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Objective: Despite the increasing reports of blaNDM in Enterobacterales in Brazil, comprehensive whole genome sequencing (WGS) data remain scarce. To address this knowledge gap, our study focuses on the characterization of the genome of an New Delhi Metallo-β-lactamase (NDM)-1-producing Klebsiella quasipneumoniae subsp. quasipneumoniae (KQPN) clinical strain isolated in Brazil. Methods: The antimicrobial susceptibility profile of the A-73.113 strain was performed by agar dilution or broth microdilution following the Brazilian Antimicrobial Susceptibility Testing Committee/European Committee on Antimicrobial Susceptibility Testing recommendations. WGS was performed using the Illumina® NextSeq platform and the generated reads were assembled using the SPAdes software. The sequences obtained were submitted to the bioinformatics pipelines to determine the sequence type, resistome, plasmidome, and virulome. Results: The A-73.113 strain was identified as KQPN and was susceptible to polymyxins (MICs, ≤0.25 µg/mL), tigecycline (MIC, 0.5 µg/mL), ciprofloxacin (MIC, 0.5 µg/mL), and levofloxacin (MIC, 1 µg/mL). WGS analysis revealed the presence of genes conferring resistance to β-lactams (blaNDM-1, blaCTX-M-15, blaOXA-9, blaOKP-A-5, blaTEM-1), aminoglycosides [aph(3′)-VI, aadA1, aac(6′)-Ib], and fluoroquinolones (oqxAB, qnrS1, aac(6′)-Ib-cr]. Additionally, the presence of the plasmid replicons Col(pHAD28), IncFIA(HI1), IncFIB(K) (pCAV1099-114), IncFIB(pQil), and IncFII(K), as well as virulence-encoding genes fimABCDEFGHIK (type 1 fimbria), pilW (type IV pili), iutA (aerobactin), entABCDEFS/fepABCDG/fes (Ent siderophores), iroE (salmochelin), and allABCDRS (allantoin utilization) was verified. Furthermore, we found that the A-73.113 strain belongs to ST1040. Conclusions: Here we report the genomic characteristics of an NDM-1-producing KQPN ST1040 strain isolated from blood cultures in Brazil. These data will enhance our comprehension of how this species contributes to the acquisition and dissemination of blaNDM-1 in Brazilian nosocomial settings.

Abstract Brazil is recognized for its biodiversity and the genetic variability of its organisms. This genetic variability becomes even more valuable when it is properly documented and accessible. Understanding bacterial diversity through molecular characterization is necessary as it can improve patient treatment, reduce the length of hospital stays and the selection of resistant bacteria, and generate data for health and epidemiological surveillance. In this sense, in this study, we aimed to understand the biodiversity and molecular epidemiology of carbapenem-resistant bacteria in clinical samples recovered in the state of Rondônia, located in the Southwest Amazon region. Retrospective data from the Central Public Health Laboratories (LACEN/RO) between 2018 and 2021 were analysed using the Laboratory Environment Manager Platform (GAL). Seventy-two species with carbapenem resistance profiles were identified, of which 25 species carried at least one gene encoding carbapenemases of classes A (bla KPC-like), B (bla NDM-like, bla SPM-like or bla VIM-like) and D (bla OXA-23-like, bla OXA-24-like, bla OXA-48-like, bla OXA-58-like or bla OXA-143-like), among which we will highlight Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Serratia marcescens, and Providencia spp. With these results, we hope to contribute to the field by providing epidemiological molecular data for state surveillance on bacterial resistance and assisting in public policy decision-making.

The occurrence of carbapenemases encoding genes in Providencia rettgeri is a critical public health concern since this species has intrinsic resistance to several antimicrobials, including polymyxins. The identification of this multidrug-resistant (MDR) pathogen outside the hospital setting has become increasingly frequent, and raises an alert for the global health agencies, as they indicate a possible spread of such pathogens. Herein, we described three MDR P. rettgeri isolates carrying a diversity of antimicrobial resistance genes (ARGs) isolated from stool samples of swine and bovine in Brazil. Molecular analysis revealed that all isolates belonged to the same clone. The whole genome sequencing (WGS) of a representative isolate (PVR-188) was performed by MiSeq Illumina® platform, while the assembling and annotation was achieved using SPAdes and Prooka, respectively. The WGS analyses indicated the presence of ARGs that confer resistance to β-lactams (blaNDM-1, blaCTX-M-2), quinolones (qnrD1), aminoglycosides (aadA2, aadA1, aph(3′)-Via), phenicol (catB2), sulfonamides (sul1, sul2), and trimethoprim (dfrA12, dfrA1). The presence of three plasmid replicons (Col3M, IncQ1, and IncT) was detected, but no phage sequences were found. The phylogenetic analyses confirmed the genomic relationship of the PVR-188 with P. rettgeri isolates recovered from animals and humans in the USA and Malaysia. In conclusion, we report the occurrence of MDR P. rettgeri clone colonizing the gut microbiota of food-producing animals in Brazil, revealing the spread of this pathogen beyond hospital boundaries.

Serratia marcescens is a Gram-negative bacterium presenting intrinsic resistance to polymyxins that has emerged as an important human pathogen. Although previous studies reported the occurrence of multidrug-resistance (MDR) S. marcescens isolates in the nosocomial settings, herein, we described isolates of this extensively drug-resistant (XDR) species recovered from stool samples of food-producing animals in the Brazilian Amazon region. Three carbapenem-resistant S. marcescens strains were recovered from stool samples of poultry and cattle. Genetic similarity analysis showed that these strains belonged to the same clone. Whole-genome sequencing of a representative strain (SMA412) revealed a resistome composed of genes encoding resistance to β-lactams [blaKPC-2, blaSRT-2], aminoglycosides [aac(6′)-Ib3, aac(6′)-Ic, aph(3′)-VIa], quinolones [aac(6′)-Ib-cr], sulfonamides [sul2], and tetracyclines [tet(41)]. In addition, the analysis of the virulome demonstrated the presence of important genes involved in the pathogenicity of this species (lipBCD, pigP, flhC, flhD, phlA, shlA, and shlB). Our data demonstrate that food-animal production can act as reservoirs for MDR and virulent strains of S. marcescens.

Introdução: Streptococcus constellatus é um coco gram-positivo anaeróbio facultativo do grupo Anginosus (previamente grupo milleri), que inclui também S. anginosus e intermedius. São frequentemente encontrados na microbiota dos tratos respiratório superior, digestivo e genital de pessoas saudáveis. Pode causar infecções piogênicas, principalmente na cavidade oral, garganta e seios paranasais, no entanto após disseminação hematogênica é capaz de formar abscessos em órgãos e cavidades. Objetivo: Relatar três casos de infecção por S. constellatus associados à formação de abscesso. Dois dos casos ocorreram em pacientes em tratamento para tuberculose. Método: Caso 1: Mulher, 37 anos, no quinto mês de tratamento para tuberculose renal, é hospitalizada por dor lombar, disúria e febre há 2 meses. A tomografia computadorizada evidenciou múltiplos abscessos renais. É liberada após tratamento com ceftriaxona por 7 dias e cefuroxima por mais 21 dias. Retorna após 2 meses febril e hipotensa e imagem revela coleção renal de 660 mL. É submetida à drenagem, sendo isolado em cultura S. constellatus. Evoluiu com melhora gradativa após receber ceftriaxona 2 g ao dia por 30 dias. Caso 2: Mulher, 41 anos, no segundo mês de tratamento por tuberculose peritoneal, interna por dor abdominal difusa e febre. Tomografia de abdome indicou volumosa coleção em cavidade, com níveis hidroaéreos. Foi submetida à drenagem de 2050 mL e S. constellatus foi identificado na cultura do líquido peritoneal. Foi tratada com ceftriaxona 2 g ao dia por 7 dias. Caso 3: Homem, 71 anos, ex-tabagista, admitido por tosse, febre e perda ponderal há 4 meses. Realizou dois tratamentos com antibiótico, sem melhora. A tomografia de tórax evidenciou múltiplas consolidações bilaterais, além de derrame pleural loculado. Realizada toracocentese com saída de líquido purulento, cuja análise identificou S. constellatus. Apesar do tratamento com antibióticos e drenagem do empiema, o paciente faleceu devido a complicações relacionadas à internação. Discussão/Conclusão: O grupo S. anginosus pode causar infecções graves com formação de abscessos em vários sítios e seus fatores de virulência são pouco conhecidos. Dois dos pacientes desenvolveram infecção por S. constellatus durante tratamento para tuberculose, na mesma topografia da infecção primária, levantando suspeita para uma possível relação entre os microrganismos. Há poucos relatos acerca dessa coinfecção e pouco se sabe sobre a capacidade de interação entre os patógenos.

