Long-term occurrence of multiple antimicrobial drug resistant Klebsiella pneumoniae isolates harboring virulent potential in a tertiary hospital from Brazil.

Klebsiella pneumoniae strains are globally associated with a plethora of opportunistic and severe human infections and are known to spread genes conferring antimicrobial resistance. Some strains harbor virulence determinants that enable them to cause serious disease in any patient, both in the hospital and in the community. The aim of this study was to determine the frequency of antimicrobial resistance and virulence traits (by gene detection and string test) among 83 K. pneumoniae isolates obtained from patient cultures of a scholar tertiary hospital in the Midwestern Brazil (Brasília, DF). Antimicrobial susceptibility analysis showed that 94% (78/83) of the isolates presented one of the following resistance profiles: resistant (R, 39), multidrug-resistant (MDR, 29), or extensively drug-resistant (XDR, 10). Several MDR and XDR strains harbored multiple virulence genes and displayed hypermucoviscous phenotype. These characteristics were observed among isolates obtained throughout all the sample collection period (2013 - 2017). The K2 serotype gene, a molecular marker of hypervirulence, was detected in three isolates, one of which classified as XDR. Sequence typing revealed the occurrence of isolates belonged to high-risk (ST13) and multiple resistance-spreading clones (ST105). Thus, our findings showed the occurrence of virulent potential isolates that also presented MDR/XDR phenotypes from 2013 to 2015. This study also indicates the probable convergence of virulence and resistance since at least 2013 in Brazil.

fim3-24/ptxP-3 genotype is associated to whooping cough outbreak in Brazilian Midwest: The selection of Bordetella pertussis strains driven by vaccine immunization.

Whopping cough (or Pertussis) is an acute infectious respiratory disease caused by Bordetella pertussis bacteria. The disease is highly transmissible and can be fatal in children under two years old. Since the introduction of vaccine immunization in 1940, Pertussis incidence decreased worldwide. In Brazil, the immunization was introduced in 1977 using the whole cell (wP) vaccine. Despite the high vaccination coverage, an unexpected increase in the number of observed Pertussis cases was observed in 2012. In this year, 2257 cases were reported exceeding the average incidence rate of <1000 cases per year until 2010. This outbreak reached a peak level in 2014 and ended in 2018 according to the Brazilian National Surveillance System (SINAN). To understand the relationship between the outbreak and the vaccination, bacterial isolates (n = 136) from the Brazilian Midwest region obtained during the outbreak were submitted to genotyping of two vaccine loci: ptxP and fim3. Most of isolates (102) were obtained from nursing children (29 days to 2 years old). Genotyping of 94 isolates revealed that fim3-24/ptxP-3 was the most prevalent genotype (68%) associated with the outbreak peak. Two additional genotypes were also observed: fim3-1/ptxP-3 (15%) and fim3-3/ptxP-3 (17%). Conversely, the fim3-1/ptxP-2 genotype, which is harbored by the strain used in the wP vaccine (Bp137), was not observed. These results showed that B. pertussis circulating strains in the outbreak analyzed were different from the strain used for Pertussis immunization in Brazil. These observations provide insights that could be used to target vaccination programs to prevent future whooping cough outbreaks in Brazil.

Scalable and versatile container-based pipelines for de novo genome assembly and bacterial annotation.

Background: Advancements in DNA sequencing technology have transformed the field of bacterial genomics, allowing for faster and more cost effective chromosome level assemblies compared to a decade ago. However, transforming raw reads into a complete genome model is a significant computational challenge due to the varying quality and quantity of data obtained from different sequencing instruments, as well as intrinsic characteristics of the genome and desired analyses. To address this issue, we have developed a set of container-based pipelines using Nextflow, offering both common workflows for inexperienced users and high levels of customization for experienced ones. Their processing strategies are adaptable based on the sequencing data type, and their modularity enables the incorporation of new components to address the community's evolving needs. Methods: These pipelines consist of three parts: quality control, de novo genome assembly, and bacterial genome annotation. In particular, the genome annotation pipeline provides a comprehensive overview of the genome, including standard gene prediction and functional inference, as well as predictions relevant to clinical applications such as virulence and resistance gene annotation, secondary metabolite detection, prophage and plasmid prediction, and more. Results: The annotation results are presented in reports, genome browsers, and a web-based application that enables users to explore and interact with the genome annotation results. Conclusions: Overall, our user-friendly pipelines offer a seamless integration of computational tools to facilitate routine bacterial genomics research. The effectiveness of these is illustrated by examining the sequencing data of a clinical sample of Klebsiella pneumoniae.

Outbreak of extensively drug-resistant Serratia marcescens in an intensive care unit.

