Bloodstream infection (BSI) organisms were consecutively collected from >200 medical centers in 45 nations between 1997 and 2016. Species identification and susceptibility testing followed Clinical and Laboratory Standards Institute broth microdilution methods at a central laboratory. Clinical data and isolates from 264,901 BSI episodes were collected. The most common pathogen overall was Staphylococcus aureus (20.7%), followed by Escherichia coli (20.5%), Klebsiella pneumoniae (7.7%), Pseudomonas aeruginosa (5.3%), and Enterococcus faecalis (5.2%). S. aureus was the most frequently isolated pathogen overall in the 1997-to-2004 period, but E. coli was the most common after 2005. Pathogen frequency varied by geographic region, hospital-onset or community-onset status, and patient age. The prevalence of S. aureus isolates resistant to oxacillin (ORSA) increased until 2005 to 2008 and then declined among hospital-onset and community-acquired BSI in all regions. The prevalence of vancomycin-resistant enterococci (VRE) was stable after 2012 (16.4% overall). Daptomycin resistance among S. aureus and enterococci (DRE) remained rare (<0.1%). In contrast, the prevalence of multidrug-resistant (MDR) Enterobacteriaceae increased from 6.2% in 1997 to 2000 to 15.8% in 2013 to 2016. MDR rates were highest among nonfermentative Gram-negative bacilli (GNB), and colistin was the only agent with predictable activity against Acinetobacter baumannii-Acinetobacter calcoaceticus complex (97% susceptible). In conclusion, S. aureus and E. coli were the predominant causes of BSI worldwide during this 20-year surveillance period. Important resistant phenotypes among Gram-positive pathogens (MRSA, VRE, or DRE) were stable or declining, whereas the prevalence of MDR-GNB increased continuously during the monitored period. MDR-GNB represent the greatest therapeutic challenge among common bacterial BSI pathogens.

Among 39 carbapenem-resistant Enterobacteriaceae (2.7% overall; Escherichia coli, Enterobacter cloacae, and Klebsiella pneumoniae strains) isolated in 2006 and 2007 in India, 15 strains carried bla NDM-1 and 10 harbored a gene encoding a variant of the carbapenemase OXA-48, named bla OXA-181 . One E. cloacae strain harbored bla VIM-6 , and one K. pneumoniae strain carrying bla OXA-181 also possessed bla VIM-5 . Multiple pulsed-field gel electrophoresis patterns and clonal dissemination within and among sites were observed. Isolates producing NDM-1 were disseminated in Indian health care facilities as early as 2006.

Metallo-␤-lactamase enzymes (M␤L) 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 M␤L-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 M␤L-harboring clinical isolates, and the T m -assigned genotypes were 100% coincident with previous sequencing results. This assay could be suitable for identification of M␤L-producing gram-negative bacteria by molecular diagnostic laboratories.Since the first report of acquired metallo-␤-lactamase (M␤L) in Japan in 1994 (15), genes encoding IMP-and VIMtype enzymes have spread rapidly among Pseudomonas spp. (1,5,10,13,14,16,18,(22)(23)(24), Acinetobacter spp. (3,4,17,21,29), and strains of Enterobacteriaceae (6,8,11,12,20,28). Moreover, new M␤L types have been described, such as SPM (25), GIM (2), and, more recently, SIM (9).The prevalence of M␤L-producing gram-negative bacilli has increased in some hospitals, particularly among clinical isolates of Pseudomonas aeruginosa and Acinetobacter spp. (21,23,27). Since M␤L production may confer phenotypic resistance to virtually all clinically available ␤-lactams, the continued spread of M␤L is a major clinical concern (26). The aim 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 the M␤L-type enzymes so far described. The M␤L type identification was based on the characteristic amplicon melting peak. MATERIALS AND METHODS M␤L-harboring clinical isolates and M␤L-negative control strains.The strains used in this study are listed in Tables 1 and 2. The M␤L genotypes of the clinical isolates of gram-negative nonfermentative and fermentative bacteria harboring M␤L were previously characterized by PCR and sequencing. When applicable, these clinical isolates were also previously molecularly typed to ensure that genetically unrelated strains were used. Additionally, several American Type Culture Collection (ATCC; Manassas, VA) reference strains and laboratory strains were used as M␤L-negative controls (Table 2).DNA preparation. The microorganisms were grown on blood agar plates overnight at 37°C to ensure colony purity. Three or four bacterial colonies were taken from the blood agar plates and suspended in 200 l of DNase/RNase-free distilled water (Invitrogen, CA). Two microliters of this suspension was used as templates for further amplification.Primer design. The currently available reference sequences of the M␤L-encoding IMP-and VIM-type (http://www.lahey.org/studies/), SPM-1 (AJ492820), GIM-1 (AJ620678), and SIM-1 (AY887066) genes were downloaded from GenBank (National...