Introdução/Objetivo: Surtos por Legionella spp são comumente associados a edifícios ou estruturas que possuem sistemas de água com torres de resfriamento e aquecimento central, como hospitais. As fontes mais comuns incluem água usada para banho, fontes decorativas e torres de resfriamento. Surtos por esse agente podem ser de difícil identificação. Este estudo objetiva descrever a investigação de um surto de legionelose em uma unidade de Transplante de Medula Óssea (TMO). Métodos: Estudo realizado na unidade de TMO de um hospital de ensino terciário, na cidade de São Paulo. Foram incluídos todos os pacientes com diagnóstico laboratorial confirmado para legionelose de aquisição hospitalar (critérios do CDC/NHSN). A investigação inicial do ambiente foi realizada através de cultura de água e semeada em meio específico. Na impossibilidade de identificação do patógeno e persistência do surto, nova coleta de água e swabs dos metais sanitários foi realizada utilizando pesquisa molecular com metagenômica, pela técnica de detecção de amplicon. Resultados: O primeiro caso de Legionella spp. foi detectado em agosto/2020, totalizando 8 casos até abril/2023. Todos os diagnósticos foram realizados por teste de antígeno urinário. Seis eram do sexo feminino e a média de idade 51,3 (26-65) anos. A principal doença de base era LMA (62,5%) e 75% eram receptores de TCTH alogênico. Após o primeiro caso, foi coletada água dos chuveiros e torneiras dos banheiros da unidade e caixa d’água e realizada cultura, porém sem identificação do agente. Em 2022, após novos casos, foi identificado vínculo epidemiológico com dois quartos específicos, sendo realizadas novas culturas de água de todos os pontos de água dos banheiros, sem isolamento do patógeno. Em 2023, novas coletas de água e swabs de superfície foram realizadas nos dois quartos (53 amostras) e realizado sequenciamento de amplicon. Foi identificada Legionella pneumophila em swab de superfície interna de chuveiro/chuveirinho do quarto com maior número de casos (n = 7). Amostra clínica respiratória obtida de um dos pacientes com diagnóstico, foi submetida ao sequenciamento com identificação do agente, 100% compatível com isolado ambiental. Após bloqueio do quarto, não observamos novos casos na unidade. Conclusão: A metagenômica possibilitou a confirmação do vínculo epidemiológico do surto de Legionella pneumophila com ambiente, sendo possivelmente superior aos métodos tradicionais para identificação do patógeno de amostras ambientais.

Measurement(s) Metagenome Technology Type(s) Illumina Sequencing

ABSTRACT The epidemiology of antimicrobial resistance (AMR) is complex, with multiple interfaces (human-animal-environment). In this context, One Health surveillance is essential for understanding the distribution of microorganisms and antimicrobial resistance genes (ARGs). This report describes a multicentric study undertaken to evaluate the bacterial communities and resistomes of food-producing animals (cattle, poultry, and swine) and healthy humans sampled simultaneously from five Brazilian regions. Metagenomic analysis showed that a total of 21,029 unique species were identified in 107 rectal swabs collected from distinct hosts, the highest numbers of which belonged to the domain Bacteria, mainly Ruminiclostridium spp. and Bacteroides spp., and the order Enterobacterales. We detected 405 ARGs for 12 distinct antimicrobial classes. Genes encoding antibiotic-modifying enzymes were the most frequent, followed by genes related to target alteration and efflux systems. Interestingly, carbapenemase-encoding genes such as blaAIM-1, blaCAM-1, blaGIM-2, and blaHMB-1 were identified in distinct hosts. Our results revealed that, in general, the bacterial communities from humans were present in isolated clusters, except for the Northeastern region, where an overlap of the bacterial species from humans and food-producing animals was observed. Additionally, a large resistome was observed among all analyzed hosts, with emphasis on the presence of carbapenemase-encoding genes not previously reported in Latin America. IMPORTANCE Humans and food production animals have been reported to be important reservoirs of antimicrobial resistance (AMR) genes (ARGs). The frequency of these multidrug-resistant (MDR) bacteria tends to be higher in low- and middle-income countries (LMICs), due mainly to a lack of public health policies. Although studies on AMR in humans or animals have been carried out in Brazil, this is the first multicenter study that simultaneously collected rectal swabs from humans and food-producing animals for metagenomics. Our results indicate high microbial diversity among all analyzed hosts, and several ARGs for different antimicrobial classes were also found. As far as we know, we have detected for the first time ARGs encoding carbapenemases, such as blaAIM-1, blaCAM-1, blaGIM-2, and blaHMB-1, in Latin America. Thus, our results support the importance of metagenomics as a tool to track the colonization of food-producing animals and humans by antimicrobial-resistant bacteria. In addition, a network surveillance system called GUARANI, created for this study, is ready to be expanded and to collect additional data.

Introdução: Na última década, a emergência de Pseudomonas do grupo putida (PPG) vêm recebendo um maior destaque por conta de sua capacidade de causar infecções nosocomiais e de sua plasticidade genética, sendo capazes de adquirir elementos genéticos móveis (EGM) contendo genes de resistência aos antibióticos. Objetivo: Neste estudo investigamos o resistoma e o contexto genético de espécies distintas de isolados clínicos pertencentes a PPG e sua relevância no cenário brasileiro atual. Método: Um total de 12 isolados de PPG resistentes aos carbapenêmicos foram recuperados de uma coleção de micro-organismos (2010-2019) provenientes de pacientes internados em três hospitais brasileiros. A determinação das espécies e suas relações filogenéticas, bem como a caracterização de seus resistomas, foram realizadas utilizando sequências obtidas através do sequenciamento de genoma completo (Illumina MiSeq). As concentrações inibitórias mínimas de 13 antimicrobianos foram determinadas por microdiluição em caldo (BrCAST, 2021). Adicionalmente, a localização dos genes codificadores de carbapenemases foi confirmada por Southern-Blot e Hibridização. Resultados: Os isolados foram identificados como P. juntendi (n = 7), P. monteilii (n = 2), P. asiatica (n = 1), P. mosselii (n = 1) e P. putida (n = 1), pertencentes a 11 STs diferentes. Todos isolados foram resistentes ao meropenem, ao imipenem, à ceftazidima, à piperacilina-tazobactam, à cefepima e ao aztreonam. Além disso, a maioria (n = 11) foi resistente à levofloxacina e à ciprofloxacina. Por outro lado, a maior parte dos isolados foi suscetível à amicacina (MIC90 16 µg/mL) e à polimixina B (MIC90 0,5 µg/mL). Os isolados carregavam ao menos um gene codificador de β-lactamases (VIM-2 [n=6]; IMP-16 [n = 3]; IMP-74 [n = 2]; BKC-1 [n = 1]; KPC-2 [n = 1]) associados a diversos MGEs incorporados em plasmídeos de 34 até 210 kb. Adicionalmente, observamos a persistência de integrons de classe 1 entre isolados de espécies distintas, recuperados com um intervalo de sete anos. Conclusão: A maioria dos isolados carreavam genes codificadores de MβLs (IMP e/ou VIM) inseridos em integrons de classe 1 carreados por plasmídeos. Este também foi o primeiro relato de um bacilo gram-negativo não-fermentador produtor de BKC-1. Este estudo demonstrou a variabilidade de espécies de PpG presentes no ambiente nosocomial e a diversidade de mecanismos adquiridos de resistência aos carbapenêmicos, ressaltando o potencial dessas espécies como emergentes patógenos nosocomiais.Ag. Financiadora: CNPQ.Nr. Processo: 134434/2018-8.

