Rodrigo E. Mendes

Early Dissemination of NDM-1- and OXA-181-Producing Enterobacteriaceae in Indian Hospitals: Report from the SENTRY Antimicrobial Surveillance Program, 2006-2007

ABSTRACT 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.

Rapid Detection and Identification of Metallo-β-Lactamase-Encoding Genes by Multiplex Real-Time PCR Assay and Melt Curve Analysis

ABSTRACT 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 (Tm). The real-time PCR assay was able to detect all MβL-harboring clinical isolates, and the Tm-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.

Pathogen frequency and resistance patterns in Brazilian hospitals: summary of results from three years of the SENTRY antimicrobial surveillance program

BACKGROUND Pathogen frequency and resistance patterns may vary significantly from country to country and also in different hospitals within a country. Thus, regional surveillance programs are essential to guide empirical therapy and infection control measures. METHODS Rank order of occurrence and antimicrobial susceptibility of pathogenic species causing bloodstream infections (BSI), lower respiratory tract infections (LRTI), wound or skin and soft tissue infections (WSSTI), and urinary tract infections (UTI) in hospitalized patients were determined by collecting consecutive isolates over a specified period of time, as part of the SENTRY Antimicrobial Resistance Surveillance Program (SENTRY). All isolates were tested by reference broth microdilution. RESULTS AND CONCLUSIONS A total of 3,728 bacterial strains were obtained from January, 1997, to December, 1999, from 12 Brazilian hospitals located in 4 states. The largest number of isolates were obtained from patients with BSI (2,008), followed by LRTI (822 cases), UTI (468 cases), and WSSTI (430 cases). Staphylococcus aureus was the most frequently isolated pathogen in general (22.8% - 852 isolates), followed by E. coli (13.8% - 516 cases) and Pseudomonas aeruginosa (13.3% - 496 cases). Staphylococcus aureus was also the most common species isolated from BSI (23.6%) and WSSTI (45.8%), and P. aeruginosa was the most frequent species isolated from patients with LRTI (29.4%). The main bacterial resistance problems found in this study were: imipenem resistance among P. aeruginosa (69.8% susceptibility) and Acinetobacter spp. (88.1% susceptibility); ESBL production among K. pneumoniae (48.4%) and E. coli (8.9%); resistance to third generation cephalosporins among Enterobacter spp. (68.1% susceptible to ceftazidime) and oxacillin resistance among S. aureus (34.0%) and coagulase negative staphylococci (80.1%). Only the carbapenems (88.1% to 89.3% susceptibility) showed reasonable activity against the Acinetobacter spp. isolates evaluated.

Emergence and widespread dissemination of OXA-23, -24/40 and -58 carbapenemases among Acinetobacter spp. in Asia-Pacific nations: report from the SENTRY Surveillance Program

OBJECTIVES The aim of this study was to evaluate the occurrence and dissemination of acquired carbapenem-hydrolysing class D beta-lactamase (class D carbapenemase)- and metallo-beta-lactamase (MBL)-encoding genes among Acinetobacter spp. isolates recovered from medical centres in the Asia-Pacific (APAC) region. METHODS During 2006-07, 41 medical centres located in 10 countries in the APAC region forwarded to a central monitoring site 544 Acinetobacter spp. isolates, which were tested for susceptibility by the reference broth microdilution method. Isolates non-susceptible to imipenem or meropenem (MIC>or=8 mg/L) were screened for OXA-23-, OXA-24/40-, OXA-58- and MBL-encoding genes and confirmed by sequencing. Clonality was assessed by ribotyping and PFGE. RESULTS Polymyxins (99.1% susceptible) and tigecycline (98.9% susceptible) were the most active antimicrobial agents tested. Among the isolates, 230 (42.3%) were non-susceptible to imipenem or meropenem, and class D carbapenemase- or MBL-encoding genes were detected in 162 (70.4%). blaOXA-23 was found in isolates recovered from six countries, while blaOXA-24/40 and blaOXA-58 were less common. Several isolates harboured more than one class D carbapenemase, and MBL-encoding genes were detected in one Acinetobacter johnsonii from the Philippines (blaIMP-4) and one Acinetobacter baumannii from Korea (blaVIM-2)). Overall, clonal dissemination was noted within medical centres; however, genetic relatedness was also noted among class D carbapenemase-producing A. baumannii isolates recovered from different countries. CONCLUSIONS This study shows a high distribution of class D carbapenemase-encoding genes, mainly blaOXA-23, in Acinetobacter spp. isolates. In addition, clonal dissemination among medical centres located in different countries in the APAC region, previously documented in many regions of Europe, emphasizes the epidemic potential of these bacteria.

Metallo-beta-lactamase detection: comparative evaluation of double-disk synergy versus combined disk tests for IMP-, GIM-, SIM-, SPM-, or VIM-producing isolates.

