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.

An updated gene regulatory network reconstruction of multidrug-resistant Pseudomonas aeruginosa CCBH4851.

BACKGROUND: Healthcare-associated infections due to multidrug-resistant (MDR) bacteria such as Pseudomonas aeruginosa are significant public health issues worldwide. A system biology approach can help understand bacterial behaviour and provide novel ways to identify potential therapeutic targets and develop new drugs. Gene regulatory networks (GRN) are examples of in silico representation of interaction between regulatory genes and their targets. OBJECTIVES: In this work, we update the MDR P. aeruginosa CCBH4851 GRN reconstruction and analyse and discuss its structural properties. METHODS: We based this study on the gene orthology inference methodology using the reciprocal best hit method. The P. aeruginosa CCBH4851 genome and GRN, published in 2019, and the P. aeruginosa PAO1 GRN, published in 2020, were used for this update reconstruction process. FINDINGS: Our result is a GRN with a greater number of regulatory genes, target genes, and interactions compared to the previous networks, and its structural properties are consistent with the complexity of biological networks and the biological features of P. aeruginosa. MAIN CONCLUSIONS: Here, we present the largest and most complete version of P. aeruginosa GRN published to this date, to the best of our knowledge.

Carbapenem-Resistant Pseudomonas aeruginosa in Chronic Lung Infection: Current Resistance Profile and Hypermutability in Patients with Cystic Fibrosis.

Pseudomonas aeruginosa is associated with chronic and progressive lung disease and is closely related to increased morbidity and mortality in cystic fibrosis (CF) patients. Hypermutable (HPM) P. aeruginosa isolates have been described in these patients and are usually associated with antibiotic resistance. This study aimed to investigate the occurrence of carbapenem resistance and hypermutable phenotype in 179 P. aeruginosa isolates from 8 chronically CF patients assisted at two reference centers in Rio de Janeiro, Brazil. Using disk diffusion test, non-susceptible (NS) rates higher than 40% were observed for imipenem, amikacin, and gentamicin. A total of 79 isolates (44.1%), 71 (39.6%), and 8 (4.4%) were classified as carbapenem-resistant (CR resistance to at least one carbapenem), multidrug-resistant (MDR), and extensively drug-resistant (XDR), respectively. Minimal inhibitory concentration was determined for 79 CR P. aeruginosa and showed the following variations: 4 and 128 µg/mL to imipenem, 4 and 64 µg/mL to meropenem, and 4 and ≥ 32 µg/mL to doripenem. We have found only four (2.23%) HPM isolates from 4 patients. Analyzing the genetic relationship among the HPM isolates, 3 pulsed-field gel electrophoresis/pulsotypes (D, M, and J) were observed. Only M pulsotype was recovered from two patients in different years. Polymerase chain reaction screening for blaGES, blaIMP, blaKPC, blaNDM, blaOXA-48, blaSPM, and blaVIM genes was performed for all CR isolates and none of them were positive. Our results demonstrate a high occurrence of CR and MDR P. aeruginosa of CF patients follow-up in both centers studied, while the presence of HPM is still unusual.

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.

Gene regulatory network inference and analysis of multidrug-resistant Pseudomonas aeruginosa.

BACKGROUND: Healthcare-associated infections caused by bacteria such as Pseudomonas aeruginosa are a major public health problem worldwide. Gene regulatory networks (GRN) computationally represent interactions among regulatory genes and their targets. They are an important approach to help understand bacterial behaviour and to provide novel ways of overcoming scientific challenges, including the identification of potential therapeutic targets and the development of new drugs. OBJECTIVES: The goal of this study was to reconstruct the multidrug-resistant (MDR) P. aeruginosa GRN and to analyse its topological properties. METHODS: The methodology used in this study was based on gene orthology inference using the reciprocal best hit method. We used the genome of P. aeruginosa CCBH4851 as the basis of the reconstruction process. This MDR strain is representative of the sequence type 277, which was involved in an endemic outbreak in Brazil. FINDINGS: We obtained a network with a larger number of regulatory genes, target genes and interactions as compared to the previously reported network. Topological analysis results are in accordance with the complex network representation of biological processes. MAIN CONCLUSIONS: The properties of the network were consistent with the biological features of P. aeruginosa. To the best of our knowledge, the P. aeruginosa GRN presented here is the most complete version available to date.

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.

CTX-M-15-producing uropathogenic Escherichia coli isolates at Rio de Janeiro, Brazil: Molecular epidemiology and MALDI-TOF MS.

