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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.

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.

Carbapenem-resistant Acinetobacter baumannii from Brazil: role of carO alleles expression and blaOXA-23 gene

BackgroundCarbapenems are the antibiotics of choice to treat infections caused by Acinetobacter baumannii, and resistance to this class can be determined by loss of membrane permeability and enzymatic mechanisms. Here, we analyzed the basis of carbapenem resistance in clinical A. baumannii isolates from different Brazilian regions.ResultsThe analyses addressed the carbapenemase activity of OXA-23, CarO expression and alterations in its primary structure. Susceptibility test revealed that the strains presented the COS (Colistin-Only-Sensitive) profile. PCR and sequencing showed the presence of the chromosomally-encoded blaOXA-51 in all isolates. The majority of strains (53%) carried the carbapenemase blaOXA-23 gene associated with ISAba1. The Hodge test indicated that these strains are carbapenemase producers. PFGE revealed 14 genotypes among strains from Rio de Janeiro and Maranhão. The influence of carO on imipenem resistance was evaluated considering two aspects: the composition of the primary amino acid sequence; and the expression level of this porin. Sequencing and in silico analyses showed the occurrence of CarOa, CarOb and undefined CarO types, and Real Time RT-PCR revealed basal and reduced carO transcription levels among isolates.ConclusionsWe concluded that, in general, for these Brazilian isolates, the major carbapenem resistance mechanism was due to OXA-23 carbapenemase activity and that loss of CarO porin plays a minor role in this phenotype. However, it was possible to associate the carO alleles and their expression with imipenem resistance. Therefore, these findings underline the complexity in addressing the role of different mechanisms in carbapenem resistance and highlight the possible influence of CarO type in this phenotype.

The colistin-only-sensitive Brazilian Pseudomonas aeruginosa clone SP (sequence type 277) is spread worldwide.

We read with great interest the recent publication by Salabi et al. (8), which reported the first case of a blaSPM-1 metallo-β-lactamase gene outside Brazil. The authors stated that an SPM-1-positive Pseudomonas aeruginosa strain, BH121, recovered from a Swiss inpatient coming from Recife, Brazil, was genetically related to the Brazilian P. aeruginosa clone SP, which is endemic in Brazil (1, 2, 6). In fact, such study showed that, even though the bacterium isolation had occurred in Switzerland, this patient had probably been infected in Brazil. Here, we applied the P. aeruginosa multilocus sequence typing (MLST) scheme (3) for determining the epidemiology of colistin-only-sensitive (COS) Brazilian P. aeruginosa clone SP harboring blaSPM-1, and it was demonstrated that this clone had already been circulating outside Brazil before the BH121 identification in Europe (8). Thirty-seven SPM-1 (n = 27) and non-SPM-1 (n = 10) isolates presenting the COS phenotype were recovered from different inpatients between 2001 and 2006 from Rio de Janeiro and Sao Luis, two Brazilian cities 3,000 km apart. These strains were analyzed by MLST and pulsed-field gel electrophoresis (PFGE) in order to characterize and compare both approaches. The P. aeruginosa MLST scheme was applied as proposed (3). PFGE was performed as described previously (5), and the macrorestriction profile was analyzed according to the criteria of Tenover et al. (9). PFGE and MLST analysis showed that the COS P. aeruginosa strains harboring blaSPM-1 clustered together in clone SP and belonged to sequence type (ST) 277, while COS SPM-1-negative strains grouped in another clone, and they were assigned to ST 244. Therefore, in our study, PFGE and MLST demonstrated the same resolution power as reported previously (4). Since the Brazilian clone was previously named clone SP (6), we decided to use the same nomenclature to define all strains from ST 277. Interestingly, according to the P. aeruginosa MLST database, ST 277 had already been assigned to a strain isolated in Austria in 2006, showing the presence of clone SP in Europe before the description by Salabi et al. (8). Moreover, ST 277 was also assigned to isolates from China and Australia, showing its potential to be widespread, its high fitness, and its being established as an international clone. Nevertheless, there are no data concerning the presence of blaSPM-1 in those isolates from clone SP/ST 277 identified outside Brazil. However, considering that the blaSPM-1 gene is a marker of clone SP in Brazil, it would be of interest to investigate its presence also in ST 277 isolates from other countries. ST 244 was assigned to isolates from outbreaks in Poland (2003 to 2007) (4) and from surveillance studies in China (2006) and the United States (2001 to 2004) (7), as well as with an environmental isolate from the United Kingdom (1998), showing that this clone is also widespread. The majority of the 932 STs available in December 2009 in the MLST database belonged to unique isolates. Few STs were assigned to more than one strain; however, they were restricted to one country. The exceptions, so far, have been STs 17, 27, 111, 155, 175, 179, 235, 244, 253, 277, 649, and 712, which have been reported in different countries over several years. None of these widespread STs formed clonal complexes with clone SP ST 277. The present study associated ST 277 with Brazilian clone SP, which has been revealed as a public health problem since it presents a COS resistance profile, beyond its persistence and dissemination in hospitals from a country with continental dimensions such as Brazil. Our analysis showed that this clone has an epidemic/pandemic potential. Human beings can be vectors promoting its spread, as clearly demonstrated in the study by Salabi et al. (8).

