Diagnostic and vaccine potential of Zika virus envelope protein (E) derivates produced in bacterial and insect cells.

Introduction: In the present study we evaluated the features of different recombinant forms of Zika virus (ZIKV) proteins produced in either bacterial (Eschericha coli) or insect cells (Drosophila melanogaster). The ZIKV-envelope glycoprotein (EZIKV) is responsible for virus entry into host cells, is the main target of neutralizing antibodies and has been used as a target antigen either for serological tests or for the development of subunit vaccines. The EZIKV is composed of three structural and functional domains (EDI, EDII, and EDIII), which share extensive sequence conservation with the corresponding counterparts expressed by other flaviviruses, particularly the different dengue virus (DENV) subtypes. Methods: In this study, we carried out a systematic comparison of the antigenicity and immunogenicity of recombinant EZIKV, EDI/IIZIKV and EDIIIZIKV produced in E. coli BL21 and Drosophila S2 cells. For the antigenicity analysis we collected 88 serum samples from ZIKV-infected participants and 57 serum samples from DENV-infected. For immunogenicity, C57BL/6 mice were immunized with two doses of EZIKV, EDI/IIZIKV and EDIIIZIKV produced in E. coli BL21 and Drosophila S2 cells to evaluate humoral and cellular immune response. In addition, AG129 mice were immunized with EZIKV and then challenge with ZIKV. Results: Testing of samples collected from ZIKV-infected and DENV-infected participants demonstrated that the EZIKV and EDIIIZIKV produced in BL21 cells presented better sensitivity and specificity compared to proteins produced in S2 cells. In vivo analyses were carried out with C57BL/6 mice and the results indicated that, despite similar immunogenicity, antigens produced in S2 cells, particularly EZIKV and EDIIIZIKV, induced higher ZIKV-neutralizing antibody levels in vaccinated mice. In addition, immunization with EZIKV expressed in S2 cells delayed the onset of symptoms and increased survival rates in immunocompromised mice. All recombinant antigens, either produced in bacteria or insect cells, induced antigen-specific CD4+ and CD8+ T cell responses. Conclusion: In conclusion, the present study highlights the differences in antigenicity and immunogenicity of recombinant ZIKV antigens produced in two heterologous protein expression systems.

Genome and plasmid context of two rmtG-carrying Enterobacter hormaechei isolated from urinary tract infections in Brazil.

OBJECTIVES: Enterobacter hormaechei is an important causative agent of severe infections in critically ill patients. Aminoglycosides are among the main antibiotics for the treatment of E. hormaechei infections, however the development of antimicrobial resistance is an increasing problem. RmtG is a 16S rRNA methyltransferase, a class of enzymes conferring high-level resistance to clinically relevant aminoglycosides. The aim of this study was to characterise the full genetic context of plasmids harbouring the rmtG gene in two aminoglycoside-resistant E. hormaechei isolated in Brazil. METHODS: ThermtG-harbouring plasmids were transferred to an Escherichia coli J53 recipient strain and were fully sequenced using a MiSeq sequencing system. Complete genome assemblies were accomplished using a combination of Newbler v.3.0, SPAdes 3.10.0 and phrap/cross_match programs. Plasmid sequences were annotated using RAST server and were then manually curated using BLAST databases and ISfinder. Easyfig 2.0 was used to map and compare regions of interest containing rmtG in both plasmids. RESULTS: Both isolates carried thermtG gene on an IncA/C plasmid of ˜152kb and ˜235kb, respectively, associated with a Tn3 transposon. The plasmids contain a transfer region as well as genes involved in plasmid stability and resistance to ß-lactams, sulfonamides and quaternary ammonium compounds. One of the plasmids also carried the mrk operon encoding type 3 fimbriae. CONCLUSION: This first detection ofrmtG in E. hormaechei supports the ability for horizontal transfer. The location in complex genetic platforms carried by Tn3 transposons in IncA/C plasmids may facilitate dissemination to other Gram-negative pathogens, further limiting treatment options.

The role of therapy with aminoglycoside in the outcomes of kidney transplant recipients infected with polymyxin- and carbapenem-resistant Enterobacteriaceae.