We present the investigation and control of an extensively drug-resistant Serratia marcescens outbreak in a 30-bed intensive care unit (ICU). Within 6 weeks, 4 critically ill trauma patients were infected by the same strain. Intensive containment measures limited the spread of this strain while sustaining the capacity of the trauma ICU.

A Novel Multidrug Resistant, Non-Tn4401 Genetic Element-Bearing, Strain of Klebsiella pneumoniae Isolated From an Urban Lake With Drinking and Recreational Water Reuse.

Antimicrobial resistance (AMR) is an increasing and urgent issue for human health worldwide, as it leads to the reduction of available antibiotics to treat bacterial infections, in turn increasing hospital stays and lethality. Therefore, the study and genomic surveillance of bacterial carriers of resistance in and outside of clinical settings is of utter importance. A colony of multidrug resistant (MDR) bacteria identified as Klebsiella spp., by 16S rDNA amplicon sequencing, has been isolated from an urban lake in Brazil, during a drug-degrading bacterial prospection. Genomic analyses revealed the bacteria as Klebsiella pneumoniae species. Furthermore, the in silico Multilocus Sequence Typing (MLST) identified the genome as a new sequence type, ST5236. The search for antimicrobial resistance genes (ARGs) detected the presence of genes against beta-lactams, fosfomycin, acriflavine and efflux pumps, as well as genes for heavy metal resistance. Of particular note, an extended-spectrum beta-lactamase gene (blaCTX-M-15) has been detected in close proximity to siphoviridae genes, while a carbapenemase gene (KPC-2) has been found in an extrachromosomal contig, within a novel non-Tn4401 genetic element (NTEKPC). An extrachromosomal contig found in the V3 isolate is identical to a contig of a K. pneumoniae isolate from a nearby hospital, which indicates a putative gene flow from the hospital network into Paranoá lake. The discovery of a MDR isolate in this lake is worrisome, as the region has recently undergone periods of water scarcity causing the lake, which receives treated wastewater effluent, and is already used for recreational purposes, to be used as an environmental buffer for drinking water reuse. Altogether, our results indicate an underrepresentation of environmental K. pneumoniae among available genomes, which may hamper the understanding of the population dynamics of the species in the environment and its consequences in the spread of ARGs and virulence genes.

Multidrug-Resistant (MDR) Klebsiella variicola Strains Isolated in a Brazilian Hospital Belong to New Clones.

Klebsiella variicola is mainly associated with opportunistic infections and frequently identified as Klebsiella pneumoniae. This misidentification implies a wrong epidemiology result as well as incorrect attribution to K. pneumoniae as the etiology of some severe infections. Recently, huge efforts have been made to study K. variicola, however, the biological aspects of this species are still unclear. Here we characterized five K. variicola strains initially identified as K. pneumoniae, with a Vitek-2 System and 16S rRNA sequencing. One-step multiplex polymerase chain reaction and Whole Genome Sequencing (WGS) identified them as K. variicola. Additionally, WGS analysis showed that all the strains are closely related with K. variicola genomes, forming a clustered group, apart from K. pneumoniae and K. quasipneumoniae. Multilocus sequence typing analysis showed four different sequence types (STs) among the strains and for two of them (Kv97 and Kv104) the same ST was assigned. All strains were multidrug-resistant (MDR) and three showed virulence phenotypes including invasion capacity to epithelial cells, and survival in human blood and serum. These results showed the emergence of new K. variicola clones with pathogenic potential to colonize and cause infection in different tissues. These characteristics associated with MDR strains raise great concern for human health.

High Level of Resistance to Antimicrobials and Heavy Metals in Multidrug-Resistant Pseudomonas sp. Isolated from Water Sources.

Antimicrobial-resistant bacteria (ARB) carrying antimicrobial resistance genes (ARGs) have been increasingly detected in water sources. Pseudomonas sp. are opportunistic pathogens commonly reported in water samples and different antimicrobial resistance mechanisms have been described in Pseudomonas sp., producing multidrug-resistant (MDR) phenotype. Besides, the presence of heavy metal resistance genes (HMRGs) may select ARB, which is worrying. Therefore, this study aimed to characterize the genotypes of Pseudomonas sp. isolated from different water sources. Water samples (i.e., rivers, streams, lakes and sewage treatment plants) were collected from different cities in Brazil. The bacterial identification was performed by sequencing the 16S rDNA and the resistance profile to antimicrobials and heavy metals were determined by minimum inhibitory concentration (MIC). Several ARGs, HMRGs, and plasmids were researched by PCR and the amplicons were sequenced for confirmation. A total of 23 Pseudomonas sp. isolates were obtained and identified as Pseudomonas saponiphila, Pseudomonas hunanensis, Pseudomonas aeruginosa, and Pseudomonas asiatica. These isolates presented high MICs to antimicrobials and heavy metals, being the great majority (n = 21; 91%) classified as MDR. Different clinically important ARGs were detected, such as blaGES, qnrS, qepA, tetB, aac(3')-IIa, and ant(2″)-Ia. The czcA gene was the only HMRG detected and no plasmids were found. To the best of our knowledge, this is the first report of the world of P. saponiphila carrying ARGs (i.e., blaGES, qnrS, aac(3')-IIa, tetB) and QepA-producing P. hunanensis and the first time of P. saponiphila, P. asiatica, and P. hunanensis in Brazil.

