Emergence of KPC-3- and OXA-181-producing ST13 and ST17 Klebsiella pneumoniae in Portugal: genomic insights on national and international dissemination.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) strains are of particular concern, especially strains with mobilizable carbapenemase genes such as blaKPC, blaNDM or blaOXA-48, given that carbapenems are usually the last line drugs in the ß-lactam class and, resistance to this sub-class is associated with increased mortality and frequently co-occurs with resistance to other antimicrobial classes. OBJECTIVES: To characterize the genomic diversity and international dissemination of CRKP strains from tertiary care hospitals in Lisbon, Portugal. METHODS: Twenty CRKP isolates obtained from different patients were subjected to WGS for species confirmation, typing, drug resistance gene detection and phylogenetic reconstruction. Two additional genomic datasets were included for comparative purposes: 26 isolates (ST13, ST17 and ST231) from our collection and 64 internationally available genomic assemblies (ST13). RESULTS: By imposing a 21 SNP cut-off on pairwise comparisons we identified two genomic clusters (GCs): ST13/GC1 (n = 11), all bearing blaKPC-3, and ST17/GC2 (n = 4) harbouring blaOXA-181 and blaCTX-M-15 genes. The inclusion of the additional datasets allowed the expansion of GC1/ST13/KPC-3 to 23 isolates, all exclusively from Portugal, France and the Netherlands. The phylogenetic tree reinforced the importance of the GC1/KPC-3-producing clones along with their rapid emergence and expansion across these countries. The data obtained suggest that the ST13 branch emerged over a decade ago and only more recently did it underpin a stronger pulse of transmission in the studied population. CONCLUSIONS: This study identifies an emerging OXA-181/ST17-producing strain in Portugal and highlights the ongoing international dissemination of a KPC-3/ST13-producing clone from Portugal.

Spread of Mycobacterium tuberculosis in Southern Brazilian persons deprived of liberty: a molecular epidemiology study.

To evaluate the genetic diversity and clustering rates of M. tuberculosis strains to better understand transmission among persons deprived of liberty (PDL) in Rio Grande do Sul (RS), southern Brazil. This is a cross-sectional study, including strains of M. tuberculosis isolated from PDL, stored at the Central Laboratory of RS, in the period from 2013 to 2018. The molecular characterization was performed using the MIRU-VNTR 15 loci method. A total of 598 M. tuberculosis strains were genotyped, and 37.5% were grouped into 53 clusters. Cluster sizes ranged from 2 to 34 strains. The largest cluster of the study had strains from 34 PDL, and 58.8% of the PDL of this cluster were in P01. Among the clusters formed, in 60.3%, there was at least one strain from P01. The most common strains in RS were LAM (53.2%) and Haarlem (31.1%). The LAM strain was the most likely to form clusters, and Haarlem was associated with anti-TB drug resistance. This was translational research, and the results can collaborate with the TB control programs, leading to improved strategies that allow the reduction of the TB burden in prisons.

Insight into pathogenomics and phylogeography of hypervirulent and highly-lethal Mycobacterium tuberculosis strain cluster.

BACKGROUND: . The Mycobacterium tuberculosis Beijing genotype is globally spread lineage with important medical properties that however vary among its subtypes. M. tuberculosis Beijing 14717-15-cluster was recently discovered as both multidrug-resistant, hypervirulent, and highly-lethal strain circulating in the Far Eastern region of Russia. Here, we aimed to analyze its pathogenomic features and phylogeographic pattern. RESULTS: . The study collection included M. tuberculosis DNA collected between 1996 and 2020 in different world regions. The bacterial DNA was subjected to genotyping and whole genome sequencing followed by bioinformatics and phylogenetic analysis. The PCR-based assay to detect specific SNPs of the Beijing 14717-15-cluster was developed and used for its screening in the global collections. Phylogenomic and phylogeographic analysis confirmed endemic prevalence of the Beijing 14717-15-cluster in the Asian part of Russia, and distant common ancestor with isolates from Korea (> 115 SNPs). The Beijing 14717-15-cluster isolates had two common resistance mutations RpsL Lys88Arg and KatG Ser315Thr and belonged to spoligotype SIT269. The Russian isolates of this cluster were from the Asian Russia while 4 isolates were from the Netherlands and Spain. The cluster-specific SNPs that significantly affect the protein function were identified in silico in genes within different categories (lipid metabolism, regulatory proteins, intermediary metabolism and respiration, PE/PPE, cell wall and cell processes). CONCLUSIONS: . We developed a simple method based on real-time PCR to detect clinically significant MDR and hypervirulent Beijing 14717-15-cluster. Most of the identified cluster-specific mutations were previously unreported and could potentially be associated with increased pathogenic properties of this hypervirulent M. tuberculosis strain. Further experimental study to assess the pathobiological role of these mutations is warranted.

