5 isolates from inpatients (I-1 to I-5). Thai MRSA isolates from a healthcare worker (N-1) and a pig (P-1) were also included as ST9 MRSA strains from other sources.ResultsAll MRSA isolates from the outpatients and inpatients were multidrug-resistant (resistant to ≥3 classes of antimicrobials). All of them except strains O-2 and I-3 carried type III SCCmec and belonged to agrI, coagulase IV, spa type t037 or t233, which related to ST239. The strain O-2 (JCSC6690) carried type IX SCCmec and belonged to agrII, coagulaseXIc, spa type t337 and ST9, whereas the strain I-3 carried a type III SCCmec and belonged to ST1429. Nucleotide sequence determination revealed that the type IX SCCmec element in strain O-2 was distinct from that in a Thai ST398 strain (JCSC6943) previously identified in 2011; nucleotide identities of ccrA and ccrB were 93 and 91%, respectively and several open reading frames (ORFs) at the joining regions were different. PCR experiments suggested that strain O-2 and N-1 carried similar SCCmec element, whereas that of strain P-1 was different, suggesting that distinct ST9-MRSA–IX clones might be spreading in this province.ConclusionsThe SCCmecIX-ST9 MRSA clones of distinct SCCmec subtypes might have emerged in the Thai community and might also have disseminated into the hospital.

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.

Phenotypic Characteristics of Vancomycin-Non-Susceptible Staphylococcus aureus

Background: Staphylococcus aureus, with reduced vancomycin susceptibility, is probably under the regulation of several genes and various express phenotypes. Objectives: This study aimed to investigate the phenotypic differences between vancomycin-susceptible S. aureus (VSSA), vancomycin-intermediate S. aureus (VISA), and heterogeneous VISA (hVISA) isolates. Materials and Methods: A total of 130 methicillin-resistant S. aureus (MRSA) isolates were studied, including 49 VSSA, 28 hVISA, and 5 VISA isolates from blood cultures and 48 isolates (two VSSA, six hVISA, and 40 VISA) derived in vitro (laboratory-induced/sub-passaged). Their phenotypes were examined using a coagulase tube test, colony spreading on soft agar, and urease activity. The SCCmec and agr typing were performed using multiplex PCR. Results: Most of the MRSA isolates were SCCmec III-agr I (84.5%), followed by SCCmec II-agr II (11.8%). The average plasma coagulation time of vancomycin-non-susceptible isolates was longer than that of the susceptible isolates (12 vs. 2.6 hours). Four hVISA (P = 0.023) and nine VISA (P < 0.001) isolates yielded a negative coagulase test after 24-hour incubation. The percentage of VSSA isolates showing non-spreading colonies (accessory gene regulator (agr) dysfunction) was significantly lower than in the VISA group (P = 0.013), but no significant difference was found between VSSA and hVISA. The VISA group showed higher urease activity than that of the VSSA and hVISA groups (P = 0.002). Conclusions: There were diverse phenotypic changes among vancomycin-non-susceptible S. aureus isolates. This may be due to the variety of related regulatory systems. The diversity of phenotypic expression may result in its misidentification in routine laboratory checks.

Performance of vancomycin and teicoplanin disk diffusion test in isogenic vancomycin non-susceptible Staphylococcus aureus

INTRODUCTION Detection of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) is currently problematic. Although the population analysis profile with area under the curve (PAP-AUC) is the gold standard for detecting hVISA strains, this method is time consuming. This study aimed to induce vancomycin non-susceptible Staphylococcus aureus isolates in methicillin-resistant S. aureus (MRSA) and to determine the performance of the vancomycin and teicoplanin disk diffusion test for screening of induced and natural vancomycin non-susceptible isolates. METHODOLOGY Vancomycin resistance was induced in vitro in methicillin-resistant S. aureus by serial passage in media with increasing vancomycin concentrations. All test isolates were confirmed for their susceptibility to vancomycin by using a PAP-AUC method. The performance of the vancomycin and teicoplanin disk diffusion test for detecting both induced and natural hVISA/VISA isolates was analyzed using the MedCal program version 10.2.0. RESULTS The induction test revealed that 42 of 78 MRSA isolates (53.8%) became hVISA and/or VISA. Using 10, 15, 20, 30 µg vancomycin disks and a 30 µg teicoplanin disk, the highest performance (88.9%) for hVISA/VISA detection (71.1%), sensitivity, 100% specificity, 100% positive predictive value, and 75% negative predictive value) was obtained when a 20 µg vancomycin disk was used at 1.0 McFarland inoculum for a 24-hour incubation. CONCLUSIONS The results indicated that using a 20 µg vancomycin disk and bacterial inoculum of 1.0 McFarland is simple to perform and provides a primary result for hVISA/VISA screening within 24 hours.

A novel GoldNano Carb test for rapid phenotypic detection of carbapenemases, particularly OXA type, in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter spp.

Objectives: To develop a simple gold nanoparticle (AuNP)‐based colorimetric test, GoldNano Carb (GoldC), for detecting carbapenemase production in Gram‐negative bacteria, compared with updated Carba NP (CNP) and CarbAcineto NP (CAcNP) tests by using PCR methods as gold standard. Methods: Ninety‐nine carbapenemase‐producing Enterobacteriaceae (CPE), Pseudomonas spp. and Acinetobacter spp. isolates and 89 non‐CPE isolates were tested by the GoldC and CNP. Additionally, the CAcNP was performed in the Acinetobacter spp. isolates. The final imipenem (imipenem/cilastatin form) concentration was 5 mg/mL for all three tests. For the GoldC, the imipenem powder was added directly to bacterial cell suspension in distilled water prior to detection of acid product by the citrate‐capped AuNP solution. An AuNP change from red to purple, blue or green indicates carbapenemase activity. Results: The GoldC detected all carbapenemase producers except one OXA‐23‐like producer (99.0% sensitivity), whereas 11 carbapenemase producers (10 Acinetobacter and 1 P. aeruginosa) were CNP negative (88.9% sensitivity). However, the GoldC and CNP provided 100% and 98.6% sensitivity, respectively, for the CPE and Pseudomonas spp. Both tests gave one false positive from CTX‐M‐1‐like‐producing Enterobacter spp. (98.9% specificity). The GoldC and CAcNP detected 96.7% and 93.3% of the Acinetobacter spp. isolates, respectively. Interestingly, times to positivity by the GoldC were markedly shorter than those by the CNP (76.8% versus 36.2% positive at 5 min) and CAcNP (43.3% at 5 min versus 20% within 30 min). Conclusions: The GoldC is fast, easy, highly sensitive and inexpensive (˜

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

0.25 per test), suggesting that it may be suitable for routine carbapenemase detection in low‐resource settings for infection control or epidemiological purposes.