In Brazil, the spread of bla(KPC-2) is occurring due to dispersion of Tn4401 'b', carried by IncN plasmids of 40 kb, and mainly the dissemination of CC11, with ST437 and ST11 playing an important role.

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Ninety-three isolates were screened as ESBL positive and 85 (91%) were found to carry CTX-M-type, as follows: K. pneumoniae 59 (49%), E. cloacae 15 (42%), and E. coli 11 (15%). Ten isolates resistant for carbapenems in K. pneumoniae were blaKPC-2 gene positive. Among CTX-M type isolates, CTX-M-15 was predominant in more than 50% of isolates for K. pneumoniae, E. coli, and E. cloacae. PFGE analysis of CTX-M producing isolates showed the predominance of CTX-M-15 in 10 of 24 pulsotypes in K. pneumoniae, 6 of 13 in E. cloacae and 3 of 6 in E. coli. CTX-M-15 was also predominant among KPC producing isolates. In conclusion, this study showed that CTX-M-15 was circulating in Rio de Janeiro state in 2007-2008. This data reinforce the need for continuing surveillance because this scenario may have changed over the years.

We aimed to investigate polymyxin B (PMB) resistance and its molecular mechanisms in 126 Klebsiella pneumoniae isolates from rectal swabs in Brazil. Ten isolates exhibited PMB resistance with interruption of mgrB gene by insertion sequences or missense mutations. Most of the PMB-resistant isolates harbored bla KPC-2 (n ‫؍‬ 8) and belonged to clonal complex 258 (CC258) (n ‫؍‬ 7). These results highlight the importance of monitoring the spread of polymyxin-resistant bacteria in hospitals, since few options remain to treat multidrug-resistant isolates. P olymyxin has been widely used to treat infections caused by multidrug-resistant (MDR) Gram-negative bacteria, including Klebsiella pneumoniae. However, reports of polymyxin-resistant K. pneumoniae (PRKP) isolates have increased worldwide, becoming a great public health concern (1).Most studies on PRKP have focused on patients with infections. However, there have been few reports assessing data on PRKP carriage in patients around the world (2). Some studies have described a remarkable and concerning number of patients who developed infection by PRKP after previous colonization, resulting in elevated mortality rates (3, 4). Colonization by KPCproducing K. pneumoniae and polymyxin therapy are considered important risk factors for PRKP infection (5, 6).Studies have demonstrated that modifications of PmrA/PmrB and PhoP/PhoQ two-component systems and inactivation of the mgrB gene (a regulator of the PhoP/PhoQ system) led to polymyxin resistance by modification of the lipopolysaccharide target (7). Recently, the plasmid-mediated transferable polymyxin resistance mcr-1 gene, causing resistance by modification of lipid A, was identified in China in Escherichia coli and K. pneumoniae strains (8).Here, we searched for molecular mechanisms associated with polymyxin resistance in K. pneumoniae isolates from Brazil. A first-step screening for polymyxin B (PMB) resistance was conducted using Etest (bioMérieux, France) in 126 isolates randomly selected from approximately 850 K. pneumoniae isolates with reduced susceptibility to carbapenems recovered from rectal swabs from 11 Brazilian states during 2007 to 2013. The bacterial identification was confirmed by conventional biochemical techniques. Considering the PRKP strains showing a MIC of Ͼ2.0 mg/liter (9), 10 (8%) PRKP isolates were observed and included in this study. These 10 PRKP isolates were collected between 2009 and 2013 from five Brazilian states (Fig. 1).To confirm the resistance phenotype, the PMB MIC was retested in duplicate by microdilution with cation-adjusted Mueller-Hinton broth (10). The isolates showed a MIC 50 of 64 mg/liter, a MIC 90 of Ͼ128 mg/liter, and a MIC range of 16 to Ͼ128 mg/liter (Table 1). Concordant Etest and microdilution results were found for only three isolates. The Etest MICs tended to be 1.3-fold to 4.0-fold lower than the microdilution MICs. Discrepancy between the two methodologies demonstrated that the Etest provided a

In Brazil, since 2009, there has been an ever increasing widespread of the bla(KPC-2) gene, mainly in Klebsiella pneumoniae. This study aims to assess the molecular epidemiology and genetic background of this gene in Enterobacteriaceae (non-K. pneumoniae) species from 9 Brazilian states between 2009 and 2011. Three hundred eighty-seven isolates were analyzed exhibiting nonsusceptibility to carbapenems, in which the bla(KPC-2) gene was detected in 21.4%. By disk diffusion and E-test, these isolates exhibited high rates of resistance to most of the antimicrobials tested, including tigecycline (45.6% nonsusceptible) and polymyxin B (16.5%), the most resistant species being Enterobacter aerogenes and Enterobacter cloacae. We found great clonal diversity and a variety of bla(KPC-2)-carrying plasmids, all of them exhibiting a partial Tn4401 structure. Therefore, this study demonstrates the dissemination of KPC-2 in 9 Enterobacteriaceae species, including species that were not previously described such as Pantoea agglomerans and Providencia stuartii.

