Characterization of CTX-M-140, a Variant of CTX-M-14 Extended-Spectrum ß-Lactamase with Decreased Cephalosporin Hydrolytic Activity, from Cephalosporin-Resistant Proteus mirabilis.

CTX-M-140, a novel CTX-M-type extended-spectrum ß-lactamase (ESBL), was identified in cephalosporin-resistant clinical isolates of Proteus mirabilis CTX-M-140 contained an alanine-to-threonine substitution at position 109 compared to its putative progenitor, CTX-M-14. When it was expressed in an Escherichia coli isogenic background, CTX-M-140 conferred 4- to 32-fold lower MICs of cephalosporins than those with CTX-M-14, indicating that the phenotype was attributable to this single substitution. For four mutants of CTX-M-14 that were constructed by site-directed mutagenesis (A109E, A109D, A109K, and A109R mutants), MICs of cephalosporins were similar to those for the E. coli host strain, which suggested that the alanine at position 109 was essential for cephalosporin hydrolysis. The kinetic properties of native CTX-M-14 and CTX-M-140 were consistent with the MICs for the E. coli clones. Compared with that of CTX-M-14, a lower hydrolytic activity against cephalosporins was observed for CTX-M-140. blaCTX-M-140 is located on the chromosome as determined by I-CeuI pulsed-field gel electrophoresis (I-CeuI-PFGE) and Southern hybridization. The genetic environment surrounding blaCTX-M-140 is identical to the sequence found in different plasmids with blaCTX-M-9-group genes among the Enterobacteriaceae Genome sequencing and analysis showed that P. mirabilis strains with blaCTX-M-140 have a genome size of ∼4 Mbp, with a GC content of 38.7% and 23 putative antibiotic resistance genes. Our results indicate that alanine at position 109 is critical for the hydrolytic activity of CTX-M-14 against oxyimino-cephalosporins.

NDM-1-Producing Citrobacter freundii, Escherichia coli, and Acinetobacter baumannii Identified from a Single Patient in China.

We identified New Delhi metallo-ß-lactamase (NDM-1)-producing Citrobacter freundii GB032, Escherichia coli GB102, and Acinetobacter baumannii GB661 in urine and stool samples from a single patient in China. Plasmid profiling and Southern blotting indicated that blaNDM-1 from GB032 and that from GB102 were likely located on the same plasmid, while blaNDM-1 from GB661 was located on a very large (>400-kb) plasmid. This case underscores the broad host range of blaNDM-1 and its potential to spread between members of the family Enterobacteriaceae and A. baumannii.

CTX-M-137, a hybrid of CTX-M-14-like and CTX-M-15-like ß-lactamases identified in an Escherichia coli clinical isolate.

OBJECTIVES: To characterize a novel CTX-M chimera, CTX-M-137, from Escherichia coli clinical isolates in China. METHODS: Isolates were collected from five hospitals between 22 February 2009 and 20 December 2011. Resistance genes were investigated by PCR. blaCTX-M-137 was cloned and purified for kinetic measurements. Conjugation experiments, S1-PFGE and Southern blotting were performed to study the plasmid harbouring blaCTX-M-137. The genetic environment of blaCTX-M-137 was determined by genomic cloning and sequencing. RESULTS: A total of 247 cephalosporin-resistant E. coli were identified. blaCTX-M group genes were the most prevalent extended-spectrum ß-lactamase (ESBL) genes, with 71 isolates harbouring blaCTX-M-1 group genes and 137 isolates harbouring blaCTX-M-9 group genes. A novel chimera of CTX-M-14-like and CTX-M-15-like ESBLs, designated CTX-M-137, was identified from a 60-year-old man with a urinary tract infection. The N-terminus of CTX-M-137 matched CTX-M-14 and the C-terminus matched CTX-M-15. CTX-M-137 conferred resistance to ceftazidime, cefotaxime and aztreonam. Purified CTX-M-137 showed good hydrolytic activity against ceftazidime and cefotaxime, and was inhibited by clavulanic acid. The blaCTX-M-137 was carried on an ∼83 kb IncI1 plasmid. blaCTX-M-137 was carried on a complete transposition unit ISEcp1-blaCTX-M-137-Δorf477 inserted into yagA, which is part of the IncI1 plasmid backbone. CONCLUSIONS: We identified a novel CTX-M chimera, CTX-M-137, with a CTX-M-14-like N-terminus and a CTX-M-15-like C-terminus. Our findings suggest an ongoing diversification of CTX-M-type ESBLs through recombination events.

