Acinetobacter baumannii infections in Amazon Region driven by extensively drug resistant international clones, 2016-2018.

BACKGROUND: Acinetobacter baumannii is a leading cause of nosocomial infections. This species is characterised by the presence of pandemic lineages (International Clones) that present a broad antimicrobial resistance profile. OBJECTIVE: To perform the molecular epidemiology of carbapenem-resistant A. baumannii from a clinical setting in the Amazon Basin, and to characterise their antimicrobial resistance determinants. METHODS: The genetic relationship of carbapenem-resistant A. baumannii were assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Class A, B and D ß-lactamase genes were screened by polymerase chain reaction (PCR) and sequencing. The antimicrobial susceptibility profile was obtained by Disc-diffusion method and minimum inhibitory concentration (MIC) determination. FINDINGS: All carbapenem-resistant A. baumannii strains belonged to three international clones, IC-1, IC-5 and IC-6, the latter recently reported by the first time in Brazil. The major determinant of carbapenem resistance in IC-1 and IC-5 strains was bla OXA-23, associated with ISAba1 and ISAba3, respectively, while IC-6 harboured the bla OXA-72. CONCLUSIONS: The A. baumannii epidemiology in Brazilian Amazon Region was unknown. It was demonstrated that A. baumannii XDR international clones were responsible for nosocomial infections in Boa Vista during 2016-2018, revealing that the epidemiological scenario of A. baumannii infections in Amazon Region resembles those from the cosmopolitan regions worldwide.

The invasive MenC cc103 lineage with penicillin reduced susceptibility persisting in Brazil.

Penicillin is the antibiotic of choice for the treatment of meningococcal infections, and mutations in penA gene are involved with reduced susceptibility (penI) emergence to this antibiotic. This study aimed to characterize the penA allelic diversity, their association with penI phenotype and distribution among prevalent meningococci serogroups in Brazil. The entire penA from 49 invasive strains of distinct serogroups circulating in Brazil for more than two decades were obtained by PCR and sequencing. Additionally, the penA from 22 publicly available complete Neisseria meningitidis genomes from Brazil were included in the study. The allelic diversity was determined and a genetic tree was built using the penA sequence alignment. The penicillin MIC was obtained by the E-Test method. In general, the identified penA alleles correlated with the observed penI phenotype. The canonical penA1 was the most prevalent allele, however, several altered penA were also identified in strains presenting increased penicillin MICs. It was identified a new penA amino acid position (residue 480) that possibly influence the penicillin MIC in some strains. Interestingly, the altered penA14 was found in penI invasive MenC cc103 strains spread in Brazil and persisting since 2011, indicating that the biological cost imposed by penI phenotype can be ameliorated by particular features present in this lineage, which represents an additional public health threat.

Full characterization of the integrative and conjugative element carrying the metallo-ß-lactamase bla SPM-1 and bicyclomycin bcr1 resistance genes found in the pandemic Pseudomonas aeruginosa clone SP/ST277.

OBJECTIVES: This study aimed to characterize the genomic context of the bla SPM-1 gene in Brazilian strains belonging to the pandemic Pseudomonas aeruginosa clone SP/ST277. METHODS: WGS of clone SP/ST277 strains was performed using a Nextera paired-end library in an Illumina HiSeq 2500 sequencer. bla SPM-1 context was assessed by de novo assembly and gene prediction and annotation tools. bla SPM-1 was screened in P. aeruginosa genomes through BlastN, and comparative genomics were performed. RESULTS: The metallo-ß-lactamase bla SPM-1 has been disseminated by the pandemic Brazilian P. aeruginosa clone SP/ST277. In spite of its association with the CR4 element and with the Tn4371 element, the overall bla SPM-1 genomic context remains uncharacterized and its determination is valuable to understanding gene dispersion dynamics and the consequent emergence of carbapenem resistance. In this study, bla SPM-1 and its surrounding sequences (CR4-groEL-bla SPM-1-CR4-groEL) were found in the variable region of an ICE-like element resembling Tn4371 (where ICE stands for integrative and conjugative element). This element, named ICETn4371 6061, had 46 ORFs, including the bicyclomycin resistance bcr1 gene. An integrase gene and a set of conjugative transfer genes were identified. Gene content and order were shared with other Tn4371-ICEs, presenting remarkable amino acid identities. bla SPM-1 and surrounding sequences were missing in ICETn4371 6061 of PS600-MA, another isolate belonging to clone SP/ST277, indicating their mobilization. Eight/nine P. aeruginosa genomes assigned to clone SP/ST277, by in silico MLST, harboured bla SPM-1 inserted into ICETn4371 6061. CONCLUSIONS: The presence of bla SPM-1 in a Tn4371-ICE with intact integration/conjugation modules demonstrated that, besides gene dispersion by clonal expansion of the pandemic SP/ST277 lineage, bla SPM-1 may be spread through ICE conjugation.

