Biodiversity of carbapenem-resistant bacteria in clinical samples from the Southwest Amazon region (Rondônia/Brazil).

Brazil is recognized for its biodiversity and the genetic variability of its organisms. This genetic variability becomes even more valuable when it is properly documented and accessible. Understanding bacterial diversity through molecular characterization is necessary as it can improve patient treatment, reduce the length of hospital stays and the selection of resistant bacteria, and generate data for health and epidemiological surveillance. In this sense, in this study, we aimed to understand the biodiversity and molecular epidemiology of carbapenem-resistant bacteria in clinical samples recovered in the state of Rondônia, located in the Southwest Amazon region. Retrospective data from the Central Public Health Laboratories (LACEN/RO) between 2018 and 2021 were analysed using the Laboratory Environment Manager Platform (GAL). Seventy-two species with carbapenem resistance profiles were identified, of which 25 species carried at least one gene encoding carbapenemases of classes A (blaKPC-like), B (blaNDM-like, blaSPM-like or blaVIM-like) and D (blaOXA-23-like, blaOXA-24-like, blaOXA-48-like, blaOXA-58-like or blaOXA-143-like), among which we will highlight Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Serratia marcescens, and Providencia spp. With these results, we hope to contribute to the field by providing epidemiological molecular data for state surveillance on bacterial resistance and assisting in public policy decision-making.

Clonal dissemination of highly virulent Serratia marcescens strains producing KPC-2 in food-producing animals.

Serratia marcescens is a Gram-negative bacterium presenting intrinsic resistance to polymyxins that has emerged as an important human pathogen. Although previous studies reported the occurrence of multidrug-resistance (MDR) S. marcescens isolates in the nosocomial settings, herein, we described isolates of this extensively drug-resistant (XDR) species recovered from stool samples of food-producing animals in the Brazilian Amazon region. Three carbapenem-resistant S. marcescens strains were recovered from stool samples of poultry and cattle. Genetic similarity analysis showed that these strains belonged to the same clone. Whole-genome sequencing of a representative strain (SMA412) revealed a resistome composed of genes encoding resistance to ß-lactams [blaKPC-2, blaSRT-2], aminoglycosides [aac(6')-Ib3, aac(6')-Ic, aph(3')-VIa], quinolones [aac(6')-Ib-cr], sulfonamides [sul2], and tetracyclines [tet(41)]. In addition, the analysis of the virulome demonstrated the presence of important genes involved in the pathogenicity of this species (lipBCD, pigP, flhC, flhD, phlA, shlA, and shlB). Our data demonstrate that food-animal production can act as reservoirs for MDR and virulent strains of S. marcescens.

In Vitro and In Vivo Synergism of Fosfomycin in Combination with Meropenem or Polymyxin B against KPC-2-Producing Klebsiella pneumoniae Clinical Isolates.

Fosfomycin disodium is a potential therapeutic option to manage difficult-to-treat infections, especially when combined with other antimicrobials. In this study, we evaluated the activity of fosfomycin in combination with meropenem or polymyxin B against contemporaneous KPC-2-producing K. pneumoniae clinical isolates (KPC-KPN). Synergistic activity was assessed by checkerboard (CKA) and time-kill (TKA) assays. TKA was performed using serum peak and trough concentrations. The activity of these combinations was also assessed in the Galleria mellonella model. Biofilm disruption was assessed by the microtiter plate technique. CKA resulted in an 8- to 2048-fold decrease in meropenem MIC, restoring meropenem activity for 82.4% of the isolates when combined with fosfomycin. For the fosfomycin + polymyxin B combination, a 2- to 128-fold reduction in polymyxin B MIC was achieved, restoring polymyxin B activity for 47% of the isolates. TKA resulted in the synergism of fosfomycin + meropenem (3.0-6.7 log10 CFU/mL decrease) and fosfomycin + polymyxin B (6.0-6.2 log10 CFU/mL decrease) at peak concentrations. All larvae treated with fosfomycin + meropenem survived. Larvae survival rate was higher with fosfomycin monotherapy (95%) than that observed for fosfomycin + polymyxin B (75%) (p-value < 0.0001). Finally, a higher biofilm disruption was observed under exposure to fosfomycin + polymyxin B (2.4-3.4-fold reduction). In summary, we observed a synergistic effect of fosfomycin + meropenem and fosfomycin + polymyxin B combinations, in vitro and in vivo, against KPC-KPN, as well as biofilm disruption.

