Hybrid genome assembly of colistin-resistant mcr-1.5-producing Escherichia coli ST354 reveals phylogenomic pattern associated with urinary tract infections in Brazil.

BACKGROUND: The rapid and global spread of Escherichia coli carrying mcr-type genes at the human-animal-environmental interface has become a serious global public health problem. OBJECTIVE: To perform a genomic investigation of a colistin-resistant E. coli strain (14005RM) causing urinary tract infection, using a hybrid de novo assembly of Illumina/Nanopore sequence data, presenting phylogenomic insights into the relationship with mcr-1-positive strains circulating at the human-animal-environmental interface, in Brazil. METHODS: Genomic DNA was sequenced using both the Illumina NexSeq and Nanopore MinION platforms. De novo hybrid assembly was performed by Unicycler. Genomic data were assessed by in silico prediction and bioinformatic tools. RESULTS: The genome assembly size was 5 333 039 bp. The mcr-1.5-positive E. coli strain 14005RM belongs to the sequence type ST354 and presented a broad resistome (antibiotics, heavy metals, disinfectants, and glyphosate) and virulome. The mcr-1.5 gene was carried by an IncI2 plasmid (p14005RM, sizing 65,458 kb). Full genome SNP-based phylogenetic analysis reveals that mcr-1.5-producing E. coli strain 14005RM is highly related (> 98% identity) to colistin-resistant mcr-1.1-positive ST354 lineages associated with urinary tract infections in Brazil since 2015. CONCLUSION: Mobile colistin resistance within the Brazilian One Health microbiosphere is mediated by mcr gene variants propagated by IncX4, IncHI2, and IncI2 plasmids, circulating among global clones of E. coli.

Beyond the identifiable proteome: Delving into the proteomics of polymyxin-resistant and non-resistant Acinetobacter baumannii from Brazilian hospitals.

This work discloses a unique, comprehensive proteomic dataset of Acinetobacter baumannii strains, both resistant and non-resistant to polymyxin B, isolated in Brazil generated using Orbitrap Fusion Lumos. From nearly 4 million tandem mass spectra, the software DiagnoMass produced 240,685 quality-filtered mass spectral clusters, of which PatternLab for proteomics identified 44,553 peptides mapping to 3479 proteins. Crucially, DiagnoMass shortlisted 3550 and 1408 unique mass spectral clusters for the resistant and non-resistant strains, respectively, with only about a third with sequences (and PTMs) identified by PatternLab. Further open-search attempts via FragPipe yielded an additional ∼20% identifications, suggesting the remaining unidentified spectra likely arise from complex combinations of post-translational modifications and amino-acid substitutions. This highlights the untapped potential of the dataset for future discoveries, particularly given the importance of PTMs, which remain elusive to nucleotide sequencing approaches but are crucial for understanding biological mechanisms. Our innovative approach extends beyond the identifications that are typically subjected to the bias of a search engine; we discern which spectral clusters are differential and subject them to increased scrutiny, akin to spectral library matching by comparing captured spectra to themselves. Our analysis reveals adaptations in the resistant strain, including enhanced detoxification, altered protein synthesis, and metabolic adjustments. SIGNIFICANCE: We present comprehensive proteomic profiles of non-resistant and resistant Acinetobacter baumannii from Brazilian Hospitals strains, and highlight the presence of discriminative and yet unidentified mass spectral clusters. Our work emphasizes the importance of exploring this overlooked data, as it could hold the key to understanding the complex dynamics of antibiotic resistance. This approach not only informs antimicrobial stewardship efforts but also paves the way for the development of innovative diagnostic tools. Thus, our findings have profound implications for the field, as far as methods for providing a new perspective on diagnosing antibiotic resistance as well as classifying proteomes in general.

A 7-Year Brazilian National Perspective on Plasmid-Mediated Carbapenem Resistance in Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii Complex and the Impact of the Coronavirus Disease 2019 Pandemic on Their Occurrence.

