In healthcare settings and beyond, the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) among other pathogens exchange antibiotic resistance and virulence factors and emerge as new infectious clones. According to the Saudi General Authority for Statistics (stats.gov.sa), Saudi Arabia is a country where more than 27 million pilgrims meet in annual continual mass-gathering events. This massive influx of people could introduce novel pathogens to the community that could not necessarily be detected with traditional culture-dependent clinical microbiological tests. Conventional clinical microbiology and environmental pathogen detection methods have had many limitations and narrow search scope. These methods can only target known and culturable pathogens. Over the past decade, applications of next-generation sequencing (NGS) and bioinformatics tools have revolutionized the way pathogens are detected and their relevant phenotypes such as clonal types, antibiotic resistance are predicted to aid in clinical decision making as additional practice to traditional clinical microbiology-based testing protocols. The aim of this study was to apply whole-genome sequencing (WGS) and bioinformatic analysis tools on clinical samples and bacterial isolates in order to pave the way for transforming current clinical microbiology practices in a tertiary referral hospital in Jeddah, Saudi Arabia. My attempt to utilize WGS as a tool on pathogenic strains in this study combined with the clinical data has resulted in discovering a silent outbreak of an emerging hypervirulent strain of Klebsiella pneumoniae (Chapter 2). Analysis of the strains antimicrobial profiles genetically has yielded the first characterization of a misidentified Klebsiella quasipneumoniae harboring plasmid-mediated carbapenemases of Klebsiella pneumoniae carbapenemases (KPC) (Chapter 3). Similarly, I was able to study mobile colistin resistance genes in the isolates and identify a novel occurrence of mcr-1 and mcr-8 (Chapter 4). I applied clinical metagenomic protocol on an intestinal biopsy of an inflammatory bowel disease patient with Crohn’s disease, where I identified an association of three co-occurring and an actively replicating non-tuberculosis mycobacteria (Chapter 5). The deployment of whole-genome sequencing and metagenomic in infectious disease surveillance and diagnostics could prove beneficial in limiting epidemics and detect transmission patterns of antimicrobial-resistant genes.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21819号 / 農博第2332号 / 新制||農||1067(附属図書館) / 学位論文||H31||N5191(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 三上 文三, 教授 植田 充美, 教授 宮川 恒 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

This master thesis deals with analysis of structural variation of genome and with methods of its sequencing across all generations. Subsequently it contains a description of copy number variation and methods of its detection. The experimental part focuses on algorithm proposal for CNV detection according analysis and testing of uneven coverage in genome, variable representation of GC content and distance of sequence reads. Finally, the algorithm for detecting copy number variation is tested on genomic data of bacteria Klebsiella pneumoniae.

Orientador: Cristiane Angélica Ottoni / Resumo: A utilização de nanopartículas de prata (AgNP) biológicas nos dias atuais é uma alternativa promissora frente as obtidas por via sintética (VS), uma vez que, não geram resíduos em seu processo de síntese e possuem superfície revestida por proteínas que viabilizam sua incorporação em diversos compostos comerciais, sendo os fármacos os de maior destaque. O efeito do descarte de AgNP sintéticas no meio ambiente é atualmente fonte de investigação de diversos grupos de pesquisa, entretanto, são restritas as informações associadas as AgNP biológicas. Diante deste contexto, o presente estudo utilizou a AgNP IBCLP20 biossintetizada pelo fungo Aspergillus niger para avaliar a sua ação antimicrobiana e efeito tóxico em ambiente aquático dulcícula utilizando organismos pertencentes a diferentes níveis tróficos. Nos ensaios antimicrobianos a AgNP IBCLP20 apresentou excelente ação antibacteriana em uma faixa de concentração de 5 a 100 µg/mL e ação antifúngica na faixa de concentração de 20 a 100 µg/mL. A densidade celular da microalga Chrorella vulgaris, quando exposta a AgNP IBCLP20 e ao sal precursor AgNO3, na maior concentração analisada (100 uM/mL) e após 96 horas de incubação, apresentou uma taxa de redução de 34,4% e 85,71%, respectivamente. Nas mesmas condições supracitadas, só foi detectada viabilidade celular da microalga quando exposta a AgNP biológica (65,38%). A letalidade média em D. silimis causada pelas AgNP IBCLP20 foi estimada a uma concentração de 4,06 μg/L (2,29 -6,42).... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The use of biological silver nanoparticles (AgNP) today is a promising alternative compared to those obtained synthetically (VS), since they do not generate residues in their synthesis process and have a surface coated with proteins that enable their incorporation in several commercial compounds, with drugs being the most prominent. The effect of discarding synthetic AgNP in the environment is currently a source of investigation for several research groups, however, information associated with biological AgNP is restricted. In this context, the present study used the AgNP IBCLP20 biosynthesized by the fungus Aspergillus niger to evaluate its antimicrobial action and toxic effect in aquatic dulcicle environment using organisms belonging to different trophic levels. In antimicrobial tests, AgNP IBCLP20 showed excellent antibacterial action in a concentration range of 5 to 100 µg / mL and antifungal action in the concentration range of 20 to 100 µg / mL. The cell density of the microalgae Chrorella vulgaris, when exposed to AgNP IBCLP20 and the precursor salt AgNO3, in the highest concentration analyzed (100 uM / mL) and after 96 hours of incubation, showed a reduction rate of 34.4% and 85.71 %, respectively. Under the same conditions mentioned above, cell viability of the microalgae was only detected when exposed to biological AgNP (65.38%). The average lethality in D. silimis caused by AgNP IBCLP20 was estimated at a concentration of 4.06μg/L (2.29 -6.42). , The physiological ... (Complete abstract click electronic access below) / Mestre

Genetic typing of Klebsiella pneumoniae is used for epidemiological referencing. In the clinical setting it can be useful in outbreak investigations, understanding transmission and managing hospital infections. Multi-drug resistant bacteria exist and proliferate either due to natural selection of clonal lineages or the transfer of mobile genetic elements, sometimes in response to antibiotic-use selective pressure. Pulsed-field gel electrophoresis (PFGE) is highly discriminatory and the gold standard typing method for the characterisation of K. pneumoniae isolates. The aim of the study was to genetically characterise K. pneumoniae isolates by PFGE and multilocus sequence typing (MLST). One hundred unrepeated ESBL-producing K. pneumoniae isolates were collected from the National Health Laboratory Service (NHLS). The PFGE was performed on a Rotaphor VI system (Biometra, Germany). Clonal representatives were further characterised by MLST. All the strains were typeable by PFGE using XbaI, which discerned multiple pulsotypes and MLST identified 10 different STs including a novel sequence type, ST1632. The diverse pulsotypes of K. pneumoniae isolates are not suggestive of clonal spread of particular strains. The MLST results further confirmed the variability among isolates tested and elucidated several STs, some of which have been identified internationally and often associated with carbapenem-resistance. Data on K. pneumoniae STs is still limited in the South African clinical setting, although the close monitoring of resistance profiles and characterisation of isolates is imperative for outbreak analysis, identification of prominent STs in clinical settings as compared to international counterparts and surveillance of expanding resistance. / Dissertation (MSc (Medical Microbiology))--University of Pretoria, 2015. / Medical Microbiology / MSc (Medical Microbiology) / Unrestricted

Bacteriophages (phages) are the smallest biological entity on the planet. They provide vast amounts of valuable knowledge to biologists. Phage genomes are relatively simple compared to the organisms they infect (prokaryotes) and yet continually point to the complexity surrounding molecular- and microbiological mechanisms of life. By studying phages we can learn of the systems of gene expression, protein interaction and DNA organization. Phages are useful not only from an academic perspective, but may also have useful clinical applications. In the face of the rise of antibiotic-resistant bacterial “super pathogens”, scientists and researchers turn to phages as alternative treatments to these types of infections. Phages are capable of infecting and killing even the deadliest of bacterial pathogens, such as carbapenem-resistant Enterobacteriaceae (CRE) or Bacillus anthracis, and may prove increasingly useful in the future for combatting harmful pathogens. This thesis looks at several aspects of phage biology—from the underlying genetics contributing to phage virulence, to the clinical application of phage therapy to treat infections. First, a look at CRE-Klebsiella pneumoniae isolates and phages capable of infecting some strains may reveal a potential therapeutic approach in the future. Additionally, genomic analysis reveals interesting features that may explain aspects of phage virulence and evolutionary history. Then, a collection of genetically diverse phages is used in infection assays on pathogenic strains of Bacillus anthracis to establish the first-reported phages capable of infecting these strains. Finally, the process of preparing phage samples for therapeutic application is explored in-depth to conclude with discussion of clinical application. During the course of these projects over 25 phages were isolated, as many phage genomes were assembled and annotated, resulting in the preparation of two genome announcements and near-completion of two publishable first-author papers (chapters II and III). In addition, participation in a variety of collaborative efforts may lead to a handful of co-author papers and on various topics, including phage biology and application.