Introdução: Delafloxacino é uma fluoroquinolona recentemente aprovada para o tratamento de infecções bacterianas agudas de pele e partes moles complicadas (IPPMc). Objetivo: Esse estudo teve como objetivo avaliar a frequência de espécies isoladas de pacientes com IPPMc e osteomielite bem como atividade antimicrobiana do delafloxacino e agentes comparadores. Método: Cem isolados bacterianos foram consecutivamente coletados de 77 pacientes com IPPMc e osteomielite assistidos em hospital terciário de São Paulo. Os isolados foram identificados por MALDI-TOF MS (Biotyper, versão 3.1). O teste de sensibilidade aos antimicrobianos (TSA) e sua interpretação foram realizados seguindo as recomendações do BrCAST, utilizando-se a técnica de microdiluição em caldo. Na ausência de pontos de corte do BrCAST, utilizaram-se os da FDA para interpretação dos TSA. Avaliou-se a produção de biofilme em superfície abiótica pela técnica do cristal violeta. A análise de mutações nos genes gyrA e parC e a detecção do gene mecA foram realizadas em isolados selecionados de bacilos gram-negativos (BGN) e Staphylococcus spp., respectivamente. Resultados: Staphylococcus coagulase negativos (SCoN; 18%) e Staphylococcus aureus (SA; 18%) foram as espécies mais frequentemente isoladas, seguidas por P. aeruginosa (PSA; 14%), K. pneumoniae (KPN; 9%) e Enterobacter cloacae (7%). Delafloxacino apresentou excelente atividade in vitro contra SA (MIC50, ≤0,008 mg/L) e SCoN (MIC50, 0,06 mg/L), sendo pelo menos 64 vezes mais potente que levofloxacino contra SA (MIC50, 0,5 mg/L) e SCoN (MIC50, 4mg/L). Entre os BGN, delafloxacino (MIC50, 0,25 mg/L; 78,7% S) foi pelo menos quatro vezes mais potente e apresentou maior taxa de sensibilidade que ciprofloxacino (MIC50, 1 mg/L; 42,9% I) contra as amostras de PSA. Ainda, 74 isolados foram produtores de biofilme, sendo 11 classificados como fortemente aderentes [SA (n=4), PSA (n=4), SCoN (n=2) e KPN (n=1)]. Todos os isolados resistentes ao delafloxacino testados apresentaram mutações em gyrA e/ou parC. O gene mecA foi detectado em 25/27 (92,6%) dos isolados de Staplylococcus spp. resistentes à oxacilina. Conclusão: O delafloxacino demonstrou excelente atividade in vitro contra Staphylococcus spp. e PSA, apresentando maior potência do que as outras fluoroquinolonas contra esses gêneros.

Introdução: Infecções do Trato Respiratório (RTI) causadas por Pseudomonas aeruginosa são comuns entre os pacientes hospitalizados, o que leva à alta morbidade e mortalidade. Este patógeno exibe múltiplos mecanismos de resistência aos antibióticos. Ceftolozane-tazobactam (C/T) é um agente inibidor beta-lactam-beta-lactamase que pode ser considerado uma opção clínica para pneumonia nosocomial em pacientes graves principalmente pelo risco aumentado de isolados resistentes à meropenem e piperacilina-tazobactam (P/T). Objetivo: Avaliar as taxas de susceptibilidade de Pseudomonas aeruginosa em isolados de trato respiratório no Brasil entre 2016-2020. Método: Foram coletados 515 isolados de Pseudomonas aeruginosa de infecções do trato respiratório de pacientes entre 2016 e 2020 no Brasil, utilizando o programa SMART. Os isolados foram testados em laboratório central utilizando metodologia do Instituto de Normas Clínicas e Laboratoriais. As amostras foram consecutivas e não duplicadas. As concentrações inibitórias mínimas foram determinadas pela microdiluição de caldo para C/T e vários antibióticos comparativos, incluindo P/T e meropenem. Os resultados de suscetibilidade antimicrobiana foram interpretados pelos critérios. Resultados: Entre todos os isolados de P. aeruginosa, C/T [MIC50/90, 1/4 μg/mL; 91,5% suscetível (S)], foi o antimicrobiano testado com maior atividade seguido do meropenem (MIC50/90, 1/ > 16 μg/mL; 63,3% S), P/T (MIC50/90, 8/ > 64 μg/mL; 67,2% I - Suscetível, maior exposição). A suscetibilidade do C/T oscilou entre 87,9% e 96,3% no período de 5 anos, com a maior suscetibilidade relatada no ano de 2020. Comparamos isolados resistentes a P/T (N=169); observou-se uma suscetibilidade de 76,3% C/T e 36,1% do meropenem. Ao avaliar a amostra resistente ao meropenem (N=110) a suscetibilidade nessa população foi de 70,0% para C/T e 22,7% para P/T (I - Suscetível, maior exposição). Entre os isolados resistentes a ambos os fármacos (N=85), a suscetibilidade para o C/T de 62,4%. Conclusão: O Ceftolozane-Tazobactam foi o beta-lactamico mais ativo in vitro testado contra P. aeruginosa mesmo entre as cepas resistentes a meropenem e P/T. C/T manteve a suscetibilidade à Pseudomonas aeruginosa no Brasil ao longo dos 5 anos de estudo. O C/T pode representar uma boa opção para o tratamento empírico em casos de infecção do trato respiratório.Ag. Financiadora: Funding for this study has been provide by MSD.

Objective: : Herein, this study aimed to perform the genomic characterization of a blaKPC-2 positive Klebsiella pneumoniae (KP1.1JP) strain isolated from the surface water of river located the Brazilian Amazon region. Methods: : Antimicrobial susceptibility testing was performed following BrCAST/EUCAST recommendations. Genomic DNA was extracted and sequenced using the Illumina® NextSeq platform and the assembly of the generated reads was performed using the SPAdes software. Research on the sequence type, resistance and virulence encoding genes, and plasmid replicon typing was carried out. Results: : The KP1.1JP strain was resistant to all β-lactams, aminoglycosides, and fluoroquinolones tested. The genome size was 5 626 346 bp, distributed in 203 contigs and a guanine and cytosine content of 57.02%. The values of N50 and N75 were 285 583 bp and 173 927 bp, respectively. We verified that KP1.1JP belongs to ST101 and carries genes encoding resistance to β-lactams (blaCTX-M-15, blaTEM-1B, blaOXA-1, blaSVH-182, and blaKPC-2), aminoglycosides [aac(3′)-IIa, aph(3′)-Vla], fluoroquinolones [aac(6′)-Ib-cr], phenicol (catA1, catA2, catB3), tetracycline [tet(D)], trimethoprim (dfrA14), and fosfomycin (fosA). Additionally, the following virulence encoding genes were also detected: mrkABCDFHIJ (Fimbria type 3); fimABCDRFGHIK (Fimbria type 1); entABCDEFS and fepABCDG (siderophores); iroN, irp1, and irp2 (salmochelins); fyuA and ybtAEPQSTUX (yersiniabactin); and iutA (aerobactin). Conclusions: : We report the occurrence of a K. pneumoniae ST101 strain carrying blaKPC-2 gene in an Amazon river in Brazil. The genomic characteristics of this strain will contribute to a better understanding of the spread of pathogens of clinical importance in the environment based on a One Health perspective.

The detection of KPC-type carbapenemases is necessary for guiding appropriate antibiotic therapy and the implementation of antimicrobial stewardship and infection control measures. Currently, few tests are capable of differentiating carbapenemase types, restricting the lab reports to their presence or not. The aim of this work was to raise antibodies and develop an ELISA test to detect KPC-2 and its D179 mutants. The ELISA-KPC test was designed using rabbit and mouse polyclonal antibodies. Four different protocols were tested to select the bacterial inoculum with the highest sensitivity and specificity rates. The standardisation procedure was performed using 109 previously characterised clinical isolates, showing 100% of sensitivity and 89% of specificity. The ELISA-KPC detected all isolates producing carbapenemases, including KPC variants displaying the ESBL phenotype such as KPC-33 and -66.