BACKGROUND: Carbapenemase production is a global public health threat. Antimicrobial resistance (AMR) data analysis is critical to public health policy. Here we analyzed carbapenemase detection trends using the AMR Brazilian Surveillance Network. METHODS: Carbapenemase detection data from Brazilian hospitals included in the public laboratory information system dataset were evaluated. The detection rate (DR) was defined as carbapenemase detected by gene tested per isolate per year. The temporal trends were estimated using the Prais-Winsten regression model. The impact of COVID-19 on carbapenemase genes in Brazil was determined for the period 2015-2022. Detection pre- (October 2017 to March 2020) and post-pandemic onset (April 2020 to September 2022) was compared using the χ2 test. Analyses were performed with Stata 17.0 (StataCorp, College Station, TX). RESULTS: 83 282 blaKPC and 86 038 blaNDM were tested for all microorganisms. Enterobacterales DR for blaKPC and blaNDM was 68.6% (41 301/60 205) and 14.4% (8377/58 172), respectively. P. aeruginosa DR for blaNDM was 2.5% (313/12 528). An annual percent increase for blaNDM of 41.1% was observed, and a decrease for blaKPC of -4.0% in Enterobacterales, and an annual increase for blaNDM of 71.6% and for blaKPC of 22.2% in P. aeruginosa. From 2020 to 2022, overall increases of 65.2% for Enterobacterales, 77.7% for ABC, and 61.3% for P. aeruginosa were observed in the total isolates. CONCLUSIONS: This study shows the strengths of the AMR Brazilian Surveillance Network with robust data related to carbapenemases in Brazil and the impact of COVID-19 with a change in carbapenemase profiles with blaNDM rising over the years.

Increased Detection of Carbapenemase-Producing Enterobacterales Bacteria in Latin America and the Caribbean during the COVID-19 Pandemic.

During 2020-2021, countries in Latin America and the Caribbean reported clinical emergence of carbapenemase-producing Enterobacterales that had not been previously characterized locally, increased prevalence of carbapenemases that had previously been detected, and co-production of multiple carbapenemases in some isolates. These increases were likely fueled by changes related to the COVID-19 pandemic, including empirical antibiotic use for potential COVID-19-related bacterial infections and healthcare limitations resulting from the rapid rise in COVID-19 cases. Strengthening antimicrobial resistance surveillance, epidemiologic research, and infection prevention and control programs and antimicrobial stewardship in clinical settings can help prevent emergence and transmission of carbapenemase-producing Enterobacterales.

Escherichia coli ST224 and IncF/blaCTX-M-55 plasmids drive resistance to extended-spectrum cephalosporins in poultry flocks in Parana, Brazil.

Resistant Enterobacterales of avian intestinal origin can contaminate carcasses during broiler processing and thereby spread through the human food chain. This study aimed at assessing the prevalence, diversity and genomic characteristics of ESBL/AmpC Enterobacterales in poultry flocks from different farms and cities in the state of Paraná, Brazil. Enterobacterales isolated from cloacal samples were subjected to antimicrobial susceptibility testing (AST). ESBL/AmpC isolates were whole-genome sequenced and subjected to S1-nuclease pulsed-field gel electrophoresis (S1-PFGE) followed by Southern blotting to determine the location of resistant genes on plasmids. A surprisingly high proportion of E. coli (40.6 %) collected on non-selective plates presented an ESBL/AmpC phenotype. Multidrug resistance was statistically not higher in ESBL/AmpC E. coli having the potential to be Avian Pathogenic (APEC-like) compared to non-APEC-like ESBL/AmpC E. coli isolates. Resistance to antibiotics not authorized for use in poultry in the State of Paraná was observed, suggesting that antimicrobial resistance (AMR) is co-selected by the use of veterinary-licensed antibiotics. Phylogenetic analyzes revealed the presence of identical or highly similar ESBL/AmpC E. coli clones on farms distant up to 100 km of each other; this strongly suggests that the centralization and verticalization of the poultry industry can facilitate the spread of resistant bacteria among different farms, companies, and cities. The molecular characterization of clones and plasmids proved the dominance of the ST224 E. coli lineage and the IncF/blaCTX-M-55 plasmid, possibly indicating the emergence of successful clones and plasmids adapted to the chicken host. Our data contribute to the epidemiological tracking of resistance mechanisms in Enterobacterales from poultry and to knowledge for further One Health studies to control the spread of resistant bacteria from food animals to humans.

