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.

Staphylococcus nasal colonization in three species of non-human primates.

Bacterial nasal colonization is common in many mammals and Staphylococcus represents the main pathogen isolated. Staphylococcus nasal carriage in humans constitutes a risk factor for Staphylococcus infections pointing out the need for animal experimentation for nasal colonization studies, especially for vaccine development. A limitation in addressing this hypothesis has been a lack of appropriate animal model. Murine models do not mimic human nasal colonization studies. Non-human primates (NHP) remain the best classical models for nasal colonization studies. In this study, we analyzed nasal colonization between two species of Old World monkeys (cynomolgus and rhesus) and a New World monkey (squirrel monkey) from breeding colony at Fiocruz (Brazil). Sixty male and female NHP with the average age of 1-21 years old, comprising twenty animals of each species, were analyzed. Nine different Staphylococcus species (S. aureus, S. cohnii, S. saprophyticus, S. haemolyticus, S. xylosus, S. warneri, S. nepalensis, S. simiae, and S. kloosi) were identified by MALDI-TOF and 16S rRNA gene sequence analyses. Antibiotic resistance was not detected among the isolated bacterial population. S. aureus was the main isolate (19 strains), present in all species, predominant in cynomolgus monkeys (9/20) and squirrel monkeys (7/20). spa typing was used to examine the clonal structure and genetic profile of Staphylococcus aureus isolates. Eight (8) spa types were identified among the S. aureus strains. A major cluster was identified, corresponding to a new spa type t20455, and no spa types found in this study were seen before in Brazil.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Non-clonal occurrence of pmrB mutations associated with polymyxin resistance in carbapenem-resistant Klebsiella pneumoniae in Brazil.

BACKGROUND: Polymyxins are currently used as a "last-line" treatment for multidrug-resistant Gram-negative infections. OBJECTIVES: To identify the major mechanisms of resistance to polymyxin and compare the genetic similarity between multi-drug resistant Klebsiella pneumoniae strains recovered from inpatients of public hospitals in the Mid-West of Brazil. METHODS: 97 carbapenems non-susceptible K. pneumoniae were studied. ß-lactamases (bla OXA-48, bla KPC, bla NDM, bla CTX-M, bla SHV, bla TEM, bla IMP, bla VIM) and mcr-1 to mcr-5 genes were investigated by polymerase chain reaction (PCR). Mutations in chromosomal genes (pmrA, pmrB, phoP, phoQ, and mgrB) were screened by PCR and DNA sequencing. Clonal relatedness was established by using pulsed-field gel electrophoresis and multilocus sequence typing. FINDINGS: K. pneumoniae isolates harbored bla KPC (93.3%), bla SHV (86.6%), bla TEM (80.0%), bla CTX-M (60%) genes. Of 15 K. pneumoniae resistant to polymyxin B the authors identified deleterious mutations in pmrB gene, mainly in T157P. None K. pneumoniae presented mcr gene variants. Genetic polymorphism analyses revealed 12 different pulsotypes. MAIN CONCLUSIONS: Deleterious mutations in pmrB gene is the main chromosomal target for induction of polymyxin resistance in carbapenem-resistant K. pneumoniae in public hospitals in the Mid-West of Brazil.

Epidemiology and antibiotic resistance trends in clinical isolates of Pseudomonas aeruginosa from Rio de janeiro - Brazil: Importance of mutational mechanisms over the years (1995-2015).

Pseudomonas aeruginosa is a major health concern globally and treating infections caused by MDR-isolates unarguably a humongous challenge that remains an unmet need in modern medicine. To determine patterns and mechanisms of antimicrobial resistance and its spread over the years in Rio de Janeiro, Brazil, 88 P. aeruginosa isolates were selected from 1995 to 2015. Phenotypic and genotypic characterization of antimicrobial resistance was evaluated and isolates were submitted to clonality by PFGE and MLST. PFGE analysis showed a great variability of clonal groups mainly over the past 10 years of this study. STs predominant in the early years (ST804, ST1860, ST487 and ST1602) associated to multidrug resistance (MDR) phenotype were replaced by ST277, ST244, ST1945, ST1791 with extensive drug resistance (XDR) in last years, with significant increase in resistance to carbapenems, fluoroquinolones and aminoglycosides. Colistin resistance was detected in 3.5%. The main mechanisms of antimicrobial resistance were mutational mechanisms (mutations in oprD, mexT and gyrA genes). We found the ESBL genes blaTEM (n = 2), blaSHV (n = 3) and blaCTX (n = 1).The carbapenemases genes was present in ST277 (blaSPM, n = 3), ST1560 (blaKPC, n = 3) and ST1944 (blaKPC, n = 2). The 16S RNA methylase gene (rmtD) was found in five isolates belonged to ST277. In conclusion, molecular epidemiological investigation reveals an increase of antimicrobial resistance in P. aeruginosa over 21 years in Rio de Janeiro with higher population structure and occurrence of high risk clone in the last years. The mutational mechanisms of resistance were present in all XDR isolates.

Distribution of Clinical NDM-1-Producing Gram-Negative Bacteria in Brazil.

New Delhi metallo-ß-lactamase (NDM)-producing bacteria have been identified at a worrying rate in Brazil since 2013. Owing to the need to understand the extent of their spread, this study reports the dissemination of blaNDM in different species of Gram-negative bacilli in different regions and states of Brazil. A total of 81 isolates from nine states were studied, including 11 species. All isolates carried blaNDM-1 variant and were considered multidrug resistant. Colistin and amikacin were the agents with higher activity compared with the other drugs tested. The findings indicate that the NDM-1 enzyme is already widespread in the country.