Lower respiratory tract infectious diseases are a world-wide healthcare burden with bacterial pathogens accounting for a large portion of primary and secondary infections. The human respiratory tract is home to hundreds of species of microbes that comprise the human airway microbiome. These commensals play a crucial role in human health in part by providing colonization resistance against pathogens. In a previous study from the Surette lab it was shown that specific bacterial isolates from the respiratory microbiome inhibits the growth of pathogens aerobically. This included an isolate of Staphylococcus aureus which inhibited the growth of Enterococcus faecium. This activity was further characterized in this thesis and the underlying mechanism was explored through comparative genomics. As well, this observation provided proof-of-concept for a large-scale screen for additional isolates which inhibit pathogen growth. I hypothesized that the respiratory tract microbiota included many other bacteria capable of inhibiting the growth of respiratory tract pathogens in both aerobic and anaerobic environments, and that anaerobic conditions will identify new activities not detected aerobically. To examine and identify potential beneficial bacteria, I have screened ~5000 respiratory tract bacteria from the Surette lab’s airway isolate collection against four pathogens: Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae. The respiratory tract commensals were pinned onto the pathogen-lawn and their interaction was expressed as zones of clearing or altered growth phenotypes of the pathogen. The results of the screen showed that anti-pathogen activity was a common feature of respiratory tract commensals. In particular, S. pneumoniae was inhibited by taxonomically diverse members of the microbiota representing three phyla (Proteobacteria, Firmicutes and Actinobacteria). Many of the facultative anaerobes that inhibited S. pneumoniae expressed their activity in anerobic conditions. / Thesis / Master of Science (MSc) / The human respiratory tract harbours commensal and pathogenic bacteria, and the latter cause most of the lower respiratory tract infections. The commensal bacteria help to train the immune system and impede the growth of pathogens through colonization resistance. A previous study by the Surette lab identified bacterial isolates from the respiratory tract that inhibit the growth of select pathogens, among them, a particular strain of Staphylococcus aureus. Based on the results of the earlier study, I hypothesized that the respiratory tract bacteria is a good source of commensals that can inhibit the growth of S. aureus and other respiratory pathogens, such as Streptococcus pneumoniae, Pseudomonas aeruginosa and Klebsiella pneumoniae. To find potential therapeutic bacteria, I screened ~5000 respiratory tract isolates from the Surette lab’s strain collection for the ability to impair growth of target pathogens. Additionally, I further characterized the activity of the previously identified S. aureus strain against various Lactobacillalles strains and used comparative genomics to identify potential biosynthetic genes required for biosynthesis of molecules with antibacterial activity within the genome of S. aureus. The research reported in this thesis demonstrates that many commensal bacteria that live within our airways have the ability to inhibit the growth of bacterial pathogens. This work may provide a new source of antibiotics against respiratory infections and new strategies to reduce susceptibility to infections in vulnerable populations.

