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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 β-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 β-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., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

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 bla BKC-1 and a class 1 integron (In 1996 ) 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.

Objectives: We characterised the complex surrounding regions of bla GES-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 bla GES-16 , which was found as a gene cassette inserted into a novel class I integron, In1992 (aadB-bla OXA-56 -bla GES-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 (bla GES-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 bla GES-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., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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 β-lactamase, the real contribution of OXA-50, a second chromosomally encoded β-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 bla OXA-50 , bla OXA-488 , and bla OXA-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.

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., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Acinetobacter baumannii is a successful nosocomial pathogen due to its genomic plasticity. Homologous recombination allows genetic exchange and allelic variation among different clonal lineages and is one of the mechanisms associated with horizontal gene transfer (HGT) of resistance determinants. The main mechanism of colistin resistance in A. baumannii is mediated through mutations in the pmrCAB operon. Here, we describe two A. baumannii clinical isolates belonging to International Clone 7 (IC7) that have undergone recombination in the pmrCAB operon and evaluate the contribution of mobile genetic elements (MGE) to this phenomenon. Isolates 67569 and 72554 were colistin susceptible and resistant, respectively, and were submitted for short- and long-read genome sequencing using Illumina MiSeq and MinION platforms. Hybrid assemblies were built with Unicycler, and the assembled genomes were compared to reference genomes using NUCmer, Cortex, and SplitsTree. Genomes were annotated using Prokka, and MGEs were identified with ISfinder and repeat match. Both isolates presented a 21.5-kb recombining region encompassing pmrCAB . In isolate 67659, this region originated from IC5, while in isolate 72554 multiple recombination events might have happened, with the 5-kb recombining region encompassing pmrCAB associated with an isolate representing IC4. We could not identify MGEs involved in the mobilization of pmrCAB in these isolates. In summary, A. baumannii belonging to IC7 can present additional sequence divergence due to homologous recombination across clonal lineages. Such variation does not seem to be driven by antibiotic pressure but could contribute to HGT mediating colistin resistance. IMPORTANCE Colistin resistance rates among Acinetobacter baumannii clinical isolates have increased over the last 20 years. Despite reports of the spread of plasmid-mediated colistin resistance among Enterobacterales , the presence of mcr -type genes in Acinetobacter spp. remains rare, and reduced colistin susceptibility is mainly associated with the acquisition of nonsynonymous mutations in pmrCAB . We have recently demonstrated that distinct pmrCAB sequences are associated with different A. baumannii International Clones (IC). In this study, we identified the presence of homologous recombination as an additional cause of genetic variation in this operon, which, to the best of our knowledge, was not mediated by mobile genetic elements. Even though this phenomenon was observed in both colistin-susceptible and -resistant isolates, it has the potential to contribute to the spread of resistance-conferring alleles, leading to reduced susceptibility to this last-resort antimicrobial agent.

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 β-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 β-lactamase genes bla OXA-488 , bla PIB-1 and bla PDC-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 β-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., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest G.T.C. has recently received research funding and/or consultation fees from MSD, Eurofarma, Pfizer and Sanofi. A.C.G. has recently received research funding and/or consultation fees from Bayer, Cristália, InfectoPharm, Eurofarma, Pfizer, MSD, and Zambon. D.P.N. consultant, speaker bureau member or have received research funding from Allergan, Bayer, Cepheid, Merck, Melinta, Pfizer, Wockhardt, Shionogi, and Tetraphase. C.R.V.K. consultant, speaker bureau member or have received research funding from Pfizer, Eurofarma, Johnson & Johnson, Biotest AG and New Objective outside the submitted work; Other authors have nothing to declare. This study was not financially supported by any Diagnostic/Pharmaceutical company. This work was partially presented as a poster (P2800) during the 29th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID) in Amsterdam, 2019., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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 IS Aba1 - bla OXA-23 structure was found in the chromosome of five isolates, whereas bla OXA-72 was inserted in a 16.6-kb plasmid in two isolates. The presence of IS Aba1 - bla ADC -like justified the high broad-spectrum cephalosporins minimal inhibitory concentrations (MICs) (MIC 50 , > 512 mg/L) verified in all isolates. Only minocycline (MIC 50 , ≤ 0.5 μg/mL), polymyxin B (MIC 50 , 0.5 μg/mL), and tigecycline (MIC 50 , 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 bla OXA-23 and bla OXA-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, bla OXA-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.