Resolving taxonomic confusion: establishing the genus Phytobacter on the list of clinically relevant Enterobacteriaceae.

Although many clinically significant strains belonging to the family Enterobacteriaceae fall into a restricted number of genera and species, there is still a substantial number of isolates that elude this classification and for which proper identification remains challenging. With the current improvements in the field of genomics, it is not only possible to generate high-quality data to accurately identify individual nosocomial isolates at the species level and understand their pathogenic potential but also to analyse retrospectively the genome sequence databases to identify past recurrences of a specific organism, particularly those originally published under an incorrect or outdated taxonomy. We propose a general use of this approach to classify further clinically relevant taxa, i.e., Phytobacter spp., that have so far gone unrecognised due to unsatisfactory identification procedures in clinical diagnostics. Here, we present a genomics and literature-based approach to establish the importance of the genus Phytobacter as a clinically relevant member of the Enterobacteriaceae family.

Direct detection of microorganisms in sonicated orthopedic devices after in vitro biofilm production and different processing conditions.

BACKGROUND: The gold standard for microbial detection in prosthetic joint infections is the multiple culture of the peri-prosthetic tissue. The fluid cultures after sonication can improve the recovery of the microorganisms. OBJECTIVE: The aim of this study was to evaluate the sonication technique with a plastic bag and the effect of refrigeration on microorganism detection with conventional culturing, MALDI-TOF MS and qPCR assay on an orthopedic screw model. METHODS: We produced biofilms of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans on orthopedic screws, which were stored under different conditions and temperatures before sonication. After sonication, the mass spectrometry by MALDI-TOF, qPCR and culture protocols was performed using the sonicated fluid, for detecting the microorganisms involved in the biofilm. RESULTS: The bacterial bioburden decreased by approximately one log after the refrigeration period, in the screws containing P. aeruginosa and S. aureus biofilms. All the microorganisms involved in the screw biofilms were detected with MALDI-TOF and qPCR. Significant reductions in CFU counts occurred only in groups stored in the plastic bag, indicating that changes in temperature and humidity may favor cell death. However, this variation is not important for this model as it did not affect the detection owing to the high counts obtained. CONCLUSION: Microbial identification by MALDI-TOF in sonicated fluid is feasible. With qPCR, there were no differences between the detection in the screws processed immediately or after refrigeration. It is necessary to consider whether or not the refrigeration period would affect microbial recovery in an explanted prosthesis.

The Experience of Implementing a National Antimicrobial Resistance Surveillance System in Brazil.

