Clinical utilization of bacteriophages: a new perspective to combat the antimicrobial resistance in Brazil

Due to the emergence of multi-drug resistant bacteria, and the evident limitation in therapeutic options, alternatives to combat bacterial infections have been sought. One of these is phage therapy, which is the use of bacterial viruses to kill pathogenic bacteria responsible for the infection. These viruses called bacteriophages are very abundant organisms in the world and are harmless to humans. There are several advantages in using phage therapy, especially against multi-drug resistant pathogens, which tend to be dominated by individual strains. The advantages include fewer collateral effects such as lower disturbance of gut microbiota and less antimicrobials consumption, which itself leads to reducing antibiotic resistance rates. Unfortunately, few clinical studies have been initiated in Brazil and this area is little explored in our country. This manuscript describes clinical evidence of successful phage utilization on pathogens considered a threat in Brazil, highlighting the benefits of a possible phage utilization as an important tool to combat antimicrobial resistance in our country

First Description of KPC-2-Producing Klebsiella oxytoca in Brazil

The present work reports the detection of the first case of nosocomial Klebsiella oxytoca producing class A carbapenemase KPC-2 in Brazil. the isolate KPN106 carried a 65-kb IncW-type plasmid that harbors the bla(KPC) gene and Tn4401b. Moreover, we detected the presence of a class 1 integron containing a new allele, arr-8, followed by a 5'-truncated dhfrIIIc gene. in view of the recent results, we emphasize the high variability of the bacterial and genetic hosts of this resistance determinant.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)FACEPEPFA/UPEUniv Pernambuco, Inst Ciencias Biol, Lab Resistencia Microbiana, Recife, PE, BrazilUniv Fed Pernambuco, Dept Genet, Lab Genet Microrganismos, Recife, PE, BrazilUniversidade Federal de São Paulo, Lab Alerta, São Paulo, BrazilCPqAM Fiocruz, Ctr Pesquisa Aggeu Magalhaes, Recife, PE, BrazilUniversidade Federal de São Paulo, Lab Alerta, São Paulo, BrazilWeb of Scienc

Characterization of amino acid substitution W20S in MgrB involved in polymyxin resistance in Klebsiella pneumoniae

In the major human pathogen Klebsiella pneumoniae, MgrB inactivation by disruptive insertion sequence (IS) elements and mutations leading to early termination are known to play an important role in polymyxin resistance. In this study, we examined a collection of invasive blaKPC-2-producing K. pneumoniae isolates belonging to the high-risk clone sequence type 258 (ST258) displaying high rates of resistance to many antimicrobials, including polymyxins. We identified a deleterious substitution (W20S) in MgrB and confirmed by genetic complementation analysis that this variant was inactive, leading to increased polymyxin B and colistin MICs

BKC-2, a new BKC variant detected in MCR-9.1-producing Enterobacter hormaechei subsp. xiangfangensis

We performed the characterization of a multidrug-resistant (MDR) Enterobacter spp. isolate highlighting the genetic aspects of the antimicrobial resistance genes. An Enterobacter spp. isolate (Ec61) was recovered in 2014 from a transtracheal aspirate sample from a patient admitted to a Brazilian tertiary hospital and submitted to further microbiological and genomic characterization. Ec61 was identified as Enterobacter hormaechei subsp. xiangfangensis ST451, showed a MDR profile and the presence of genes codifying new β-lactamase variants, BKC-2 and ACT-84, and the mobile colistin resistance gene mcr-9.1

Clinical and molecular description of a high-copy IncQ1 KPC-2 plasmid harbored by the international ST15 Klebsiella pneumoniae clone