Kidney transplant recipients are at risk for infections due to carbapenem-resistant Enterobacteriaceae (CRE). Polymyxin-resistant CRE (PR-CRE) infections are especially difficult to treat. The aim of this study was to characterize PR-CRE infections among kidney transplant recipients and identify risk factors for treatment failure. This retrospective cohort study involved all kidney transplant recipients with PR-CRE infection between 2013 and 2017 at our center. Minimal inhibitory concentrations for polymyxin B were determined by broth microdilution. Carbapenem-resistant genes (blaKPC, blaNDM, and blaOXA-48), aminoglycoside-resistance genes, and polymyxin-resistant gene mcr-1 were identified by polymerase chain reaction. All but one of the 47PR-CRE infections identified were due to Klebsiella pneumoniae. The most common type of infection (in 54.3%) was urinary tract infection (UTI). Monotherapy was used in 10 cases. Combined treatment regimens included double-carbapenem therapy in 19 cases, oral fosfomycin in 19, and amikacin in 13. Treatment failure occurred in 21 cases (45.7%). Clinical success was achieved 78.9% of patients who used aminoglycosides versus 37.0% of those who not used this drug (p = 0.007). Multivariate analysis showed diabetes mellitus to be a risk factor for treatment failure; amikacin use and UTI were found to be protective. Nine strains were RmtB producers. Although aminoglycosides constitute an important therapeutic option for PR-CRE infection, the emergence of aminoglycoside resistance could have a major impact on the management of CRE infection.

Endemicity of the High-Risk Clone Klebsiella pneumoniae ST340 Coproducing QnrB, CTX-M-15, and KPC-2 in a Brazilian Hospital.

The dissemination of multiresistant Klebsiella pneumoniae carbapenemase (KPC)-2-producing Klebsiella pneumoniae isolates belonging to international high-risk clones poses a major health care threat. In this study, 48 nonduplicated, carbapenem-resistant K. pneumoniae isolated from 2011 to 2014 in a tertiary hospital were investigated. The blaKPC-2 gene was the only determinant for carbapenem resistance. The blaCTX-M-15 gene was the main determinant for the production of extended-spectrum beta-lactamase (ESBL), whereas aph(3')-Ia and qnrB were the most common genes associated with resistance to aminoglycosides and quinolones, respectively. Nine different sequence types (STs) were identified. The most common was ST340. Molecular typing by enterobacterial repetitive intergenic consensus-PCR placed 48 strains among 10 different clusters. In the studied hospital, the high-risk clone of KPC-2-producing K. pneumoniae ST340, harboring genes that codify aminoglycoside modifying enzymes, QnrB and CTX-M-15 plus CTXM-2-type ESBLs, is disseminated and acts as a major agent of infections in critically ill patients.

Draft genome sequence of a KPC-2-producing Klebsiella pneumoniae ST340 carrying blaCTX-M-15 and blaCTX-M-59 genes: a rich genome of mobile genetic elements and genes encoding antibiotic resistance.

OBJECTIVES: Klebsiella pneumoniae is considered an opportunistic pathogen and an important agent of nosocomial and community infections. It presents the ability to capture and harbour several antimicrobial resistance genes and, in this context, the extensive use of carbapenems to treat serious infections has been responsible for the selection of several resistance genes. This study reports the draft genome sequence of a KPC-2-producing K. pneumoniae strain (Kp10) simultaneously harbouring blaCTX-M-15 and blaCTX-M-59 genes isolated from urine culture of a patient with Parkinson's disease. METHODS: Classical microbiological methods were applied to isolate and identify the strain, and PCR and sequencing were used to identify and characterise the genes and the genetic environment. Whole-genome sequencing (WGS) was performed using a Nextera XT DNA library and a NextSeq platform. RESULTS: WGS analysis revealed the presence of 5915 coding genes, 46 RNA-encoding genes and 255 pseudogenes. Kp10 belonged to sequence type 340 (ST340) of clonal complex 258 (CC258) and carried 20 transferable genes associated with antimicrobial resistance, comprising seven drug classes. Although the simultaneous presence of different blaCTX-M genes in the same strain is rarely reported, the blaKPC-2, blaCTX-M-15 and blaCTX-M-59 genes were not associated with the same genetic mobile structure in Kp10. CONCLUSIONS: These results confirm the capacity of K. pneumoniae to harbour several antimicrobial resistance genes. Thus, this draft genome could help in future epidemiological studies regarding the dissemination of clinically relevant resistance genes.

Draft genome sequence of an aminoglycoside-resistant RmtD2-producing Enterobacter cloacae subsp. cloacae ST395 in Brazil.