Prevalence and characterization of an integrative and conjugative element carrying tet(X) gene in Elizabethkingia meningoseptica.

OBJECTIVES: To investigate the tet(X) gene, a determinant of tigecycline resistance, in the emerging pathogen Elizabethkingia meningoseptica and its association with an integrative and conjugative element (ICE). METHODS: All E. meningoseptica genomes from the National Center for Biotechnology Information (n = 87) were retrieved and annotated for resistome searches using the CARD database. A phylogenic analysis was performed based on the E. meningoseptica core genome. The ICE was identified through comparative genomics with other ICEs occurring in Elizabethkingia spp. RESULTS: Phylogenetic analysis revealed E. meningoseptica genomes from six countries distributed across different lineages, some of which persisted for years. The common resistome of these genomes included blaBlaB, blaCME, blaGOB, ranA/B, aadS, and catB (genes associated with resistance to ß-lactams, aminoglycosides, and chloramphenicol). Some genomes also presented additional resistance genes (dfrA, ereD, blaVEB, aadS, and tet(X)). Interestingly, tet(X) and aadS were located in an ICE of 49 769 bp (ICEEmSQ101), which was fully obtained from the E. meningoseptica SQ101 genome. We also showed evidence that the other 27 genomes harboured this ICE. The distribution of ICEEmSQ101, carrying tet(X), was restricted to a single Chinese lineage. CONCLUSIONS: The tet(X) gene is not prevalent in the species E. meningoseptica, as previously stated for the genus Elizabethkingia, since it is present only in a single Chinese lineage. We identified that several E. meningoseptica genomes harboured an ICE that mobilized the Elizabethkingia tet(X) gene and exhibited characteristics similar to the ICEs of other Flavobacteria, which would favour their transmission in this bacterial family.

Three outbreak-causing Neisseria meningitidis serogroup C clones, Brazil(1.).

During 2003-2012, 8 clusters of meningococcal disease were identified in Rio de Janeiro State, Brazil, all caused by serogroup C Neisseria meningitidis. The isolates were assigned to 3 clonal complexes (cc): cc11, cc32, and cc103. These hyperinvasive disease lineages were associated with endemic disease, outbreaks, and high case-fatality rates.

Outbreak of high-risk XDR CRAB of international clone 2 (IC2) in Rio Janeiro, Brazil.