Because of the high prevalence of CTX-M-15-producing Escherichia coli isolates causing urinary tract infections in Rio de Janeiro, we have investigated bla-CTX-M-15 gene presence, as well as CTX-M-15 production, in 32 E. coli isolates recovered from the urine of outpatients assisted at a public hospital located in the west zone of Rio. Molecular epidemiology was assessed by PFGE and phylo-typing methods. The work highlights the good performance of MALDI-TOF MS as an alternative tool to detect extended-spectrum beta-lactamases among CTX-M-15-producing E. coli isolates.

Multiclonal Expansion of Klebsiella pneumoniae Isolates Producing NDM-1 in Rio de Janeiro, Brazil.

We characterized NDM-1-producing Klebsiella isolates from Rio de Janeiro, Brazil. PCR was applied for resistance and virulence determinants. The genetic context of blaNDM was determined by S1 nuclease pulsed-field gel electrophoresis (PFGE) and hybridization. Genotyping was performed by PFGE and multilocus sequence typing (MLST). Most isolates carried multiple resistance genes and remained susceptible to amikacin, fosfomycin-trometamol, polymyxin B, and tigecycline. The spread of NDM-1-producing Klebsiella pneumoniae was not associated with clonal expansion and appears to be associated with Tn3000.

mgrB Mutations Mediating Polymyxin B Resistance in Klebsiella pneumoniae Isolates from Rectal Surveillance Swabs in Brazil.

We aimed to investigate polymyxin B (PMB) resistance and its molecular mechanisms in 126 Klebsiella pneumoniae isolates from rectal swabs in Brazil. Ten isolates exhibited PMB resistance with interruption of mgrB gene by insertion sequences or missense mutations. Most of the PMB-resistant isolates harbored blaKPC-2 (n = 8) and belonged to clonal complex 258 (CC258) (n = 7). These results highlight the importance of monitoring the spread of polymyxin-resistant bacteria in hospitals, since few options remain to treat multidrug-resistant isolates.

Detection of Carbapenemase Genes in Aquatic Environments in Rio de Janeiro, Brazil.

This study reveals the presence of different carbapenemase genes (blaKPC, blaNDM, blaGES, and blaOXA48-like genes) detected directly from water samples and clonal dispersion (by pulsed-field gel electrophoresis [PFGE] and multilocus sequence typing [MLST]) of KPC-2-producing Enterobacteriaceae in two important urban aquatic matrixes from Rio de Janeiro, Brazil, highlighting the role of aquatic environments as gene pools and the possibility of community spreading.

Draft genome sequence of Acinetobacter pittii ST643 shared by cystic fibrosis patients.

Acinetobacter pittii has emerged as an important hospital pathogen that is associated with outbreaks and drug resistance. In cystic fibrosis (CF) patients, the detection of Acinetobacter spp. is rare; however, we isolated the A. pittii sequence type ST643 in several Brazilian CF patients treated in the same centre. The current study describes the draft genome of A. pittii ST643.

Clonal Dissemination of OXA-370-Producing Klebsiella pneumoniae in Rio de Janeiro, Brazil.

Enzymes of the OXA-48 family have become some of the most important beta-lactamases in the world. A new OXA-48 variant (OXA-370) was first described for an Enterobacter hormaechei strain isolated in Rio Grande do Sul (southern region of Brazil) in 2013. Here we report detection of the blaOXA-370 gene in 24 isolates belonging to three Enterobacteriaceae species (22 Klebsiella pneumoniae isolates, 1 Enterobacter cloacae isolate, and 1 Enterobacter aerogenes isolate) collected from five hospitals in Rio de Janeiro, Brazil, in 2013 and 2014. The isolates showed a multidrug resistance profile, and 12.5% were resistant to polymyxin B. Besides blaOXA-370, no other carbapenemase genes were observed by PCR, whereas blaOXA-1 was found in all isolates and 22 isolates (91.6%) possessed blaCTX-M-15. Molecular typing of the K. pneumoniae isolates by pulsed-field gel electrophoresis (PFGE) showed the presence of two clonal groups, i.e., KpA (21 isolates) and KpB (1 isolate). KpA was characterized as sequence type 16 (ST16) and KpB as ST1041 by multilocus sequence typing (MLST). ST16 has been observed for KPC-producing K. pneumoniae in Rio de Janeiro. Plasmid analysis performed with six representative OXA-370-producing isolates showed plasmids harboring the blaOXA-370 gene in all strains, ranging from 25 kb to 150 kb. This study suggests that there is an urgent need to investigate the presence of OXA-370 and dissemination of the K. pneumoniae ST16 clone carrying this gene in Brazil.

