Evaluation of phenotypic detection of carbapenemase-producing Pseudomonas spp. from clinical isolates.

Carbapenems are considered last-resort antibiotics for the treatment of infections caused by multidrug-resistant Gram-negative bacteria. Although the main mechanism of carbapenem-resistance in Pseudomonas aeruginosa is the loss of OprD porin, carbapenemases continue to be a problem worldwide. The aim of this study was to evaluate the performance of phenotypic tests (Carba NP, Blue Carba, and mCIM/eCIM) for detection of carbapenemase-producing Pseudomonas spp. in Brazil. One hundred twenty-seven Pseudomonas spp. clinical isolates from different Brazilian states were submitted to phenotypic and molecular carbapenemase detection. A total of 90 carbapenemase-producing P. aeruginosa and 5 Pseudomonas putida (35, blaVIM-2; 17, blaSPM-1; 2, blaIMP-10; 1, blaVIM-24; 1, blaNDM-1; 39, blaKPC-2). The phenotypic Carba NP, Blue Carba, and mCIM/eCIM showed sensitivity of 94.7%, 93.6%, and 93.6%, and specificity of 90.6%, 100%, and 96.8%, respectively. However, only the Carba NP presented the highest sensitivity and showed the ability in differentiating the carbapenemases between class A and class B using EDTA. Blue Carba failed to detect most of the class B carbapenemases, having the worst performance using EDTA. Our results show changes in the epidemiology of the spread of carbapenemases and the importance of their detection by phenotypic and genotypic tests. Such, it is essential to use analytical tools that faithfully detect bacterial resistance in vitro in a simple, sensitive, rapid, and cost-effective way. Much effort must be done to improve the current tests and for the development of new ones.

Polymyxin Resistance in Clinical Isolates of K. pneumoniae in Brazil: Update on Molecular Mechanisms, Clonal Dissemination and Relationship With KPC-Producing Strains.

In Brazil, the production of KPC-type carbapenemases in Enterobacteriales is endemic, leading to widespread use of polymyxins. In the present study, 502 Klebsiella pneumoniae isolates were evaluated for resistance to polymyxins, their genetic determinants and clonality, in addition to the presence of carbapenem resistance genes and evaluation of antimicrobial resistance. Resistance to colistin (polymyxin E) was evaluated through initial selection on EMB agar containing 4% colistin sulfate, followed by Minimal Inhibitory Concentration (MIC) determination by broth microdilution. The susceptibility to 17 antimicrobials was assessed by disk diffusion. The presence of blaKPC, blaNDM and blaOXA-48-like carbapenemases was investigated by phenotypic methods and conventional PCR. Molecular typing was performed by PFGE and MLST. Allelic variants of the mcr gene were screened by PCR and chromosomal mutations in the pmrA, pmrB, phoP, phoQ and mgrB genes were investigated by sequencing. Our work showed a colistin resistance frequency of 29.5% (n = 148/502) in K. pneumoniae isolates. Colistin MICs from 4 to >128 µg/mL were identified (MIC50 = 64 µg/mL; MIC90 >128 µg/mL). All isolates were considered MDR, with the lowest resistance rates observed for amikacin (34.4%), and 19.6% of the isolates were resistant to all tested antimicrobials. The blaKPC gene was identified in 77% of the isolates, in consonance with the high rate of resistance to polymyxins related to its use as a therapeutic alternative. Through XbaI-PFGE, 51 pulsotypes were identified. MLST showed 21 STs, with ST437, ST258 and ST11 (CC11) being the most prevalent, and two new STs were determined: ST4868 and ST4869. The mcr-1 gene was identified in 3 K. pneumoniae isolates. Missense mutations in chromosomal genes were identified, as well as insertion sequences in mgrB. Furthermore, the identification of chromosomal mutations in K. pneumoniae isolates belonging from CC11 ensures its success as a high-risk epidemic clone in Brazil and worldwide.

CABGen: A Web Application for the Bioinformatic Analysis of Bacterial Genomes.