S. pseudintermedius is a known resident of the skin and mucous membranes and a constituent of the normal microbiota of dogs. It has also been recognized as an opportunistic and zoonotic pathogen that is able to colonize humans and cause severe diseases, especially in immunocompromised hosts. Most importantly, methicillin-resistant S. pseudintermedius (MRSP), which is intrinsically multidrug-resistant, has emerged with serious public health consequences. The epidemiological situation is further exacerbated with reports of its zoonotic transmission and human infections which have been mostly attributed to the increasing frequency of dog ownership and close contact between dogs and humans. Evidence on the zoonotic transmission of MRSP from pet dogs to humans (such as dog owners, small-animal veterinarians, and other people in close proximity to dogs) is limited, especially due to the misidentification of S. pseudintermedius as S. aureus. Despite this fact, reports on the increasing emergence and spread of MRSP in humans have been increasing steadily over the years since its first documented report in 2006 in Belgium. The emergence of MRSP strains has further compromised treatment outcomes in both veterinary and human medicine as these strains are resistant to beta-lactam antimicrobials usually prescribed as first line treatment. Frustratingly, the limited awareness and surveillance of the zoonotic transmission of S. pseudintermedius have underestimated their extent of transmission, prevalence, epidemiology, and public health significance. In order to fill this gap of information, this review focused on detailed reports on zoonotic transmission, human colonization, and infections by S. pseudintermedius, their pathogenic features, antimicrobial resistance profiles, epidemiology, risk factors, and treatment. In writing this review, we searched Web of Science, PubMed, and SCOPUS databases using the keyword “Staphylococcus pseudintermedius AND humans”. A phylogenetic tree to determine the genetic relatedness/diversity of publicly available genomes of S. pseudintermedius was also constructed.

Objectives: This study aimed to evaluate the persistence of the plasmid-mediated quinolone resistance (PMQR) among uropathogenic Escherichia coli strains grown under or without exposure to subinhibitory concentrations of ciprofloxacin. Based on that, we evaluated the possible spontaneous loss or maintenance of PMQR and the possible appearance of compensatory mutations in gyrA and parC genes. Methods: Three uropathogenic E. coli strains harbouring chromosomal mutations in the gyrA and/or parC genes coupled with qnrS1 or qnrB2 determinants carried by distinct plasmid sizes and incompatibility N groups (IncN/ST1, IncN/ST5) were evaluated using in vitro and in vivo assays. Results: PMQRs remained stable in all strains throughout the generations evaluated, independently of exposure to ciprofloxacin in both in vivo and in vitro assays. Analysis of gyrA and parC genes after in vivo and in vitro assays revealed that no changes occurred in quinolone-resistance determining regions (QRDR). Conclusion: We demonstrated that IncN plasmids were persistent over 14 days in E. coli clinical strains independently of exposure to ciprofloxacin, as well as previous mutations in QRDR.

Multi-drug resistant Gram-negative bacilli (GNB) have been reported as cause of serious hospital-acquired infections worldwide. The aim of this study was to investigate the in vitro activity of ceftolozane-tazobactam compared to other agents against GNB isolated from patients admitted to Brazilian medical centers between the years 2016 and 2017. Presence of β-lactamase encoding genes was also evaluated. Methods: Antimicrobial susceptibility testing of GNB isolated from intra-abdominal (IAI), respiratory (RTI), and urinary tract infections (UTI) was performed according to ISO 227-1 guidelines and interpreted following CLSI and BrCAST/EUCAST guidelines. Qualifying Enterobacteriaceae isolates were screened for the presence of β-lactamase genes by PCR followed by DNA sequencing. Results: 1748 GNB collected from UTI (45.2%), IAI (25.7%) and RTI (29.1%) were evaluated. Ceftolozane-tazobactam remained highly active (94.7%) against E. coli isolates. Among K. pneumoniae, susceptibility rates were 85.9% and 85.4% for amikacin and colistin, whereas ceftolozane-tazobactam (44.1% susceptible) and carbapenems (55.2-62.2% susceptible) showed poor activity due to blaKPC-2. Against E. cloacae amikacin, imipenem, and meropenem retained good activity (>90%). Ceftolozane-tazobactam was the most potent β-lactam agent tested against P. aeruginosa (90.9% susceptible), including ceftazidime and imipenem resistant isolates. β-lactamase encoding genes testing was carried out in 433 isolates. blaCTX-M variants were predominant in E. coli, P. mirabilis and E. cloacae. Among the K. pneumoniae molecularly tested, most carried blaKPC (68.5%), with all harboring blaKPC-2, except two isolates carrying blaKPC-3 or blaKPC-30. ESBL encoding genes, mainly CTX-M family, were frequently detected in K. pneumoniae, plasmid-mediated AmpC were rare. A variety of PDC encoding genes were detected in P. aeruginosa isolates with five isolates harboring MBL and one KPC encoding genes. Conclusion: Ceftolozane-tazobactam was very active against E. coli, P. mirabilis and P. aeruginosa isolates and could constitute an excellent therapeutic option including for those isolates resistant to extended-spectrum cephalosporins and carbapenems but not producers of carbapenemases.

Urinary tract infection (UTI) is a common condition in women. There is an increased concern on reduction of bacterial susceptibility resulting from wrongly prescribing antimicrobials. This paper summarizes the recommendations of four Brazilian medical societies (SBI – Brazilian Society of Infectious Diseases, FEBRASGO – Brazilian Federation of Gynecology and Obstetrics Associations, SBU – Brazilian Society of Urology, and SBPC/ML – Brazilian Society of Clinical Pathology/Laboratory Medicine) on the management of urinary tract infection in women.Asymptomatic bacteriuria should be screened at least twice during pregnancy (early and in the 3rd trimester). All cases of significant bacteriuria (≥105 CFU/mL in middle stream sample) should be treated with antimicrobials considering safety and susceptibility profile. In women with typical symptoms of cystitis, dipsticks are not necessary for diagnosis. Urine cultures should be collected in pregnant women, recurrent UTI, atypical cases, and if there is suspicion of pyelonephritis. First line antimicrobials for cystitis are fosfomycin trometamol in a single dose and nitrofurantoin, 100 mg every 6 hours for five days. Second line drugs are cefuroxime or amoxicillin-clavulanate for seven days. During pregnancy, amoxicillin and other cephalosporins may be used, but with a higher chance of therapeutic failure.In recurrent UTI, all episodes should be confirmed by urine culture. Treatment should be initiated only after urine sampling and with the same regimens indicated for isolated episodes. Prophylaxis options of recurrent UTI are behavioral measures, non-antimicrobial and antimicrobial prophylaxis. Vaginal estrogens may be recommended for postmenopausal women. Other non-antimicrobial prophylaxis, including cranberry and immunoprophylaxis, have weak evidence supporting their use. Antimicrobial prophylaxis may be offered as a continuous or postcoital scheme. In pregnant women, options are cephalexin, 250–500 mg and nitrofurantoin, 100 mg (contraindicated after 37 weeks of pregnancy). Nonpregnant women may use fosfomycin trometamol, 3 g every 10 days, or nitrofurantoin, 100 mg (continuous or postcoital).

Introdução/Objetivos: Stenotrophomonas maltophilia tem mostrado participação significativa em infecções nosocomiais, especialmente em pacientes imunossuprimidos, como os que fazem hemodiálise e aqueles com fibrose cística. Falhas clínicas em infecções causadas por bactérias suscetíveis têm chamado a atenção para o fenômeno da tolerância antimicrobiana. Assim, considerando a pressão seletiva imposta pela terapia empírica, especialmente em pacientes imunossuprimidos, buscamos detectar tolerância a antimicrobianos em isolados clínicos de S. maltophilia. Métodos: Cinquenta amostras de S. maltophilia confirmadas por MALDI-TOF MS foram testadas para suscetibilidade aos antimicrobianos por microdiluição ou disco-difusão. Apenas as amostras sensíveis foram testadas para tolerância à ceftazidime (CAZ), ciprofloxacina (CIP), levofloxacina (LVX) e ticarcilina-clavulanato (TCC) utilizando uma técnica de disco difusão modificada. Amostras com redução média de halo de inibição menor ou igual ao controle positivo para tolerância (D25) foram consideradas tolerantes. Resultados: A frequência de resistência antimicrobiana foi de 64% (n = 32), 24% (n = 12) e 2% (n = 1) para CAZ, CIP e TCC, respectivamente. Todas as amostras foram sensíveis à LVX. A tolerância a pelo menos um antimicrobiano foi detectada em 80% das amostras. A frequência de tolerância foi de 40%, 79,5%, 52% e 16,3% para CAZ, CIP, LVX e TCC, respectivamente. Conclusão: A maioria das amostras clínicas de S. maltophilia apresentaram altas taxas de tolerância aos antimicrobianos, especialmente para CIP, LVX e CAZ, o que pode ser uma preocupação, dado o arsenal antimicrobiano limitado contra esse patógeno.