Characterization of Achromobacter Species in Cystic Fibrosis Patients: Comparison of bla(OXA-114) PCR Amplification, Multilocus Sequence Typing, and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Molecular methodologies were used to identify 28 Achromobacter spp. from patients with cystic fibrosis (CF). Multilocus sequence typing (MLST) identified 17 Achromobacter xylosoxidans isolates (all bla(OXA-114) positive), nine Achromobacter ruhlandii isolates (all bla(OXA-114) positive), one Achromobacter dolens isolate, and one Achromobacter insuavis isolate. All less common species were misidentified as A. xylosoxidans by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Chronic colonization by clonally related A. ruhlandii isolates was demonstrated.

Draft genome sequences of three NDM-1-producing Enterobacteriaceae species isolated from Brazil.

The emergence of multidrug-resistant Enterobacteriaceae strains producing carbapenemases, such as NDM-1, has become a major public health issue due to a high dissemination capacity and limited treatment options. Here we describe the draft genome of three NDM-1-producing isolates: Providencia rettgeri (CCBH11880), Enterobacter hormaechei subsp. oharae (CCBH10892) and Klebsiella pneumoniae (CCBH13327), isolated in Brazil. Besides blaNDM-1, resistance genes to aminoglycosides [aadA1, aadA2, aac(6')-Ib-cr] and quinolones (qnrA1, qnrB4) were observed which contributed to the multidrug resistance profile. The element ISAba125 was found associated to the blaNDM-1 gene in all strains.

Draft genome sequence of a multidrug-resistant Acinetobacter baumannii ST15 (CC15) isolated from Brazil.

Acinetobacter baumannii is an important pathogen frequently associated with nosocomial outbreaks around the world. In Brazil, A. baumannii has become particularly problematic because of its prevalence and the carbapenems resistance. Here, we report the draft genome sequence of a multidrug-resistant A. baumannii(ST15/CC15) isolated in 2009 from the state of Espírito Santo (Southeast Brazil). We observed important resistance determinant genes in an estimated genome size of 4,102,788 bp with 3,862 predicted coding regions. A detailed report of the genomic data analysis might help to understand the specific features of highly successful strains belonged to a relevant complex clonal in different Brazilian geographical regions.

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.

Comparative analysis of the antimicrobial resistance and virulence traits in ESBL-producing-Klebsiella pneumoniae ST307 strains colonizing the gastrointestinal tract and causing a fatal bloodstream infection in a leukemia patient.

Klebsiella pneumoniae is an opportunistic pathogen that can colonize the gastrointestinal tract (GIT) of humans. The mechanisms underlying the successful translocation of this pathogen to cause extra-intestinal infections remain unknown, although virulence and antimicrobial resistance traits likely play significant roles in the establishment of infections. We investigated K. pneumoniae strains isolated from GIT colonization (strains Kp_FZcol-1, Kp_FZcol-2 and Kp_FZcro-1) and from a fatal bloodstream infection (strain Kp_HM-1) in a leukemia patient. All strains belonged to ST307, carried a transferable IncF plasmid containing the blaCTX-M-15 gene (pKPN3-307 TypeA-like plasmid) and showed a multidrug-resistance phenotype. Phylogenetic analysis demonstrated that Kp_HM-1 was more closely related to Kp_FZcro-1 than to the other colonizing strains. The Kp_FZcol-2 genome showed 81 % coverage with the Kp_HM-1 246,730 bp plasmid (pKp_HM-1), lacking most of its putative virulence genes. Searching public genomes with similar coverage, we observed the occurrence of this deletion in K. pneumoniae ST307 strains recovered from human colonization and infection in different countries. Our findings suggest that strains lacking the putative virulence genes found in the pKPN3-307 TypeA plasmid are still able to colonize and infect humans, highlighting the need to further investigate the role of these genes for the adaptation of K. pneumoniae ST307 in distinct human body sites.

Polymyxin resistance in Enterobacter cloacae complex in Brazil: phenotypic and molecular characterization.