Genome-wide analysis of Mycobacterium tuberculosis polymorphisms reveals lineage-specific associations with drug resistance.

BACKGROUND: Continuing evolution of the Mycobacterium tuberculosis (Mtb) complex genomes associated with resistance to anti-tuberculosis drugs is threatening tuberculosis disease control efforts. Both multi- and extensively drug resistant Mtb (MDR and XDR, respectively) are increasing in prevalence, but the full set of Mtb genes involved are not known. There is a need for increased sensitivity of genome-wide approaches in order to elucidate the genetic basis of anti-microbial drug resistance and gain a more detailed understanding of Mtb genome evolution in a context of widespread antimicrobial therapy. Population structure within the Mtb complex, due to clonal expansion, lack of lateral gene transfer and low levels of recombination between lineages, may be reducing statistical power to detect drug resistance associated variants. RESULTS: To investigate the effect of lineage-specific effects on the identification of drug resistance associations, we applied genome-wide association study (GWAS) and convergence-based (PhyC) methods to multiple drug resistance phenotypes of a global dataset of Mtb lineages 2 and 4, using both lineage-wise and combined approaches. We identify both well-established drug resistance variants and novel associations; uniquely identifying associations for both lineage-specific and -combined GWAS analyses. We report 17 potential novel associations between antimicrobial resistance phenotypes and Mtb genomic variants. CONCLUSIONS: For GWAS, both lineage-specific and -combined analyses are useful, whereas PhyC may perform better in contexts of greater diversity. Unique associations with XDR in lineage-specific analyses provide evidence of diverging evolutionary trajectories between lineages 2 and 4 in response to antimicrobial drug therapy.

Tuberculosis caused by Mycobacterium pinnipedii in a wild South American sea lion Otaria flavescens stranded in southern Brazil.

Tuberculosis (TB) in pinnipeds is typically caused by Mycobacterium pinnipedii, which has also been associated with infections in other species, such as cattle and humans. As a result, this pathogen has zoonotic potential and is a public health concern. In 2016, a female South American sea lion Otaria flavescens in southern Brazil presented with emaciation and severe dyspnea and died within 3 h of capture. Gross pathology identified pulmonary granulomas, and Ziehl-Neelsen stain identified acid-fast bacilli. M. tuberculosis complex bacteria were confirmed by a BD BACTEC™ MGIT™ 320 detection system using fibrinous exudate, lung granulomas and thoracic fluid. Molecular characterization by spoligotyping showed a hybridization pattern characteristic of M. pinnipedii (SIT593/PINI1). Currently, there is a paucity of data concerning the transmission and epidemiology of M. pinnipedii in pinniped populations in South America. The case report shows that the disease appeared in a free-ranging beached sea lion on the coast, and further surveillance is needed to determine the origin of this TB because of its potential impact on public health.

Role of Alanine Racemase Mutations in Mycobacterium tuberculosis d-Cycloserine Resistance.

A screening of more than 1,500 drug-resistant strains of Mycobacterium tuberculosis revealed evolutionary patterns characteristic of positive selection for three alanine racemase (Alr) mutations. We investigated these mutations using molecular modeling, in vitro MIC testing, as well as direct measurements of enzymatic activity, which demonstrated that these mutations likely confer resistance to d-cycloserine.

Associated aneurysmal bone cyst and cemento-osseous dysplasia: a case report and review of the literature.