Due to recent developments in NGS technologies, genome sequencing is generating large volumes of new data containing a wealth of biological information. Understanding sequenced genomes in a biologically meaningful way and delineating their functional and metabolic landscapes is a first-level challenge. Considering the global antimicrobial resistance (AMR) problem, investments to expand surveillance and improve existing genome analysis technologies are pressing. In addition, the speed at which new genomic data is generated surpasses our capacity to analyze it with available bioinformatics methods, thus creating a need to develop new, user-friendly and comprehensive analytical tools. To this end, we propose a new web application, CABGen, developed with open-source software. CABGen allows storing, organizing, analyzing, and interpreting bioinformatics data in a friendly, scalable, easy-to-use environment and can process data from bacterial isolates of different species and origins. CABGen has three modules: Upload Sequences, Analyze Sequences, and Verify Results. Functionalities include coverage estimation, species identification, de novo genome assembly, and assembly quality, genome annotation, MLST mapping, searches for genes related to AMR, virulence, and plasmids, and detection of point mutations in specific AMR genes. Visualization tools are also available, greatly facilitating the handling of biological data. The reports include those results that are clinically relevant. To illustrate the use of CABGen, whole-genome shotgun data from 181 bacterial isolates of different species collected in 5 Brazilian regions between 2018 and 2020 were uploaded and submitted to the platform's modules.

Description of a novel IncP plasmid harboring blaKPC-2 recovered from a SPM-1-producing Pseudomonas aeruginosa from ST277.

The high rates of carbapenem resistance among Brazilian Pseudomonas aeruginosa isolates are mainly associated with the clone ST277 producing the carbapenemase SPM-1. Here, the complete genetic composition of a IncP plasmid harboring blaKPC-2 in isolates of this endemic clone carrying chromosomal blaSPM-1 was described using whole genome sequencing. These results confirm the association of these two carbapenemases in ST277 and also describe the genetic composition of a novel blaKPC-2-plasmid. Considering the fact that this association occurs in a high-risk clone, monitoring the dissemination of this plasmid should be a public health concern.

Genetic Basis of Antimicrobial Resistant Gram-Negative Bacteria Isolated From Bloodstream in Brazil.

Multidrug-resistant microorganisms are a well-known global problem, and gram-negative bacilli are top-ranking. When these pathogens are associated with bloodstream infections (BSI), outcomes become even worse. Here we applied whole-genome sequencing to access information about clonal distribution, resistance mechanism diversity and other molecular aspects of gram-negative bacilli (GNB) isolated from bloodstream infections in Brazil. It was possible to highlight international high-risk clones circulating in the Brazilian territory, such as CC258 for Klebsiella pneumoniae, ST79 for Acinetobacter baumannii and ST233 for Pseudomonas aeruginosa. Important associations can be made such as a negative correlation between CRISPR-Cas and K. pneumoniae CC258, while the genes bla TEM, bla KPC and bla CTX-M are highly associated with this clone. Specific relationships between A. baumannii clones and bla OXA-51 variants were also observed. All P. aeruginosa ST233 isolates showed the genes bla VIM and bla OXA486. In addition, some trends could be identified, where a new P. aeruginosa MDR clone (ST3079), a novel A. baumannii clonal profile circulating in Brazil (ST848), and important resistance associations in the form of bla VIM-2 and bla IMP-56 being found together in one ST233 strain, stand out. Such findings may help to develop approaches to deal with BSI and even other nosocomial infections caused by these important GNB.

Letter to the Editor: Escherichia fergusonii Harboring IncHI2 Plasmid Containing mcr-1 Gene-A Novel Reservoir for Colistin Resistance in Brazil.

Emergence of colistin-resistant bacteria harboring mobile colistin resistance genes (mcr genes) pose a threat for food-producing animals and humans. In this article, we aim to highlight the emergence of Escherichia fergusonii as an important new reservoir to mcr-1-harboring plasmid in poultry production. Three strains closely related were isolated from cloacal swabs. Their genome contains four plasmids, including a 182,869 bp IncHI2 plasmid harboring the colistin resistance gene mcr-1. These results will contribute to our understanding of plasmid-mediated mcr-1 gene presence and transmission in E. fergusonii.

Exploring the success of Brazilian endemic clone Pseudomonas aeruginosa ST277 and its association with the CRISPR-Cas system type I-C.

