Genetic and biochemical characterization of BIM-1, a novel acquired subgroup B1 MBL found in a Pseudomonas sp. strain from the Brazilian Amazon region.

OBJECTIVES: To characterize a novel acquired MBL, BIM-1, in a Pseudomonas #2 (subgroup P. guariconensis) strain isolated from the Aurá river located in the Brazilian Amazon hydrographic basin. METHODS: WGS using an Illumina® MiSeq System was used to characterize the genome of Pseudomonas sp. IEC33019 strain. Southern blotting/hybridization assays were performed to confirm the location of the MBL-encoding gene, blaBIM-1 (Belém Imipenemase). Antimicrobial susceptibility testing, cloning, and biochemical and phenotypic characterization were performed to determine BIM-1 kinetics. RESULTS: The IEC33019 strain showed high resistance rates to ß-lactams, ciprofloxacin and aminoglycosides, being susceptible only to polymyxins and susceptible, increased exposure to aztreonam. WGS analysis revealed a novel acquired MBL-encoding gene, blaBIM-1, found as a gene cassette inserted into a class 1 integron (In1326) that also carried qnrVC1 and aadA11e. In1326 was located in a complex transposon, Tn7122, carried by a 52.7 kb conjugative plasmid (pIEC33019) with a toxin/antitoxin system (vapB/vapC). BIM-1 belongs to the molecular subgroup B1 and shares 70.2% and 64.9% similarity with SIM-1 and IMP-1, respectively. Kinetics analysis of BIM-1 showed hydrolytic activity against all ß-lactams tested. CONCLUSIONS: BIM-1 is a novel acquired MBL encoded by a gene carried by mobile genetic elements, which can be transferred to other Gram-negative bacilli (GNB). Because the IEC33019 strain was recovered from a river impacted by a populous metropolitan region with poor basic sanitation and served by limited potable freshwater, it would be important to establish the role of the BIM-1-producing GNB as nosocomial pathogens and/or as colonizers of the riverside population in this geographical region.

Genomic analyses of ciprofloxacin-resistant Neisseria gonorrhoeae isolates recovered from the largest South American metropolitan area.

We sequenced 13 Neisseria gonorrhoeae isolates exhibiting distinct susceptibility profiles and which were recovered over 12 years in the metropolitan region of São Paulo, Brazil. Whole Genome Sequencing (WGS) was performed on an Illumina MiSeq™ 2 × 300 bp paired-end reads. Bioinformatics analyses were carried out using CGE, PATRIC, and BLAST databases for manual curation of obtained genomes. Multilocus sequence typing (MLST) analysis identified seven STs, namely ST1580, ST1590, ST1901, ST1902, ST8161, ST9363, and ST15640. Moreover, a diversity of mutations was observed in MtrR/G45D-A39T, PIB/G120K-A121S, and PBP1/L421P. Mutations associated with sulfonamides (DHPS/R228S) and rifampicin (RNAP/H552N) were also detected, as well as tetracycline resistance determinants, namely rpsJ/V57M and tet(M). The results presented herein can contribute to the knowledge of N. gonorrhoeae strains circulating in Sao Paulo, Brazil.

Characterization of a Carbapenem-Resistant BKC-1-Producing Clinical Isolate Belonging to the Pseudomonas putida Group from Brazil.

Since its first report, the class A Brazilian Klebsiella carbapenemase (BKC) has been detected only among Enterobacterales isolates from Brazilian hospitals. In this study, we characterized a multidrug-resistant Pseudomonas juntendi clinical isolate and identified a 43.3-kb plasmid carrying blaBKC-1 and a class 1 integron (In1996) containing the arr-2, qnrVC1, dfrA21, and aac(6')-Ib' gene cassettes. Our results confirm the ability of Pseudomonas putida group isolates to acquire antimicrobial resistance determinants and further act as resistance reservoirs.

In vitro synergy of ceftolozane/tazobactam in combination with fosfomycin or aztreonam against MDR Pseudomonas aeruginosa.

