Leonardo Neves Andrade

Dissemination of blaKPC-2 by the Spread of Klebsiella pneumoniae Clonal Complex 258 Clones (ST258, ST11, ST437) and Plasmids (IncFII, IncN, IncL/M) among Enterobacteriaceae Species in Brazil

ABSTRACT This article reports the spread of blaKPC-2 in the Sao Paulo and Rio de Janeiro states, facilitated by globally spread K. pneumoniae clonal complex 258 (CC258) clones (ST258, ST11, and ST437) and a diversity of plasmids (IncFII, IncN, and IncL/M, two untypeable plasmids carrying Tn4401a or Tn4401b) successfully disseminated among species of the Enterobacteriaceae (Enterobacter cloacae, Serratia marcescens, and Citrobacter freundii). It also constitutes the first description of sequence type 258 (ST258) in Brazil, which was associated with a nosocomial hospital outbreak in Ribeirao Preto city.

Expansion and Evolution of a Virulent, Extensively Drug-Resistant (Polymyxin B-Resistant), QnrS1-, CTX-M-2- and KPC-2-producing Klebsiella pneumoniae ST11 International High-risk Clone

ABSTRACT In this study, we report the early expansion, evolution, and characterization of a multiresistant Klebsiella pneumoniae clone that was isolated with increasing frequency from inpatients in a tertiary-care university hospital in Brazil. Seven carbapenem- and quinolone-resistant and polymyxin B-susceptible or -resistant K. pneumoniae isolates isolated between December 2012 and February 2013 were investigated. Beta-lactamase- and plasmid-mediated quinolone resistance (PMQR)-encoding genes and the genetic environment were investigated using PCR, sequencing, and restriction fragment length polymorphism (RFLP). Clonal relatedness was established using XbaI–pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and phylogenetic group characterization. Plasmid analyses included PCR-based replicon typing (PBRT) and hybridization of the S1-PFGE product, plasmid MLST, and conjugation experiments. Virulence potential was assessed by PCR by searching for 10 virulence factor-encoding genes (ureA, fimH, kfuBC, uge, wabG, magA, mrkD, allS, rmpA, and cf29a) and by phenotypic tests to analyze the hypermucoviscous phenotype. The genetic context of a multidrug-resistant and extensively drug-resistant K. pneumoniae ST11-KpI clone harboring IncFIIk-Tn4401a-bla KPC-2, qnrS1, and bla CTX-M-2 was found. Moreover, three isolates displayed high resistance to polymyxin B (MICs = 32, 32, and 128 mg/liter) as well as mucous and hypermucoviscous phenotypes. These bacteria also harbored ureA, fimH, uge, wabG, and mrkD, which code for virulence factors associated with binding, biofilm formation, and the ability to colonize and escape from phagocytosis. Our study describes the association of important coresistance and virulence factors in the K. pneumoniae ST11 international high-risk clone, which makes this pathogen successful at infections and points to the quick expansion and evolution of this multiresistant and virulent clone, leading to a pandrug-resistant phenotype and persistent bacteria in a Brazilian hospital.

Detection of chromosomal blaCTX-M-2 in diverse Escherichia coli isolates from healthy broiler chickens

The rise of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in food-producing animals is a growing concern for public health. We investigated ESBL producers isolated from broiler chickens in Brazil and characterized 19 CTX-M-2-producing E. coli. The ISCR1 was detected upstream of the chromosome-located gene bla(CTX-M-2), associated with sul-1 type integron structure. CTX-M-2-producing E. coli exhibited different PFGE-types and phylogenetic groups, showing a non-clonal dissemination. The sequence types found (ST93, ST155 and ST2309) have been associated with humans and animals worldwide. Herein, we report the chromosomal location of bla(CTX-M-2) on E. coli, highlighting the risks of multidrug-resistant bacteria in food-producing animals.

Molecular characterization of Klebsiella pneumoniae carbapenemase-producing isolates in southern Brazil.

Carbapenem-resistant Enterobacteriaceae have been frequently reported worldwide. They represent a serious concern because of the limited therapeutic options. The aim of this study was to investigate the molecular epidemiology of 14 Klebsiella pneumoniae carbapenemase (KPC) producers among 345 clinical isolates of Enterobacteriaceae with reduced susceptibility to carbapenems recovered from 11 separate hospitals in southern Brazil. The blaKPC-2 gene was detected in 14 isolates (4 %): six Enterobacter cloacae, five K. pneumoniae and three Serratia marcescens. Most of these isolates exhibited high-level resistance against β-lactams and ciprofloxacin, while the most active drugs were polymyxin B and amikacin. Genetic environment analysis, based on the classical Tn4401 structure, revealed six distinct platforms. Plasmids carrying blaKPC-2 were not typable and most were approximately 20 kb. Only KPC carbapenemases were found among the isolates studied, highlighting the local relevance of these enzymes in acquired resistance to carbapenems in Enterobacteriaceae. Our results contribute to the understanding of carbapenem resistance in Enterobacteriaceae and to the molecular characterization of KPC-2-producing isolates in Brazil.