Fosfomycin disodium is a potential therapeutic option to manage difficult-to-treat infections, especially when combined with other antimicrobials. In this study, we evaluated the activity of fosfomycin in combination with meropenem or polymyxin B against contemporaneous KPC-2-producing K. pneumoniae clinical isolates (KPC-KPN). Synergistic activity was assessed by checkerboard (CKA) and time–kill (TKA) assays. TKA was performed using serum peak and trough concentrations. The activity of these combinations was also assessed in the Galleria mellonella model. Biofilm disruption was assessed by the microtiter plate technique. CKA resulted in an 8- to 2048-fold decrease in meropenem MIC, restoring meropenem activity for 82.4% of the isolates when combined with fosfomycin. For the fosfomycin + polymyxin B combination, a 2- to 128-fold reduction in polymyxin B MIC was achieved, restoring polymyxin B activity for 47% of the isolates. TKA resulted in the synergism of fosfomycin + meropenem (3.0–6.7 log10 CFU/mL decrease) and fosfomycin + polymyxin B (6.0–6.2 log10 CFU/mL decrease) at peak concentrations. All larvae treated with fosfomycin + meropenem survived. Larvae survival rate was higher with fosfomycin monotherapy (95%) than that observed for fosfomycin + polymyxin B (75%) (p-value < 0.0001). Finally, a higher biofilm disruption was observed under exposure to fosfomycin + polymyxin B (2.4–3.4-fold reduction). In summary, we observed a synergistic effect of fosfomycin + meropenem and fosfomycin + polymyxin B combinations, in vitro and in vivo, against KPC-KPN, as well as biofilm disruption.

ABSTRACT 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.

This study aimed to characterize a Klebsiella pneumoniae strain (KP411) recovered from the stool samples of poultry (Gallus gallus) in the Brazilian Amazon Region. The whole-genome sequencing of KP411 revealed the presence of an important arsenal of antimicrobial resistance genes to β-lactams (blaCTX-M-14, blaTEM-1B, blaKPC-2, blaSVH-11), aminoglycosides [aph(3″)- Ib, aph(6)-Id, aph(3′)-Ia], sulfonamides (sul1, sul2), quinolones (oqxAB), fosfomycin (fosAKP), and macrolides [mph(A)]. Furthermore, our analyses revealed that the KP411 strain belongs to the ST258 clonal lineage, which is one of the main epidemic clones responsible for the dissemination of KPC-2 worldwide. Our data suggest that food-producing animals may act as reservoirs of multidrug-resistant K. pneumoniae belonging to the ST258 clone, and, consequently, contribute to their dissemination to humans and the environment.

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) 15Pas/103Ox (n = 4) and 79Pas/113Ox (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 blaOXA–23 or blaOXA–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.

Abstract INTRODUCTION: In this study, we report a clonal dissemination of carbapenem resistant Acinetobacter baumannii isolates due to the acquisition of blaOXA-23 in a regional hospital located in Brazilian Amazon Region. METHODS: The isolates were identified by MALDI-TOF and the carbapenemase-encoding genes were detected by multiplex-PCR. The genetic similarity was investigated by pulsed-field gel electrophoresis (PFGE). RESULTS: Only 10 (55.6%) isolates harbored the gene bla OXA-23. PFGE analysis revealed that these isolates belong to a single clone. CONCLUSIONS: This dissemination strategy indicates the need for surveillance, adoption of control procedures defined in guidelines, and the careful administration of antimicrobials should be reinforced.

The mechanisms involved in the uncommon resistance phenotype, carbapenem resistance and broad-spectrum cephalosporin susceptibility, were investigated in 25 Pseudomonas aeruginosa clinical isolates that exhibited this phenotype, which were recovered from three different hospitals located in São Paulo, Brazil. The antimicrobial susceptibility profile was determined by CLSI broth microdilution. β-lactamase-encoding genes were investigated by PCR followed by DNA sequencing. Carbapenem hydrolysis activity was investigated by spectrophotometer and MALDI-TOF assays. The mRNA transcription level of oprD was assessed by qRT-PCR and the outer membrane proteins profile was evaluated by SDS-PAGE. Genetic relationship among P. aeruginosa isolates was assessed by PFGE. Carbapenems hydrolysis was not detected by carbapenemase assay in the carbapenem-resistant and cephalosporin-susceptible P. aueruginosa clinical isolates. OprD decreased expression was observed in all P. aeruginosa isolates by qRT-PCR. The outer membrane protein profile by SDS-PAGE suggested a change in the expression of the 46 kDa porin that could correspond to OprD porin. The isolates were clustered into 17 genotypes without predominance of a specific PFGE pattern. These results emphasize the involvement of multiple chromosomal mechanisms in carbapenem-resistance among clinical isolates of P. aeruginosa, alert for adaptation of P. aeruginosa clinical isolates under antimicrobial selective pressure and make aware of the emergence of an uncommon phenotype among P. aeruginosa clinical isolates. Keywords: Gram-negative bacilli, Carbapenem resistance, OprD porin

Abstract Background The emergence of multidrug-resistant Klebsiella pneumoniae is a major public health concern. Many K. pneumoniae infections can only be treated when resorting to last-line drugs such as polymyxin B (PB). However, resistance to this antibiotic is also observed, although insufficient information is described on its mode of action as well as the mechanisms used by resistant bacteria to evade its effects. We aimed to study PB resistance and the influence of abiotic stresses in a clinical K. pneumoniae strain using whole transcriptome profiling. Results We sequenced 12 cDNA libraries of K. pneumoniae Kp13 bacteria, from two biological replicates of the original strain Kp13 (Kp13) and five derivative strains: induced high-level PB resistance in acidic pH (Kp13pH), magnesium deprivation (Kp13Mg), high concentrations of calcium (Kp13Ca) and iron (Kp13Fe), and a control condition with PB (Kp13PolB). Our results show the involvement of multiple regulatory loci that differentially respond to each condition as well as a shared gene expression response elicited by PB treatment, and indicate the participation of two-regulatory components such as ArcA-ArcB, which could be involved in re-routing the K. pneumoniae metabolism following PB treatment. Modules of co-expressed genes could be determined, which correlated to growth in acid stress and PB exposure. We hypothesize that polymyxin B induces metabolic shifts in K. pneumoniae that could relate to surviving against the action of this antibiotic. Conclusions We obtained whole transcriptome data for K. pneumoniae under different environmental conditions and PB treatment. Our results supports the notion that the K. pneumoniae response to PB exposure goes beyond damaged membrane reconstruction and involves recruitment of multiple gene modules and intracellular targets.