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.

Epidemiology of qnrVC alleles and emergence out of the Vibrionaceae family

The quinolones are antibiotics effective in the treatment of several current nosocomial infections. Bacteria carrying qnr genes present with decreased susceptibility to fluoroquinolones. In addition to this low-level resistance, qnr genes are associated with reduced bactericidal activity of ciprofloxacin in vitro and in vivo, which represents a therapeutic threat (Allou et al., 2009). Moreover, in association with mutations in gyrA/parC and with efflux pump regulatory systems, a full fluoroquinolone resistance phenotype can easily emerge, resulting in treatment failure (Rodrı́guezMartı́nez et al., 2011). Recently, due to increasing reports of qnrVC alleles in different genetic contexts, these genes were classified within a new transferable qnr family (Pons et al., 2013). Here, using in silico analysis, we show that qnrVC has already emerged worldwide out of the Vibrionaceae family in bacterial species of public health relevance, and in association with mobile genetic elements.

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.

Full characterization of the integrative and conjugative element carrying the metallo-β-lactamase blaSPM-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-β-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.

Functional Characterization of a Cassette-Specific Promoter in the Class 1 Integron-Associated qnrVC1 Gene

ABSTRACT Integrons are natural expression vectors due to the presence of an intrinsic promoter (Pc). Although rare, gene cassettes can harbor their own promoter. This study determined the functionality of an internal promoter in the qnrVC1 cassette whose presence was suggested by a level of transcription similar to that of the preceding cassette (aadA2) and confirmed by in silico analysis. Its functionality was determined by 5′ rapid amplification of cDNA ends (RACE) and cloning into promoter-probe vectors. PqnrVC was found in the qnrVC cassette family, stressing its role in contributing to resistance manifestation.

Occurrence and composition of class 1 and class 2 integrons in clinical and environmental O1 and non-O1/non-O139 Vibrio cholerae strains from the Brazilian Amazon

This study identified and characterised class 1 and 2 integrons in clinical and environmental Vibrio cholerae O1 and non-O1/non-O139 strains isolated from the Brazilian Amazon. The aadA2 and aadA7 gene cassettes were found in class 1 integrons in two genotypes of environmental V. cholerae non-O1/non-O139. Empty integrons were found in strains from the Brazilian cholera epidemic. A class 2 integron was detected in one strain from the V. cholerae Amazonia lineage harbouring sat1 and aadA1 genes. All isolates were resistant to aminoglycosides, indicating aadA functionality. These findings suggest that environmental bacteria act as cassette reservoirs that favour the emergence of resistant pathogens.