IncI1/ST113 and IncI1/ST114 conjugative plasmids carrying blaCTX-M-8 in Escherichia coli isolated from poultry in Brazil.

Extended-spectrum beta-lactamase-producing Escherichia coli isolated from poultry in Brazil showed blaCTX-M-8 gene. IS10 was found upstream of blaCTX-M-8, harbored on plasmids IncI1, ST113/ST114 subtypes. Genomic relationship revealed a heterogeneous E. coli population. The gene blaCTX-M-8 is established in South America in food-producing animals, which represent risk of dissemination for other countries.

Reply to “Clonal Complex 258, the Most Frequently Found Multilocus Sequence Type Complex in KPC-2-Producing Klebsiella pneumoniae Isolated in Brazilian Hospitals”

We acknowledge the letter by Nicoletti et al. (6) in response to the study in which different genetic platforms carrying the blaKPC-2 gene among several species of Enterobacteriaceae from Brazil were identified (2). Nicoletti et al. (6) confirm the circulation of diverse KPC-2-producing Klebsiella pneumoniae clones (2, 3, 7) from six Brazilian states and from the city of Brasilia in the Federal District of Brazil and further document the predominance of ST437, followed by ST11, and the description of a new sequence type, ST617. While some CC258 clones, such as ST258 or ST11, are globally spread, others, such as ST437, have been documented to occur in only a few locations. It would be of interest to analyze the continental spread of such clones during nonoutbreak situations and to evaluate whether such clonal expansion is independent of the emergence of blaKPC. We also observed the presence of blaCTX-M-2, a gene that encodes resistance to extended-spectrum cephalosporins and is widespread in Brazil and other South American countries, such as Argentina, where K. pneumoniae ST437-producing KPC-2 has been detected (3) among different Klebsiella backgrounds (2). It could be hypothesized that blaCTX-M-2 acquisition by ST437 would have facilitated the spread of ST437, with a resulting increased connectivity with other bacterial (donor) populations, in a way that facilitates the acquisition of different plasmids or genetic elements in some areas. Obviously, ST437 might have increased its connectivity for other reasons, and the capture of either blaCTX-M-2 or blaKPC-2 could be a coincidental consequence of the clonal spread. In addition to K. pneumoniae ST437 and ST11, we identified amplification of different lineages of KPC producers, at a local level, which have not been identified in other Brazilian studies. These producers included the globally spread K. pneumoniae ST258 clone, which was associated with a nosocomial outbreak in a single hospital from Ribeirão Preto (representing the first and only description of ST258 isolated in Brazil to date), Enterobacter cloacae from Rio Grande do Sul, and Serratia marcescens and Citrobacter freundii from Rio de Janeiro. A diversity of genetic contexts for blaKPC (Tn4401a, Tn4401b, and Tn4401c located on IncFII, IncN, IncL/M, IncFI, or small untypeable plasmids) among Enterobacteriaceae and Pseudomonas may also be inferred from available studies (1, 2, 4). Similarly to the clonal distribution, some elements were amplified in different areas, such as a 130-kb IncFII plasmid carrying Tn4401a among ST258, ST11, and ST48 isolates from Ribeirão Preto and a 50to 60-kb IncL/M plasmid carrying Tn4401b to Serratia and Citrobacter species from Rio de Janeiro. Others, such as a 40-kb IncN plasmid with a replicon similar to that of the widespread plasmid pNL194 (5), were disseminated in different states linked to K. pneumoniae ST437 and ST327 and Enterobacter cloacae. All these studies show that the blaKPC-2 gene is “moving through” among different backgrounds. This complex epidemiological scenario highlights the relevance of comprehensive local studies and the need to perform them to understand the evolvability of clones and bacterial populations under different ecological conditions. The diversity of highly transmissible clonal and also plasmid backgrounds carrying blaKPC-2 found in Brazil is of great concern, as these backgrounds can drive a rapid spread of blaKPC and other adaptive traits in South America, a continent highly connected with North America and South Europe for different social and economical links. Besides detection of both clones and plasmids, it would be necessary to differentiate between endemic and imported high-risk clonal complexes and plasmids involved in the spread of blaKPC-2.