The purpose of this case report is to describe a previously unpublished association between focal cemento-osseous dysplasia (FCOD) and an aneurysmal bone cyst (ABC) and review the literature with regard to associated benign fibro-osseous lesions and cysts. A 41-year-old woman without a history of trauma presented with asymptomatic swelling in the right side of the mandible. Radiographs of the region revealed a unilocular radiolucent area with radiopaque foci. After aspiration of the lesion was positive for serosanguineous fluid, complete excision of the lesion was performed. Microscopic examination revealed a hybrid ABC and FCOD. The 12-month follow-up showed significant bone repair and no signs of recurrence. A review of the English-language literature from 1980 to 2012 revealed 1 retrospective study, 4 case series, and 18 single-case reports on the topic of cemento-osseous dysplasias, fibro-osseous lesions, and aneurysmal bone cysts. Of 59 cases, none reported an association between an ABC and FCOD. Although fibro-osseous lesions do not require intervention, surgical excision is recommended when they are associated with cysts. This case, in which an ABC and FCOD were associated, reinforces the need for a careful diagnostic process in radiographically mixed lesions that respond positively to aspiration biopsy.

Dental care in patients with antiphospholipid syndrome: two case reports.

Antiphospholipid syndrome (APS) is a prothrombotic autoimmune disease that may be classified as primary or secondary. Treatment consists of oral anticoagulant, antiplatelet, and/or immunosuppressant drugs. This report describes the dental treatment of 2 women with APS and multiple dental concerns, including periodontal disease, caries, and missing teeth. The invasive dental procedures were performed in an outpatient setting with hematologic monitoring and use of local hemostatic measures. Neither interruption of anticoagulant medications nor administration of blood products was necessary. All of the procedures were performed without complications. To date, no recommendations for the dental care of patients with APS have been established, demonstrating a need to investigate the risks for bleeding and infection, among other concerns, during dental treatment of these patients.

Recombination in pe/ppe genes contributes to genetic variation in Mycobacterium tuberculosis lineages.

BACKGROUND: Approximately 10% of the Mycobacterium tuberculosis genome is made up of two families of genes that are poorly characterized due to their high GC content and highly repetitive nature. The PE and PPE families are typified by their highly conserved N-terminal domains that incorporate proline-glutamate (PE) and proline-proline-glutamate (PPE) signature motifs. They are hypothesised to be important virulence factors involved with host-pathogen interactions, but their high genetic variability and complexity of analysis means they are typically disregarded in genome studies. RESULTS: To elucidate the structure of these genes, 518 genomes from a diverse international collection of clinical isolates were de novo assembled. A further 21 reference M. tuberculosis complex genomes and long read sequence data were used to validate the approach. SNP analysis revealed that variation in the majority of the 168 pe/ppe genes studied was consistent with lineage. Several recombination hotspots were identified, notably pe_pgrs3 and pe_pgrs17. Evidence of positive selection was revealed in 65 pe/ppe genes, including epitopes potentially binding to major histocompatibility complex molecules. CONCLUSIONS: This, the first comprehensive study of the pe and ppe genes, provides important insight into M. tuberculosis diversity and has significant implications for vaccine development.

Uprighting Bilateral Impacted Mandibular Permanent Second Molars with the Brass Wire Technique: A Case Report.

UNLABELLED: Revalence of impaction of mandibular permanent second molars is between 0.06 and 2.3 percent. In order to reduce treatment time and complications associated with tooth impaction, intervention should take place once the problem is detected. The usual treatment options consists of surgical exposure, luxation of the impacted tooth, extraction of adjacent third molar, orthodontic treatment, and uprighting with brass wires or mini-screws. AIM: The present paper reports a case of bilateral impaction of mandibular permanent second molars ' (MM2s) treated with extraction of the mandibular third molars (MM3s) and surgical-orthodontic uprighting with the brass wire technique. METHOD: The MM3s were removed, and the impacted MM2s were surgically exposed. Brass wire was placed apicaly to the mesial of the MM2 from the lingual tissue out toward the buccal. The lingual end of the wire was bent over the area of contact and twisted with the buccal end Monthly wire tightening gradually moved the MM2s distally and towards the occlusal plane. RESULTS: Uprighting was achieved in 4-5 months, with discrete pain caused by activation of the wire. CONCLUSION: This technique proved to be a simple, low-cost, and quick treatment option for uprighting impacted mandibular permanent second molars.