Carbapenem-resistant Acinetobacter baumannii (CRAB) are emerging worldwide. In South America, clinical isolates presenting such a phenotype usually do not belong to the globally distributed international clone 2 (IC2). The majority of these isolates are also resistant to multiple other antimicrobials and are often designated extremely drug-resistant (XDR). The aim of this study was to characterize the resistance mechanisms presented by 18 carbapenem-resistant A. baumannii isolates from five different Brazilian hospitals. Species identification was determined by rpoB sequencing, and antimicrobial susceptibility was determined by broth microdilution. Isolates were submitted to whole genome sequencing using Illumina platform and genetic similarity was determined by PFGE, MLST, and cgMLST. Genome analysis was used to identify intrinsic and acquired resistance determinants, including mutations in the AdeRSABC efflux system and in outer membrane proteins (OMPs). All isolates were identified as A. baumannii and grouped into 4 pulsotypes by PFGE, which belonged to clonal complexes (CC) 15 Pas /103 Ox ( n = 4) and 79 Pas /113 Ox ( n = 14), corresponding to IC4 and IC5, respectively. High MIC values to carbapenems, broad-spectrum cephalosporins, amikacin, and ciprofloxacin were observed in all isolates, while MICs of ampicillin/sulbactam, gentamicin, and tigecycline varied among the isolates. Minocycline was the most active antimicrobial agent tested. Moreover, 12 isolates (66.7%) were considered resistant to polymyxins. Besides intrinsic OXA-51 and ADC variants, all isolates harbored an acquired carbapenem-hydrolyzing class D β-lactamase (CHDL) encoding gene, either bla OXA- 23 or bla OXA- 72 . A diversity of aminoglycoside modifying enzymes and resistance determinants to other antimicrobial classes were found, as well as mutations in gyrA and parC . Non-synonymous mutations have also been identified in the AdeRSABC efflux system and in most OMPs, but they were considered natural polymorphisms. Moreover, resistance to polymyxins among isolates belonging to IC5 were associated to non-synonymous mutations in pmrB , but no known polymyxin resistance mechanism was identified in isolates belonging to IC4. In conclusion, A. baumannii clinical isolates belonging to South America's major clones present a myriad of antimicrobial resistance determinants. Special attention should be paid to natural polymorphisms observed in each clonal lineage, especially regarding non-synonymous mutations in constitutive genes associated with distinct resistance phenotypes., Competing Interests: AG has recently received research funding and/or consultation fees from Bayer, Cristália, InfectoPharm, Eurofarma, MSD, Pfizer and Zambon. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Nodari, Cayô, Streling, Lei, Wille, Almeida, de Paula, Pignatari, Seifert, Higgins and Gales.)

Objectives: Carbapenem-resistant Pseudomonas aeruginosa (CR-PSA) imposes great limitations on empirical therapeutic choices, which are further complicated by metallo-β-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 β-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., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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 bla CTX-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., Competing Interests: A.C.G. has recently received research funding and/or consultation fees from Eurofarma, MSD, Pfizer, and Zambon. This study was not financially supported by any diagnostic/pharmaceutical company. Other authors declare no conflict of interest.

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., (Copyright © 2019 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

We evaluated the performance of five phenotypic tests [Modified Hodge Test (MHT); combined-disk test (CDT) using phenylboronic acid, EDTA, and cloxacillin; CarbaNP and CarbAcinetoNP; Blue-Carba, Carbapenembac™ and Carbapenembac Metallo™] for carbapenemase detection in Gram-negative bacilli (GNB). A total of 73 carbapenemase producers and 27 non-carbapenemase producers were tested. All GNB were subcultured onto Müeller-Hinton agar (MHA), MacConkey agar (MAC), and sheep blood agar (SBA). High sensitivity (100%) and specificity (100%) was observed for MHA using CarbaNP, Blue-Carba, and Carbapenembac™. The sensitivity and specificity of CarbaNP (98.6%/100%), Blue-Carba (97.1%/91.0%), and Carbapenembac™ (100%/96.5%) were slightly lower for SBA. In contrast, unacceptable sensitivity rates of CarbaNP (71.1%) and Blue-Carba (66.6%), but not Carbapenembac™ (97.3%), were observed for MAC. The colorimetric methods showed high sensitivity and specificity to detect carbapenemase production from isolates grown on MHA or SBA. However, colonies obtained from MAC must not be tested for carbapenemase detection by colorimetric methods., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We characterize by whole-plasmid-sequence (WPS) two-plasmid-borne bla OXA-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 bla OXA-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 bla OXA-58 revealed that it was flanked by two intact IS Aba3 copies on pAb45063_b, which differed from pAs1069_a. In the latter, the upstream IS Aba3 copy was truncated by insertion of IS Aba825 element. Although Re27-specific recombination sites were found adjacent to IS Aba3-bla OXA-58 -IS Aba3 arrangement on pAb45063_b, such structures were absent on pAs1069_a. The conserved IS Aba125 - araC1 - lysE arrangement was disrupted by Tn aphA6 harboring the aminoglycosides resistance gene aphA6 on pAs1069_a, while an IS 26 - bla TEM-1 - aac( 3 )-IIa -IS 26 genetic structure was found upstream from IS Aba3-bla OXA-58 -IS Aba3 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 bla OXA-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 bla OXA-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 bla OXA-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., (Copyright © 2019 Matos et al.)