Outbreak of PER-1 and diversity of ß-lactamases among ceftazidime-resistant Pseudomonas aeruginosa clinical isolates.

A growing number of ß-lactamases have been reported in Pseudomonas aeruginosa clinical isolates. The aim of this study was to investigate the diversity of ß-lactamases in the collection of 51 ceftazidime-resistant P. aeruginosa clinical isolates in four hospitals of southern China. Among these isolates, variable degrees of resistance to other ß-lactam and non-ß-lactam agents were observed. Pulsed-field gel electrophoresis (PFGE) revealed a high degree of clonality with five main genotypes. Of the 51 isolates tested, 35 (68.6%) were identified as extended-spectrum ß-lactamase (ESBL) producers, with 35 producing PER-1, 1 CTX-M-3, 7 CTX-M-15 and 1 CTX-M-14. Most (82.9%, 29/35) PER-1-producing isolates were collected from two hospitals between January and April in 2008 and belonged to the same PFGE pattern (pattern B) with similar antibiogram and ß-lactamase profiles, which suggested an outbreak of this clone at the time. The prevalence of CTX-M-type ESBL (17.6%, 9/51) was unexpectedly high. One isolate was identified as producing VIM-2. Furthermore, we also reported an occurrence of a novel OXA-10 variant, OXA-246, in 14 P. aeruginosa isolates. In addition, AmpC overproduction was found to be the ß-lactamase-mediated mechanism responsible for ceftazidime resistance in 6 isolates (11.8%). Our results revealed an overall diversity of ß-lactamases and outbreak of a PER-1-producing clone among ceftazidime-resistant P. aeruginosa in southern China.

OprD mutations and inactivation in imipenem-resistant Pseudomonas aeruginosa isolates from China.

To investigate the mechanisms involved in imipenem resistance of Pseudomonas aeruginosa in southern China, 61 imipenem-resistant P. aeruginosa clinical isolates were collected from 4 hospitals between October 2011 and June 2012. All isolates were resistant to imipenem, whereas 21.3% were susceptible or intermediate to meropenem. Variable degrees of resistance to other ß-lactam and non-ß-lactam antimicrobials were observed. PFGE revealed high-level of clonal diversity. Among the 61 isolates, 50 isolates had OprD loss by disrupted oprD mutations, including 43 with frameshift mutations of oprD and 7 with a premature stop codon by single point mutation. Six isolates were oprD-negative by PCR, suggestive of a major disruption of oprD genes. Five isolates had intact oprD but had reduced expression of oprD genes. In addition, only one isolate with disrupted oprD mutation by a premature stop codon was confirmed to be a metallo-ß-lactamase producer (IMP-9). Our results show that the loss of OprD, as well as reduced expression of oprD and MBL production, were the predominant mechanisms of imipenem resistance in P. aeruginosa in southern China.

Pre- and post-exposure protection against Western equine encephalitis virus after single inoculation with adenovirus vector expressing interferon alpha.

Western equine encephalitis virus (WEEV) is a positive-sense, single-stranded RNA virus which is transmitted to equines and humans through mosquito bites. WEEV infects the central nervous system with severe complications and even death. There are no human vaccine and antiviral drugs. We investigated whether adenovirus-mediated expression of interferon alpha could be used for pre- and post-exposure protection against a lethal WEEV challenge in mice. A human adenoviral vector (Ad5-mIFNalpha) expressing mouse interferon alpha was constructed. We found that Ad5-mIFNalpha provided 100% protection against various WEEV strains in mice after a single intramuscular inoculation at 24 h, 48 h or 1 week before the challenge. When given as a single inoculation at 6 h after the challenge, Ad5-mIFNalpha delayed the progress of WEEV infection and provided about 60% protection. Our findings suggest that adenovirus-mediated expression of interferon alpha can be an alternative approach for the prevention and treatment of WEEV infection.