OBJECTIVES: Carbapenem-resistant Pseudomonas aeruginosa (CR-PSA) imposes great limitations on empirical therapeutic choices, which are further complicated by metallo-ß-lactamase production. This study evaluated in vitro antimicrobial synergy of ceftolozane/tazobactam in combination with aztreonam and fosfomycin against MDR PSA. METHODS: MICs were determined by broth microdilution and gradient strips. The effect of ceftolozane/tazobactam+aztreonam and ceftolozane/tazobactam+fosfomycin combinations were tested against 27 MDR PSA isolates carrying blaSPM-1 (n = 13), blaIMP (n = 4), blaVIM (n = 3), blaGES-1 (n = 2) and blaCTX-M-like (n = 2), and 3 isolates with no acquired ß-lactamase production detected by gradient diffusion strip crossing (GDSC). Six genetically unrelated SPM-1-producing isolates were also evaluated by time-kill analysis (TKA). RESULTS: All CR-PSA isolates harbouring blaSPM-1, blaGES-1 and blaIMP-1 were categorized as resistant to ceftolozane/tazobactam, meropenem and fosfomycin, with 70% being susceptible to aztreonam. Synergism for ceftolozane/tazobactam+fosfomycin and ceftolozane/tazobactam+aztreonam combinations was observed for 88.9% (24/27) and 18.5% (5/27) of the isolates by GDSC, respectively. A 3- to 9-fold reduction in ceftolozane/tazobactam MICs was observed, depending on the combination. Ceftolozane/tazobactam+fosfomycin was synergistic by TKA against one of six SPM-1-producing isolates, with additional non-synergistic bacterial density reduction for another isolate. Aztreonam peak concentrations alone demonstrated a ≥3 log10 cfu/mL reduction against all six isolates, but all strains were within the susceptible range for the drug. No antagonism was observed. CONCLUSIONS: In the context of increasing CR-PSA and the genetic diversity of resistance mechanisms, new combinations and stewardship strategies may need to be explored in the face of increasingly difficult to treat pathogens.

Diversity of metallo-ß-lactamase-encoding genes found in distinct species of Acinetobacter isolated from the Brazilian Amazon Region.

BACKGROUND: The multidrug resistance (MDR) phenotype is frequently observed in Acinetobacter baumannii, the most clinically relevant pathogenic species of its genus; recently, other species belonging to the A. calcoaceticus-A. baumannii complex have emerged as important MDR nosocomial pathogens. OBJECTIVES: The present study aimed to verify the occurrence of metallo-ß-lactamase genes among distinct Acinetobacter species in a hospital located in the Brazilian Amazon Region. METHODS: Antimicrobial susceptibility profiles were determined by broth microdilution. The genetic relationships among these isolates were assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Pyrosequencing reads of plasmids carrying the bla NDM-1 gene were generated using the Ion Torrent™ platform sequencing. FINDINGS: A total of six isolates carried bla NDM-1: A. baumannii (n = 2), A. nosocomialis (n = 3), and A. pittii (n = 1); three carried bla IMP-1: A. baumannii, A. nosocomialis, and A. bereziniae. Resistance to colistin was observed for an NDM-1-producing A. nosocomialis isolate. Diverse PFGE patterns and sequence types were found among A. nosocomialis and A. baumannii isolates. The bla NDM-1 sequence was inserted in a Tn125 transposon, while the bla IMP-1 was found as a gene cassette of the class 1 integron In86. MAIN CONCLUSIONS: To the best of our knowledge, this is the first report describing the dissemination of bla NDM-1 among distinct Acinetobacter species recovered from the same hospital in South America.

Kinetics Analysis of ß-Lactams Hydrolysis by OXA-50 Variants of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunist pathogen usually associated with life threatening infections and exhibits a set of intrinsic and acquired antimicrobial mechanisms. Although resistance to penicillins-like compounds is commonly associated with the chromosomal Pseudomonas-derived cephalosporinases ß-lactamase, the real contribution of OXA-50, a second chromosomally encoded ß-lactamase, remains unclear. In this study, we characterized the biochemical properties of OXA-50, OXA-488, and OXA-494. Both oxacilinases differ from OXA-50 in two amino acids each. The blaOXA-50, blaOXA-488, and blaOXA-494 were cloned into pET26b+ that was transformed into Escherichia coli DH5α strain, expressed in E. coli BL21 strain, and then purified for obtaining the hydrolytic parameters. Benzylpenicillin was the preferential substrate instead of oxacillin. Besides, OXA-488 showed a threefold increase in catalytic efficiency for benzylpenicillin, and it was twofold more efficient in hydrolyzing imipenem, compared with OXA-50, although such carbapenemase activity was considered weak. In addition, OXA-488 and OXA-494 showed an increased affinity for penicillins, which contributed to the increased catalytic efficiency against ampicillin, especially OXA-488. Chromosomally encoded resistance mechanisms are usually overshadowed by acquired mechanisms. However, understanding their real contribution is essential to comprehend the versatile profiles verified in P. aeruginosa isolates. Such information can help to choose the best therapy in a scenario of limited options.

Unravelling complex transposable elements surrounding blaGES-16 in a Pseudomonas aeruginosa ExoU strain.