This study provides the genomic characterization and clinical description of bloodstream infections (BSI) cases due to ST15 KPC-2 producer Klebsiella pneumoniae. Six KPC-K. pneumoniae isolates were recovered in 2015 in a tertiary Brazilian hospital and were analyzed by whole-genome sequencing (WGS) (Illumina MiSeq short reads). Of these, two isolates were further analyzed by Nanopore MinION sequencing, allowing complete chromosome and plasmid circularization (hybrid assembly), using Unicycler software. The clinical analysis showed that the 30-day overall mortality for these BSI cases was high (83%). The isolates exhibited meropenem resistance (MICs, 32 to 128 mg/liter), with 3/6 isolates resistant to polymyxin B. The conjugative properties of the blaKPC-2 plasmid and its copy number were assessed by standard conjugation experiments and sequence copy number analysis. We identified in all six isolates a small (8.3-kb), high-copy-number (20 copies/cell) non-self-conjugative IncQ plasmid harboring blaKPC-2 in a non-Tn4401 transposon. This plasmid backbone was previously reported to harbor blaKPC-2 only in Brazil, and it could be comobilized at a high frequency (10−4) into Escherichia coli J53 and into several high-risk K. pneumoniae clones (ST258, ST15, and ST101) by a common IncL/M helper plasmid, suggesting the potential of international spread. This study thus identified the international K. pneumoniae ST15 clone as a carrier of blaKPC-2 in a high-copy-number IncQ1 plasmid that is easily transmissible among other common Klebsiella strains. This finding is of concern since IncQ1 plasmids are efficient antimicrobial resistance determinant carriers across Gram-negative species. The spread of such carbapenemase-encoding IncQ1 plasmids should therefore be closely monitored. IMPORTANCE In many parts of the world, carbapenem resistance is a serious public health concern. In Brazil, carbapenem resistance in Enterobacterales is mostly driven by the dissemination of KPC-2-producing K. pneumoniae clones. Despite being endemic in this country, only a few reports providing both clinical and genomic data are available in Brazil, which limit the understanding of the real clinical impact caused by the dissemination of different clones carrying blaKPC-2 in Brazilian hospitals. Although several of these KPC-2-producer K. pneumoniae isolates belong to the clonal complex 258 and carry Tn4401 transposons located on large plasmids, a concomitant emergence and silent dissemination of small high-copy-number blaKPC-2 plasmids are of importance, as described in this study. Our data identify a small high-copy-number IncQ1 KPC plasmid, its clinical relevance, and the potential for conjugative transfer into several K. pneumoniae isolates, belonging to different international lineages, such as ST258, ST101, and ST15

The polymyxin B-induced transcriptomic response of a clinical, multidrug-resistant Klebsiella pneumoniae involves multiple regulatory elements and intracellular targets

Background: The emergence of multidrug-resistant Klebsiella pneumoniae is a major public health concern. Many K. pneumoniae infections can only be treated when resorting to last-line drugs such as polymyxin B (PB). However, resistance to this antibiotic is also observed, although insufficient information is described on its mode of action as well as the mechanisms used by resistant bacteria to evade its effects. We aimed to study PB resistance and the influence of abiotic stresses in a clinical K. pneumoniae strain using whole transcriptome profiling. Results: We sequenced 12 cDNA libraries of K. pneumoniae Kp13 bacteria, from two biological replicates of the original strain Kp13 (Kp13) and five derivative strains: induced high-level PB resistance in acidic pH (Kp13(pH)), magnesium deprivation (Kp13(Mg)), high concentrations of calcium (Kp13(Ca)) and iron (Kp13(Fe)), and a control condition with PB (Kp13(PolB)). Our results show the involvement of multiple regulatory loci that differentially respond to each condition as well as a shared gene expression response elicited by PB treatment, and indicate the participation of two-regulatory components such as ArcA-ArcB, which could be involved in re-routing the K. pneumoniae metabolism following PB treatment. Modules of co-expressed genes could be determined, which correlated to growth in acid stress and PB exposure. We hypothesize that polymyxin B induces metabolic shifts in K. pneumoniae that could relate to surviving against the action of this antibiotic. Conclusions: We obtained whole transcriptome data for K. pneumoniae under different environmental conditions and PB treatment. Our results supports the notion that the K. pneumoniae response to PB exposure goes beyond damaged membrane reconstruction and involves recruitment of multiple gene modules and intracellular targets.Fundacao de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Lab Nacl Comp Cient, Petropolis, RJ, BrazilFiocruz MS, Ctr Pesquisas Goncalo Moniz, Salvador, BA, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Internal Med, Lab Alerta,Div Infect Dis, Sao Paulo, SP, BrazilUniv Catolica Cordoba, Fac Ingn, CONICET, Cordoba, ArgentinaUniv Fed Sao Paulo, Escola Paulista Med, Dept Internal Med, Lab Alerta,Div Infect Dis, Sao Paulo, SP, BrazilFAPERJ: E-26/110.315/2014FAPESP: 2010/12891-9CAPES: 23038.010041/2013-13Web of Scienc