Genomic Characterization of a Multidrug-Resistant and Hypermucoviscous/Hypervirulent Klebsiella quasipneumoniae subsp. similipneumoniae ST4417 Isolated from a Sewage Treatment Plant.

Aim: Clinical strains of Klebsiella quasipneumoniae subsp. similipneumoniae have been reported worldwide. Multidrug-resistant (MDR) hypermucoviscous (hm)/hypervirulent (hv) lineages have become a global problem for public health worldwide. Therefore, this study aimed to characterize by whole-genome sequencing a MDR-hm/hv K. quasipneumoniae subsp. similipneumoniae SWT10 strain belonging to the new sequence type ST4417 isolated from a sewage treatment plant. Results: The SWT10 strain was recovered from a sewage treatment plant in Brazil and presented the hm and MDR phenotypes. Resistome analysis showed antimicrobial resistance genes associated with resistance to fluoroquinolones, ß-lactams, tetracyclines, trimethoprim, aminoglycosides, sulfonamides, macrolides, and fosfomycin as well as several heavy metal resistance genes. Virulome analysis showed virulence factors related to hv lineages. Multilocus sequence typing analysis revealed the new ST4417, which was grouped in CC1584 by the minimum-spanning tree. Besides, five plasmid incompatibility groups, two prophage-related sequences, and 66 genomic islands were detected. Conclusion: This study reports for the first time the genome sequence of a MDR-hm/hv K. quasipneumoniae subsp. similipneumoniae recovered from the environment, which contributes to a better understanding about these lineages as well as for surveillance studies worldwide.

RR2 dye adsorption to Hymenaea courbaril L. bark activated carbon associated with biofilm.

This study addressed the removal performance of RR2 from aqueous solutions in adsorption columns experiments by comparing the potential of activated carbon alone (ACA) and microbially inoculated (MIAC), prepared from barks of a largely available tree in Brazilian Cerrado biome, Hymenaea courbaril L. or "Jatobá," presenting the kinetics, isotherms, breakthrough curves, and dissolved organic carbon removal. ACA presented strong interaction to RR2 dye, evidenced at the first 20 min when absorbance already attained 66.4%. The removal percentage gradually increased with time and the equilibrium occurred around 91.7% within 120 min. Langmuir model best fitted the isotherm data, indicating a maximum adsorption capacity of 4.068 mg g-1 for the amount of 0.5 g of adsorbent. The Langmuir's model parameters KL, RL, and R2 corresponded to 0.0234 L mg-1, 0.4159, and 0.9663, respectively, indicating a favorable adsorption process (0 < RL < 1). The experiments in adsorption columns revealed maximum adsorption capacities of 14.38 and 11.43 mg g-1 for MIAC and ACA, respectively, where the microbial activity favorably retarded the adsorption breakpoint in approximately 20 min and enhanced the RR2 consumption in 25.8%. Effectiveness of DOC removal attained above 90% for both ACA and MIAC, reducing the content from 86.1 to 7.84 mg L-1 and 4.82 mg L-1, respectively.

A Fatal Bacteremia Caused by Hypermucousviscous KPC-2 Producing Extensively Drug-Resistant K64-ST11 Klebsiella pneumoniae in Brazil.

We report a fatal bacteremia caused by Klebsiella pneumoniae in a 60-70-year-old patient from Brazil. The genomic analysis of three isolates (from blood culture, nasal and anal swabs) showed that the bacteremia was caused by a KPC-2 producing extensively drug-resistant K64-ST11 hypermucousviscous K. pneumoniae (hmKP) harboring several virulence and antimicrobial resistance genes. Although the isolates did not present virulence markers associated with hypervirulent K. pneumoniae (hvKP), they showed invasion and toxicity to epithelial Hep-2 cells; resistance to cell microbicidal mechanisms; and blood and human serum survival, evidencing their pathogenic potential. This study highlights the risk of infection caused by hmKp strains not characterized as hvKP as well as the clinical implications and difficulty of treatment, especially in elderly or immunocompromised patients.