OBJECTIVES: We characterised the complex surrounding regions of blaGES-16 in a Pseudomonas aeruginosa exoU+ strain (P-10.226) in Brazil. METHODS: Species identification was performed by MALDI-TOF MS, and the antimicrobial susceptibility profile was determined by broth microdilution based on European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. The whole genome sequencing (WGS) of P-10.226 strain was performed using both short-read paired-end sequencing on the Illumina MiSeq platform as well as the long-read Oxford Nanopore MinION. RESULTS: WGS analysis showed that P-10.226 carried blaGES-16, which was found as a gene cassette inserted into a novel class I integron, In1992 (aadB-blaOXA-56-blaGES-16-aadB-aadA6c), whose 3'-CS was truncated by a nested transposable element, IS5564::ISPa157. The structure was even more complex since IS6100-ΔIS6100 structure and a TnAs2-like harbouring the operon merRTPADE was found downstream In1992. Fragments of TnAs3 harbouring 25-bp imperfect inverted repeats were identified bordering the intl1 of In1992 and also flanking IS6100-ΔIS6100, which might be genetic marks of its previous presence in the genome. Interestingly, In1992 also shows a distinct cassette array from In581 (blaGES-16-dfrA22-aacA27-aadA1), which was previously reported in Serratia marcescens strains recovered in Brazil. Finally, exoU gene, which encodes a potent cytotoxin of type III secretion systems (T3SS) effector proteins from P. aeruginosa and is associated to severe infections, was also detected. CONCLUSION: We described the novel In1992 carrying blaGES-16 surrounded by complex transposition events in a XDR P. aeruginosa strain. The identification of many sets of direct repeats adjacent to TnAs3 fragments indicates a major past of transposition events that shaped the current genetic environment of In1992.

Genomic analysis of carbapenem-resistant Pseudomonas aeruginosa ST143 clone showing susceptibility to broad-spectrum cephalosporins.

OBJECTIVES: Using whole-genome sequencing (WGS), we aimed to characterise a Pseudomonas aeruginosa ST143 clinical strain (Pb9) that presented resistance to meropenem and imipenem and susceptibility to piperacillin/tazobactam and broad-spectrum cephalosporins. METHODS: The antimicrobial susceptibility profile was confirmed by broth microdilution. WGS was performed using an Illumina MiSeq platform to identify possible genetic determinants of ß-lactam resistance. Transcription levels of chromosomally encoded efflux systems and oprD were evaluated by RT-qPCR. RESULTS: WGS analysis showed that no acquired carbapenemase-encoding gene was found in isolate Pb9, although mutations in the chromosomally encoded ß-lactamase genes blaOXA-488, blaPIB-1 and blaPDC-5 were observed. In addition, we detected a premature stop codon in the major porin-encoding gene oprD coupled with hyperexpression of MexAB-OprM and MexEF-OprN. CONCLUSION: Our results suggest that the ß-lactam resistance phenotype presented by strain Pb9 might be related to an association of OprD loss with hyperexpression of the efflux pump systems MexAB-OprM and MexEF-OprN. However, the contribution of OXA-488, PDC-5 and PIB-1 to this phenotype remains unclear and warrants further investigation.

In vitro synergy of ticarcillin/clavulanate in combination with aztreonam and ceftolozane/tazobactam against SPM-1-producing Pseudomonas aeruginosa strains.

Minimal inhibitory concentrations (MICs) of ticarcillin/clavulanic acid (TLc), ceftolozane/tazobactam (C/T), and aztreonam (AT) were determined for 6 SPM-1-producing Pseudomonas aeruginosa (PSA) using Etest® strips and the synergistic effect of such antimicrobials against was evaluated by gradient diffusion strip crossing (GDSC) test. The fraction inhibitory concentration indexes (FICI) were calculated and showed a synergistic (n = 3) and additive (n = 2) effects of TLc + AT against SPM-1 producers, while TLc + C/T combination caused no effect. Average MIC reduction of TLc and AT by GDSC was 3-fold and 2-fold dilutions, respectively. Thus, TLc + AT might be a candidate as a combination therapy to treat SPM-1-producing PSA infections.

Dynamic of High-Risk Acinetobacter baumannii Major Clones in a Brazilian Tertiary Hospital During a Short Time Period.