Multidrug-Resistant Mycobacterium tuberculosis of the Latin American Mediterranean Lineage, Wrongly Identified as Mycobacterium pinnipedii (Spoligotype International Type 863 [SIT863]), Causing Active Tuberculosis in South Brazil.

We recently detected the spoligotype patterns of strains of Mycobacterium pinnipedii, a species of the Mycobacterium tuberculosis complex, in sputum samples from nine cases with pulmonary tuberculosis residing in Porto Alegre, South Brazil. Because this species is rarely encountered in humans, we further characterized these nine isolates by additional genotyping techniques, including 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing, verification of the loci TbD1, RD9, pks15/1, RD(Rio), and fbpC, the insertion of IS6110 at a site specific to the M. tuberculosis Latin American Mediterranean (LAM) lineage, and whole-genome sequencing. The combined analysis of these markers revealed that the isolates are in fact M. tuberculosis and more specifically belong to the LAM genotype. Most of these isolates (n8) were shown to be multidrug resistant (MDR), which prompted us to perform partial sequencing of the rpoA, rpoB, rpoC, katG, and inhA genes. Seven isolates (77.8%) carried the S315T mutation in katG, and one of these (11%) also presented the C((-17)T single-nucleotide polymorphism (SNP) in inhA. Interestingly, six of the MDR isolates also presented an undescribed insertion of 12 nucleotides (CCA GAA CAA CCC) in codon 516 of rpoB. No putative compensatory mutation was found in either rpoA or rpoC. This is the first report of an M. tuberculosis LAM family strain with a convergent M. pinnipedii spoligotype. These spoligotypes are observed in genotype databases at a modest frequency, highlighting that care must be taken when identifying isolates in the M. tuberculosis complex on the basis of single genetic markers.

Unraveling Mycobacterium tuberculosis genomic diversity and evolution in Lisbon, Portugal, a highly drug resistant setting.

BACKGROUND: Multidrug- (MDR) and extensively drug resistant (XDR) tuberculosis (TB) presents a challenge to disease control and elimination goals. In Lisbon, Portugal, specific and successful XDR-TB strains have been found in circulation for almost two decades. RESULTS: In the present study we have genotyped and sequenced the genomes of 56 Mycobacterium tuberculosis isolates recovered mostly from Lisbon. The genotyping data revealed three major clusters associated with MDR-TB, two of which are associated with XDR-TB. Whilst the genomic data contributed to elucidate the phylogenetic positioning of circulating MDR-TB strains, showing a high predominance of a single SNP cluster group 5. Furthermore, a genome-wide phylogeny analysis from these strains, together with 19 publicly available genomes of Mycobacterium tuberculosis clinical isolates, revealed two major clades responsible for M/XDR-TB in the region: Lisboa3 and Q1 (LAM).The data presented by this study yielded insights on microevolution and identification of novel compensatory mutations associated with rifampicin resistance in rpoB and rpoC. The screening for other structural variations revealed putative clade-defining variants. One deletion in PPE41, found among Lisboa3 isolates, is proposed to contribute to immune evasion and as a selective advantage. Insertion sequence (IS) mapping has also demonstrated the role of IS6110 as a major driver in mycobacterial evolution by affecting gene integrity and regulation. CONCLUSIONS: Globally, this study contributes with novel genome-wide phylogenetic data and has led to the identification of new genomic variants that support the notion of a growing genomic diversity facing both setting and host adaptation.

Pre-multidrug-resistant Mycobacterium tuberculosis Beijing strain associated with disseminated tuberculosis in a pet dog.

Resistance to isoniazid, ethambutol, and streptomycin was detected in a Mycobacterium tuberculosis strain, belonging to the Beijing family lineage, isolated from two nodule exudates of a Yorkshire terrier with generalized tuberculosis. This report alerts medical practitioners to the risk of dissemination of pre-multidrug-resistant tuberculosis (preMDR-TB) through exposure to M. tuberculosis-shedding pets.

