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.

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.

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.

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.

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 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.

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.

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.

Streptococcal taxonomy based on genome sequence analyses.

The identification of the clinically relevant viridans streptococci group, at species level, is still problematic. The aim of this study was to extract taxonomic information from the complete genome sequences of 67 streptococci, comprising 19 species, by means of genomic analyses, multilocus sequence analysis (MLSA), average amino acid identity (AAI), genomic signatures, genome-to-genome distances (GGD) and codon usage bias. We then attempted to determine the usefulness of these genomic tools for species identification in streptococci. Our results showed that MLSA, AAI and GGD analyses are robust markers to identify streptococci at the species level, for instance, S. pneumoniae, S. mitis, and S. oralis. A Streptococcus species can be defined as a group of strains that share ≥ 95% DNA similarity in MLSA and AAI, and > 70% DNA identity in GGD. This approach allows an advanced understanding of bacterial diversity.

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.

Cholera outbreaks in Nigeria are associated with multidrug resistant atypical El Tor and non-O1/non-O139 Vibrio cholerae.

BACKGROUND: The current millennium has seen a steep rise in the number, size and case-fatalities of cholera outbreaks in many African countries. Over 40,000 cases of cholera were reported from Nigeria in 2010. Variants of Vibrio cholerae O1 El Tor biotype have emerged but very little is known about strains causing cholera outbreaks in West Africa, which is crucial for the implementation of interventions to control epidemic cholera. METHODOLOGY/PRINCIPAL FINDINGS: V. cholerae isolates from outbreaks of acute watery diarrhea in Nigeria from December, 2009 to October, 2010 were identified by standard culture methods. Fifteen O1 and five non-O1/non-O139 strains were analyzed; PCR and sequencing targeted regions associated with virulence, resistance and biotype were performed. We also studied genetic interrelatedness among the strains by multilocus sequence analysis and pulsed-field gel electrophoresis. The antibiotic susceptibility was tested by the disk diffusion method and E-test. We found that multidrug resistant atypical El Tor strains, with reduced susceptibility to ciprofloxacin and chloramphenicol, characterized by the presence of the SXT element, and gyrA(Ser83Ile)/parC(Ser85Leu) alleles as well CTX phage and TCP cluster characterized by rstR(ElTor), ctxB-7 and tcpA(CIRS) alleles, respectively, were largely responsible for cholera outbreaks in 2009 and 2010. We also identified and characterized a V. cholerae non-O1/non-O139 lineage from cholera-like diarrhea cases in Nigeria. CONCLUSIONS/SIGNIFICANCE: The recent Nigeria outbreaks have been determined by multidrug resistant atypical El Tor and non-O1/non-O139 V. cholerae strains, and it seems that the typical El Tor, from the beginning of seventh cholera pandemic, is no longer epidemic/endemic in this country. This scenario is similar to the East Africa, Asia and Caribbean countries. The detection of a highly virulent, antimicrobial resistant lineage in Nigeria is worrisome and points to a need for vaccine-based control of the disease. This study has also revealed the putative importance of non-O1/non-O139 V. cholerae in diarrheal disease in Nigeria.

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.

Vibrio cholerae O1 lineages driving cholera outbreaks during seventh cholera pandemic in Ghana.

In recent years, the frequency of cholera epidemics across Africa has increased significantly with thousands of people dying each year. However, there still exists a lack of information concerning the Vibrio cholerae O1 lineages driving early and contemporary epidemics since the seventh cholera pandemic started in the continent. This compromises the understanding of the forces determining the epidemiology of cholera in Africa and its control. This study aimed to analyze a collection of V. cholerae O1 strains from the beginning of the seventh cholera pandemic in Ghana and to compare them with recent isolates to understand the evolution of the cholera epidemic in Ghana. V. cholerae O1 strains were characterized by means of Multilocus Sequence Analysis (MLSA), genes from the virulence core genome (VCG), and genes related to the choleragenic phenotype. Our results revealed two major clusters of Ghanaian V. cholerae O1 strains, El Tor and Amazonia/Ghana. Concerning the virulence genes, all strains harbored the set of VCG and most were positive for VSP-II genomic island. The ctxB gene of the contemporary strains was characterized as Altered El Tor. The strains from 1970 to 1980 were susceptible to all antibiotics tested, except for the Amazonia/Ghana cluster that was resistant to aminoglycosides and carried the class 2 integron with the sat2-aadA1 arrangement. This study showed that distinct V. cholerae O1 were the determinants of cholera outbreaks in Ghana. Thus, in endemic regions, such as Africa, cholera can be caused by various V. cholerae O1 genotypes.

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.