Colistin resistance in Acinetobacter baumannii is of great concern and is a threat to human health. In this study, we investigate the mechanisms of colistin resistance in four isogenic pairs of A. baumannii isolates displaying an increase in colistin MICs. A mutation in pmrB was detected in each colistin-resistant isolate, three of which were novel (A28V, I232T, and ΔL9-G12). Increased expression of pmrC was shown by semi-quantitative reverse transcription-PCR (qRT-PCR) for three colistin-resistant isolates, and the addition of phosphoethanolamine (PEtN) to lipid A by PmrC was revealed by mass spectrometry. Interestingly, PEtN addition was also observed in some colistin-susceptible isolates, indicating that this resistance mechanism might be strain specific and that other factors could contribute to colistin resistance. Furthermore, the introduction of pmrAB carrying the short amino acid deletion ΔL9-G12 into a pmrAB knockout strain resulted in increased pmrC expression and lipid A modification, but colistin MICs remained unchanged, further supporting the strain specificity of this colistin resistance mechanism. Of note, a mutation in the pmrC homologue eptA and a point mutation in IS Aba1 upstream of eptA were associated with colistin resistance and increased eptA expression, which is a hitherto undescribed resistance mechanism. Moreover, no cost of fitness was observed for colistin-resistant isolates, while the virulence of these isolates was increased in a Galleria mellonella infection model. Although the mutations in pmrB were associated with colistin resistance, PEtN addition appears not to be the sole factor leading to colistin resistance, indicating that the mechanism of colistin resistance is far more complex than previously suspected and is potentially strain specific., (Copyright © 2019 American Society for Microbiology.)

A total of 124 Neisseria gonorrhoeae isolates recovered during a 12-year period (2003-2015) from outpatients assisted at Centro de Referência e Treinamento DST/AIDS-CRT of São Paulo city, Brazil, were analysed. The following resistance rates were observed: penicillin-59.6%, ciprofloxacin-15.3%, and azithromycin-6.7%. Although reduced susceptibility to these drugs was observed since 2003, no ceftriaxone-resistant isolates were detected. Ciprofloxacin- and azithromycin non-susceptible isolates were grouped in 11 clusters. Mutations were detected in GyrA and ParC of isolates 124 and 260, and a C2611T substitution on 23S rRNA alleles was also observed in isolate 260. Both isolates belonged to ST1901/ST6210 (MSLT/NG-MAST schemes).

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.

The global emergence of carbapenemases led to the need of developing new methods for their rapid detection. The aim of this study was to evaluate the performance of the rapid tests for carbapenemase-producing and non-producing Enterobacteriaceae. Carbapenem non-susceptible Enterobacteriaceae from a surveillance study submitted to a multiplex real time PCR for carbapenemase detection were included in this study. The isolates were subjected to the rapid phenotypic tests Carba NP, Blue-Carba and Carbapenem Inactivation Method (CIM). A total of 83 carbapenemase-producing (43) and non-producing (40) isolates were included in the study. The sensitivity/specificity were 62.7%/97.5%, 95.3%/100%, and 74.4%/97.5% for Carba NP, Blue-Carba and CIM, respectively. Both Carba NP and Blue-Carba presented their final results after 75min of incubation; the final results for CIM were obtained only after 8h. Failure to detect OXA-370 carbapenemase was the main problem for Carba NP and CIM assays. As the Blue-Carba presented the highest sensitivity, it can be considered the best screening test. Conversely, CIM might be the easiest to perform, as it does not require special reagents. The early detection of carbapenemases aids to establish infection control measures and prevent carbapenemases to spread reducing the risk of healthcare associated infections and therapeutic failure., (Copyright © 2018 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

The aim of this study was to characterize the presence of carbapenemase-encoding genes in distinct species of Acinetobacter spp. isolated from Brazilian hospitals. Five carbapenem-resistant Acinetobacter spp. isolates (two Acinetobacter pittii, two Acinetobacter bereziniae and one Acinetobacter junii) recovered from two distinct hospitals between 2000 and 2016 were included in this study. All of the isolates harboured blaIMP-1, which was inserted into In86, a class 1 integron. Pulsed field gel eletrophoresis analysis showed that both A. pittii were identical, while the two A. berezinae isolates were considered to be clonally related. In this study, we demonstrated that mobile elements carrying carbapenemase-encoding genes such as In86 may persist for a long period, allowing their mobilization from A. baumannii to other Acinetobacter spp. that are usually susceptible to multiple antimicrobials.

We evaluated the performance of OXA-48 K-SeT assay for detecting OXA-370 directly from spiked rectal swabs and blood culture vials. The limit of detection of this test was 10 4 UFC/mL for rectal swabs. Detection of the OXA-370-producing isolates was successfully achieved directly from positive blood culture vials independent of growing conditions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The intrinsic polymyxin resistance displayed by Serratia marcescens makes the acquisition of carbapenemase encoding genes a worrisome event. This study report a SME-4-producing S. marcescens isolate causing septic shock in Brazil. The insertion of novel resistance determinants and their consequent spread in our territory is noteworthy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Serratia marcescens is a Gram-negative rod intrinsically resistant to polymyxins and usually associated with wound, respiratory and urinary tract infections. The whole genome of the first GES-5-producing S. marcescens isolated from a Brazilian patient was sequenced using Ion Torrent PGM System. Besides bla GES-5 , we were able to identify genes encoding for other β-lactamases, for aminoglycoside modifying enzymes and for an efflux pump to tetracyclines., (Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)