Summary: Background: The Global Point Prevalence Survey (Global-PPS) established an international network of hospitals to measure antimicrobial prescribing and resistance worldwide. We aimed to assess antimicrobial prescribing and resistance in hospital inpatients. Methods: We used a standardised surveillance method to collect detailed data about antimicrobial prescribing and resistance from hospitals worldwide, which were grouped by UN region. The internet-based survey included all inpatients (adults, children, and neonates) receiving an antimicrobial who were on the ward at 0800 h on one specific day between January and September, 2015. Hospitals were classified as primary, secondary, tertiary (including infectious diseases hospitals), and paediatric hospitals. Five main ward types were defined: medical wards, surgical wards, intensive-care units, haematology oncology wards, and medical transplantation (bone marrow or solid transplants) wards. Data recorded included patient characteristics, antimicrobials received, diagnosis, therapeutic indication according to predefined lists, and markers of prescribing quality (eg, whether a stop or review date were recorded, and whether local prescribing guidelines existed and were adhered to). We report findings for adult inpatients. Findings: The Global-PPS for 2015 included adult data from 303 hospitals in 53 countries, including eight lower-middle-income and 17 upper-middle-income countries. 86 776 inpatients were admitted to 3315 adult wards, of whom 29 891 (34·4%) received at least one antimicrobial. 41 213 antimicrobial prescriptions were issued, of which 36 792 (89·3%) were antibacterial agents for systemic use. The top three antibiotics prescribed worldwide were penicillins with β-lactamase inhibitors, third-generation cephalosporins, and fluoroquinolones. Carbapenems were most frequently prescribed in Latin America and west and central Asia. Of patients who received at least one antimicrobial, 5926 (19·8%) received a targeted antibacterial treatment for systemic use, and 1769 (5·9%) received a treatment targeting at least one multidrug-resistant organism. The frequency of health-care-associated infections was highest in Latin America (1518 [11·9%]) and east and south Asia (5363 [10·1%]). Overall, the reason for treatment was recorded in 31 694 (76·9%) of antimicrobial prescriptions, and a stop or review date in 15 778 (38·3%). Local antibiotic guidelines were missing for 7050 (19·2%) of the 36 792 antibiotic prescriptions, and guideline compliance was 77·4%. Interpretation: The Global-PPS showed that worldwide surveillance can be accomplished with voluntary participation. It provided quantifiable measures to assess and compare the quantity and quality of antibiotic prescribing and resistance in hospital patients worldwide. These data will help to improve the quality of antibiotic prescribing through education and practice changes, particularly in low-income and middle-income countries that have no tools to monitor antibiotic prescribing in hospitals. Funding: bioMérieux.

The global spread of carbapenem-resistant Acinetobacter baumannii (A. baumannii) strains has restricted the therapeutic options available to treat infections due to this pathogen. Understanding the prevalence of such infections and the underlying genetic mechanisms of resistance may help in the implementation of adequate measures to control and prevent acquisition of nosocomial infections, especially in an intensive care unit setting. This study describes the molecular characteristics and risk factors associated with OXA-23-producing A. baumannii infections. A case-control study was undertaken from September/2013 to April/2015. Acquisition of OXA-23-producing A. baumannii was found to be associated with the use of nasogastric tubes, haemodialysis, and the use of cephalosporins. These isolates were only susceptible to amikacin, gentamicin, tigecycline, and colistin, and contained the ISAba1 insertion sequence upstream ofblaOXA-23 and blaOXA-51 genes. Twenty-six OXA-23-producing A. baumannii strains belonged to the ST79 (CC79) clonal group,and patients infected or colonised by these isolates had a higher mortality rate (34.6%). In conclusion, this study showed a dissemination of OXA-23-producing A. baumannii strains that was associated with several healthcare-related risk factors and high mortality rates among intensive care unit patients.

Rahnella aquatilis is an environmental Gram-negative bacillus that is rarely reported as human pathogen, being mainly associated with infections in immunocompromised patients. Herein we describe two cases of R. aquatilis isolates recovered from endotracheal aspirate cultures of different patients in a tertiary hospital located in the city of São Paulo, Brazil. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and 16S rDNA gene sequencing were performed to confirm bacterial identification after the isolates being erroneously identified as Pantoea spp. by automated system. Both isolates showed the same PFGE pattern and presented the β-lactamase encoding gene blaRAHN-1, responsible for resistance to cephalothin. The isolates were susceptible to broad-spectrum cephalosporins, carbapenems, fluoroquinolones, aminoglycosides, and polymyxin B. This report shows the presence and transmission of uncommon bacteria in the nosocomial environment and alerts us about the need for new tools of correct microbiologic diagnosis. Keywords: Rahnella spp., Bacteria identification, MALDI-TOF MS, Environmental bacteria

Carbapenems represent the mainstay therapy for the treatment of serious P. aeruginosa infections. However, the emergence of carbapenem resistance has jeopardized the clinical use of this important class of compounds. The production of SPM-1 metallo-β-lactamase has been the most common mechanism of carbapenem resistance identified in P. aeruginosa isolated from Brazilian medical centres. Interestingly, a single SPM-1-producing P. aeruginosa clone belonging to the ST277 has been widely spread within the Brazilian territory. In the current study, we performed a next-generation sequencing of six SPM-1-producing P. aeruginosa ST277 isolates. The core genome contains 5 899 coding genes relative to the reference strain P. aeruginosa PAO1. A total of 26 genomic islands were detected in these isolates. We identified remarkable elements inside these genomic islands, such as copies of the blaSPM-1 gene conferring resistance to carbapenems and a type I-C CRISPR-Cas system, which is involved in protection of the chromosome against foreign DNA. In addition, we identified single nucleotide polymorphisms causing amino acid changes in antimicrobial resistance and virulence-related genes. Together, these factors could contribute to the marked resistance and persistence of the SPM-1-producing P. aeruginosa ST277 clone. A comparison of the SPM-1-producing P. aeruginosa ST277 genomes showed that their core genome has a high level nucleotide similarity and synteny conservation. The variability observed was mainly due to acquisition of genomic islands carrying several antibiotic resistance genes.

Nosocomial infections caused by carbapenem-resistant Acinetobacter baumannii isolates have reached epidemic levels in past decades. Currently this microorganism is responsible for outbreaks of difficult eradication and with high mortality rates worldwide. We herein report a rare case of an OXA-72-producing A. baumannii isolate colonizing a 47-year-old male patient with peritonitis due to abdominal stab wound, four years earlier than the first report of this carbapenemase in Acinetobacter pittii in Colombia. Although OXA-72 presents a low prevalence compared with OXA-23, our study demonstrated that A. baumannii isolates carrying the blaOXA-72 gene were present in the hospital environment in Colombia and could act as a reservoir for further spread to other Acinetobacter species, like A. pittii, causing carbapenem-resistance. Keywords: Acinetobacter baumannii, Carbapenem-resistance, Oxacillinase, Colombia

Rapid identification of microorganisms by the clinical microbiology laboratory is of crucial importance for optimal patients’ management and treatment. In general, bacterial identification by conventional methods requires 18-24 hours for colony isolation and at least 24 additional hours for species identification. New technologies in microbiology have focused on the rapid diagnosis of bloodstream infections, since they are associated with high morbidity and mortality rates.

The aim of this study was to characterize two metallo-β-lactamases (MBLs)-producing Pseudomonas aeruginosa clinical isolates showing meropenem susceptibility. Antimicrobial susceptibility was assessed by automated testing and Clinical and Laboratory Standards Institute agar dilution method. MBL production was investigated by phenotypic tests. Molecular typing was determined by pulsed field gel electrophoresis (PFGE). MBL-encoding genes, as well as their genetic context, were identified by polymerase chain reaction (PCR) and sequencing. The location of blaIMP-16 was determined by plasmid electrophoresis, Southern blot and hybridization. Transcriptional levels of blaIMP-16, mexB, mexD, mexF, mexY, ampC and oprD were determined by semi-quantitative real time PCR. The P. aeruginosa isolates studied, Pa30 and Pa43, showed imipenem and meropenem susceptibility by automated testing. Agar dilution assays confirmed meropenem susceptibility whereas both isolates showed low level of imipenem resistance. Pa30 and Pa43 were phenotypically detected as MBL producers. PFGE revealed their clonal relatedness. blaIMP-16 was identified in both isolates, carried as a single cassette in a class 1 integron that was embedded in a plasmid of about 60-Kb. Pa30 and Pa43 overexpressed MexAB-OprM, MexCD-OprJ and MexXY-OprM efflux systems and showed basal transcriptional levels of ampC and oprD. MBL-producing P. aeruginosa that are not resistant to meropenem may represent a risk for therapeutic failure and act as silent reservoirs of MBL-encoding genes.