Diversity, functional classification and genotyping of SHV ß-lactamases in Klebsiella pneumoniae.

Interpreting the phenotypes of bla SHV alleles in Klebsiella pneumoniae genomes is complex. Whilst all strains are expected to carry a chromosomal copy conferring resistance to ampicillin, they may also carry mutations in chromosomal bla SHV alleles or additional plasmid-borne bla SHV alleles that have extended-spectrum ß-lactamase (ESBL) activity and/or ß-lactamase inhibitor (BLI) resistance activity. In addition, the role of individual mutations/a changes is not completely documented or understood. This has led to confusion in the literature and in antimicrobial resistance (AMR) gene databases [e.g. the National Center for Biotechnology Information (NCBI) Reference Gene Catalog and the ß-lactamase database (BLDB)] over the specific functionality of individual sulfhydryl variable (SHV) protein variants. Therefore, the identification of ESBL-producing strains from K. pneumoniae genome data is complicated. Here, we reviewed the experimental evidence for the expansion of SHV enzyme function associated with specific aa substitutions. We then systematically assigned SHV alleles to functional classes (WT, ESBL and BLI resistant) based on the presence of these mutations. This resulted in the re-classification of 37 SHV alleles compared with the current assignments in the NCBI's Reference Gene Catalog and/or BLDB (21 to WT, 12 to ESBL and 4 to BLI resistant). Phylogenetic and comparative genomic analyses support that (i) SHV-1 (encoded by bla SHV-1) is the ancestral chromosomal variant, (ii) ESBL- and BLI-resistant variants have evolved multiple times through parallel substitution mutations, (iii) ESBL variants are mostly mobilized to plasmids and (iv) BLI-resistant variants mostly result from mutations in chromosomal bla SHV. We used matched genome-phenotype data from the KlebNET-GSP AMR Genotype-Phenotype Group to identify 3999 K. pneumoniae isolates carrying one or more bla SHV alleles but no other acquired ß-lactamases to assess genotype-phenotype relationships for bla SHV. This collection includes human, animal and environmental isolates collected between 2001 and 2021 from 24 countries. Our analysis supports that mutations at Ambler sites 238 and 179 confer ESBL activity, whilst most omega-loop substitutions do not. Our data also provide support for the WT assignment of 67 protein variants, including 8 that were noted in public databases as ESBL. These eight variants were reclassified as WT because they lack ESBL-associated mutations, and our phenotype data support susceptibility to third-generation cephalosporins (SHV-27, SHV-38, SHV-40, SHV-41, SHV-42, SHV-65, SHV-164 and SHV-187). The approach and results outlined here have been implemented in Kleborate v2.4.1 (a software tool for genotyping K. pneumoniae), whereby known and novel bla SHV alleles are classified based on causative mutations. Kleborate v2.4.1 was updated to include ten novel protein variants from the KlebNET-GSP dataset and all alleles in public databases as of November 2023. This study demonstrates the power of sharing AMR phenotypes alongside genome data to improve the understanding of resistance mechanisms.

From multidrug-resistant to extensively drug-resistant tuberculosis in Lisbon, Portugal: the stepwise mode of resistance acquisition.

OBJECTIVES: The development and transmission of extensively drug-resistant (XDR) tuberculosis (TB) constitutes a serious threat to the effective control of TB in several countries. Here, in an attempt to further elucidate the dynamics of the acquisition of resistance to second-line drugs and investigate an eventual role for eis promoter mutations in aminoglycoside resistance, we have studied a set of multidrug-resistant (MDR)/XDR-TB isolates circulating in Lisbon, Portugal. METHODS: Forty-four MDR-TB or XDR-TB isolates were genotyped and screened for mutations in genes associated with second-line drug resistance, namely tlyA, gyrA, rrs and eis. RESULTS: The most prevalent mutations found in each gene were Ins755GT in tlyA, A1401G in rrs, G-10A in eis and S91P in gyrA. Additionally, two genetic clusters were found in this study: Lisboa3 and Q1. The characteristic mutational profile found among recent XDR-TB circulating in Lisbon was also found in MDR-TB strains isolated in the 1990s. Also investigated was the resistance level conferred by eis G-10A mutations, revealing that eis G-10A mutations may result in amikacin resistance undetectable by widely used phenotypic assays. CONCLUSIONS: The analysis of the distribution of the mutations found by genetic clustering showed that in the Q1 cluster, two mutations, gyrA D94A and rrs A1401G, were enough to ensure development of XDR-TB from an MDR strain. Moreover, in the Lisboa3 cluster it was possible to elaborate a model in which the development of low-level kanamycin resistance was at the origin of the emergence of XDR-TB strains that can be discriminated by tlyA mutations.