<p>Escherichia coli i Klebsiella pneumoniae su među najznačajnijim uzročnicima infekcija kod ljudi. Problem predstavljaju multirezistentni sojevi koji se javljaju ne samo u bolničkom nego i u vanbolničkom okruženju. Karbapenemi, beta-laktami sa naj&scaron;irim spektrom delovanja, spadaju u lekove poslednje linije odbrane. Rezistencija na karbapeneme među enterobakterijama je u porastu &scaron;irom sveta. Može nastati usled prisustva karbapenemaza, enzima koji degradiraju karbapeneme, ili usled hiperprodukcije AmpC cefalosporinaza ili beta-laktamaza pro&scaron;irenog spektra uz gubitak porina. Geni koji kodiraju karbapenemaze se nalaze na mobilnim genetičkim elementima koji im omogućavaju brz prenos. Najče&scaron;će karbapenemaze su KPC, NDM, VIM, IMP i OXA-48 enzimi. Detekcija sojeva koji produkuju karbapenemaze nije moguća samo na osnovu profila rezistencije izolata, s obzirom da minimalne inhibitorne koncentracije karbapenema mogu biti u referentnom opsegu. Svaki izolat sa smanjenom osetljivo&scaron;ću na karbapeneme bi trebalo ispitati kako bi se sprečilo njihovo &scaron;irenje. Detekcija karbapenemaza može da se zasniva na fenotipskim i genotipskim metodama. Ciljevi istraživanja su bili da se utvrdi postojanje rezistencije na karbapeneme kod multirezistentnih izolata Escherichia coli i Klebsiella pneumoniae iz kliničkih uzoraka, da se dokaže produkcija karbapenemaza kori&scaron;ćenjem fenotipskih i genotipskih testova, kao i da se analizira osetljivost izolata Escherichia coli i Klebsiella pneumoniae sa molekularno dokazanim karbapenemazama. Istraživanje je sprovedeno kao prospektivna studija u periodu 01.11.2013. do 01.11.2014. godine u Centru za mikrobiologiju Instituta za javno zdravlje Vojvodine u Novom Sadu. U istraživanje je bilo uključeno 300 multirezistentnih izolata Escherichia coli i Klebsiella pneumoniae konsekutivno izolovanih iz kliničkih uzoraka (krv, punktat, sekret iz donjeg respiratornog trakta, urin i sekret rana) hospitalizovanih pacijenata. Identifikacija do nivoa vrste je vr&scaron;ena klasičnim bakteriolo&scaron;kim metodama. Za ispitivanje osetljivosti kori&scaron;ćeni su disk difuziona metoda i gradijent testovi. Vrednosti minimalnih inhibitornih koncentracija su ispitane automatizovanim Vitek 2 sistemom (BioM&eacute;rieux, Francuska), a interpretacija izvr&scaron;ena u skladu sa preporukama CLSI (Clinical Laboratory Standards Institute). Za fenotipsko testiranje prisustva betalaktamaza pro&scaron;irenog spektra kori&scaron;ćen je kombinovani disk test. Za fenotipsko testiranje prisustva karbapenemaza kod sojeva rezistentnih na karbapeneme kori&scaron;ćen je kombinovani disk test i test sinergizma sa dva diska. Detekcija gena za beta-laktamaze blaCTXM, gena za karbapenemaze blaKPC, blaVIM, blaNDM, blaIMP i blaOXA-48-like izvr&scaron;ena je metodom lančane reakcije polimeraze. Genotipizacija odabranih izolata Klebsiella pneumoniae izvr&scaron;ena pomoću repetitivne lančane reakcije polimeraze kori&scaron;ćenjem DiversiLab sistema (BioM&eacute;rieux, Francuska). Od 300 multirezistetntnih izolata, bilo je 242 (80,7%) Klebsiella pneumoniae i 58 (19,3%) Escherichia coli izolovanih iz kliničkih uzoraka. Smanjenu osetljivost na bar jedan karbapenem (imipenem, meropenem, ertapenem) pokazalo je 179 (59,7%) izolata. Fenotipski test za dokazivanje produkcije betalaktamaza pro&scaron;irenog spektra bio je pozitivan kod 87/171 (50,9%) izolata. Gen blaCTX-M je dokazan kod 111/121 (91,7%) izolata. Fenotipski test za dokazivanje karbapenemaza bio je pozitivan kod 65/179 (36,3%) izolata, kod 63 (96,9%) je ukazivao na prisustvo metalo-beta laktamaza, a kod 2 (3,1%) na prisustvo karbapenemaza iz grupe A. Senzitivnost fenotipskog testa za dokazivanje karbapenemaza klase A i B iznosila je 100,0%, specifičnost 96,6%, a ukupna tačnost 97,6%. Karbapenemaze su nađene kod 79/179 (44,1%) izolata rezistentnih na karbapeneme. Gen blaNDM nađen je kod 58 (32,4%) izolata, blaOXA- 48-like kod 11 (6,1%), a blaKPC kod 2 (1,1%) izolata. Geni blaVIM i blaIMP nisu detektovani. Kod 8 (4,5%) izolata nađena su 2 gena koja kodiraju karbapenemaze, blaNDM i blaOXA-48-like. Određivanjem osetljivosti disk difuzionom metodom i automatizovanim Vitek 2 sistemom, izolati koji produkuju karbapenemaze pokazivali su smanjenu osetljivost na sve testirane beta-laktame i gentamicin, odnosno tobramicin. Visok procenat rezistenicije izolati su pokazali u odnosu na