Linezolid resistance in vancomycin-resistant enterococci (VRE) strains has been rarely reported, with Enterococcus faecium being the species most commonly associated with these few cases (1, 2, 3). Here, we report infections due to linezolid-and vancomycin-resistant Enterococcus (LRVRE) strains in patients who were treated with linezolid in a tertiary-care hospital in Brazil. To our knowledge, this is the first report of LRVRE strains in Brazil.From August 2009 to December 2011, five E. faecalis strains and one E. faecium strain exhibiting high-level resistance to both vancomycin (MIC, Ͼ256 mg/liter) and linezolid (MIC, 8 to 64 mg/liter) were isolated from blood and urine cultures from different inpatients in a Brazilian hospital (Table 1).All five subjects included were severely ill patients from intensive care units (ICUs). Patient 1 received linezolid for sepsis caused by VRE 10 days before an E. faecalis strain (18/755) was isolated from a blood culture. The regimen was changed to ampicillin, but the patient died 5 days later. Patient 2 data were not available. Patient 3 received linezolid for a period of 27 days. Two E. faecalis strains that were indistinguishable by pulsed-field gel electrophoresis (PFGE) were isolated from two blood cultures of this patient. The first strain (37/245), resistant to linezolid, was isolated at the 27th day of the total course of treatment with this drug, and the other (38/443), with intermediate resistance to linezolid, on day 53. Patient 4 received linezolid for 30 days. Eight days after the end of treatment, two strains from different species at different sites, one E. faecalis strain from a urine culture (40/ 1258) and a S. hominis strain from a blood culture, were obtained (4). Patient 6 received linezolid for a total of 32 days of treatment.The E. faecium 42/448 strain was isolated 9 days after the end of treatment.The strains were multidrug resistant, except the E. faecalis ST525 clone, which presented intermediate erythromycin and chloramphenicol MIC values, and the E. faecalis ST526 and E. faecium ST412 clones, which were susceptible to tetracycline. All E. faecalis strains were susceptible to ampicillin. Regarding glycopeptide resistance, the vanA gene was identified in all isolates. The G2576T mutation, which confers resistance to linezolid, was identified in the 23S rRNA gene in all linezolid-resistant strains, and the incomplete digestion of domain V with NheI suggested the presence of fragments with both G2576T mutant and wild-type sequences in these strains. The cfr gene was not identified in any isolate. Multilocus sequence typing (MLST) analysis revealed that the linezolid resistance was found to occur in two novel sequence types (STs) of E. faecalis (ST525 and ST526) and in ST412 of E. faecium. Strains 50/515 and 51/426 corresponding to the linezolid-susceptible E. faecalis and E. faecium control strains showed ST62 and ST838, respectively.

Plasmid-mediated AmpC β-lactamases (PMACBLs) in Enterobacteriaceae encode resistance to third-generation cephalosporins, and these can mediate carbapenem resistance when associated with porin loss. However, no standardized phenotypic method is available for detecting these enzymes in the clinical microbiology laboratory. Limited data are available concerning the frequency of PMACBLs in Enterobacteriaceae in Brazil. This study was conducted in response to an increased cefoxitin (CFO) resistance rate of 3.7% in Escherichia coli isolates from urine samples from patients with suspected urinary tract infections during 2010. We collected 2,266 E. coli isolates prospectively during January 2012. A total of 109 (4.8%) isolates were nonsusceptible to CFO. These strains were further examined using multiplex PCR for the presence of genes encoding PMACBLs and using inhibitor assays with CFO and ceftazidime (CAZ) disks with and without phenylboronic acid. Pulsed-field gel electrophoresis was used to evaluate clonal dissemination. Genes encoding PMACBLs were detected in 1.8% of the isolates from inpatients and 0.46% of isolates from outpatients. The most prevalent gene was blaCMY-2 and blaCMY-4 was also detected. The phenotypic analysis showed 100% sensitivity and specificity for CMY-2 and CMY-4 when CFO-resistant isolates with a minimum zone diameter difference of 5 mm for CAZ or CAZ and CFO were considered positive. Although most of the isolates were nonclonal, one clonal group with two isolates was observed. Thus, the most frequent PMACBL in E. coli from São Paulo, Brazil is CMY-2, and both clonal and plasmid-mediated dissemination occur.

In Enterobacteriaceae, the bla genes have been identified on plasmids in different regions of the world. The OXA-370 is a plasmid-encoded OXA- enzyme reported in two distinct regions of Brazil. Recently, we demonstrate that the bla gene is disseminated among several Enterobacteriaceae species and clones, indicating a high potential for dissemination. In this work, we described for the first time the complete nucleotide sequence of six plasmids harboring the bla gene. Complete DNA sequencing using the Illumina platform and annotation of the plasmids showed that they belonged to incompatibility groups IncX and had in average 70 kbp. The bla gene is located in a composite transposon containing four genes encoding transposases, named Tn6435. In this study, highly similar plasmids were detected in different Enterobacteriaceae genera.

Carba-NP original report for blood cultures described the need of subculture and mechanical lysis before testing, reaching the turnaround time of approximately 4 hours for sample preparation. We tested 100 consecutive blood cultures positive for Gram-negative bacilli on the Gram stain from a large clinical laboratory. Bacterial pellets were prepared by centrifugation and submitted to Carba-NP and Blue-Carba tests and used further to prepare smears for Vitek MS. Results obtained with colonies grown on sheep blood agar using the same methodologies were used as the gold standard. Carbapenemase genes were confirmed by PCR and DNA sequencing. Vitek MS identified correctly 86% of the samples. Of note, 7% of the samples were incorrectly reported by the instrument as containing a single isolate. KPC-2 was the predominant carbapenemase detected. There was 100% concordance for both negative and positive results for Carba-NP. In contrast, for Blue-Carba the concordance for positive results was 92.8%, and 41% of strains negative for carbapenemases presented a yellowish color on control well turning the test non-interpretable. The turnaround time for sample preparation for preparing the pellet was 13 min, and no subculture or mechanical lysis is needed when detecting KPC production in Enterobacterales.

fIn this work, we demonstrate that the fosI gene encodes a predicted small protein with 134 amino acids and determines reduced susceptibility to fosfomycin. It raised the MIC from 0.125 to 6 g/ml when the pBRA100 plasmid was introduced into Escherichia coli TOP10 and to 16 g/ml when the gene was cloned into the pBC_SK(؊) vector and expressed in E. coli TOP10.