High-level resistance to isoniazid and ethionamide in multidrug-resistant Mycobacterium tuberculosis of the Lisboa family is associated with inhA double mutations.

OBJECTIVES: The purpose of this study was to determine the levels of isoniazid and ethionamide resistance and to identify associated mutations in endemic multidrug-resistant (MDR) strains of Mycobacterium tuberculosis from the Lisbon metropolitan area, Portugal. METHODS: Seventeen clinical MDR tuberculosis (TB) strains were characterized by standard and semi-quantitative drug susceptibility testing to assess the level of isoniazid and ethionamide resistance. The genes katG, inhA, ethA and ndh were screened for mutations. All strains were genotyped by 24 loci mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) analysis. RESULTS: All strains showed high-level resistance to both isoniazid (>1 mg/L) and ethionamide (>25 mg/L). MIRU-VNTR typing revealed the presence of two main clusters, Lisboa3 and Q1, in 16/17 strains, all of which showed the C-15T mutation in the promoter region of the inhA gene. The 16 strains belong to the Latino-American-Mediterranean (LAM) genotype and the other strain belongs to the Beijing genotype. Sequencing of the inhA open reading frame revealed that the 16 strains also had mutations in the structural region of the gene, leading to the S94A substitution in 9 strains and the I194T substitution in 7 strains. CONCLUSIONS: The results reveal that the presence of a mutation in the inhA regulatory region together with a mutation in the inhA coding region can lead to the development of high-level isoniazid resistance and cross-resistance to ethionamide among the MDR-TB strains circulating in Lisbon. This mutational pattern also hints to a possible involvement of strain-specific factors that could be a feature of the Portuguese MDR-TB strains where the LAM family is the major circulating genotype.

Exploring the contribution of mycobacteria characteristics in their interaction with human macrophages.

Tuberculosis (TB) is a major health problem. The emergence of multidrug resistant (MDR) Mycobacterium tuberculosis (Mtb) isolates confounds treatment strategies. In Portugal, cases of MDR-TB are reported annually with an increased incidence noted in Lisbon. The majority of these MDR-TB cases are due to closely related mycobacteria known collectively as the Lisboa family and Q1 cluster. Genetic determinants linked to drug resistance have been exhaustively studied resulting in the identification of family and cluster specific mutations. Nevertheless, little is known about other factors involved in development of mycobacteria drug resistance. Here, we complement genetic analysis with the study of morphological and structural features of the Lisboa family and Q1 cluster isolates by using scanning and transmission electron microscopy. This analysis allowed the identification of structural differences, such as cell envelope thickness, between Mtb clinical isolates that are correlated with antibiotic resistance. The infection of human monocyte derived macrophages allowed us to document the relative selective advantage of the Lisboa family isolates over other circulating Mtb isolates.

Large-scale genomic analysis of global Klebsiella pneumoniae plasmids reveals multiple simultaneous clusters of carbapenem-resistant hypervirulent strains.