ciprofloksacin, levofloksacin i trimetoprim/sulfametoksazol. Najefikasniji antibiotski lekovi su bili amikacin, tigeciklin, fosfomicin i kolistin. Poređenjem minimalnih inhibitornih koncentracija izolata koji produkuju i izolata koji ne produkuju karbapenemaze utvrđena je statistički značajna razlika za meropenem, imipenem, ertapenem, amikacin, gentamicin. Genotipizacijom odabranih izolata Klebsiella pneumoniae kori&scaron;ćenjem DiversiLab sistema klonalno &scaron;irenje je dokazano među izolatima koji produkuju NDM i OXA-48-like karbapenemaze u okviru iste zdravstvene institucije, ali i među različitim zdravstvenim ustanovama. Među izolatima rezistentnim na karbapeneme Klebsiella pneumoniae se če&scaron;će izoluje od Escherichia coli. Kod izolata koji su pokazali smanjenu osetljivost prema bar jednom karbapenemu, karbapenemaze su detektovane u manje od polovine izolata. Kod ostalih izolata dokazane su beta-laktamaze pro&scaron;irenog spektra koje uz gubitak porina mogu uzrokovati rezistenciju na karbapeneme. Kod izolata Klebsiella pneumoniae sa dokazanim genima koji kodiraju karbapenemaze detektovani su pojedinačni blaKPC, blaNDM i blaOXA-48-like geni, kao i kombinacija gena blaNDM i blaOXA-48-like. Kod izolata Escherichia coli nađeni su samo blaNDM geni. Najefikasniji antibiotski lekovi za izolate koji produkuju karbapenemaze su amikacin, tigeciklin, fosfomicin i kolistin. Izolati sa dokazanim karbapenemazama pokazuju rezistenciju na veći broj antibiotika u odnosu na izolate koji ne produkuju karbapenemaze. Dokazano je klonalno &scaron;irenje izolata Klebsiella pneumoniae koji produkuju karbapenemaze. Testove za fenotipsku detekciju karbapenemaza bi trebalo koristiti i u rutinskim mikrobiolo&scaron;kim laboratorijama u skladu sa EUCAST (European Committee on Antimicrobial Susceptibility Testing) preporukama, a konačnu potvrdu treba izvr&scaron;iti molekularnim metodama u referentnoj laboratoriji.</p> / <p>Escherichia coli and Klebsiella pneumoniae are among the most common human pathogens. Multiresistant strains are emerging not only in hospital settings, but also in the community representing a major concern. Carbapenems, beta-lactams with the broadest spectrum of activity are considered to be antibiotics of last resort. Resistance to carbapenems among enterobacteria is spreading worldwide. It is mainly caused by carbapenemases, enzymes capable of degrading carbapenems or by hyperproduction/overexpression of AmpC betalactamases or extended spectrum betalactamases with porin loss. Carbapenemaseencoding genes are usually located on mobile genetic elements providing their fast transfer. The most common carbapenemases are KPC, NDM, VIM, IMP and OXA-48. The detection of carbapenemase-producer cannot rely only on the resistance profile as their minimal inhibitory concentration values may sometimes lay within the susceptibility range. Therefore, every multidrug-resistant isolates with lower susceptibility to carbapenems should be tested for the presence of carbapenemases in order to prevent further spreading. The detection of carbapenemases is based on phenotypic and genotypic methods. The aims of the study were to determine the occurrence of carbapenem resistance in multidrug-resistant Escherichia coli and Klebsiella pneumoniae isolated from clinical samples, to detect carbapenemase production using both phenotypic and genotypic methods and to analyze the susceptibility of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae. The study was conducted from 1st November 2013 to 1st November 2014 at the Center for Microbiology in the Institute for Public Health of Vojvodina, Novi Sad, Serbia. The study included 300 nonrepetitive multidrug-resistant strains of Escherichia coli and Klebsiella pneumoniae isolated from clinical specimen (blood, aspirates, lower respiratory tract secretions, urine and wound secretion) of hospitalized patients. Identification of isolated strains was done using conventional bacteriological methods. Antimicrobial susceptibility was tested using the disk diffusion method and MIC test strips. Minimal inhibitory concentrations were determined using Vitek 2 Compact automated system (BioM&eacute;rieux, France), interpreted according to the CLSI (Clinical and Laboratory Standards Institute) recommendations. Phenotypic testing of extended-spectrum beta-lactamases production was done using combined disk test. Phenotypic testing of carbapenemase