As part of the SENTRY Antimicrobial Surveillance Program, a total of 1078 Acinetobacter species and 842 Stenotrophomonas maltophilia isolates were co

To comparatively evaluate the antimicrobial activities of colistin and polymyxin B with those of other antimicrobials against a worldwide collection of 40 625 Gram-negative bacilli. Antimicrobial susceptibility testing was performed and interpreted using the CLSI broth microdilution method except for colistin against Enterobacteriaceae. The polymyxins showed potent in vitro activities (MIC₉₀, ≤0.5–1 mg/L) against this large collection of clinical isolates, with very low resistance rates (<0.1%–1.5%). Resistance to the polymyxins remained stable among organisms tested except for Klebsiella spp. isolates collected from the Asia-Pacific and Latin American regions, where a trend towards greater resistance was observed (P ≤ 0.05). In addition, an important reduction in imipenem susceptibility among Acinetobacter spp. and Klebsiella spp. was demonstrated in most geographical regions. Although the polymyxins showed excellent in vitro activity against the vast majority of Gram-negative bacilli evaluated, a trend to greater resistance was observed in the Asia-Pacific and Latin American regions. Therefore, the clinical use of polymyxins must be cautious and surveillance monitored.

The emergence of infections caused by multidrug-resistant Pseudomonas aeruginosa and Acinetobacter spp. has necessitated the search for alternative parenteral agents such as the polymyxins. The National Committee for Clinical Laboratory Standards (NCCLS) documents do not currently provide interpretative criteria for the testing of the polymyxins, colistin and polymyxin B. Therefore, an evaluation of the antimicrobial activity of colistin and polymyxin B was initiated using 200 bloodstream infection pathogens collected through the SENTRY Antimicrobial Surveillance Program. All susceptibility tests were performed according to the NCCLS recommendations. Polymyxin B and colistin displayed a nearly identical spectrum of activity, exhibiting excellent potency against P. aeruginosa (MIC ₉₀ , 2 μg/ml) and Acinetobacter sp. (MIC ₉₀ , 2 μg/ml). In contrast, they showed limited activity against some other nonfermentative bacilli such as Burkholderia cepacia (MIC ₉₀ , ≥128 μg/ml). Excellent correlation was achieved between broth microdilution and agar dilution tests ( r = 0.96 to 0.98); 94.3% of the results were ±1 log ₂ dilution between the methods used for both compounds. At a resistance breakpoint of ≥4 μg/ml for both agents, unacceptable false-susceptible or very major errors were noted for colistin (5%) and polymyxin B (6%). Modified zone criteria for colistin (≤11 and ≥14 mm) and polymyxin B (≤10 and ≥14 mm) were suggested, but some degree of error persisted (≥3.5%). It is recommended that all susceptible disk diffusion results be confirmed by MIC tests using the preferred reference NCCLS method. The quality control (QC) ranges listed in the product package insert require an adjusted range by approximately 3 mm for both NCCLS gram-negative quality control strains. This evaluation of in vitro susceptibility test methods for the polymyxin class drugs confirmed continued serious testing error with the disk diffusion method, the possible need for breakpoint adjustments, and the recalculation of disk diffusion QC ranges. Clinical laboratories should exclusively use MIC methods to assist the therapeutic application of colistin or polymyxin B until disk diffusion test modifications are sanctioned and published by the NCCLS.

Klebsiella pneumoniae isolates that produce carbapenemases (KPCs) are rapidly disseminating worldwide. To determine their genetic background, we investigated 16 blaKPC-2-harboring K. pneumoniae isolates from 5 countries. The isolates were multidrug resistant, possessed the blaKPC-2 gene, and differed by additional β-lactamase content. They harbored a naturally chromosome-encoded bla gene (blaSHV-1 [12.5%], blaSHV-11 [68.7%], or blaOKP-A/B [18.8%]) and several acquired and plasmid-encoded genes (blaTEM-1 [81.3%], blaCTX-M-2 [31.3%], blaCTX-M-12 [12.5%], blaCTX-M-15 [18.7%], and blaOXA-9 [37.5%]). The blaKPC-2 gene was always associated with 1 of the Tn4401 isoforms (a, b, or c). Tn4401 was inserted on different-sized plasmids that belonged to different incompatibility groups. Several blaKPC-containing K. pneumoniae clones were found: 9 different pulsotypes with 1 major (sequence type 258) and 7 minor distinct allelic profiles. Different clones harboring different plasmids but having identical genetic structure, Tn4401, could be at the origin of the worldwide spread of this emerging resistance gene.

Metallo-β-lactamase enzymes (MβL) are encoded by transferable genes, which appear to spread rapidly among gram-negative bacteria. The objective of this study was to develop a multiplex real-time PCR assay followed by a melt curve step for rapid detection and identification of genes encoding MβL-type enzymes based on the amplicon melting peak. The reference sequences of all genes encoding IMP and VIM types, SPM-1, GIM-1, and SIM-1 were downloaded from GenBank, and primers were designed to obtain amplicons showing different sizes and melting peak temperatures ( T _m ). The real-time PCR assay was able to detect all MβL-harboring clinical isolates, and the T _m -assigned genotypes were 100% coincident with previous sequencing results. This assay could be suitable for identification of MβL-producing gram-negative bacteria by molecular diagnostic laboratories.

The gene encoding the metallo-β-lactamase SPM-1 was cloned from a genomic library of Pseudomonas aeruginosa strain 48-1997A. The insert carrying spm-1 possessed a GC content of 47%, indicating that it is of non-Pseudomonas origin. Upstream of spm-1 there is a small open reading frame (ORF), which is homologous to the LysR family of proteins (69% identity to the LysR protein from Salmonella enterica serovar Typhimurium). Downstream of spm-1 there is the start of an ORF, the product of which shows close homology with the GroEL-type proteins from Xanthomonas campestris. No transmissible element could be identified upstream or downstream of spm-1. The spm-1 gene is carried on a plasmid that can transform both Escherichia coli and P. aeruginosa to ceftazidime resistance. SPM-1 contains the classic metallo-β-lactamase zinc-binding motif HXHXD and shows the highest identity (35.5%) to IMP-1. SPM-1 is a distinctly different metallo-β-lactamase from VIM and IMP and, accordingly, represents a new subfamily of mobile metallo-β-lactamases. The predicted molecular weight of the protein was 27 515 Da, significantly higher than that of IMP (25 041 Da) or VIM (25 322 Da). SPM-1 possesses a unique loop of 23 residues that accounts for the higher molecular mass.