Polymyxins are polypeptide antimicrobials that act in the cell membranes and promote decrease of the cell wall integrity. These antimicrobials are used in the clinical practice for treatment of the multi-drug resistant Gram negative bacilli infections as the last therapeutic option. The polymyxin resistance involves lipopolysaccharide modifi cations that decrease the affi nity of the antimicrobial with the cell surface. These modifi cations are regulated by two component systems that are active by environmental infl uences as cation presence, pH or polymyxin exposure. The environmental infl uences initiate the action of the genes that develop the polymyxins resistant phenotype. The polymyxins viability maintenance is essential for the treatment for multi-drug resistant bacilli infections, while new therapeutic options are not available.KEYWORDS polimixins antimicrobial resistance

Polimixinas são antimicrobianos polipeptídicos que agem nas membranas celulares promovendo a diminuição da integridade da parede celular e consequente morte celular bacteriana. Esses antimicrobianos são utilizados na prática clínica para o tratamento de infecções por bacilos Gram negativos multirresistentes, como última opção terapêutica. A resistência às polimixinas envolve modificações no lipopolissacarídeo da parede celular bacteriana, que diminuem aafinidade do antimicrobiano pela superfície celular. Essas modificações são reguladas por diferentes sistemas de doiscomponentes que são ativados por fatores ambientais como presença de cátions, pH ou presença do antimicrobiano. Esses fatores desencadeiam a ação uma cascata de genes que, por sua vez, desenvolvem o fenótipo de resistência às polimixinas. A manutenção da viabilidade das polimixinas é essencial para o tratamento de infecções por bactériasmultirresistentes, enquanto novas opções terapêutica não estejam disponíveis.

In vitro activity of doripenem and comparator antimicrobial agents was evaluated against Gram-negative bacilli recently isolated from Brazilian private hospitals that were enrolled in the INVITA-A-DORI Brazilian Study. A total of 805 unique Gram-negative bacilli were collected from patients hospitalized at 18 medical centers between May/08 and March/09. Each hospital was asked to submit 50 single Gram-negative bacilli isolated from blood, lower respiratory tract or intraabdominal secretions. Bacterial identification was confirmed and antimicrobial susceptibility testing was performed using Clinical Laboratory Standards Institute (CLSI) microdilution method at a central laboratory. CLSI M100-S21 (2011) or US-FDA package insert criteria (tigecycline) was used for interpretation of the antimicrobial susceptibility results. Doripenem was as active as meropenem and more active than imipenem against E. coli and K. pneumoniae isolates. A total of 50.0% of Enterobacter spp. isolates were resistant to ceftazidime but 85.7% of them were inhibited at doripenem MICs ≤ 1 μg/mL. Polymyxin B was the only agent to show potent activity against Acinetobacter spp. (MIC50/90, ≤ 0.5/1 μg/mL) and P. aeruginosa (MIC50/90, 1/2 μg/mL). Although high rates of imipenem (53.1%) and meropenem (44.5%) resistance were detected among P. aeruginosa, doripenem showed MIC50 of 16 μg/mL against imipenem-resistant P. aeruginosa and inhibited a greater number of imipenem-resistant P. aeruginosa (10.5%) at MIC values of ≤ 4 μg/mL than did meropenem (0.0%). In this study, doripenem showed similar in vitro activity to that of meropenem and retained some activity against imipenem-resistant P. aeruginosa isolated from Brazilian medical centers. Keywords: carbapenems, Gram-negative bacteria, Brazil

INTRODUÇÃO: As fitas Oxoid® M.I.C.Evaluator® (M.I.C.E., Thermo Fisher Scientific, Basingstoke, UK), recém-lançadas no mercado brasileiro, representam uma alternativa rápida para a realização de testes de sensibilidade a antimicrobianos (TSA). OBJETIVO: Avaliar o desempenho da metodologia M.I.C.E. em relação à microdiluição em caldo (teste de referência) e ao Etest® (BioMérieux, Marcy l'Étoile, France). Material e métodos: Foram selecionados 160 isolados bacterianos, sendo P. aeruginosa (20), Acinetobacter spp. (20), K. pneumoniae (20), E. coli (20), S. aureus (20), Staphylococcus coagulase-negativa (20), E. faecalis (20) e E. faecium (20). Os TSAs foram realizados por microdiluição em caldo, Etest e M.I.C.E., seguindo-se as recomendações do Clinical Laboratory Standards Institute (CLSI, 2009) e dos respectivos fabricantes. Os resultados foram interpretados segundo os critérios estabelecidos pelo CLSI e comparados por análise de regressão. RESULTADOS: Avaliando-se todas as combinações de antimicrobianos vs. a espécie bacteriana, o desempenho da metodologia M.I.C.E. foi muito bom, apresentando uma concordância geral (variação na concentração inibitória mínima [CIM] ± 1-log2) > 90%, exceto para cefotaxima (85%) e vancomicina (76,3%), quando em comparação com os resultados da metodologia de referência. Quando comparado com o Etest, a metodologia M.I.C.E. apresentou concordância geral > 96%, com exceção para a combinação amoxicilina/ácido clavulânico (67,5%). CONCLUSÃO: Os resultados do TSA obtidos pela metodologia M.I.C.E. apresentaram boa correlação com aqueles obtidos pela microdiluição em caldo e pelo Etest, indicando que essa metodologia é uma alternativa rápida para a determinação da CIM pelos laboratórios de microbiologia clínica. Atenção especial deve ser dada á determinação da CIM para a combinação amoxicilina/ácido clavulânico.INTRODUCTION: The Oxoid® M.I.C.EvaluatorTM methodology (M.I.C.E., Thermo Fisher Scientific, Basingstoke, UK), recently released into the market, represents a rapid alternative to antimicrobial susceptibility testing. OBJECTIVE: The objective of this study was to evaluate the performance of M.I.C.E. methodology in relation to broth microdilution (reference test) and Etest® (BioMérieux, Marcy l'Étoile, France). Material and method: A total of 160 bacterial isolates were collected comprising the following species: P. aeruginosa (20), Acinetobacter spp. (20), K. pneumoniae (20), E. coli (20), S. aureus (20), coagulase-negative Staphylococcus (20), E. faecalis (20) and E. faecium (20). Following Clinical Laboratory Standands Institute (CLSI) standards (2009) and the manufacturers' recommendations, antimicrobial susceptibility testing was performed using broth microdilution method, Etest and M.I.C.E. The results were interpreted according to the criteria established by CLSI and compared through regression analysis. RESULTS: All antimicrobial combinations vs. bacterial species were evaluated and M.I.C.E. methodology yielded good results with general correlation (MIC variation ± 1-log2) > 90%, except for cefotaxime (85%) and vancomycin (76.3%) when compared with the reference method. The M.I.C.E. results compared to Etest showed general correlation (> 96%), except for amoxicillin/clavulanic acid (67.5%) combination. CONCLUSION: AST results obtained from M.I.C.E. methodology showed a good correlation with those from broth microdilution and Etest, which corroborates its time effectiveness in the determination of MIC. However, the combination of amoxicillin/clavulanic acid requires further attention.