OBJECTIVES: Among the high-risk clones of Acinetobacter baumannii, called international clones (ICs), IC2 represents the main lineage causing outbreaks worldwide. Despite the successful global spread of IC2, the occurrence of IC2 is rarely reported in Latin America. Here, we aimed to evaluate the susceptibility and genetic relatedness of isolates from a nosocomial outbreak in Rio de Janeiro/Brazil (2022) and perform genomic epidemiology analyses of the available genomes of A. baumannii. METHODS: Sixteen strains of A. baumannii were subjected to antimicrobial susceptibility tests and genome sequencing. These genomes were compared phylogenetically with other IC2 genomes from the NCBI database, and virulence and antibiotic resistance genes were searched. RESULTS: The 16 strains represented carbapenem-resistant A. baumannii (CRAB) with an extensively drug-resistant profile. In silico analysis established the relationship between the Brazilian CRAB genomes and IC2/ST2 genomes in the world. The Brazilian strains belonged to three sub-lineages, associated with genomes from countries in Europe, North America, and Asia. These sub-lineages presented three distinct capsules, KL7, KL9, and KL56. The Brazilian strains were characterised by the co-presence of blaOXA-23 and blaOXA-66, in addition to the genes APH(6), APH(3"), ANT(3"), AAC(6'), armA, and the efflux pumps adeABC and adeIJK. A large set of virulence genes was also identified: adeFGH/efflux pump; the siderophores barAB, basABCDFGHIJ, and bauBCDEF; lpxABCDLM/capsule; tssABCDEFGIKLM/T6SS; and pgaABCD/biofilm. CONCLUSION: Widespread extensively drug-resistant CRAB IC2/ST2 is currently causing outbreaks in clinical settings in southeastern Brazil. This is due to at least three sub-lineages characterised by an enormous apparatus of virulence and resistance to antibiotics, both intrinsic and mobile.

Integron Functionality and Genome Innovation: An Update on the Subtle and Smart Strategy of Integrase and Gene Cassette Expression Regulation.

Integrons are considered hot spots for bacterial evolution, since these platforms allow one-step genomic innovation by capturing and expressing genes that provide advantageous novelties, such as antibiotic resistance. The acquisition and shuffling of gene cassettes featured by integrons enable the population to rapidly respond to changing selective pressures. However, in order to avoid deleterious effects and fitness burden, the integron activity must be tightly controlled, which happens in an elegant and elaborate fashion, as discussed in detail in the present review. Here, we aimed to provide an up-to-date overview of the complex regulatory networks that permeate the expression and functionality of integrons at both transcriptional and translational levels. It was possible to compile strong shreds of evidence clearly proving that these versatile platforms include functions other than acquiring and expressing gene cassettes. The well-balanced mechanism of integron expression is intricately related with environmental signals, host cell physiology, fitness, and survival, ultimately leading to adaptation on the demand.

Global Genomic Epidemiology of Escherichia coli (ExPEC) ST38 Lineage Revealed a Virulome Associated with Human Infections.

BACKGROUND: Most of the extraintestinal human infections worldwide are caused by specific extraintestinal pathogenic Escherichia coli (ExPEC) lineages, which also present a zoonotic character. One of these lineages belongs to ST38, a high-risk globally disseminated ExPEC. To get insights on the aspects of the global ST38 epidemiology and evolution as a multidrug-resistant and pathogenic lineage concerning the three axes of the One Health concept (humans, animals, and natural environments), this study performed a global phylogenomic analysis on ST38 genomes. METHODS: A phylogenetic reconstruction based on 376 ST38 genomes recovered from environments, humans, livestock, and wild and domestic animals in all continents throughout three decades was performed. The global information concerning the ST38 resistome and virulome was also approached by in silico analyses. RESULTS: In general, the phylogenomic analyses corroborated the zoonotic character of the ExPEC ST38, since clonal strains were recovered from both animal and human sources distributed worldwide. Moreover, our findings revealed that, independent of host sources and geographic origin, the genomes were distributed in two major clades (Clades 1 and 2). However, the ST38 accessory genome was not strictly associated with clades and sub-clades, as found for the type 2 T3SS ETT2 that was evenly distributed throughout Clades 1 and 2. Of note was the presence of the Yersinia pestis-like high-pathogenicity island (HPI) exclusively in the major Clade 2, in which prevails most of the genomes from human origin recovered worldwide (2000 to 2020). CONCLUSIONS: This evidence corroborates the HPI association with successful E. coli ST38 establishment in human infections.

Emergence of a VIM-2-producing extensively drug-resistant (XDR) Pseudomonas aeruginosa ST309 in South America: a comparative genomic analysis.