Antimicrobial resistance (AMR) is a major public health threat of global proportions, which has the potential to lead to approximately ten million deaths per year by 2050. Pressured by this wicked problem, in 2014, the World Health Organization launched a call for member states to share AMR data through the implementation of the Global Antimicrobial Resistance Surveillance System (GLASS), to appropriately scale and monitor the general situation world-widely. In 2017, Brazil joined GLASS and, in 2018, started its own national antimicrobial surveillance program (BR-GLASS) to understand the impact of resistance in the country. We compiled data obtained from the complete routine of three hospitals' microbiology labs during the year of 2018. This pilot data sums up to 200,874 antimicrobial susceptibility test results from 11,347 isolates. It represents 119 different microorganisms recovered from 44 distinct types of clinical samples. Specimens came from patients originating from 301 Brazilian cities, with 4,950 of these isolates from presumed Healthcare-Associated Infections (HAIs) and the other 6,397 community-acquired cases. The female population offered 58% of the collected samples, while the other 42% were of male origin. The urinary tract was the most common topography (6,372/11,347 isolates), followed by blood samples (2,072/11,347). Gram-negative predominated the bacterial isolates: Escherichia coli was the most prevalent in general, representing 4,030 isolates (89.0% of these from the urinary tract). Coagulase-negative Staphylococci were the most prevalent bacteria in blood samples. Besides these two species, the ESKAPE group have consolidated their prevalence. Regarding drug susceptibility results, 141,648 (70.5%) were susceptible, 9,950 (4.9%) intermediate, and 49,276 (24.5%) resistant. Acinetobacter baumannii was the most worrisome microorganism, with 65.3% of the overall antimicrobial susceptibility tests showing resistance, followed by ESBL-producing Klebsiella pneumoniae, with a global resistance rate of 59%. Although this is a pilot project (still limited to one state), this database shows the importance of a nation-wide surveillance program,[153mm][-12mm] Q14 especially considering it already had patients coming from 301 distinct counties and 18 different states. The BR-GLASS Program is an ongoing project that intends to encompass at least 95 hospitals distributed in all five geographical regions in Brazil within the next 5 years.

Elution methods to evaluate colistin susceptibility of Gram-negative rods.

Recently it was developed the Colistin Broth Disk Elution test which uses colistin disks as a source of these antibiotics. The aim of this study was to evaluate the performance of protocols that used diminished volumes of the reagents: the Colistin Broth Microelution (CBM) (1 mL) and the Microelution-Plates Test (MPT) (200 µL), as well as the Colistin Susceptibility Test Tube (CSTT), which uses only one colistin disk added to a tube containing broth. The tests were performed with 85 Gram-negative isolates collected from surveillance studies. The CBM, MPT, and CSTT tests presented a good Categorical Agreement (CA), Essential Agreement (EA), sensitivity and specificity to Enterobacterales isolates, however the ME and VME were less satisfactory. The results for non-fermentative isolates were not satisfactory. In conclusion, the proposed methods, mainly the CSTT, can be used as screening tests to detect colistin resistant among Enterobacterales, as they are an easy and inexpensive option to the reference method.

Earliest identification of New Delhi metallo-ß-lactamase 1 (NDM-1) in Acinetobacter pittii in Brazil.

We report the occurrence in Brazil of the bla NDM-1 gene in Acinetobacter pittii, prior to the previously described first reports regarding the species Providencia rettgeri and Enterobacter hormaechei. Clinical isolates were investigated by polymerase chain reaction followed by bidirectional sequencing, and species was confirmed by 16S rDNA sequencing and matrix-assisted laser desorption-ionization time-of-flight spectrometry. A. pittii carrying bla NDM-1 was confirmed in a patient with no national or international travel history, or transfer from another hospital. The findings warn of the possibility of silent spread of bla NDM-1 to the community.

Low level of polymyxin resistance among nonclonal mcr-1-positive Escherichia coli from human sources in Brazil.

We report 26 human isolates of mcr-1-positive Escherichia coli, most of them (65.4%) with a polymyxin B MIC of 2 mg/L. Seventeen out of the 24 mcr-1-positive E. coli proved to be nonclonal by rep-PCR which strengthens the hypothesis of environmental or animal origin of these strains and reinforces the one health context of antimicrobial resistance.

Earliest identification of New Delhi metallo-ß-lactamase 1 (NDM-1) in Acinetobacter pittii in Brazil

Abstract We report the occurrence in Brazil of the bla NDM-1 gene in Acinetobacter pittii, prior to the previously described first reports regarding the species Providencia rettgeri and Enterobacter hormaechei. Clinical isolates were investigated by polymerase chain reaction followed by bidirectional sequencing, and species was confirmed by 16S rDNA sequencing and matrix-assisted laser desorption-ionization time-of-flight spectrometry. A. pittii carrying bla NDM-1 was confirmed in a patient with no national or international travel history, or transfer from another hospital. The findings warn of the possibility of silent spread of bla NDM-1 to the community.