In 2008 isolates of KPC-producing Klebsiella pneumoniae (KPC-KPN) were detected for the first time at Hospital Heliópolis, São Paulo, Brazil. The aim of this study was to characterize the clinical and microbiological outcomes of infections caused by KPC-KPN. A historical cohort of patients from whom KPC-KPN strains were isolated was performed. Isolates were identified as resistant to ertapenem by automated broth microdilution system and screened as carbapenemase producers by the modified Hodge test. The beta-lactamase resistance gene blaKPC was detected by PCR. The genetic relatedness of isolates was determined by PFGE. The study provides early clinical experience in treating KPC-KPN infections in a Brazilian tertiary center. Keyword: Klebsiella pneumonia, KPC, hospital infection, carbapenemase

Nos últimos anos tem sido observada maior incidência de bacilos Gram-negativos resistentes a cefalosporinas de espectro ampliado no ambiente hospitalar, ocasionando, assim, maior uso de betalactâmicos mais potentes, como os carbapenens. A utilização de carbapenens exerce maior pressão seletiva sobre a microbiota hospitalar, o que pode ocasionar aumento da resistência a esses agentes. Entre os mecanismos de resistência a carbapenens mais comumente identificados estão a produção de betalactamases, como, por exemplo, as pertencentes à classe D de Ambler e as que pertencem à classe B de Ambler, ou metalo-beta-lactamases (MbetaL). Essas últimas hidrolisam todos betalactâmicos comercialmente disponíveis, sendo a única exceção o monobactam aztreonam. Desde o início da década de 1990, novos genes que codificam MbetaLs têm sido descritos em microrganismos clinicamente importantes, como Pseudomonas spp., Acinetobacter spp. e membros da família Enterobacteriaceae. O encontro desses microrganismos não-sensíveis a carbapenens pode ser submetido a metodologias fenotípicas para detecção da produção de MbetaL com o intuito de auxiliar a Comissão de Controle de Infecção Hospitalar (CCIH) e prevenir a disseminação desses determinantes de resistência, uma vez que genes que codificam MbetaLs estão contidos em estruturas genéticas que propiciam sua mobilidade de forma muito efetiva, sendo então facilmente disseminados.Increase isolation of Gram-negative bacilli resistant to broad-spectrum cephalosporin has been observed during the last few years, thus determining the use of more potent beta-lactams, such as carbapenems. The use of these antimicrobial agents may lead to the emergence of carbapenem resistant Gram-negative bacilli in the nosocomial environment. Carbapenem resistance may be due to the production of Ambler class D beta-lactamase or Ambler class B beta-lactamase, also called metallo-beta-lactamase (MbetaL). Apart from the monobactam aztreonam, this class of enzyme virtually hydrolyze all the commercially available beta-lactams. Since 90s, several clinical important nosocomial microorganisms, including members of Enterobacteriaceae family, Pseudomonas spp. and Acinetobacter spp., have been found to produce MbetaLs enzymes. When these carbapenem non-susceptible strains are found, they may be submitted to phenotypic MbetaL detection test in the microbiology laboratory in order to help infection control practioners and prevent the MbetaL gene dissemination, once these genes are embedded in mobile genetic elements, which can spread rapidly to other Gram-negative species.

INTRODUCTION: Pseudomonas aeruginosa is a leading cause of ventilator-associated pneumonia (VAP) and exhibits high rates of resistance to several antimicrobial drugs. The carbapenens are usually the drugs of choice against this microorganism. However, the carbapenem resistance has increased among these strains worldwide. The presence of metallo-β-lactamases (MBL) has been pointed out as a major mechanism of resistance among these strains. No previous study addressed outcomes of respiratory infections caused by these strains. METHODS: Our group sought to analyze the epidemiology and clinical outcomes of patients with VAP caused by imipenem-resistant P. aeruginosa. A total of 29 clinical isolates of carbapenem-resistant Pseudomonas aeruginosa were screened for metallo-β-lactamase (MBL) genes. RESULTS: Demographic and clinical variables were similar between the SPM-1-producing and non-SPM-1-producing group. Five (17.2%) isolates were positive for blaSPM-1. No other MBL gene was found. All patients were treated with polymyxin B. The infection-related mortality was 40% and 54.2% for SPM-1-producing and -non-producing isolates, respectively. CONCLUSIONS: There were no differences in epidemiological and clinical outcomes between the two groups.

The objective of this study was to evaluate the susceptibility to cefepime of a large group of ESBL-producing enterobacteria recently isolated in a Brazilian teaching hospital. The study included 280 strains of ESBL-producing enterobacteria, isolated between 2005 and 2008. The presence of the genes blaCTX-M, blaTEM and blaSHV was determined by PCR and confirmed by nucleotide sequencing. Susceptibility testing for cefepime was performed by disc-diffusion, agar dilution method and E-test®. Among the isolates, 34 (12.1%) presented a cefepime inhibition zone ≥ 21 and MIC ≤ 8 mg/L by agar dilution and E-strip methods. The use of cefepime for the treatment of infections caused by ESBL-producing bacteria has been controversial. Some studies of PD/PK show the probability of achieving the required PD parameters for cefepime, when the MICs were

SUMMARY In this brief communication we describe the occurrence of a KPC-producing Serratia marcescensisolate in a home-care patient from Recife, Brazil. The blaKPC, blaSPM, blaIMP, blaVIMblaOXA, blaCTX-M, blaSHV, blaTEM and blaGES genes were investigated by Polymerase Chain Reaction (PCR) and DNA sequencing. The isolate was positive for blaKPC-2 and blaTEM-1 and was resistant to aztreonam, cefepime, cefotaxime, imipenem, meropenem, gentamicin, ciprofloxacin and cefazidime, and susceptible only to amikacin, tigecycline and gatifloxacin. This is the first report in Brazil of KPC-producing S. marcescens clinical isolate outside of a hospital environment. Caregivers should be alert for the presence of this isolate in the community setting.

Nevirapine-induced DRESS syndrome is uncommon but a potentially life-threatening condition, with significant morbidity and mortality rates due to multiple-organ involvement. The authors report a case of a 47-year-old HIV-infected female patient who presented with fever, right hypochondrium pain, jaundice, and skin rash. The Nevirapine-induced DRESS syndrome was suspected based on clinical presentation and RegiSCAR scoring system. This case highlights the need for a high index of clinical suspicion among HIV-infected patients with severe skin eruption and systemic symptoms.

A Escherichia coli é, entre os bacilos Gram-negativos, o principal agente etiológico de infecções da corrente sanguínea, seja comunitárias seja as nosocomiais. Uma vez que são poucos os trabalhos que pesquisaram a virulência de amostras de E. coli isoladas de bacteremia no Brasil, este trabalho teve por objetivo investigar os fatores de virulência frequentes em amostras isoladas de seres humanos hospitalizados bem como a origem filogenética destas amostras, relacionando essas características bacterianas com características do hospedeiro como sexo e idade. A pesquisa da origem filogenética das amostras foi realizada utilizando-se a técnica da PCR e a pesquisa de fatores de virulência por hibridação de colônias com sondas radioativas. O resultado obtido com a pesquisa de 20 fatores de virulência demonstrou que os fatores relacionados à fimbria tipo 1, ao sistema yersiniabactina de aquisição de ferro e à produção de cápsula, foram os mais frequentes nas amostras estudadas. Além disso, a grande maioria das amostras foi classificada nos grupos filogenéticos B2 e D. Em relação às características do hospedeiro, foi possível estabelecer uma relação positiva entre amostras mais virulentas e infecções em pacientes do sexo feminino. Contudo, não houve diferença na virulência das amostras em relação à idade do hospedeiro. Estes dados sugerem que as amostras que causam infecções extra-intestinais em pacientes do sexo feminino são, em geral, mais virulentas do que aquelas que causam infecções nos pacientes do sexo masculino. Por outro lado, a idade não parece ter correlação com a virulência da amostra infectante.

This study evaluated the clonal spread of carbapenem-resistant P. aeruginosa producing SPM-1 type metallo-β-lactamase (MBL), at the university hospital of Florianópolis, Santa Catarina, Brazil, compared to an epidemic clone previously reported, as well as strains collected in other three Brazilian states. Among the isolates, 17 (62%) were clonal and highly related to strains from other regions of Brazil. Six clonal strains harbored the blaSPM-1 gene. The finding of a unique SPM-1 producer clone suggests that its dissemination has contributed to the high resistance to carbapenems in Brazilian hospitals. Keywords: P. aeruginosa, carbapenem resistance, metallo-β-lactamase

In 2008 isolates of KPC-producing Klebsiella pneumoniae (KPC-KPN) were detected for the first time at Hospital Heliópolis, São Paulo, Brazil. The aim of this study was to characterize the clinical and microbiological outcomes of infections caused by KPC-KPN. A historical cohort of patients from whom KPC-KPN strains were isolated was performed. Isolates were identified as resistant to ertapenem by automated broth microdilution system and screened as carbapenemase producers by the modified Hodge test. The beta-lactamase resistance gene blaKPC was detected by PCR. The genetic relatedness of isolates was determined by PFGE. The study provides early clinical experience in treating KPC-KPN infections in a Brazilian tertiary center.