Induction and nosocomial dissemination of carbapenem and polymyxin-resistant Klebsiella pneumoniae

INTRODUCTION Polymyxins are antimicrobial agents capable of controlling carbapenemase-producing Klebsiella pneumoniae infection. METHODS We report a cluster of four patients colonized or infected by polymyxin-resistant and Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae. RESULTS Three patients were hospitalized in adjacent wards, and two were admitted to the intensive care unit. The index case maintained prolonged intestinal colonization by KPC-producing K. pneumoniae. Three patients received polymyxin B before the isolation of polymyxin-resistant K. pneumoniae. CONCLUSIONS Colonization by KPC-producing K. pneumoniae and previous use of polymyxin B may be causally related to the development of polymyxin-resistant microorganisms.

Evaluation and characterization of plasmids carrying CTX-M genes in a non-clonal population of multidrug-resistant Enterobacteriaceae isolated from poultry in Brazil

The increasing presence of ESBL-producing bacteria in food-producing animals might impact on public health. In this study, ESBL-producing enterobacteria were investigated in the microbiota of chickens produced in Brazil. We detected blaCTX-M-2, blaCTX-M-8 and blaCTX-M-15 in 13 Escherichia coli isolates, within 9 different PFGE-types. Escherichia fergusonii and Klebsiella pneumoniae were found carrying blaCTX-M-2. Plasmid Inc groups found included repF, FIB, FIC, I1, Y, B/O, A/C, K and HI1. F plasmids were present in 87.5% of the isolates, however, no resistance gene was harbored in this replicon. The pMLST for IncI1 showed ST113 and the novel ST130, ST131 and ST132 harboring blaCTX-M-8. IncK plasmids carried blaCTX-M-2 in one E. coli isolate. Non-typeable plasmids carrying blaCTX-M-2 or blaCTX-M-15 had up to 260kb. blaCTX-M-2 was also associated with class I integron and ISCR1 and blaCTX-M-8 with IS10. Overall, similar resistance elements were disseminated among a diverse population of ESBL-producing enterobacteria.

Antimicrobial resistance and plasmid replicons in Yersinia enterocolitica strains isolated in Brazil in 30 years

Some studies evaluated the resistance profile of the Y. enterocolitica strains isolated in diverse countries. However, in Brazil the isolation and the study of Y. enterocolitica are not common and therefore information about the antimicrobial resistance profile of this species in this country is scarce. Therefore, the aim of this study was to evaluate the antimicrobial resistance of Y. enterocolitica of biotypes 1A, 2 and 4 isolated from clinical and non-clinical sources between 1979 and 2012, in Brazil. This study showed that some Yersinia enterocolitica of different biotypes remain susceptible to antimicrobials used for gastroenteritis treatment. Moreover, neither acquired resistance genes nor diversity of plasmids replicons were found; however, variation in the in vitro intrinsic resistant pattern was detected, except the non-resistance to cefoxitin in all strains. Notwithstanding, due to epidemiological link between Y. enterocolitica and the pork production chain, monitoring plasmid acquired resistance in Y. enterocolitica could also be considered for antimicrobial resistance control purposes and food safety measures.

Genomic diversification and virulence features in SPM-1–producing Pseudomonas aeruginosa 13 years later

inBrazil by P.aeruginosa hasbeen broadly reported;howev-er, no virulence factor encoding genes were searched in these isolates,and we investigate sample from 3 different cities that are at least500 km away from each other. Important virulence factor andquorum-sensing systems that contribute to successful bacterialcolonization and infection were identified in the studied isolates. Theknowledge regarding the genetic context of resistance and virulencecontributes to followingthe evolution of thisbacterialpathogen,routesof dissemination, and future establishment of epidemiologicalcontrol measures.Several virulence factors (VFs) and quorum-sensing (QS) systemshave been well characterized in P.aeruginosa isolated from cysticfibro-sis patients (Sousa and Pereira, 2014). However, there are little dataregarding virulence traits in nosocomial infections due to multidrug-resistant (MDR)P. aeruginosa. In this study, we investigated the molec-ular epidemiology and genetic of resistance as well as VF and QSsystems in nosocomial SPM-1–producing P. aeruginosa, isolated in 3different Brazilian cities.Of all 12 SPM-1–producing P. aeruginosa studied, 8 were fromRibeirao Preto City; 3, from Cuiaba City; and 1, from Belo HorizonteCity. The bla