BACKGROUND: Klebsiella pneumoniae (Kp) Gram-negative bacteria cause nosocomial infections and rapidly acquire antimicrobial resistance (AMR), which makes it a global threat to human health. It also has a comparatively rare hypervirulent phenotype that can lead to severe disease in otherwise healthy individuals. Unlike classic Kp, canonical hypervirulent strains usually have limited AMR. However, after initial case reports in 2015, carbapenem-resistant hypervirulent Kp has increased in prevalence, including in China, but there is limited understanding of its burden  in other geographical regions. METHODS: Here, we examined the largest collection of publicly available sequenced Kp isolates (n=13,178), containing 1603 different sequence types (e.g. ST11 15.0%, ST258 9.5%), and 2174 (16.5%) hypervirulent strains. We analysed the plasmid replicons and carbapenemase and siderophore encoding genes to understand the movement of hypervirulence and AMR genes located on plasmids, and their convergence in carbapenem-resistant hypervirulent Kp. RESULTS: We identified and analysed 3034 unique plasmid replicons to inform the epidemiology and transmission dynamics of carbapenem-resistant hypervirulent Kp (n=1028, 7.8%). We found several outbreaks globally, including one involving ST11 strains in China and another of ST231 in Asia centred on India, Thailand, and Pakistan. There was evidence of global flow of Kp, including across multiple continents. In most cases, clusters of Kp isolates are the result of hypervirulence genes entering classic strains, instead of carbapenem resistance genes entering canonical hypervirulent ones. CONCLUSIONS: Our analysis demonstrates the importance of plasmid analysis in the monitoring of carbapenem-resistant and hypervirulent strains of Kp. With the growing adoption of omics-based technologies for clinical and surveillance applications, including in geographical regions with gaps in data and knowledge (e.g. sub-Saharan Africa), the identification of the spread of AMR will inform infection control globally.

Pluronic® F127 Hydrogel Containing Silver Nanoparticles in Skin Burn Regeneration: An Experimental Approach from Fundamental to Translational Research.

Presently, skin burns are considered one of the main public health problems and lack therapeutic options. In recent years, silver nanoparticles (AgNPs) have been widely studied, playing an increasingly important role in wound healing due to their antibacterial activity. This work is focused on the production and characterization of AgNPs loaded in a Pluronic® F127 hydrogel, as well as assessing its antimicrobial and wound-healing potential. Pluronic® F127 has been extensively explored for therapeutic applications mainly due to its appealing properties. The developed AgNPs had an average size of 48.04 ± 14.87 nm (when prepared by method C) and a negative surface charge. Macroscopically, the AgNPs solution presented a translucent yellow coloration with a characteristic absorption peak at 407 nm. Microscopically, the AgNPs presented a multiform morphology with small sizes (~50 nm). Skin permeation studies revealed that no AgNPs permeated the skin after 24 h. AgNPs further demonstrated antimicrobial activity against different bacterial species predominant in burns. A chemical burn model was developed to perform preliminary in vivo assays and the results showed that the performance of the developed AgNPs loaded in hydrogel, with smaller silver dose, was comparable with a commercial silver cream using higher doses. In conclusion, hydrogel-loaded AgNPs is potentially an important resource in the treatment of skin burns due to their proven efficacy by topical administration.

Genotyping methods and their contributions to the study of tuberculosis dynamic in Latin America.

INTRODUCTION: Mycobacterium tuberculosis genotyping has impacted evolutionary studies worldwide. Nonetheless, its application and the knowledge generated depend on the genetic marker evaluated and the detection technologies that have evolved over the years. Here we describe the timeline of main genotypic methods related to M. tuberculosis in Latin America and the main findings obtained. METHODOLOGY: Systematic searches through the PubMed database were performed from 1993 to May 2021. A total of 345 articles met the inclusion criteria and were selected. RESULTS: Spacer oligonucleotide typing (spoligotyping) was the most widely used method in Latin America, with decreasing use in parallel with increasing use of mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) and whole genome sequencing (WGS). Among the countries, Brazil, Mexico, and Argentina had the most publications, and a considerable part of the articles were in collaboration with Latin American or non-Latin American institutions; a small proportion of studies needed partnerships to perform the genotypic methods. The genotypic methods allowed the identification of M. tuberculosis genotypes with greater capacity for clonal expansion and revealed the predominance of the Euro-American lineage in Latin America. There was a notable presence of the Beijing family in Peru and Colombia. CONCLUSIONS: The data obtained demonstrated the importance of expanding collaborative networks of tuberculosis (TB) research groups to countries with low productivity in this area, the commitment of the few Latin American countries to advance TB research, as well as the inestimable value of building a Latin America database, considering ease of population mobility between countries.