ABSTRACT The emergence of metallo-β-lactamase (MBL)-producing isolates is a challenge to routine microbiology laboratories, since there are no standardized methods for detecting such isolates. The aim of this study was to evaluate the accuracy of different phenotypic methods to detect MBL production among Pseudomonas spp., Acinetobacter spp., and enterobacterial isolates, including GIM, IMP, SIM, SPM, and VIM variants. A total of 46 genetically unrelated Pseudomonas aeruginosa, Pseudomonas putida, Acinetobacter sp., and enterobacterial strains producing distinct MBLs were tested. Nineteen strains were included as negative controls. The inhibition of bacterial growth and β-lactam hydrolysis caused by MBL inhibitors (IMBL) also were evaluated. The isolates were tested for MBL production by both a double-disk synergy test (DDST) and a combined disk assay (CD) using imipenem and ceftazidime as substrates in combination with distinct IMBL. One hundred percent sensitivity and specificity were achieved by DDST using 2-mercaptopropionic acid in combination with ceftazidime and imipenem for the detection of MBL production among P. aeruginosa and Acinetobacter species isolates, respectively. The CD test showed the same results for detecting MBL-producing enterobacteria by combining imipenem and EDTA, with a 5.0-mm-breakpoint increase in the size of the inhibition zone. Our results indicate that both phenotypic methods to detect MBL-producing isolates should be based on the genera to be tested, regardless of the enzyme produced by such isolates, as well as on the local prevalence of MBL producers.

Assessment of linezolid resistance mechanisms among Staphylococcus epidermidis causing bacteraemia in Rome, Italy

OBJECTIVES To characterize linezolid resistance among blood cultured Staphylococcus epidermidis from patients at the Polyclinic Agostino Gemelli (2006-08). Isolates also showed elevated MICs of macrolide, lincosamide and streptogramin (MLS) compounds, which were investigated. METHODS Ten S. epidermidis exhibiting linezolid MICs ≥ 4 mg/L were included. Isolates were screened for cfr mutations in 23S rRNA, L3, L4 and L22, and MLS genes by PCR/sequencing. Ribosomal proteins were compared with those from a linezolid-susceptible (MIC, 1 mg/L) clinical strain and ATCC 12228. cfr location was determined by Southern blot/hybridization. The cfr strain was submitted to plasmid curing. Epidemiology was assessed by PFGE and multilocus sequence typing (MLST). RESULTS S. epidermidis displayed linezolid MICs of 4 or 8 mg/L, except for strain 4303A (MIC, 64 mg/L). These organisms and a linezolid-susceptible strain exhibited L3 Leu101Val compared with ATCC 12228. Isolates also showed L3 Phe147Leu and Ala157Arg, and L4 Asn158Ser. Strain 12375A possessed L4 Lys68Arg. Isolates were wild-type for 23S rRNA and L22. cfr was plasmid located in strain 4303A and the plasmid-cured strain exhibited a linezolid MIC (4 mg/L) similar to that for cfr-negative strains (4-8 mg/L). All organisms harboured erm(A) and msr(A), while vga(A) was detected in several isolates. All isolates were clonally related and ST-23. CONCLUSIONS L3 Phe147Leu and/or Ala157Arg appeared responsible for the elevated linezolid MIC, since adjacent alterations have been associated with resistance. L4 Asn158Ser has been reported in a linezolid-susceptible isolate and Lys68Arg detected here did not seem to provide an additive effect. Acquisition of cfr markedly increased (8- to 16-fold) the linezolid MICs. vga(A) was associated with higher MICs of quinupristin/dalfopristin and retapamulin.

Transferable Plasmid-Mediated Resistance to Linezolid Due to cfr in a Human Clinical Isolate of Enterococcus faecalis

ABSTRACT Nonmutational resistance to linezolid is due to the presence of cfr, which encodes a methyltransferase responsible for methylation of A2503 in the 23S rRNA. The cfr gene was first described in animal isolates of staphylococci, and more recently, it has been identified in Staphylococcus aureus from human clinical infections, including in an outbreak of methicillin-resistant S. aureus. In enterococci, cfr has been described in an animal isolate of Enterococcus faecalis from China. Here, we report an isolate of linezolid-resistant E. faecalis (603-50427X) recovered from a patient in Thailand who received prolonged therapy with the antibiotic for the treatment of atypical mycobacterial disease. The isolate lacked mutations in the genes coding for 23S rRNA and L3 and L4 ribosomal proteins and belonged to the multilocus sequence type (MLST) 16 (ST16), which is commonly found in enterococcal isolates from animal sources. Resistance to linezolid was associated with the presence of cfr on an ∼97-kb transferable plasmid. The cfr gene environment exhibited DNA sequences similar to those of other cfr-carrying plasmids previously identified in staphylococci (nucleotide identity, 99 to 100%). The cfr-carrying plasmid was transferable by conjugation to a laboratory strain of E. faecalis (OG1RF) but not to Enterococcus faecium or S. aureus. The cfr gene was flanked by IS256-like sequences both upstream and downstream. This is the first characterization of the potential horizontal transferability of the cfr gene from a human linezolid-resistant isolate of E. faecalis.