Enterobacter cloacae complex isolates have been reported as an important nosocomial multidrug resistance pathogen. In the present study, we investigated antimicrobial susceptibility and the colistin-resistance rates, their genetic determinants and clonality among clinical E. cloacae complex isolates from different Brazilian states. For this, an initial screening was carried out on 94 clinical isolates of E. clocacae complex received between 2016 and 2018 by LAPIH-FIOCRUZ, using EMB plates containing 4 µg/mL of colistin, followed MIC determination, resulting in the selection of 26 colistin-resistant isolates from the complex. The presence of carbapenemases encoding genes (blaKPC, blaNDM and blaOXA-48), plasmidial genes for resistance to polymyxins (mcr1-9) and mutations in chromosomal genes (pmrA, pmrB, phoP and phoQ) described as associated with resistance to polymyxin were screened by PCR and DNA sequencing. Finally, the hsp60 gene was sequenced to identify species of the E. cloacae complex and genetic diversity was evaluated by PFGE and MLST. The results have shown that among 94 E. cloacae complex isolates, 19 (20.2%) were colistin-resistant. The resistant strains exhibited MIC ranging from 4 to 128 µg / mL and E. hormaechei subsp. steigerwaltii was the prevalent species in the complex (31,6%), followed by E. cloacae subsp. cloacae (26,3%). The antimicrobials with the highest susceptibility rate were gentamicin (21%) and tigecycline (26%). Carbapenemases encoding genes (blaKPC n = 5, blaNDM n = 1) were detected in 6 isolates and mcr-9 in one. Among the modifications found in PmrA, PmrB, PhoP e PhoQ (two-component regulatory system), only the S175I substitution in PmrB found in E. cloacae subsp cloacae isolates were considered deleterious (according to the prediction of PROVEAN). By PFGE, 13 profiles were found among E. cloacae complex isolates, with EcD the most frequent. Furthermore, by MLST 10 ST's, and 1 new ST, were identified in E. cloacae. In conclusion, no prevalence of clones or association among carbapenemase production and polymyxin resistance was found between the E. cloacae. Thereby, the results suggest that the increased polymyxin-resistance is related to the selective pressure exerted by the indiscriminate use in hospitals. Lastly, this study highlights the urgent need to elucidate the mechanism involved in the resistance to polymyxin in the E. cloacae complex and the development of measures to control and prevent infections caused by these multiresistant bacteria.

Genomic study of Acinetobacter baumannii strains co-harboring bla OXA-58 and bla NDM-1 reveals a large multidrug-resistant plasmid encoding these carbapenemases in Brazil.

Introduction: Acinetobacter baumannii contributes significantly to the global issue of multidrug-resistant (MDR) nosocomial infections. Often, these strains demonstrate resistance to carbapenems (MDR-CRAB), the first-line treatment for infections instigated by MDR A. baumannii. Our study focused on the antimicrobial susceptibility and genomic sequences related to plasmids from 12 clinical isolates of A. baumannii that carry both the blaOXA-58 and bla NDM-1 carbapenemase genes. Methods: Whole-genome sequencing with long-read technology was employed for the characterization of an A. baumannii plasmid that harbors the bla OXA-58 and blaNDM-1 genes. The location of the bla OXA-58 and bla NDM-1 genes was confirmed through Southern blot hybridization assays. Antimicrobial susceptibility tests were conducted, and molecular characterization was performed using PCR and PFGE. Results: Multilocus Sequence Typing analysis revealed considerable genetic diversity among bla OXA-58 and bla NDM-1 positive strains in Brazil. It was confirmed that these genes were located on a plasmid larger than 300 kb in isolates from the same hospital, which also carry other antimicrobial resistance genes. Different genetic contexts were observed for the co-occurrence of these carbapenemase-encoding genes in Brazilian strains. Discussion: The propagation of bla OXA-58 and bla NDM-1 genes on the same plasmid, which also carries other resistance determinants, could potentially lead to the emergence of bacterial strains resistant to multiple classes of antimicrobials. Therefore, the characterization of these strains is of paramount importance for monitoring resistance evolution, curbing their rapid global dissemination, averting outbreaks, and optimizing therapy.

Update of the molecular epidemiology of KPC-2-producing Klebsiella pneumoniae in Brazil: spread of clonal complex 11 (ST11, ST437 and ST340).

OBJECTIVES: To perform molecular epidemiology for 113 KPC-producing Klebsiella pneumoniae isolated in 2010 from 12 Brazilian states. METHODS: The resistance profile was determined by disc diffusion and Etest. Genetic polymorphism was analysed by PFGE and multilocus sequence typing. The genetic environment of the bla(KPC) gene was determined by PCR and identification of the carrier plasmid was determined by hybridization. RESULTS: Most of the isolates were multidrug resistant, with 15% and 49% being resistant to polymyxin and tigecycline, respectively. Twenty-two sequence types (STs) were observed, with ST11, ST437 and ST340 [clonal complex 11 (CC11)] being the most prevalent (75% of isolates) observed in 10 states. bla(KPC-2) was associated with transposon Tn4401 'b' and in 36% this gene was found in IncN plasmids of 40 kb. CONCLUSIONS: In Brazil, the spread of bla(KPC-2) is occurring due to dispersion of Tn4401 'b', carried by IncN plasmids of 40 kb, and mainly the dissemination of CC11, with ST437 and ST11 playing an important role.