Genomic Analysis of Klebsiella pneumoniae ST258 Strain Coproducing KPC-2 and CTX-M-14 Isolated from Poultry in the Brazilian Amazon Region.

This study aimed to characterize a Klebsiella pneumoniae strain (KP411) recovered from the stool samples of poultry (Gallus gallus) in the Brazilian Amazon Region. The whole-genome sequencing of KP411 revealed the presence of an important arsenal of antimicrobial resistance genes to ß-lactams (blaCTX-M-14, blaTEM-1B, blaKPC-2, blaSVH-11), aminoglycosides [aph(3″)- Ib, aph(6)-Id, aph(3')-Ia], sulfonamides (sul1, sul2), quinolones (oqxAB), fosfomycin (fosAKP), and macrolides [mph(A)]. Furthermore, our analyses revealed that the KP411 strain belongs to the ST258 clonal lineage, which is one of the main epidemic clones responsible for the dissemination of KPC-2 worldwide. Our data suggest that food-producing animals may act as reservoirs of multidrug-resistant K. pneumoniae belonging to the ST258 clone, and, consequently, contribute to their dissemination to humans and the environment.

Genome sequencing of an XDR Klebsiella pneumoniae ST101 strain isolated from a Brazilian Amazon river.

OBJECTIVE: Herein, this study aimed to perform the genomic characterization of a blaKPC-2 positive Klebsiella pneumoniae (KP1.1JP) strain isolated from the surface water of river located the Brazilian Amazon region. METHODS: Antimicrobial susceptibility testing was performed following BrCAST/EUCAST recommendations. Genomic DNA was extracted and sequenced using the Illumina® NextSeq platform and the assembly of the generated reads was performed using the SPAdes software. Research on the sequence type, resistance and virulence encoding genes, and plasmid replicon typing was carried out. RESULTS: The KP1.1JP strain was resistant to all ß-lactams, aminoglycosides, and fluoroquinolones tested. The genome size was 5 626 346 bp, distributed in 203 contigs and a guanine and cytosine content of 57.02%. The values of N50 and N75 were 285 583 bp and 173 927 bp, respectively. We verified that KP1.1JP belongs to ST101 and carries genes encoding resistance to ß-lactams (blaCTX-M-15, blaTEM-1B, blaOXA-1, blaSVH-182, and blaKPC-2), aminoglycosides [aac(3')-IIa, aph(3')-Vla], fluoroquinolones [aac(6')-Ib-cr], phenicol (catA1, catA2, catB3), tetracycline [tet(D)], trimethoprim (dfrA14), and fosfomycin (fosA). Additionally, the following virulence encoding genes were also detected: mrkABCDFHIJ (Fimbria type 3); fimABCDRFGHIK (Fimbria type 1); entABCDEFS and fepABCDG (siderophores); iroN, irp1, and irp2 (salmochelins); fyuA and ybtAEPQSTUX (yersiniabactin); and iutA (aerobactin). CONCLUSIONS: We report the occurrence of a K. pneumoniae ST101 strain carrying blaKPC-2 gene in an Amazon river in Brazil. The genomic characteristics of this strain will contribute to a better understanding of the spread of pathogens of clinical importance in the environment based on a One Health perspective.

Exploring the Bacteriome and Resistome of Humans and Food-Producing Animals in Brazil.