Ciprofloxacin resistance in uropathogenic Escherichia coli isolates causing community-acquired urinary infections in Brasília, Brazil.

OBJECTIVES: Considering the global concern of ciprofloxacin resistance, the aim of this study was to evaluate the characteristics of ciprofloxacin-resistant (CIP-R) Escherichia coli isolated from patients with community-acquired urinary tract infections (UTIs) in Brasília, Brazil. METHODS: CIP-R E. coli isolated from different outpatients between July 2013 and April 2014 in a tertiary hospital were analysed for antibiotic resistance profile, phylotype, uropathogenic E. coli (UPEC) virulence genes, clonal relationship by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), and multilocus sequence typing (MLST). RESULTS: Among the 324 UPEC analysed, 263 (81.2%) were ciprofloxacin-sensitive and 61 (18.8%) were CIP-R. Antibiogram analysis of the 61 CIP-R strains showed that 45 (73.8%) were also multidrug-resistant. The most prevalent phylogroups were A and B2 (26/61 and 18/61, respectively). traT (53/61) and aer-traT (24/61) were the most common gene and genotype observed. Dendrogram analysis found that multidrug resistance and virulence genes were distributed among CIP-R strains independently of clonality and phylogroup. Six ERIC clusters (strains sharing ≥85% genetic similarity) were observed. MLST analysis of all strains of each cluster identified sequence types (STs) associated with worldwide antimicrobial resistance dissemination, including B2-ST131 and ST410, as well as STs not yet associated with antimicrobial resistance propagation, such as ST1725 and ST179. CONCLUSIONS: These results demonstrate that ciprofloxacin resistance dissemination by UPEC causing community-acquired UTIs was associated with multidrug resistance and was promoted by pandemic and non-pandemic STs, a concerning scenario for the local population.

Schistosome-Derived Molecules as Modulating Actors of the Immune System and Promising Candidates to Treat Autoimmune and Inflammatory Diseases.

It is long known that some parasite infections are able to modulate specific pathways of host's metabolism and immune responses. This modulation is not only important in order to understand the host-pathogen interactions and to develop treatments against the parasites themselves but also important in the development of treatments against autoimmune and inflammatory diseases. Throughout the life cycle of schistosomes the mammalian hosts are exposed to several biomolecules that are excreted/secreted from the parasite infective stage, named cercariae, from their tegument, present in adult and larval stages, and finally from their eggs. These molecules can induce the activation and modulation of innate and adaptive responses as well as enabling the evasion of the parasite from host defense mechanisms. Immunomodulatory effects of helminth infections and egg molecules are clear, as well as their ability to downregulate proinflammatory cytokines, upregulate anti-inflammatory cytokines, and drive a Th2 type of immune response. We believe that schistosomes can be used as a model to understand the potential applications of helminths and helminth-derived molecules against autoimmune and inflammatory diseases.

Multidrug resistance dissemination by extended-spectrum ß-lactamase-producing Escherichia coli causing community-acquired urinary tract infection in the Central-Western Region, Brazil.

The aim of this work was to analyse extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli strains isolated from outpatients with signs of cystitis in Hospital Universitário de Brasília (Brasília, Brazil) during the period July 2013 to April 2014. E. coli isolated from urine culture were identified and their antibiotic susceptibility profile was determined by VITEK 2. ESBL-producing strains identified were submitted to PCR for Clermont phylotyping, CTX-M group typing and virulence determinant detection, and clonal relationships were determined by enterobacterial repetitive intergenic consensus (ERIC)-PCR. One strain belonging to each cluster of the dendrogram obtained by ERIC-PCR was selected for multilocus sequence typing (MLST). Among 324 uropathogenic E. coli (UPEC) analysed, 23 (7.1%) were identified as producing ESBL. All ESBL-producing strains were multidrug-resistant (MDR), i.e. presented non-susceptibility to at least one agent in three or more antimicrobial categories. Of the 23 ESBL-producing UPEC strains, 9 were assigned to phylogenetic group B2 and 7 each belonged to phylogenetic groups D and A. Virulence genotyping showed that aer was the most prevalent gene observed among the strains (21/23), followed by traT (18/23), pap (5/23), afa (5/23), PAI (5/23), cnf (3/23) and sfa (1/23). Analysis of the dendrogram showed that multidrug resistance and CTX-M ESBL groups were distributed among all strains, independent of clonality and phylogroup. Sequence types (STs) associated with pandemic resistance clones, such as B2-ST131 and D-ST648, were observed among the isolates. In conclusion, the results showed worrisome evidence of the potential for antibiotic multiresistant dissemination among community-acquired urinary tract infection caused by UPEC.