production was done by combined disk test and double-disk synergy test. Detection of blaCTX-M, gene encoding extended-spectrum beta-lactamases and blaKPC, blaVIM, blaNDM, blaIMP i blaOXA-48-like, genes encoding carbapenemases was done using PCR. Genotyping of selected Klebsiella pneumoniae isolates was done by repPCR using DiversiLab system (BioM&eacute;rieux, France). From the total of 300 multiresistant isolates, 242 (80.7%) were Klebsiella pneumoniae and 58 (19.3%) were Escherichia coli obtained from clinical samples. Reduced susceptibility to at least one carbapenem (imipenem, meropenem, ertapenem) was found in 179 (59.7%) isolates. Phenotypic test for extended-spectrum betalactamases production was positive in 87/171 (50.9%) isolates. A total of 111/121 (91.7%) isolates harbored blaCTX-M. Phenotypic test for carbapenemase production was positive in 65/179 (36.3%) isolates, 63 (96.9%) indicating the presence of metallo-beta-lactamases and 2 (3.1%) indicating the presence of class A carbapenemases. Sensitivity of the phenotypic test for carbapenemase production of class A and B was 100.0%, specificity 96.6% and overall accuracy 97.6%. Carbapenemases were detected in 79/179 (44.1%) carbapenemresistant isolates. Gene blaNDM was found in 58 (32.4%) isolates, blaOXA-48-like in 11 (6.1%) and blaKPC in 2 (1.1%) isolates. Genes blaVIM and blaIMP were not detected. In 8 (4.5%) isolates 2 genes encoding carbapenemases were found, blaNDM and blaOXA-48-like. Using both disk diffusion method and Vitek 2 automated system for antimicrobial susceptibility testing carbapenemase-producing isolates were resistant to all beta-lactams and also to gentamicin and tobramicin respectively. Resistance rates were high for ciprofloxacin, levofloxacin and cotrimoxazole. Good activity maintained for amikacin, tigecycline, fosfomycin and colistin. Comparing minimal inhibitory concentrations of carbapenemaseproducing isolates and non-carbapenemase producers, significant difference was found for meropenem, imipenem, ertapenem, amikacin and gentamicin. Genotyping of selected Klebsiella pneumoniae isolates using DiversiLab system, revealed the clonal spread of NDM- and OXA-48-like-producers not only within one healthcare-setting, but also between different healthcare centers. Among carbapenem-resistant isolates, Klebsiella pneumoniae was found more often than Escherichia coli. Carbapenemases were detected in less than 50% of isolates resistant to at least one carbapenem. In other carbapenem resistant isolates extended-spectrum betalactamases were confirmed most likely causing carbapenem-resistance with porin deficiency or porin loss. Among carbapenemase-producing Klebsiella pneumoniae blaKPC, blaNDM and blaOXA-48-like genes were detected, as well as combination of 2 genes blaNDM and blaOXA-48-like. In carbapenemase-producing Escherichia coli only blaNDM was found. The most efficient antimicrobial drugs among tested carbapenemase-producing isolates were amikacin, tigecycline, fosfomycin and colistin. Carbapenemase-producing isolates were resistant to more antimicrobial agents compared to non-carbapenemase producers. Clonal dissemination of carbapenemase-producing Klebsiella pneumoniae was confirmed. Phenotypic detection of carbapenemase production should be done in routine microbiology laboratories according to EUCAST (European Committee on Antimicrobial Susceptibility Testing) recommendations. Final confirmation should be done by molecular methods in the reference laboratory.</p>

Os carbapenêmicos são os antimicrobianos mais amplamente utilizados no tratamento empírico de infecções graves por bacilos Gram-negativos. A pressão seletiva gerada pelo uso desses antimicrobianos ao longo das últimas três décadas contribuiu para a disseminação de enterobactérias e Gram-negativos não fermentadores produtores de carbapenemases, particularmente as do tipo KPC e NDM. Os genes que codificam essas enzimas usualmente estão localizados em plasmídeos e/ou transpósons. A hipótese atualmente mais aceita é que o gene blaNDM-1 seja uma quimera criada em Acinetobacter baumannii. A NDM-1 foi descrita em paciente proveniente da Índia e subsequentemente evidenciou-se sua ampla disseminação nesse país. A epidemiologia que tem sido observada nos casos detectados na Europa e Estados Unidos tem sido viagem à Índia, ou seja, sem casos autóctones. No Brasil, os primeiros casos foram identificados no Rio