A fter the first description of NDM-1 carbapenemase in a Providencia rettgeri isolate in Brazil in February 2013 (1), the public hospital where this isolate was recovered began an active surveillance in search of asymptomatic carriers and in the hospital environment. Furthermore, a retrospective study of carbapenem-resistant isolates stored at that hospital since 2012 was performed. Six NDM-1-producing Enterobacter hormaechei subsp. oharae isolates, identified by the Vitek2 system and hsp60 genotyping, were recovered and characterized by phenotypic assays and molecular techniques, such as PCR and DNA sequencing (2, 3). One isolate (CCBH10892) was recovered from a rectal swab of a patient at the intensive care unit (ICU) in September 2012 (a time period prior to the isolation of NDM-positive P. rettgeri). The others were isolated from March to May 2013 from rectal swabs of patients (n ϭ 4) and a sink (n ϭ 1) located in the same ICU.Pulsed-field gel electrophoresis (PFGE) of XbaI-digested DNA (4) showed that all isolates belonged to the same clone; the isolates were considered to be multidrug resistant, as they were susceptible only to amikacin (MIC range of 8 to 16 mg/liter) and polymyxin B (MIC Յ 1 mg/liter) by the Etest method (Fig. 1). Besides bla NDM-1 , all these isolates carried bla CTX-M-15 , qnrB4, and aac(6)-Ib genes, detected by PCR and sequencing. Plasmid analysis by restriction digests with S1 nuclease and Southern blotting (5) showed that the bla NDM-1 and qnrB4 genes were located on the same plasmids, ranging in size from 420 to 490 kb (Fig. 1).To obtain a comprehensive in-depth view of the genetic structure surrounding the bla NDM-1 , bla CTX-M-15 , and qnrB4 genes, the genomic sequence of the CCBH10892 isolate was determined on an Illumina MiSeq system. A total of 1,149,470 reads (5,373,710 bp) were assembled with Geneious assembler (Biomatters) to generate 56 contigs. We found bla NDM-1 in a 94,795-bp contig flanked by a truncated ISAba125 sequence at the right boundary and by a bleomycin resistance gene (ble MBL ) at the left (GenBank accession number KF727591). This region shared 99% identity with the NDM region present in a plasmid carried by a Klebsiella pneumoniae isolate from Taiwan (6). In this contig, some conjugation and plasmid transfer genes and a replication protein gene belonging to the IncF group were also observed. However, this replicon could not be detected by the PCR-based replicon typing (PBRT) scheme (7).The qnrB4 gene was found in a 16,569-bp contig in which ISCR1 and genes encoding permeases (sapA, sapB, and sapC) and phage shock proteins (pspA, pspB, pspC, and pspD) and AmpC bla DHA-1 (GenBank accession number KF646592) were also observed. This same region has been reported in different plasmids of other bacterial species (8).bla was integrated into the chromosome associated with an upstream ISEcp1 element in a 277,989-bp contig (part of it is in the sequence available at GenBank accession number KF727590). This transposition unit was inserted into the flhC gene, which enc...

bEnzymes of the OXA-48 family have become some of the most important beta-lactamases in the world. A new OXA-48 variant (OXA-370) was first described for an Enterobacter hormaechei strain isolated in Rio Grande do Sul (southern region of Brazil) in 2013. Here we report detection of the bla OXA-370 gene in 24 isolates belonging to three Enterobacteriaceae species (22 Klebsiella pneumoniae isolates, 1 Enterobacter cloacae isolate, and 1 Enterobacter aerogenes isolate) collected from five hospitals in Rio de Janeiro, Brazil, in 2013 and 2014. The isolates showed a multidrug resistance profile, and 12.5% were resistant to polymyxin B. Besides bla OXA-370 , no other carbapenemase genes were observed by PCR, whereas bla OXA-1 was found in all isolates and 22 isolates (91.6%) possessed bla CTX-M-15 . Molecular typing of the K. pneumoniae isolates by pulsed-field gel electrophoresis (PFGE) showed the presence of two clonal groups, i.e., KpA (21 isolates) and KpB (1 isolate). KpA was characterized as sequence type 16 (ST16) and KpB as ST1041 by multilocus sequence typing (MLST). ST16 has been observed for KPC-producing K. pneumoniae in Rio de Janeiro. Plasmid analysis performed with six representative OXA-370-producing isolates showed plasmids harboring the bla OXA-370 gene in all strains, ranging from 25 kb to 150 kb. This study suggests that there is an urgent need to investigate the presence of OXA-370 and dissemination of the K. pneumoniae ST16 clone carrying this gene in Brazil.