OBJECTIVES: Enterobacter cloacae has recently emerged as an important agent of nosocomial infections owing to the dissemination of extended-spectrum ß-lactamases and carbapenemases in this species. In this context, a rise in the therapeutic use of aminoglycosides was noticed, followed by the accelerated development of resistance mechanisms. In this study, we report the draft genome sequence of a multidrug-resistant E. cloacae subsp. cloacae strain (Ec2) isolated from an active surveillance culture of a patient with Chagas disease. METHODS: Whole-genome sequencing (WGS) was performed using a Nextera XT DNA library and NextSeq platform. RESULTS: WGS analysis revealed the presence of 5527 coding genes, 62 RNA-encoding genes and 275 pseudogenes. Strain Ec2 belongs to sequence type 395 (ST395) and carries 22 transferable antibiotic resistance genes, comprising eight antimicrobial classes, including the rmtD2 gene conferring high-level aminoglycoside resistance. CONCLUSIONS: This draft genome can be used in comparative genomic analyses with different E. cloacae strains. In addition, it could help at elucidating epidemiological aspects regarding the dissemination of clinically relevant resistance genes.

Transfer of KPC-2 carbapenemase from Klebsiella pneumoniae to Enterobacter cloacae in a patient receiving meropenem therapy.

The horizontal transfer of a plasmid bearing the blaKPC-2 gene from K. pneumoniae to E. cloacae infecting the respiratory tract of a patient during meropenem therapy was elucidated. This finding is particularly worrisome, since these drugs are of last resort for multidrug-resistant Gram-negative pathogens.

Prevalence of Extended-Spectrum ß-Lactamases CTX-M-8 and CTX-M-2-Producing Salmonella Serotypes from Clinical and Nonhuman Isolates in Brazil.

We characterized extended-spectrum ß-lactamases (ESBL) enzymes among Salmonella strains isolated in Brazil from 2009 to 2014. Salmonella recovered from both clinical and nonhuman (food, poultry, and environment) sources were subjected to antimicrobial susceptibility testing. ß-lactamases genes were detected by polymerase chain reaction/sequencing; plasmid profiles and transferability were assessed by S1-pulsed field gel electrophoresis (PFGE). Genetic diversity was evaluated by XbaI-PFGE. Out of 630 Salmonella strains screened, 46 displayed ESBL phenotype, distributed across 11 different serotypes. blaCTX-M-8 and blaCTX-M-2 genes were detected at frequencies of 47% and 41%, respectively. blaSHV-5 and blaSHV-2 were also detected but in lower frequencies (4%, 2%). blaTEM-1 gene was detected in 22% of the strains. Most of the ESBL genes were transferable by conjugation, and the respective blaESBL gene was detected in the recipient strain, indicating the location of ESBL determinants on transferable plasmids. XbaI-PFGE revealed genomic diversity of Salmonella Typhimurium bearing blaCTX-M-2, blaCTX-M-8, blaTEM-1, and blaSHV-2 genes. Salmonella Muenchen (harboring blaCTX-M-2) and Salmonella Corvallis (blaCTX-M-8 and blaSHV-5) showed clonal relatedness within respective serotypes. Our findings underscore the occurrence of diverse ESBL genes in several Salmonella serotypes, reinforcing the need for continuous surveillance of resistance genes circulating in human and nonhuman sources.

Caracterização de genes de resistência antimicrobiana e seu contexto genético em Klebsiella spp. produtoras de KPC isoladas de hospitais do Estado de São Paulo

Klebsiella pneumoniae produtoras de ESBL estão frequentementeenvolvidas em infecções relacionadas à assistência à saúde (IRAS). ESBLsão capazes de hidrolisar todas as penicilinas, cefalosporinas de amploespectro e aztreonam, sendo os carbapenêmicos o fármaco de escolha parainfecções por microrganismos produtores de ESBL. Porém, Klebsiella spp.produtoras de carbapenemases tem se tornado cada vez mais frequentes.Além disso, já foi descrita, no Brasil, K. pneumoniae produtoras da 16SRMTase, RmtD, uma enzima que confere resistência em nível elevado paratodos os aminoglicosídeos, tornando as opções terapêuticas bastantelimitadas. O objetivo desse trabalho foi caracterizar genes de resistênciaresponsáveis pela produção de ESBL, carbapenemases e 16S RMTases eseu contexto genético em Klebsiella spp. produtoras de KPC isoladas deamostras clínicas provenientes de hospitais do Estado de São Paulo. Cemcepas de Klebsiella spp., isoladas de 2009 a 2011, previamente confirmadascomo produtoras de KPC, foram submetidas a PCR para detecção de blaSHV,blaTEM, blaCTX-M, blaCTX-M-15, blaCTX-M-2, blaCTX-M-8, blaGES-1, blaVIM, blaIMP,blaSPM, blaNDM, blaOXA- 48, armA, rmtA, rmtB rmtC e rmtD e sequenciamentode DNA. Para as cepas negativas para os genes blaSHV foram realizadas aidentificação de Klebsiella spp.a partir de provas bioquímicas, PCR Multiplexpara detecção dos genes blaSHV, blaLEN e blaOKP e sequenciamento dosgenes 16S rRNA e rpoB. Experimentos de transformação e clonagem foramutilizados para a identificação de um novo gene de resistência aosaminoglicosídeos e plasmídeos carreadores de genes de 16S RMTasesforam sequenciados. As cepas apresentaram resistência em nível elevadopara a maioria dos antibióticos testados...