Conventional culture method and qPCR using 16S rDNA for tissue bank: a comparison using a model of cardiac tissue contamination.

Real-time polymerase chain reaction (qPCR) using 16S rDNA is an alternative to conventional culture-based tests. The aim of this study was to compare the conventional culture method with qPCR using 16S rDNA in a model of cardiac tissue contamination. Samples of cardiac tissue for artificial contamination with Escherichia coli and control samples were submitted for DNA extraction, which was conducted by selective and alkaline lysis and purification steps. A standard curve for 16S rDNA was constructed to determine the efficiency and analytical sensitivity of the assay in concentrations from 106 to 102 c.f.u. ml-1 using TaqMan Master Mix. 16S rDNA was detected in all contaminated samples; however, it was not detected in the the final washing step solution of the sample with a bioburden of 102 c.f.u. ml-1. Using qPCR is a potential alternative to conventional culture for microbiological safety testing of allograft tissues for biobanking, reducing the time and labour input required.

Molecular investigation of isolates from a multistate polymicrobial outbreak associated with contaminated total parenteral nutrition in Brazil.

BACKGROUND: Between November 2013 and June 2014, 56 cases of bacteremia (15 deaths) associated with the use of Total Parenteral Nutrition (TPN) and/or calcium gluconate (CG) were reported in four Brazilian states. METHODS: We analyzed 73 bacterial isolates from four states: 45 from blood, 25 from TPN and three from CG, originally identified as Acinetobacter baumannii, Rhizobium radiobacter, Pantoea sp. or Enterobacteriaceae using molecular methods. RESULTS: The first two bacterial species were confirmed while the third group of species could not be identified using standard identification protocols. These isolates were subsequently identified by Multi-Locus Sequence Analysis as Phytobacter diazotrophicus, a species related to strains from similar outbreaks in the United States in the 1970's. Within each species, TPN and blood isolates proved to be clonal, whereas the R. radiobacter isolates retrieved from CG were found to be unrelated. CONCLUSION: This is the first report of a three-species outbreak caused by TPN contaminated with A. baumannii, R. radiobacter and P. diazotrophicus. The concomitant presence of clonal A. baumannii and P. diazotrophicus isolates in several TPN and blood samples, as well as the case of one patient, where all three different species were isolated simultaneously, suggest that the outbreak may be ascribed to a discrete contamination of TPN. In addition, this study highlights the clinical relevance of P. diazotrophicus, which has been involved in outbreaks in the past, but was often misidentified as P. agglomerans.

Emended description of the genus Phytobacter, its type species Phytobacter diazotrophicus (Zhang 2008) and description of Phytobacter ursingii sp. nov.

The species Phytobacter diazotrophicus and the associated genus Phytobacter were originally described by Zhanget al. [Arch Microbiol189 (2008), 431-439] on the basis of few endophytic nitrogen-fixing bacteria isolated from wild rice (Oryza rufipogon) in China. In this study, we demonstrate that a number of clinical isolates that were either described in the literature, preserved in culture collections, or obtained during a 2013 multi-state sepsis outbreak in Brazil also belong to the same genus. 16S rRNA gene sequencing, multilocus sequence analysis based on gyrB, rpoB, atpD and infB genes, as well as digital DNA-DNA hybridization support the existence of a second species within the genus Phytobacter. All isolates from the recent Brazilian outbreak, along with some older American clinical strains, were found to belong to the already described species Phytobacterdiazotrophicus, whereas three clinical strains retrieved in the USA over a time span of almost four decades, could be assigned to a new Phytobacter species. Implementation of an extended set of biochemical tests showed that the two Phytobacter species could phenotypically be discriminated from each other by the ability to utilize l-sorbose and d-serine. This feature was limited to the strains of the novel species described herein, for which the name Phytobacter ursingii sp. nov. is proposed, with ATCC 27989T (=CNCTC 5729T) as the designated type strain. An emended description of the species Phytobacter diazotrophicus and of the genus Phytobacter is also provided.