BACKGROUND: The Brazilian endemic clone Pseudomonas aeruginosa ST277 carries important antibiotic resistance determinants, highlighting the gene coding for SPM-1 carbapenemase. However, the resistance and persistence of this clone is apparently restricted to the Brazilian territory. To understand the differences between Brazilian strains from those isolated in other countries, we performed a phylogenetic analysis of 47 P. aeruginosa ST277 genomes as well as analyzed the virulence and resistance gene profiles. Furthermore, we evaluated the distribution of genomic islands and assessed in detail the characteristics of the CRISPR-Cas immunity system in these isolates. RESULTS: The Brazilian genomes presented a typical set of resistance and virulence determinants, genomic islands and a high frequency of the CRISPR-Cas system type I-C. Even though the ST277 genomes are closely related, the phylogenetic analysis showed that the Brazilian strains share a great number of exclusively SNPs when compared to other ST277 genomes. We also observed a standard CRISPR spacers content for P. aeruginosa ST277, confirming a strong link between sequence type and spacer acquisition. Most CRISPR spacer targets were phage sequences. CONCLUSIONS: Based on our findings, P. aeruginosa ST277 strains circulating in Brazil characteristically acquired In163 and PAGI-25, which can distinguish them from strains that do not accumulate resistance mechanisms and can be found on the Asian, European and North American continents. The distinctive genetic elements accumulated in Brazilian samples can contribute to the resistance, pathogenicity and transmission success that characterize the ST277 in this country.

Population Structure of KPC-2-Producing Klebsiella pneumoniae Isolated from Surveillance Rectal Swabs in Brazil.

KPC-producing Klebsiella pneumoniae (KPC-Kp) has become an important public health issue. The previous intestinal colonization by KPC-Kp has been an important risk factor associated with the progression to infections. The objective of this study was to assess the genetic characterization of KPC-Kp isolates recovered from human rectal swabs in Brazil. We selected 102 KPC-Kp isolates collected during 2009-2013 in 11 states. Antimicrobial susceptibility was determined by disk diffusion, E-test, and broth microdilution. The resistance and virulence genes were investigated by PCR. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The isolates were mostly resistant to ß-lactams, sulfonamides, chloramphenicol, quinolones, and aminoglycosides but susceptible to fosfomycin/trometamol, polymyxin B, and tigecycline. The blaKPC-2 was mostly associated with Tn4401b. Besides that, the isolates carried blaCTX-M, blaSHV, blaTEM, and aac(6')-Ib in high frequency and aac(3')IIa and qnr genes in moderate frequency. The PFGE revealed 26 pulsotypes and MLST performed in representative strains revealed 23 sequence types, 45% belonging to clonal complex 258 (CC258). Isolates of CC258 were found in all states. Seventy percent of the 102 KPC-Kp isolates belonged to CC258-associated pulsotypes. We describe the dissemination of KPC-2-Kp associated with Tn4401b belonging to CC258 colonizing patients in Brazil, which is also prevalent in infected patients, suggesting a clear colonization-infection correlation.

Systematic Identification and Classification of ß-Lactamases Based on Sequence Similarity Criteria: ß-Lactamase Annotation.

ß-lactamases, the enzymes responsible for resistance to ß-lactam antibiotics, are widespread among prokaryotic genera. However, current ß-lactamase classification schemes do not represent their present diversity. Here, we propose a workflow to identify and classify ß-lactamases. Initially, a set of curated sequences was used as a model for the construction of profiles Hidden Markov Models (HMM), specific for each ß-lactamase class. An extensive, nonredundant set of ß-lactamase sequences was constructed from 7 different resistance proteins databases to test the methodology. The profiles HMM were improved for their specificity and sensitivity and then applied to fully assembled genomes. Five hierarchical classification levels are described, and a new class of ß-lactamases with fused domains is proposed. Our profiles HMM provide a better annotation of ß-lactamases, with classes and subclasses defined by objective criteria such as sequence similarity. This classification offers a solid base to the elaboration of studies on the diversity, dispersion, prevalence, and evolution of the different classes and subclasses of this critical enzymatic activity.

Genomic analysis of bifunctional Class C-Class D ß-lactamases in environmental bacteria.

ß-lactamases, which are found in several bacterial species and environments, are the main cause of resistance to ß-lactams in Gram-negative bacteria. In 2009, a protein (LRA-13) with two ß-lactamase domains (one class C domain and one class D domain) was experimentally characterised, and an extended action spectrum against ß-lactams consistent with two functional domains was found. Here, we present the results of searches in the non-redundant NCBI protein database that revealed the existence of a group of homologous bifunctional ß-lactamases in the genomes of environmental bacteria. These findings suggest that bifunctional ß-lactamases are widespread in nature; these findings also raise concern that bifunctional ß-lactamases may be transferred to bacteria of clinical importance through lateral gene transfer mechanisms.