Klebsiella pneumoniae producing ESBLs are frequently involved in hospitalinfections in intensive care unit (ICU). ESBLs are capable of hydrolyzing allpenicillins, cephalosporins and aztreonam. Carbapenems are considered theagents of choice for treatment of infections caused by ESBL-producingmicroorganisms. However, Klebsiella spp. producing carbapenemases hasbecome more frequent. Moreover, K. pneumoniae producing 16S RMTase,RmtD, which confers high level resistance to aminoglycosides, have beendescribed in Brazil, limiting the therapeutical options. The aim of this studywas to characterize resistance genes responsible to the production of ESBL,carbapenemases and 16S RMTases and their genetic context in Klebsiellaspp. producing KPC from hospitals at Sao Paulo State. A hundred K.pneumoniae strains isolated in the period of 2009 and 2011 and previouslyconfirmed as KPC producers were subjected to PCR for the detection ofblaSHV, blaTEM, blaCTX-M, blaCTX-M-15, blaCTX-M-2, blaCTX-M-8, blaGES-1, blaVIM,blaIMP, blaSPM, blaNDM, blaOXA-48, armA, rmtA, rmtB rmtC e rmtD and DNAsequencing. Identification of Klebsiella spp. by biochemical tests, detection ofblaSHV, blaLEN e blaOKP genes by Multiplex PCR and 16S rRNA gene and rpoBsequencing was performed on blaSHV negative isolates. Transformation andgenomic cloning were performed for the identification of a newaminoglycosides resistance gene. Sequencing of plasmids carrying 16SRMTases genes was performed. All strains showed high resistance levels formost antibiotics tested...

Outbreak of IMP-producing carbapenem-resistant Enterobacter gergoviae among kidney transplant recipients.

OBJECTIVES: The objective of this study was to investigate a prolonged outbreak of carbapenem-resistant Enterobacter gergoviae (CREG) involving kidney transplant recipients (KTRs) between 2009 and 2014. METHODS: A case-control study was undertaken. Controls (n = 52) were selected from CREG-negative KTRs. Surveillance cultures for CREG were collected weekly. Colonization was defined as isolation of CREG from surveillance samples or from clinical specimens, with no evidence of infection. We also investigated infection control practices at the facility. RESULTS: Of 26 identified cases, 13 had had no known contact with another CREG-positive patient before the first positive culture. Seven patients (27%) developed infection. The site most often colonized was the urinary tract. During the study period two clusters were identified, one in 2009 and another in 2013-14. DNA sequencing revealed blaIMP-1 in all CREG tested. No environmental or hand cultures tested positive for CREG. An audit of infection control practices detected flaws in the handling and cleaning of urinary tract devices. Multivariate analysis identified advanced age, ureteral stent use, retransplantation and male gender as risk factors for CREG acquisition. CONCLUSIONS: An outbreak among KTRs caused by an unusual species of MDR bacteria may have resulted from a common source of contamination related to urinary tract devices.

Microbiological characterization of Delftia acidovorans clinical isolates from patients in an intensive care unit in Brazil.

Delftia acidovorans is an opportunistic agent in several types of infections, both in immunocompromised and immune-competent individuals; its resistance to aminoglycosides and polymyxin, choice drugs for empirical treatment of Gram-negative infections, is remarkable. We report the antimicrobial susceptibility and the genetic relatedness of 24 D. acidovorans strains recovered from tracheal aspirates of 21 adult inpatients hospitalized in an intensive care unit at a Brazilian hospital, from 2012 to 2013. All of the isolates were recovered as pure cultures and in counts above 1,000,000 CFU/mL. None of them were susceptible to polymyxin B, amikacin, gentamicin, or tobramycin; quinolones and trimethoprim-sulfamethoxazole presented varied activities against the isolates, while ß-lactam resistance was not detected. Four clusters were verified in pulsed-field gel electrophoresis analysis, and a major pulsotype comprised 10 strains. A possible, but undetermined common source, can be responsible for this strain dissemination, underscoring the need of reinforcing the adherence to disinfection and infection control standard techniques.