Pseudomonas aeruginosa is considered a top priority pathogen associated with elevated morbidity and mortality. Worldwide outbreaks have been associated with a few high-risk epidemic P. aeruginosa lineages. However, the biological features involved in the persistence and spread of such lineages in clinical settings remain to be unravelled. This study reports the emergence of an extensively drug-resistant (XDR) sequence type 309 (ST309) P. aeruginosa in South America (Brazil), specifically in the Amazon region. Genomic analyses were performed with 42 complete and draft ST309 genomes, giving insights into its epidemiology, resistome and mobilome. A heterogeneous distribution of acquired antimicrobial resistance genes among ST309 genomes was observed, which included blaVIM-2, blaIMP-15 and qnrVC1, all associated with class 1 integrons. Mobilome mining showed the presence of integrative and conjugative elements (ICEs), transposons and genomic islands (GIs) harbouring a huge arsenal of heavy metal resistance determinants, which probably provided adaptive advantages to the ST309 lineage.

Emergence of extensively drug-resistant international clone IC-6 Acinetobacter baumannii carrying blaOXA-72 and blaCTX-M-115 in the Brazilian Amazon region.

OBJECTIVES: The extensively drug-resistant (XDR) Acinetobacter baumannii international clone VI (IC-6) has been identified worldwide since 2006. This study reports the emergence of IC-6 in the Brazilian Amazon region and reveals the particular genomic features considering its mobilome and resistome. METHODS: A total of 32 carbapenem-resistant A. baumannii strains recovered from Boa Vista city (Roraima, Brazil) in 2016 were characterised by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The whole genome sequences of the Brazilian IC-6 strains were obtained. The mobilome and resistome were assessed by in silico analyses. RESULTS: PFGE and MLST demonstrated that the 32 A. baumannii strains belonged to four clones. One XDR clone corresponded to the high-risk pandemic IC-6 lineage from ST944Oxf/78Pas. The IC-6 resistome was composed of aadA5, aac(3'')-IIa, aph(3')-Ia, armA, aadB, msrE, blaTEM-1, IS15DIV-blaCTX-M-115-IS15DIV, blaOXA-90, ISAba1-blaADC-152, blaOXA-72, qacEΔ1 and sul1. Mobilome prediction revealed that blaOXA-72 was embedded in a 15.5-kb plasmid and that it was flanked by putative XerC/D-binding sites, possibly involved in blaOXA-72 mobilisation. Several resistance genes were in a 48-kb multidrug resistance genomic island inserted in the chromosome, which also harboured genes involved in host pathogenicity and adaptive traits. Interestingly, the Brazilian strains shared the blaOXA-72 and blaCTX-M-115 with IC-6/ST944Oxf/78Pas recovered in a distinct spatiotemporal context, pointing to an epidemiological link among them. CONCLUSION: This study highlights the importance of surveillance of XDR A. baumannii strains, even outside of densely populated cosmopolitan regions, to reveal the epidemiology of pandemic lineages, stressing their threat to public health.

New qnr gene cassettes associated with superintegron repeats in Vibrio cholerae O1.

A novel qnr determinant emerged in ciprofloxacin-resistant Vibrio cholerae O1 from the Amazon region of Brazil. This qnrVC1 was in a typical class 1 integron. Its attC showed 89% identity with V. parahaemolyticus superintegron repeats. Analysis showed V. cholerae O1 carrying qnrVC2 associated with a V. cholerae superintegron repeat.

The Resistome of Low-Impacted Marine Environments Is Composed by Distant Metallo-ß-Lactamases Homologs.

The worldwide dispersion and sudden emergence of new antibiotic resistance genes (ARGs) determined the need in uncovering which environment participate most as their source and reservoir. ARGs closely related to those currently found in human pathogens occur in the resistome of anthropogenic impacted environments. However, the role of pristine environment as the origin and source of ARGs remains underexplored and controversy, particularly, the marine environments represented by the oceans. Here, due to the ocean nature, we hypothesized that the resistome of this pristine/low-impacted marine environment is represented by distant ARG homologs. To test this hypothesis we performed an in silico analysis on the Global Ocean Sampling (GOS) metagenomic project dataset focusing on the metallo-ß-lactamases (MßLs) as the ARG model. MßLs have been a challenge to public health, since they hydrolyze the carbapenems, one of the last therapeutic choice in clinics. Using Hidden Markov Model (HMM) profiles, we were successful in identifying a high diversity of distant MßL homologs, related to the B1, B2, and B3 subclasses. The majority of them were distributed across the Atlantic, Indian, and Pacific Oceans being related to the chromosomally encoded MßL GOB present in Elizabethkingia genus. It was observed only a reduced number of metagenomic sequence homologs related to the acquired MßL enzymes (VIM, SPM-1, and AIM-1) that currently have impact in clinics. Therefore, low antibiotic impacted marine environment, as the ocean, are unlikely the source of ARGs that have been causing enormous threat to the public health.