Polyphasic characterisation of Burkholderia cepaciacomplex species isolated from children with cystic fibrosis.

Cystic fibrosis (CF) patients with Burkholderia cepacia complex (Bcc) pulmonary infections have high morbidity and mortality. The aim of this study was to compare different methods for identification of Bcc species isolated from paediatric CF patients. Oropharyngeal swabs from children with CF were used to obtain isolates of Bcc samples to evaluate six different tests for strain identification. Conventional (CPT) and automatised (APT) phenotypic tests, polymerase chain reaction (PCR)-recA, restriction fragment length polymorphism-recA, recA sequencing, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) were applied. Bacterial isolates were also tested for antimicrobial susceptibility. PCR-recA analysis showed that 36 out of the 54 isolates were Bcc. Kappa index data indicated almost perfect agreement between CPT and APT, CPT and PCR-recA, and APT and PCR-recA to identify Bcc, and MALDI-TOF and recA sequencing to identify Bcc species. The recA sequencing data and the MALDI-TOF data agreed in 97.2% of the isolates. Based on recA sequencing, the most common species identified were Burkholderia cenocepacia IIIA (33.4%),Burkholderia vietnamiensis (30.6%), B. cenocepaciaIIIB (27.8%), Burkholderia multivorans (5.5%), and B. cepacia (2.7%). MALDI-TOF proved to be a useful tool for identification of Bcc species obtained from CF patients, although it was not able to identify B. cenocepacia subtypes.

Polyphasic characterisation of Burkholderia cepaciacomplex species isolated from children with cystic fibrosis

Cystic fibrosis (CF) patients with Burkholderia cepacia complex (Bcc) pulmonary infections have high morbidity and mortality. The aim of this study was to compare different methods for identification of Bcc species isolated from paediatric CF patients. Oropharyngeal swabs from children with CF were used to obtain isolates of Bcc samples to evaluate six different tests for strain identification. Conventional (CPT) and automatised (APT) phenotypic tests, polymerase chain reaction (PCR)-recA, restriction fragment length polymorphism-recA, recAsequencing, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) were applied. Bacterial isolates were also tested for antimicrobial susceptibility. PCR-recA analysis showed that 36 out of the 54 isolates were Bcc. Kappa index data indicated almost perfect agreement between CPT and APT, CPT and PCR-recA, and APT and PCR-recA to identify Bcc, and MALDI-TOF and recAsequencing to identify Bcc species. The recAsequencing data and the MALDI-TOF data agreed in 97.2% of the isolates. Based on recA sequencing, the most common species identified were Burkholderia cenocepacia IIIA (33.4%),Burkholderia vietnamiensis (30.6%), B. cenocepaciaIIIB (27.8%), Burkholderia multivorans (5.5%), and B. cepacia (2.7%). MALDI-TOF proved to be a useful tool for identification of Bcc species obtained from CF patients, although it was not able to identify B. cenocepacia subtypes.

First report of NDM-1-producing Acinetobacter baumannii sequence type 25 in Brazil.

New Delhi metallo-ß-lactamase 1 (NDM-1) was first identified in Brazil in Enterobacter hormaechei and Providencia rettgeri in 2013. Here, we describe the first case of NDM-1-producing Acinetobacter baumannii sequence type 25 isolated from the urinary tract of a 71-year-old man who died of multiple complications, including A. baumannii infection. The NDM-1 gene was detected by quantitative PCR, and its sequence confirmed its presence in an ∼ 100-kb plasmid.