Non-clonal occurrence of pmrB mutations associated with polymyxin resistance in carbapenem-resistant Klebsiella pneumoniae in Brazil

BACKGROUND Polymyxins are currently used as a "last-line" treatment for multidrug-resistant Gram-negative infections. OBJECTIVES To identify the major mechanisms of resistance to polymyxin and compare the genetic similarity between multi-drug resistant Klebsiella pneumoniae strains recovered from inpatients of public hospitals in the Mid-West of Brazil. METHODS 97 carbapenems non-susceptible K. pneumoniae were studied. β-lactamases (bla OXA-48, bla KPC, bla NDM, bla CTX-M, bla SHV, bla TEM, bla IMP, bla VIM) and mcr-1 to mcr-5 genes were investigated by polymerase chain reaction (PCR). Mutations in chromosomal genes (pmrA, pmrB, phoP, phoQ, and mgrB) were screened by PCR and DNA sequencing. Clonal relatedness was established by using pulsed-field gel electrophoresis and multilocus sequence typing. FINDINGS K. pneumoniae isolates harbored bla KPC (93.3%), bla SHV (86.6%), bla TEM (80.0%), bla CTX-M (60%) genes. Of 15 K. pneumoniae resistant to polymyxin B the authors identified deleterious mutations in pmrB gene, mainly in T157P. None K. pneumoniae presented mcr gene variants. Genetic polymorphism analyses revealed 12 different pulsotypes. MAIN CONCLUSIONS Deleterious mutations in pmrB gene is the main chromosomal target for induction of polymyxin resistance in carbapenem-resistant K. pneumoniae in public hospitals in the Mid-West of Brazil.

Pneumococcal Predictive Proteins Selected by Microbial Genomic Approach Are Serotype Cross-Reactive and Bind to Host Extracellular Matrix Proteins.

Streptococcus pneumoniae is a colonizer of the human nasopharynx, which accounts for most of the community-acquired pneumonia cases and can cause non-invasive and invasive diseases. Current available vaccines are serotype-specific and the use of recombinant proteins associated with virulence is an alternative to compose vaccines and to overcome these problems. In a previous work, we describe the identification of proteins in S. pneumoniae by reverse vaccinology and the genetic diversity of these proteins in clinical isolates. It was possible to purify a half of 20 selected proteins in soluble form. The expression of these proteins on the pneumococcal cells surface was confirmed by flow cytometry. We demonstrated that some of these proteins were able to bind to extracellular matrix proteins and were recognized by sera from patients with pneumococcal meningitis infection caused by several pneumococcal serotypes. In this context, our results suggest that these proteins may play a role in pneumococcal pathogenesis and might be considered as potential vaccine candidates.

Presence of leptospires on genital tract of mares with reproductive problems.

Leptospirosis is a zoonotic disease of global importance, and has a worldwide distribution. Equine leptospirosis is commonly manifested by recurrent uveitis, reproductive disorders, as abortions, embryonic absorption, stillbirth and the birth of weak foals. The aim of this study was to verify the presence of Leptospira sp or its DNA in genital tract of mares with reproductive problems. A total of 38 mares with reproductive problems were studied. All the mares were sampled for blood (for serology), urine (for culturing and qPCR), vaginal fluid-VF and endometrial biopsy-EB (for culturing, qPCR and indirect immunofluorescence). PCRs products were sequenced for secY gene. Seventeen (44.7%) serum samples were reactive, predominantly against serogroups Australis (76.4%) and Pomona (23.6%). No positive culture was obtained, but DNA was detected by qPCR on urine samples (26.3%), VF (44.7%) and EB (18.4%) collected 2 months or longer following diagnosis of early fetal death and endometritis. Leptospira cell aggregations were visible by indirect immunofluorescence on 57.1% (4/7) EBs and 17.6% (3/17) VFs. A total of 18 amplicons showed interpretable sequences. Out of those 18 amplicons, 15 presented 100% of identity with the species L. interrogans (sv Bratislava and Pomona), while three were L. borgpertersenii. This study suggests the presence of leptospires in the uterus of mares with reproductive problems. Moreover, serology was shown not to be indicated for the diagnosis of presumptive Leptospira infection in early gestation. The most common agent of the genital infection in those mares was L. interrogans, most probably sg Australis.

Invasion of endothelial cells and arthritogenic potential of endocarditis-associated Corynebacterium diphtheriae.

Although infection by Corynebacterium diphtheriae is a model of extracellular mucosal pathogenesis, different clones have been also associated with invasive infections such as sepsis, endocarditis, septic arthritis and osteomyelitis. The mechanisms that promote C. diphtheriae infection and haematogenic dissemination need further investigation. In this study we evaluated the association and invasion mechanisms with human umbilical vein endothelial cells (HUVECs) and experimental arthritis in mice of endocarditis-associated strains and control non-invasive strains. C. diphtheriae strains were able to adhere to and invade HUVECs at different levels. The endocarditis-associated strains displayed an aggregative adherence pattern and a higher number of internalized viable cells in HUVECs. Transmission electron microscopy (TEM) analysis revealed intracellular bacteria free in the cytoplasm and/or contained in a host-membrane-confined compartment as single micro-organisms. Data showed bacterial internalization dependent on microfilament and microtubule stability and involvement of protein phosphorylation in the HUVEC signalling pathway. A high number of affected joints and high arthritis index in addition to the histopathological features indicated a strain-dependent ability of C. diphtheriae to cause severe polyarthritis. A correlation between the arthritis index and increased systemic levels of IL-6 and TNF-α was observed for endocarditis-associated strains. In conclusion, higher incidence of potential mechanisms by which C. diphtheriae may access the bloodstream through the endothelial barrier and stimulate the production of pro-inflammatory cytokines such as IL-6 and TNF-α, in addition to the ability to affect the joints and induce arthritis through haematogenic spread are thought to be related to the pathogenesis of endocarditis-associated strains.