Mining of potential drug targets through the identification of essential and analogous enzymes in the genomes of pathogens of Glycine max, Zea mays and Solanum lycopersicum.

Pesticides are one of the most widely used pest and disease control measures in plant crops and their indiscriminate use poses a direct risk to the health of populations and environment around the world. As a result, there is a great need for the development of new, less toxic molecules to be employed against plant pathogens. In this work, we employed an in silico approach to study the genes coding for enzymes of the genomes of three commercially important plants, soybean (Glycine max), tomato (Solanum lycopersicum) and corn (Zea mays), as well as 15 plant pathogens (4 bacteria and 11 fungi), focusing on revealing a set of essential and non-homologous isofunctional enzymes (NISEs) that could be prioritized as drug targets. By combining sequence and structural data, we obtained an initial set of 568 cases of analogy, of which 97 were validated and further refined, revealing a subset of 29 essential enzymatic activities with a total of 119 different structural forms, most belonging to central metabolic routes, including the carbohydrate metabolism, the metabolism of amino acids, among others. Further, another subset of 26 enzymatic activities possess a tertiary structure specific for the pathogen, not present in plants, men and Apis mellifera, which may be of importance for the development of specific enzymatic inhibitors against plant diseases that are less harmful to humans and the environment.

Description of genomic islands associated to the multidrug-resistant Pseudomonas aeruginosa clone ST277.

Multidrug-resistant Pseudomonas aeruginosa clone ST277 is disseminated in Brazil where it is mainly associated with the presence of metallo-ß-lactamase SPM-1. Furthermore, it carries the class I integron In163 and a 16S rRNA methylase rmtD that confers aminoglycoside resistance. To analyze the genetic characteristics that might be responsible for the success of this endemic clone, genomes of four P. aeruginosa strains that were isolated in distinct years and in different Brazilian states were sequenced. The strains differed regarding the presence of the genes blaSPM-1 and rmtD. Genomic comparisons that included genomes of other clones that have spread worldwide from this species were also performed. These analyses revealed a 763,863bp region in the P. aeruginosa chromosome that concentrates acquired genetic structures comprising two new genomic islands (PAGI-13 and PAGI-14), a mobile element that could be used for ST277 fingerprinting and a recently reported Integrative and Conjugative Element (ICE) associated to blaSPM-1. The genetic elements rmtD and In163 are inserted in PAGI-13 while PAGI-14 has genes encoding proteins related to type III restriction system and phages. The data reported in this study provide a basis for a clearer understanding of the genetic content of clone ST277 and illustrate the mechanisms that are responsible for the success of these endemic clones.

Draft genome sequence of a multidrug-resistant Acinetobacter baumannii ST15 (CC15) isolated from Brazil

Acinetobacter baumannii is an important pathogen frequently associated with nosocomial outbreaks around the world. In Brazil, A. baumannii has become particularly problematic because of its prevalence and the carbapenems resistance. Here, we report the draft genome sequence of a multidrug-resistant A. baumannii(ST15/CC15) isolated in 2009 from the state of Espírito Santo (Southeast Brazil). We observed important resistance determinant genes in an estimated genome size of 4,102,788 bp with 3,862 predicted coding regions. A detailed report of the genomic data analysis might help to understand the specific features of highly successful strains belonged to a relevant complex clonal in different Brazilian geographical regions.

Draft genome sequence of a multidrug-resistant Acinetobacter baumannii ST15 (CC15) isolated from Brazil.

Acinetobacter baumannii is an important pathogen frequently associated with nosocomial outbreaks around the world. In Brazil, A. baumannii has become particularly problematic because of its prevalence and the carbapenems resistance. Here, we report the draft genome sequence of a multidrug-resistant A. baumannii(ST15/CC15) isolated in 2009 from the state of Espírito Santo (Southeast Brazil). We observed important resistance determinant genes in an estimated genome size of 4,102,788 bp with 3,862 predicted coding regions. A detailed report of the genomic data analysis might help to understand the specific features of highly successful strains belonged to a relevant complex clonal in different Brazilian geographical regions.