Coexistence of epidemic colistin-only-sensitive clones of Pseudomonas aeruginosa, including the blaSPM clone, spread in hospitals in a Brazilian Amazon City.

Nosocomial outbreaks caused by multidrug-resistant (MDR) Pseudomonas aeruginosa have been associated to fibrocystic patients and isolates harboring metallo-beta-lactamase (MBL) genes. Genotyping is an important tool for interpreting bacterial nosocomial outbreaks and implementing adequate control strategies. The aim of this study was to evaluate whether an outbreak of MDR P. aeruginosa occurring in different hospitals was due to a unique clone or independent isolates. From 2000 to 2003, 108 P. aeruginosa were recovered from colonized/infected inpatients in hospitals of São Luís, Maranhão, Brazil. The susceptibility test was performed with antipseudomonal drugs, and the presence of MBL genes were verified by PCR. Isolates were genotyped by pulsed-field gel electrophoresis (PFGE). The majority of strains was multiresistant including a great number presenting the colistin-only-sensitive (COS) profile. PFGE analysis revealed 54 genotypes, with predominance of three major COS clones (A, C, and E) coexisting at different moments and hospitals. Clone A harbored the bla(SPM) gene. Eight unique genotypes also had the COS profile. Other eight MDR genotypes presented isolates with differences in resistance profiles. Here we detected, for the first time, the coexistence of COS P.aeruginosa genotypes disseminated in several hospitals during long periods, attacking patients under various clinical conditions.

Class 1 integrons in Pseudomonas aeruginosa isolates from clinical settings in Amazon region, Brazil.

A hundred and six Pseudomonas aeruginosa isolates from clinical cases were screened using PCR for the presence of integrons and associated resistance gene cassettes. Forty-four isolates harboured class 1 integrons (41.5%), of which 29 isolates (66%) also carried gene cassettes. The aacA gene was most frequently found within class 1 integrons (69%), followed by blaOXA family genes (52%). From class 1 integron-positive strains, we detected a total of 15 isolates (34%) carrying no gene cassettes. Restriction fragment-length polymorphism analysis of the integrons variable region revealed some identical structures, as well as distinct profiles indicating heterogeneity among these cassette regions. Multiresistance was observed in 71% of isolates, nevertheless no strong correlation was observed between integron presence and multiresistance. This is the first report showing class 1 integron prevalence and gene cassette content in P. aeruginosa isolates from clinical settings in the Brazilian Amazon.

Conventional and molecular typing of Salmonella typhi strains from Brazil.

Phenotypic and genotypic characteristics of Salmonella Typhi were studied in 30 strains, isolated in different years, from some areas in Brazil. Conventional typing methods were performed by biochemical tests, Vi phage-typing scheme, and antimicrobial susceptibility test. Molecular typing methods were performed by analysis of plasmid DNA and by random amplified polymorphic DNA (RAPD-PCR). For the latter, an optimization step was performed to ensure the reproducibility of the process in genetic characterization of S. Typhi. The predominance of 76.7% of biotype I (xylose +, arabinose -) was noticed in all studied areas. Three phage types were recognized, with prominence for the phage types A (73.3%) and I+IV (23.3%). All the strains were susceptible to the drugs used. However, 36.7% of the strains contained plasmids, with predominance of the 105 Kb plasmid. RAPD was capable of grouping the strains in 8 genotypic patterns using primer 784, in 6, using primer 787 and in 7, using primer 797. Conventional phenotypic typing methods, as well as the DNA plasmid analysis, presented nonsignificant discriminatory power; however, RAPD-PCR analysis showed discriminatory power, reproducibility, easy interpretation and performance, being considered as a promising alternative typing method for S. Typhi.