An emerging clone, KPC-2-producing Klebsiella pneumoniae ST16, associated with high mortality rates in a CC258 endemic setting

Background Carbapenemase-producing K. pneumoniae have become a global priority, not least in low-middle income countries. Here, we report the emergence and clinical impact of a novel KPC-K. pneumoniae ST16 clone in a Clonal Complex (CC)258 endemic setting. Methods In a teaching Brazilian hospital, a retrospective cohort of adult KPC-KP bloodstream infections (BSI) cases (January 2014 to December 2016) was established to study the molecular epidemiology and its impact on outcome (30-day all-cause mortality). KPC-KP isolates were MLST-typed. Survival analysis between ST/CC groups and risk factors for fatal outcome (logistic regression) were evaluated. Representative isolates underwent whole genome sequencing (WGS), and had their virulence tested in a Galleria larvae model. Results One hundred sixty-five unique KPC-KP BSI cases were identified. CC258 was predominant (66%), followed by ST16 (12%). The overall 30-day mortality rate was 60%; in contrast, 95% of ST16 cases were fatal. Patient’s severity scores were high and baseline clinical variables were not statistically different across ST’s. In multivariate analysis, ST16 (OR 21.4; CI95% 2.3-202.8; p=0,008) and septic shock (OR 11.9; CI95% 4.2-34.1; p<0,001) were independent risk factors for fatal outcome. ST16 clone carried up to 14 resistance genes, including blaKPC-2 in an IncFIBpQIL plasmid, KL51 capsule and Yersiniabactin virulence determinants. ST16 clone was highly pathogenic in the larvae model. Conclusions Mortality rates were high in this KPC-KP BSI cohort, where CC258 is endemic. An emerging ST16 clone was associated with high mortality. Our results suggest that even in endemic settings, highly virulent clones can rapidly emerge demanding constant monitoring

Frequent Tn2 Misannotation in the Genetic Background of rmtB

Univ Fed São Paulo, Escola Paulista Med, Dept Internal Med, Lab Alerta,Div Infect Dis, São Paulo, BrazilUniv Fed São Paulo, Escola Paulista Med, Dept Internal Med, Lab Alerta,Div Infect Dis, São Paulo, BrazilWeb of Scienc

Misidentification of pan drug-resistant Klebsiella pneumoniae clinical isolates as a metallo-beta-lactamase producers by the EDTA/DDST test

Universidade Federal de São Paulo, UNIFESP, Disciplina Infectol, Dept Med, São Paulo, SP, BrazilUniv Fed Santa Catarina, Microbiol Lab, Div Anal Clin, Hosp Univ,UFSC, Florianopolis, SC, BrazilUniversidade Federal de São Paulo, UNIFESP, Disciplina Infectol, Lab ALERTA,Dept Med, São Paulo, SP, BrazilUniversidade Federal de São Paulo, UNIFESP, Disciplina Infectol, Dept Med, São Paulo, SP, BrazilUniversidade Federal de São Paulo, UNIFESP, Disciplina Infectol, Lab ALERTA,Dept Med, São Paulo, SP, BrazilWeb of Scienc