Coproduction of NDM-1 and KPC-2 in Enterobacter hormaechei from Brazil.

The most important resistance mechanism against ß-lactam drugs is the production of carbapenemases. In this study, we report the first identification of Klebsiella pneumoniae carbapenemase (KPC)-2 and New Delhi metallo-ß-lactamase (NDM)-1 in Enterobacter hormaechei subps. oharae from Brazil. The detection of carbapenemases was done by phenotypic assays, PCR, and DNA sequencing, whereas the identification was performed by conventional techniques, sequencing of the 16S rDNA gene, and hsp60-genotyping. Molecular typing was performed using pulsed-field gel electrophoresis, and antimicrobial susceptibility was surrogated by the Etest methodology. Using the whole genome sequencing approach, we searched for resistance genes, plasmid incompatibility group genes, and the genetic environment of blaNDM and blaKPC. The plasmid identification was done by restriction digests with the S1 nuclease followed by hybridization using digoxigenin labeled specific probes. The isolate was considered multiresistant, being susceptible to amikacin and polymyxin B. We observed the following resistance genes: blaCTX-M-15, blaACT-7, blaTEM-1, blaOXA-1, aadA1, aadA2, strA, strB, aac(3)-II, qnrB1, and aac(6')-Ib-cr and incompatibility group plasmid genes IncA/C, IncHI2, and IncN. The blaKPC gene was found associated to the transposon Tn4401 isoform b in plasmid with 50 kb (IncN) and blaNDM-1 was flanked by a truncated ISAba125 and bleMBL in plasmid with 160 kb (IncA/C). This study showed the coproduction of two important carbapenemases (KPC-2 and NDM-1) associated with mobile genetic elements of worldwide epidemiological importance (Tn4401 and ISAba125, respectively), reinforcing the idea that urgent measures are necessary to reduce and prevent the spreading of these carbapenemases primarily in the hospital settings.

Caracterização do ambiente genético dos genes de resistência a beta-lactâmicos e aminoglicosídeos, blaSPM-1 e rmtD, em amostras de Pseudomonas aeruginosa pertencentes ao clone ST277, epidêmico no Brasil / Characterization of the genetic environment of the beta-lactam and aminoglycoside resistance genes, blaSPM-1 and RMTD on samples of Pseudomonas aeruginosa belonging to clone ST277, epidemic in Brazil

O surgimento de bactérias Gram-negativas resistentes à drogas tem sido progressivo ealarmante. Entre os patógenos de particular importância está Pseudomonas aeruginosamultirresistentes à drogas (MDR). Um clone epidêmico de P. aeruginosa MDR produtor dametalo-beta-lactamase SPM-1, chamado clone SP (ST277), tem sido encontrado em diferentesestados brasileiros. O gene blaSPM-1 foi descrito em uma estrutura genética chamada ISCR4,responsável pela sua mobilização e expressão. Além do gene blaSPM-1, tem sido descrito, nesteclone, a presença de um integron de classe I carreando genes de resistência (In163) e umelemento genético ISCR14 carreando uma metilase de RNAr 16S (rmtD), que confereresistência a altas concentrações de aminoglicosídeos. Este tipo de associação resulta em umperfil de resistência extremamente preocupante. Assim, este estudo teve como objetivoinvestigar a contexto genético dos genes blaSPM-1 e rmtD, além de caracterizar mutações emgenes cromossomais associados a multirresistência a antimicrobianos em amostras de P.aeruginosa pertencentes ao clone ST277, através de Sourthen blot e sequenciamento de novageração. A partir de 50 amostras de P. aeruginosa MDR isoladas entre 2007 e 2010, foramselecionadas 13 amostras pertencentes ao ST277 (através de MLST) que apresentarampositividade para blaSPM-1 e/ou rmtD e In163 (através de PCR), sendo 12 do clone SP (A) euma de um clone diferente, M, através de PFGE. Através de restrição do DNA com asenzimas de restrição SpeI e XbaI, e posterior hibridação com sondas dos genes alvos (blaSPM-1,rmtD e In163) foi possível observar que os genes alvos encontravam-se em fragmentosdistintos e que a amostra do clone M apresentou um perfil de hibridação diferentes das demaisamostras demonstrando a ocorrência de vários rearranjos na mesma, indicando uma maiordistância evolutiva...