Worldwide occurrence of integrative conjugative element encoding multidrug resistance determinants in epidemic Vibrio cholerae O1.

In the last decades, there has been an increase of cholera epidemics caused by multidrug resistant strains. Particularly, the integrative and conjugative element (ICE) seems to play a major role in the emergence of multidrug resistant Vibrio cholerae. This study fully characterized, by whole genome sequencing, new ICEs carried by multidrug resistant V. cholerae O1 strains from Nigeria (2010) (ICEVchNig1) and Nepal (1994) (ICEVchNep1). The gene content and gene order of these two ICEs are the same, and identical to ICEVchInd5, ICEVchBan5 and ICEVchHai1 previously identified in multidrug resistant V. cholerae O1. This ICE is characterized by dfrA1, sul2, strAB and floR antimicrobial resistance genes, and by unique gene content in HS4 and HS5 ICE regions. Screening for ICEs, in publicly available V. cholerae genomes, revealed the occurrence and widespread distribution of this ICE among V. cholerae O1. Metagenomic analysis found segments of this ICE in marine environments far from the direct influence of the cholera epidemic. Therefore, this study revealed the epidemiology of a spatio-temporal prevalent ICE in V. cholerae O1. Its occurrence and dispersion in V. cholerae O1 strains from different continents throughout more than two decades can be indicative of its role in the fitness of the current pandemic lineage.

The genome of a clinical Klebsiella variicola strain reveals virulence-associated traits and a pl9-like plasmid.

Klebsiella species frequently cause clinically relevant human infections worldwide. We report the draft genome sequence of a Brazilian clinical isolate (Bz19) of the recently recognized species Klebsiella variicola. The comparison of Bz19 genome content with the At-22 (environmental K. variicola) and several clinical Klebsiella pneumoniae shows that these species share a set of virulence-associated determinants. Of note, this K. variicola strain harbours a plasmid-like element that shares the same backbone present in a multidrug-resistant plasmid found in a clinical K. pneumoniae isolated in USA.

Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from Pseudomonas aeruginosa.

The gene cassettes found in class 1 integrons are generally promoterless units composed by an open reading frame (ORF), a short 5' untranslated region (UTR) and a 3' recombination site ( attC). Fused gene cassettes are generated by partial or total loss of the attC from the first cassette in an array, creating, in some cases, a fusion with the ORF from the next cassette. These structures are rare and little is known about their mechanisms of mobilization and expression. The aim of this study was to evaluate the dynamic of mobilization and transcription of the gcu14-bla GES-1 /aacA4 gene cassette array, which harbours a fused gene cassette represented by bla GES-1 /aacA4. The cassette array was analyzed by Northern blot and real-time reverse transcription-polymerase chain reaction (RT-PCR) in order to assess the transcription mechanism of bla GES-1 /aacA4 fused cassette. Also, inverse polymerase chain reactions (PCR) were performed to detect the free circular forms of gcu14, bla GES-1 and aacA4. The Northern blot and real time RT-PCR revealed a polycistronic transcription, in which the fused cassette bla GES-1 /aacA4 is transcribed as a unique gene, while gcu14 (with a canonical attC recombination site) has a monocistronic transcription. The gcu14 cassette, closer to the weak configuration of cassette promoter (PcW), had a higher transcription level than bla GES-1/ aacA4, indicating that the cassette position affects the transcript amounts. The presence of ORF-11 at attI1, immediately preceding gcu14, and of a Shine-Dalgarno sequence upstream bla GES-1/ aacA4 composes a scenario for the occurrence of array translation. Inverse PCR generated amplicons corresponding to gcu14, gcu14-aacA4 and gcu14-bla GES-1/ aacA4 free circular forms, but not to bla GES-1 and aacA4 alone, indicating that the GES-1 truncated attC is not substrate of integrase activity and that these genes are mobilized together as a unique cassette. This study was original in showing the transcription of fused cassettes and in correlating cassette position with transcription.