The epidemiology of antimicrobial resistance (AMR) is complex, with multiple interfaces (human-animal-environment). In this context, One Health surveillance is essential for understanding the distribution of microorganisms and antimicrobial resistance genes (ARGs). This report describes a multicentric study undertaken to evaluate the bacterial communities and resistomes of food-producing animals (cattle, poultry, and swine) and healthy humans sampled simultaneously from five Brazilian regions. Metagenomic analysis showed that a total of 21,029 unique species were identified in 107 rectal swabs collected from distinct hosts, the highest numbers of which belonged to the domain Bacteria, mainly Ruminiclostridium spp. and Bacteroides spp., and the order Enterobacterales. We detected 405 ARGs for 12 distinct antimicrobial classes. Genes encoding antibiotic-modifying enzymes were the most frequent, followed by genes related to target alteration and efflux systems. Interestingly, carbapenemase-encoding genes such as blaAIM-1, blaCAM-1, blaGIM-2, and blaHMB-1 were identified in distinct hosts. Our results revealed that, in general, the bacterial communities from humans were present in isolated clusters, except for the Northeastern region, where an overlap of the bacterial species from humans and food-producing animals was observed. Additionally, a large resistome was observed among all analyzed hosts, with emphasis on the presence of carbapenemase-encoding genes not previously reported in Latin America. IMPORTANCE Humans and food production animals have been reported to be important reservoirs of antimicrobial resistance (AMR) genes (ARGs). The frequency of these multidrug-resistant (MDR) bacteria tends to be higher in low- and middle-income countries (LMICs), due mainly to a lack of public health policies. Although studies on AMR in humans or animals have been carried out in Brazil, this is the first multicenter study that simultaneously collected rectal swabs from humans and food-producing animals for metagenomics. Our results indicate high microbial diversity among all analyzed hosts, and several ARGs for different antimicrobial classes were also found. As far as we know, we have detected for the first time ARGs encoding carbapenemases, such as blaAIM-1, blaCAM-1, blaGIM-2, and blaHMB-1, in Latin America. Thus, our results support the importance of metagenomics as a tool to track the colonization of food-producing animals and humans by antimicrobial-resistant bacteria. In addition, a network surveillance system called GUARANI, created for this study, is ready to be expanded and to collect additional data.

Evaluation of the Pathogenic Potential of Escherichia coli Strains Isolated from Eye Infections.

While primarily Gram-positive bacteria cause bacterial eye infections, several Gram-negative species also pose eye health risks. Currently, few studies have tried to understand the pathogenic mechanisms involved in E. coli eye infections. Therefore, this study aimed to establish the pathogenic potential of E. coli strains isolated from eye infections. Twenty-two strains isolated between 2005 and 2019 from patients with keratitis or conjunctivitis were included and submitted to traditional polymerase chain reactions (PCR) to define their virulence profile, phylogeny, clonal relationship, and sequence type (ST). Phenotypic assays were employed to determine hemolytic activity, antimicrobial susceptibility, and adhesion to human primary corneal epithelial cells (PCS-700-010). The phylogenetic results indicated that groups B2 and ST131 were the most frequent. Twenty-five virulence genes were found among our strains, with ecp, sitA, fimA, and fyuA being the most prevalent. Two strains presented a hemolytic phenotype, and resistance to ciprofloxacin and ertapenem was found in six strains and one strain, respectively. Regarding adherence, all but one strains adhered in vitro to corneal cells. Our results indicate significant genetic and virulence variation among ocular strains and point to an ocular pathogenic potential related to multiple virulence mechanisms.

Comparative Phenotypic and Genomic Features of Staphylococci from Sonication Fluid of Orthopedic Implant-Associated Infections with Poor Outcome.

Staphylococcus spp. remain the leading biofilm-forming agents causing orthopedic implant-associated infections (OIAI). This is a descriptive study of phenotypic and genomic features identified in clinical isolates of S. aureus and coagulase-negative Staphylococcus (CoNS) recovered from OIAIs patients that progressed to treatment failure. Ten isolates were identified by matrix-time-of-flight laser-assisted desorption mass spectrometry (MALDI-TOF-MS) and tested for antibiotic susceptibility and biofilm formation. Genotypic characteristics, including, MLST (Multi Locus Sequence Typing), SCCmec typing, virulence and resistance genes were assessed by whole-genome sequencing (WGS). All S. aureus harbored mecA, blaZ, and multiple resistance genes for aminoglycosides and quinolones. All MRSA were strong biofilm producers harboring the complete icaADBC and icaR operon. Seven CoNS isolates comprising five species (S. epidermidis, S. haemolyticus, S. sciuri, S. capitis and S. lugdunensis) were analyzed, with mecA gene detected in five isolates. S. haemolitycus (isolate 95), and S. lugdunensis were unable to form biofilm and did not harbor the complete icaADBCR operon. High variability of adhesion genes was detected, with atl, ebp, icaADBC operon, and IS256 being the most common. In conclusion, MRSA and CoNS isolates carrying genes for biofilm production, and resistance to ß-lactam and aminoglycosides are associated with treatment failure in OIAIs.