Grande do Sul, e a seguir no Rio de Janeiro e em São Paulo. Diferentemente dos casos da Europa e América do Norte, os casos do Brasil não tem relação epidemiológica com a Índia. O sequenciamento integral dos plasmídeos e cromossomos albergando o gene blaNDM permitirá entender como ocorre a disseminação desse mecanismo de resistência no Brasil. Para isso, foi avaliado o perfil de susceptibilidade dos isolados, bem como a capacidade conjugativa e clonalidade. Das vinte e oito amostras utilizadas neste trabalho, treze delas pertencem à espécie Enterobacter hormaechei, uma à espécie Citrobacter freundii, sete à espécie Escherichia coli, quatro à Klebsiella pneumoniae e três ao gênero Acinetobacter spp. Os primeiros isolados incluídos neste estudo (Escherichia coli e Enterobacter hormaechei produzindo NDM-1) foram isolados em agosto de 2013, de uma mesma amostra de swab retal de um paciente do Rio de Janeiro que nunca viajou para o exterior. O sequenciamento completo do DNA plasmidial utilizando a plataforma Illumina e a anotação de ambos os plasmídeos albergando o gene blaNDM-1 revelou que estes pertencem a grupos de incompatibilidade diferentes, IncFIIK (E. hormaechei) e IncX3 (E. coli), e abrigam um novo transpóson composto designado Tn3000. A comparação da sequência nucleotídica do Tn3000 com aquelas disponíveis no GenBank evidencia que a mesma estrutura está presente em plasmídeos de isolados da cidade de Porto Alegre e também em diferentes continentes. As espécies de Acinetobacter (A. radioresistens, A. ursingii e A. guillouiae) isoladas em São Paulo e Porto Alegre, possuem o gene blaNDM-1 albergados em um mesmo plasmídeo não tipável de 41.087 pb. A avaliação da clonalidade dos isolados de Enterobacter hormaechei \"subsp. oharae\" mostrou dois perfis diferentes através da técnica de PFGE, sendo que todos os microrganismos foram isolados de um surto no mesmo hospital no Rio de Janeiro. Isolados de Klebsiella pneumoniae de uma mesma paciente internada em hospital em Salvador, de sítios distintos - swab retal, hemocultura e urina, em ordem cronológica - obtiveram o mesmo perfil clonal pela técnica de PFGE. O mesmo ocorreu com três isolados de Escherichia coli, de um mesmo paciente do Rio de Janeiro, em amostras de swab retal. Os achados deste estudo evidenciam que no Brasil, Nepal, Marrocos e Índia há uma disseminação do gene blaNDM-1 mediada por um novo elemento móvel designado Tn3000 em enterobactérias. A detecção de um mesmo plasmídeo em diferentes espécies de Acinetobacter evidencia que neste gênero bacteriano, no Brasil, a disseminação do gene blaNDM-1 ocorre por conjugação. / Carbapenems are the antimicrobials most widely used in the empirical treatment of severe infections caused by Gram-negative bacilli. The selective pressure generated by the use of these antibiotics over the last three decades has contributed to the spread of enterobacteria and Gram-negative non-fermenting producing carbapenemases, mainly KPC and NDM. Genes encoding these enzymes are usually located in plasmids and/or transposons. Currently the most accepted hypothesis is that the blaNDM-1 gene is a chimera created in Acinetobacter baumannii. The NDM-1 was described in a patient from India and subsequently was reported to be broadly disseminate in this country. The epidemiology that has been observed in cases detected in Europe and United States is traveling to India, but no autochthonous cases. In Brazil, the first cases were identified in Rio Grande do Sul, and then in Rio de Janeiro and São Paulo. Differently from the cases described in Europe and North America, the cases from Brazil have no epidemiological link with India. The complete sequencing of plasmids and chromosomes harboring blaNDM gene will understanding how the dissemination of this resistance mechanism in Brazil occurs. In this work we will be evaluate the susceptibility profile of the isolates, and their conjugal capacity and clonality. Of the twenty-eight samples used in this study, thirteen of them belong to the species Enterobacter hormaechei, one to Citrobacter freundii, seven to Escherichia coli, four to Klebsiella pneumoniae and three to the genus Acinetobacter sp. The first two isolates included in this study (Escherichia coli and Enterobacter hormaechei) were isolated in August 2013, from the same rectal swab sample from a patient from Rio de Janeiro that never traveled abroad. Complete sequencing of