Integron Carrying a Novel Metallo-β-Lactamase Gene, blaIMP-16, and a Fused Form of Aminoglycoside-Resistant Gene aac(6′)-30/aac(6′)-Ib′: Report from the SENTRY Antimicrobial Surveillance Program

ABSTRACT Since January 2002 Pseudomonas sp. strains resistant to carbapenems and ceftazidime have been routinely screened as part of the SENTRY Antimicrobial Surveillance Program for metallo-β-lactamase production, and their resistance determinants have been analyzed. Pseudomonas aeruginosa index strain 101-4704, which harbors a novel blaIMP variant, blaIMP-16, was isolated in April 2002 from a 60-year-old man in Brasília, Brazil. blaIMP-16 was found on the chromosome of the P. aeruginosa index strain, and the deduced amino acid sequence (IMP-16) showed the greatest identities to IMP-11 (90.3%) and IMP-8 (89.5%). Sequence analysis revealed that blaIMP-16 was associated with a class 1 integron, which also encoded aminoglycoside-modifying enzymes. Downstream of blaIMP-16 resided an open reading frame, which consisted of a new aminoglycoside-modifying gene, namely, aac(6′)-30, which was fused with aac(6′)-Ib′. The amino acid sequence of the aac(6′)-30 putative protein showed the most identity (52.7%) to the sequence of AAC(6′)-29b described previously. The fourth gene cassette constituted aadA1. The steady-state kinetics of IMP-16 demonstrated that the enzyme preferred cephalosporins and carbapenems to penicillins. The main functional difference observed among the kinetic values for IMP-16 compared to those for other IMPs was a lack of cefoxitin hydrolysis and a lower kcat/Km value for imipenem (0.36 μM−1 · s−1). This report further emphasizes the spread of metallo-β-lactamase genes and their close association with various aminoglycoside resistance genes.

Linezolid update: Stable in vitro activity following more than a decade of clinical use and summary of associated resistance mechanisms

Linezolid, approved for clinical use since 2000, has become an important addition to the anti-Gram-positive infection armamentarium. This oxazolidinone drug has in vitro and in vivo activity against essentially all Gram-positive organisms, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). The in vitro activity of linezolid was well documented prior to its clinical application, and several ongoing surveillance studies demonstrated consistent and potent results during the subsequent years of clinical use. Emergence of resistance has been limited and associated with invasive procedures, deep organ involvement, presence of foreign material and mainly prolonged therapy. Non-susceptible organisms usually demonstrate alterations in the 23S rRNA target, which remain the main resistance mechanism observed in enterococci; although a few reports have described the detection of cfr-mediated resistance in Enterococcus faecalis. S. aureus isolates non-susceptible to linezolid remain rare in large surveillance studies. Most isolates harbour 23S rRNA mutations; however, cfr-carrying MRSA isolates have been observed in the United States and elsewhere. It is still uncertain whether the occurrences of such isolates are becoming more prevalent. Coagulase-negative isolates (CoNS) resistant to linezolid were uncommon following clinical approval. Surveillance data have indicated that CoNS isolates, mainly Staphylococcus epidermidis, currently account for the majority of Gram-positive organisms displaying elevated MIC results to linezolid. In addition, these isolates frequently demonstrate complex and numerous resistance mechanisms, such as alterations in the ribosomal proteins L3 and/or L4 and/or presence of cfr and/or modifications in 23S rRNA. The knowledge acquired during the past decades on this initially used oxazolidinone has been utilized for developing new candidate agents, such as tedizolid and radezolid, and as linezolid patents soon begin to expire, generic brands will certainly become available. These events will likely establish a new chapter for this successful class of antimicrobial agents.

Rapid Emergence of blaCTX-M Among Enterobacteriaceae in U.S. Medical Centers: Molecular Evaluation from the MYSTIC Program (2007)

A total of 220 gram-negative isolates showing distinct beta-lactam resistance profiles recovered in U.S. medical centers during the MYSTIC Program 2007 were evaluated to determine the presence of selected beta-lactamase genes. CTX-M-encoding genes, considered rare in the United States, were detected in 38.8% (28/70; three species) of the extended spectrum beta-lactamase-positive isolates and were observed in 80.0% of the participating hospitals. CTX-M-14 and -15 were found in multiple institutions (eight and nine medical centers, respectively), and CTX-M-3 was detected in only one isolate. The OXA-2 and -10 were identified in nine Enterobacteriaceae strains, and plasmid-mediated AmpC enzymes CMY-2 and FOX-5 were identified in six and four isolates, respectively, displaying negative clavulanate inhibition. Genes encoding OXA-23 and -24 were detected in 30.0% (15/50) of carbapenem-resistant Acinetobacter spp. strains. Retrospective sampling showed that these OXA enzymes were present since 2004 in the MYSTIC Program isolates. The KPC serine carbapenemases were observed in the majority of the carbapenem-resistant Enterobacteriaceae (usually Klebsiella pneumoniae), confirming an epidemic problem in the New York City area. The association of beta-lactamase production and transferable quinolone resistance genes (qnr; 6.7%) in Enterobacteriaceae strains was higher than previously reported. This study illustrates the emergence and rapid dissemination of some beta-lactamases, such as CTX-M, broad-spectrum oxacillinases, and serine carbapenemases, that compromised the treatment of gram-negative infections in numerous U.S. hospitals participating in the MYSTIC Program in 2007.