Large Scale Genome-Centric Metagenomic Data from the Gut Microbiome of Food-Producing Animals and Humans.

The One Health concept is a global strategy to study the relationship between human and animal health and the transfer of pathogenic and non-pathogenic species between these systems. However, to the best of our knowledge, no data based on One Health genome-centric metagenomics are available in public repositories. Here, we present a dataset based on a pilot-study of 2,915 metagenome-assembled genomes (MAGs) of 107 samples from the human (N = 34), cattle (N = 28), swine (N = 15) and poultry (N = 30) gut microbiomes. Samples were collected from the five Brazilian geographical regions. Of the draft genomes, 1,273 were high-quality drafts (≥90% of completeness and ≤5% of contamination), and 1,642 were medium-quality drafts (≥50% of completeness and ≤10% of contamination). Taxonomic predictions were based on the alignment and concatenation of single-marker genes, and the most representative phyla were Bacteroidota, Firmicutes, and Proteobacteria. Many of these species represent potential pathogens that have already been described or potential new families, genera, and species with potential biotechnological applications. Analyses of this dataset will highlight discoveries about the ecology and functional role of pathogens and uncultivated Archaea and Bacteria from food-producing animals and humans. Furthermore, it also represents an opportunity to describe new species from underrepresented taxonomic groups.

Decreased susceptibility to imipenem and ceftazidime in early virulent Raoultella spp. strains retrieved from human intestinal infections.

The genus Raoultella spp. is comprised of four species, namely, R. electrica, R. ornithinolytica, R. planticola, and R. terrigena, which are rarely reported to cause infections in humans. This study aimed to characterize six strains of Raoultella spp. isolated from stool samples from patients with diarrhea. The strains included in the study were previously identified by biochemical methods as K. pneumoniae, during a surveillance study conducted in 1987. In the present study, the strains were re-identified by MALDI TOF and 16S rRNA sequencing and subsequently subjected to virulence gene screening by PCR, hemolytic activity, biofilm formation, hypermucoviscosity phenotype, capacity to interact with Caco-2 cells, and antimicrobial susceptibility test. Our results revealed that, among the six strains, three were identified as R. ornithinolytica and three as R. planticola. The genes related to iron uptake systems (aero1, aero2, iutA, entB, and ybtS) and adhesin (mrkD) were found in all strains. Furthermore, all strains demonstrated the ability to interact in vitro with Caco-2 cells and form biofilms. In general, the strains studied were sensitive to the antimicrobials tested; however, it was possible to observe high MICs for imipenem compared to ertapenem and meropenem and high minimal inhibitory concentrations (MICs) for ceftazidime, except for one strain. Our results show the occurrence of virulent strains of Raoultella spp. with high MICs for imipenem and ceftazidime causing diarrhea. We hope that our findings can contribute to the understanding of the evolution of this species since, as far as we know, these are the oldest isolates reported so far.