plasmid DNA using Illumina platform and annotation of both plasmids harboring the blaNDM-1 gene revealed that they belong to different incompatibility groups, IncFIIK (E. hormaechei) and IncX3 (E. coli), and are harbor to a new transposon designated Tn3000. The comparison of the Tn3000 nucleotide sequence with those available at GenBank shows that the same structure is present in plasmids from other Porto Alegre and also in different continents. The Acinetobacter species (A. radioresistens, A. ursingii and A. guillouiae) isolated in São Paulo and Porto Alegre, have the blaNDM-1 gene harbored in a single non-typing plasmid of 41,087 bp. The evaluation of clonal relationship of Enterobacter hormaechei \"subsp. oharae\" showed two different profiles by PFGE technique; of note all microorganisms were isolated from an outbreak in the same hospital in Rio de Janeiro. Isolates of Klebsiella pneumoniae from a single patient hospitalized in Salvador, from different anatomical sites - rectal swab, blood culture and urine, in chronological order - obtained the same clonal profile by the PFGE technique. The same occurred with three Escherichia coli isolates, from the same patient from Rio de Janeiro, in swab rectal strains. Our findings suggest that in Brazil, Nepal, Morocco and India there is a spread of blaNDM-1 gene mediated by Tn3000 in enterobacteria. The detection of a same plasmid in different species of Acinetobacter shows that in this bacterial genus, in Brazil, the dissemination of the blaNDM-1 gene occurs by conjugation.

Bakterielle Glykanantigene sind hoch-divers in ihrer Komposition und Verbindung. Antikörper gegen Glykanantigene können vor bakteriellen Infektionen schützen und sind wichtig um die Homöostase zwischen dem Wirt und seinem Mikrobiom aufrecht zu halten. Typischerweise lösen Glykanantigene jedoch Antikörperantworten aus, die sich durch ein vermindertes B Zell-Gedächtnis und niedrig-affine Antikörper mit geringer Spezifität auszeichnen. In dieser Arbeit konnten, mithilfe von biotinylierten Lipopolysaccharide O-Antigenen des opportunistisch-pathogenen Bakteriums Klebsiella pneumoniae (Kp), O-Antigen-spezifische B Zellen innerhalb peripherer Gedächtnis- und intestinaler Effektor-B Zellen identifiziert werden. Durch Einzel-Zell Immunoglobulin-Sequenzierung und Klonierung bzw. rekombinanter Expression von Antikörpern dieser Zellen wird gezeigt, dass, unter natürlichen Umständen, affinitätsgereifte Antikörper gegen definierte Kp Glykanantigene erzeugt werden. Diese Antikörper binden nicht nur Kp O-Serotyp-spezifisch sondern auch spezifisch an strukturell ähnliche Kp O-Antigene und taxonomisch unterschiedliche Mikroorganismen. Die Ergebnisse zeigen, dass Menschen, bei natürlicher Besiedlung, kreuz-spezifische Antikörper gegen Glykanantigene erzeugen und deuten auf einen Mechanismus hin, wie das humorale Immunsystem auf die Glykandiversität des Mikrobioms reagieren und sich anpassen kann. Weiterhin könnten die hier identifizierten Antikörper nützlich für die Behandlung von nosokomiellen Kp Infektionen sein. / Bacterial glycan antigens are highly diverse in composition and linkage. Antibodies against glycan antigens can protect against bacterial infection and are important in maintaining homeostasis between the host and its microbiome. However, glycan antigens typically elicit B cell responses that have impaired long-term memory formation and are comprised of low-affine antibodies with low specificity. In this work, the use of biotinylated Lipopolysaccharide O-antigens of the opportunistic pathogen Klebsiella pneumonia allowed to identify anti-O-antigen B cells in the peripheral memory and intestinal effector B cell pool in healthy humans. Single B cell Immunoglobulin gene sequencing, antibody cloning, and recombinant expression reveal that, under natural circumstances, humans acquire affinity-matured antibodies against defined Kp glycan antigens. Despite their O-serotype-specificity, the antibodies bind to other structurally similar Kp O-serotypes and taxonomically distinct non-Kp microbes. The findings show that humans, under natural exposure, acquire affinity-matured cross-specific anti-glycan antibodies and provide a mechanistic way how the humoral immune system could adapt to the large microbial glycan diversity present in nature. Further, the antibodies identified in this work might be beneficial in treatment of nosocomial Kp infections.