We characterized by whole-genome sequencing (WGS) six carbapenem-resistant Acinetobacter baumannii strains isolated from a Brazilian tertiary hospital during a 14-day period. The ISAba1-blaOXA-23 structure was found in the chromosome of five isolates, whereas blaOXA-72 was inserted in a 16.6-kb plasmid in two isolates. The presence of ISAba1-blaADC-like justified the high broad-spectrum cephalosporins minimal inhibitory concentrations (MICs) (MIC50, > 512 mg/L) verified in all isolates. Only minocycline (MIC50, ≤ 0.5 µg/mL), polymyxin B (MIC50, 0.5 µg/mL), and tigecycline (MIC50, 0.5 µg/mL) were in vitro active against such isolates. A diversity of other antimicrobial resistance determinants (aph(3')-VIa, aadA1, aac(3')-IIa, strA, strB, sul2, drfA1, mph(E), msr(E), tetB, and floR) was also observed, which may confer resistance to at last six distinct antimicrobial classes. Four distinct pulsed-field gel electrophoresis (PFGE) profiles were observed during the study period, which belonged to ST79/ST258 (n = 2; IC5), ST25/ST229 (n = 2; IC7), ST1 (n = 1; IC1), and ST162/ST235 (n = 1; IC4). Although the ST1 isolate that carried blaOXA-23 and blaOXA-72 was introduced in this hospital setting by a transferred patient, two clonally related ST79/ST258 isolates carrying either one of these carbapenemase encoding genes were recovered from two patients who were hospitalized within the same period of time in the same hospital unit. Finally, a good correlation between PFGE/MLST, blaOXA-51 variant, and single nucleotide polymorphisms was also observed. Here we demonstrated that distinct extensively drug-resistant A. baumannii clones can circulate in the same hospital setting during a short time period, illustrating a very complex epidemiological scenario for this priority pathogen.

Diversity of amino acid substitutions in PmrCAB associated with colistin resistance in clinical isolates of Acinetobacter baumannii.

This study aimed to investigate the mechanisms of colistin resistance in 64 Acinetobacter baumannii isolates obtained from patients with ventilator-associated pneumonia hospitalised in Greece, Italy and Spain. In total, 31 A. baumannii isolates were colistin-resistant. Several novel amino acid substitutions in PmrCAB were found in 27 colistin-resistant A. baumannii. Most substitutions were detected in PmrB, indicating the importance of the histidine kinase for colistin resistance. In two colistin-resistant isolates, 93 amino acid changes were observed in PmrCAB compared with A. baumannii ACICU, and homologous recombination across different clonal lineages was suggested. Analysis of gene expression revealed increased pmrC expression in isolates harbouring pmrCAB mutations. Complementation of A. baumannii ATCC 19606 and ATCC 17978 with a pmrAB variant revealed increased pmrC expression but unchanged colistin MICs, indicating additional unknown factors associated with colistin resistance. Moreover, a combination of PmrB and PmrC alterations was associated with very high colistin MICs, suggesting accumulation of mutations as the mechanism for high-level resistance. The pmrC homologue eptA was detected in 29 colistin-susceptible and 26 colistin-resistant isolates. ISAba1 was found upstream of eptA in eight colistin-susceptible and one colistin-resistant isolate and eptA was disrupted by ISAba125 in two colistin-resistant isolates. Whilst in most isolates an association of eptA with colistin resistance was excluded, in one isolate an amino acid substitution in EptA (R127L) combined with a point mutation in ISAba1 upstream of eptA contributed to elevated colistin MICs. This study helps to gain an insight into the diversity and complexity of colistin resistance in A. baumannii.

Virulence Potential of a Multidrug-Resistant Escherichia coli Strain Belonging to the Emerging Clonal Group ST101-B1 Isolated from Bloodstream Infection.

Escherichia coli EC121 is a multidrug-resistant (MDR) strain isolated from a bloodstream infection of an inpatient with persistent gastroenteritis and T-zone lymphoma that died due to septic shock. Despite causing an extraintestinal infection, previous studies showed that it did not have the usual characteristics of an extraintestinal pathogenic E. coli. Instead, it belonged to phylogenetic group B1 and harbored few known virulence genes. To evaluate the pathogenic potential of strain EC121, an extensive genome sequencing and in vitro characterization of various pathogenicity-associated properties were performed. The genomic analysis showed that strain EC121 harbors more than 50 complete virulence genetic clusters. It also displays the capacity to adhere to a variety of epithelial cell lineages and invade T24 bladder cells, as well as the ability to form biofilms on abiotic surfaces, and survive the bactericidal serum complement activity. Additionally, EC121 was shown to be virulent in the Galleria mellonella model. Furthermore, EC121 is an MDR strain harboring 14 antimicrobial resistance genes, including blaCTX-M-2. Completing the scenario, it belongs to serotype O154:H25 and to sequence type 101-B1, which has been epidemiologically linked to extraintestinal infections as well as to antimicrobial resistance spread. This study with E. coli strain EC121 shows that clinical isolates considered opportunistic might be true pathogens that go underestimated.