Unveiling the Virulent Genotype and Unusual Biochemical Behavior of Escherichia coli ST59.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a leading cause of human and animal infections worldwide. The utilization of selective and differential media to facilitate the isolation and identification of E. coli from complex samples, such as water, food, sediment, and gut tissue, is common in epidemiological studies. During a surveillance study, we identified an E. coli strain isolated from human blood culture that displayed atypical light cream-colored colonies in chromogenic agar and was unable to produce ß-glucuronidase and ß-galactosidase in biochemical tests. Genomic analysis showed that the strain belongs to sequence type 59 (ST59) and phylogroup F. The evaluation in silico of 104 available sequenced lineages of ST59 complex showed that most of them belong to serotype O1:K1:H7, are ß-glucuronidase negative, and harbor a virulent genotype associated with the presence of important virulence markers such as pap, kpsE, chuA, fyuA, and yfcV. Most of them were isolated from extraintestinal human infections in diverse countries worldwide and could be clustered/subgrouped based on papAF allele analysis. Considering that all analyzed strains harbor a virulent genotype and most do not exhibit biochemical behavior typical of E. coli, we report that they could be misclassified or underestimated, especially in epidemiological studies where the screening criteria rely only on typical biochemical phenotypes, as happens when chromogenic media are used. IMPORTANCE The use of selective and differential media guides presumptive bacterial identification based on specific metabolic traits that are specific to each bacterial species. When a bacterial specimen displays an unusual phenotype in these media, this characteristic may lead to bacterial misidentification or a significant delay in its identification, putting a patient at risk depending on the infection type. In the present work, we describe a virulent E. coli sequence type (ST59) that does not produce beta-glucuronidase (GUS negative), production of which is the metabolic trait widely used for E. coli presumptive identification in diverse differential media. The recognition of this unusual metabolic trait may help in the proper identification of ST59 isolates, the identification of their reservoir, and the evaluation of the frequency of these pathogens in places where automatic identification methods are not available.

Draft Whole-Genome Sequence of a Uropathogenic Escherichia coli Strain Carrying the eae Gene.

Uropathogenic Escherichia coli (UPEC) strains are responsible for most cases of urinary tract infections worldwide. We present the draft whole-genome sequence of the UPEC 252 strain, which carries the eae gene that encodes the intimin adhesin. Intimin promotes intimate adherence of enteropathogenic E. coli and enterohemorrhagic E. coli to intestinal cells.

Draft whole-genome sequence of a uropathogenic Escherichia coli strain carrying the eae gene

Uropathogenic Escherichia coli (UPEC) strains are responsible for most cases of urinary tract infections worldwide. We present the draft whole-genome sequence of the UPEC 252 strain, which carries the eae gene that encodes the intimin adhesin. Intimin promotes intimate adherence of enteropathogenic E. coli and enterohemorrhagic E. coli to intestinal cells.

Draft whole-genome sequence of a uropathogenic Escherichia coli strain carrying the eae gene

Uropathogenic Escherichia coli (UPEC) strains are responsible for most cases of urinary tract infections worldwide. We present the draft whole-genome sequence of the UPEC 252 strain, which carries the eae gene that encodes the intimin adhesin. Intimin promotes intimate adherence of enteropathogenic E. coli and enterohemorrhagic E. coli to intestinal cells.

Pesquisa de Salmonella spp. e Listeria Monocytogenes em Saladas Contendo Maionese Comercializadas em Restaurantes Localizados no Município de JI ­ Paraná, Rondônia, Brasil

O número de pessoas que realizam suas refeições em restaurantes tem aumentado exponencialmente, sendo preocupante a qualidade dos alimentos servidos, uma vez que, quando contaminados, podem acarretar graves surtos de Doenças Transmitidas por Alimentos. O presente estudo teve por objetivo analisar a presença de Salmonella spp. e Listeria monocytogenes em saladas contendo maionese servidas em restaurantes no município de Ji-Paraná, Rondônia. Foram coletadas 20 amostras de cinco restaurantes, sendo 4 amostras de cada estabelecimento. Do total de amostras analisadas, 30% apresentaram contaminação por Salmonella spp., não sendo constatada presença de Listeria monocytogenes. A contaminação por Salmonella spp. demonstra a necessidade de maior controle de qualidade no preparo desse alimento e fiscalização por parte dos órgãos responsáveis, tendo em vista o fato deste microrganismo ser o mais associado a surtos de Doenças Transmitidas por Alimento

The number of people eating in restaurants has increased, which concerns the quality of food served, since contaminated food can cause serious foodborne disease outbreaks. This study aimed to analyze the presence of Salmonella spp. and Listeria monocytogenes in salads containing mayonnaise served in restaurants in the city of Ji-Paraná, Rondônia. Twenty samples were collected in five restaurants, 4 from each establishment. Of the total, 30% were contaminated by Salmonella spp., while no contamination was found for Listeria monocytogenes. The contamination by Salmonella spp. demonstrates the need for greater quality control in food preparation, and supervision by the responsible agencies, once Salmonella is the microorganism most associated with foodborne disease outbreaks.