Genetic Characterization of Plasmid-Borne blaOXA-58 in Distinct Acinetobacter Species.

We characterize by whole-plasmid-sequence (WPS) two-plasmid-borne blaOXA-58 obtained from Acinetobacter seifertii (Asp-1069) and A. baumannii (Acb-45063) clinical strains recovered 17 years apart from distinct Brazilian regions. Multilocus sequence type (MLST) analysis showed that the Asp-1069 and Acb-45063 strains belong to ST551 and ST15/CC15, respectively. WPS analysis demonstrated that blaOXA-58 was located in two distinct plasmids named pAs1069_a (24,672 bp/44 open reading frames [ORFs]) and pAb45063_b (19,808 bp/24 ORFs), which belong to the GR8/GR23 (repAci23) and GR4 (repAci4) incompatibility groups, respectively. The genetic environments surrounding blaOXA-58 revealed that it was flanked by two intact ISAba3 copies on pAb45063_b, which differed from pAs1069_a. In the latter, the upstream ISAba3 copy was truncated by insertion of ISAba825 element. Although Re27-specific recombination sites were found adjacent to ISAba3-blaOXA-58-ISAba3 arrangement on pAb45063_b, such structures were absent on pAs1069_a. The conserved ISAba125-araC1-lysE arrangement was disrupted by TnaphA6 harboring the aminoglycosides resistance gene aphA6 on pAs1069_a, while an IS26-blaTEM-1-aac(3)-IIa-IS26 genetic structure was found upstream from ISAba3-blaOXA-58-ISAba3 on pAb45063_b. Other two plasmids, pAb45063_a (183,767 bp/209 ORFs) and pAs1069_b (13,129 bp/14 ORFs), were also found in the OXA-58-producing Acinetobacter species strains, harboring the strA and strB genes and the sul2 gene, which confer resistance to streptomycin and sulfonamides, respectively. The plasmid-mediated virulence factors corresponding to genes tonB, spl, glmM, ppa, sulP, and map were found in both strains, as well distinct toxin-antitoxin system-encoding genes stbD and relE (pAs1069_a), brnT and brnA (pAb45063_b), and xreE (pAb45063_a). Although infrequently reported in Brazil, plasmid-borne blaOXA-58 showed a complex and diverse genetic backbone that confers stability in different Acinetobacter species that have been isolated from nosocomial settings over time.IMPORTANCE Although the blaOXA-58 gene has been infrequently described in Brazil, contrasting with other bordering South American countries, we verified the maintenance of this resistance determinant over time among carbapenem-resistant Acinetobacter species isolates, not only in nosocomial settings but also in the environment. In addition, to the best of our knowledge, this is the first study to have used WPS analysis to evaluate the genetic surroundings of blaOXA-58 in Brazil. Moreover, the A. seifertii and A. baumannii clinical strains evaluated in this study were recovered 17 years apart in hospitals located in distinct Brazilian geographic regions.

Diversity of metallo-b-lactamase-encoding genes found in distinct species of Acinetobacter isolated from the Brazilian Amazon Region

BACKGROUND The multidrug resistance (MDR) phenotype is frequently observed in Acinetobacter baumannii, the most clinically relevant pathogenic species of its genus; recently, other species belonging to the A. calcoaceticus-A. baumannii complex have emerged as important MDR nosocomial pathogens. OBJECTIVES The present study aimed to verify the occurrence of metallo-β-lactamase genes among distinct Acinetobacter species in a hospital located in the Brazilian Amazon Region. METHODS Antimicrobial susceptibility profiles were determined by broth microdilution. The genetic relationships among these isolates were assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Pyrosequencing reads of plasmids carrying the bla NDM-1 gene were generated using the Ion Torrent™ platform sequencing. FINDINGS A total of six isolates carried bla NDM-1: A. baumannii (n = 2), A. nosocomialis (n = 3), and A. pittii (n = 1); three carried bla IMP-1: A. baumannii, A. nosocomialis, and A. bereziniae. Resistance to colistin was observed for an NDM-1-producing A. nosocomialis isolate. Diverse PFGE patterns and sequence types were found among A. nosocomialis and A. baumannii isolates. The bla NDM-1 sequence was inserted in a Tn125 transposon, while the bla IMP-1 was found as a gene cassette of the class 1 integron In86. MAIN CONCLUSIONS To the best of our knowledge, this is the first report describing the dissemination of bla NDM-1 among distinct Acinetobacter species recovered from the same hospital in South America.