More so than ever, Multiple Drug Resistant (MDR) bacteria are on the rise due to overuse of antibiotics along with natural selection for adaptations that enhance drug-resistant properties. One particular bacterial family, Enterobacteriaceae, has been problematic, exhibiting several bacterial members that have developed a precipitous resistance to modern antibiotics and are also primary causative agents of nosocomial, or hospital acquired, infections. Citrobacter freundii (CF) and Enterobacter cloacae (ECL) are two species of the Enterobacteriaceae family causing significant medical concern due to their role in producing numerous opportunistic infections such as bacteremia, lower respiratory tract infections, urinary tract infections, and endocarditis. Adding to the difficulty of this situation is the ability of bacteria to produce biofilms. These biofilms are communities of bacteria that exhibit increased resistance to antibiotic treatment and eradication. Previous work in the laboratory of Dr. Fox at ETSU has identified an uncharacterized product secreted by Klebsiella pneumoniae (KP), another member of the Enterobacteriaceae family, which appears to have inhibitory effects toward CF and ECL. The current study was designed to characterize the effects this secreted product has on CF and ECL biofilms. Through a high throughput microtiter plate assay, the effects of this secreted product were examined on CF and ECL phases of biofilm attachment and maturation. Based on our findings, we have concluded that this secreted product can be categorized as a possible bacteriostatic agent against biofilm cell density, biofilm mass, and cell viability for both biofilm phases of attachment and maturation.

Extended spectrum β-lactamases or ESBLs, which are derived from non-ESBL precursors by point mutation of β-lactamase genes (bla), are spreading rapidly all over the world and have caused considerable problems in the treatment of infections caused by bacteria which harbour them. The mechanism of this resistance is not fully understood and a better understanding of these mechanisms might significantly impact on choosing proper diagnostic and treatment strategies. Previous work on SHV β-lactamase gene, blaSHV, has shown that only Klebsiella pneumoniae strains which contain plasmid-borne blaSHV are able to mutate to phenotypically ESBL-positive strains and there was also evidence of an increase in blaSHV copy number. Therefore, it was hypothesised that although specific point mutation is essential for acquisition of ESBL activity, it is not yet enough, and blaSHV copy number amplification is also essential for an ESBL-positive phenotype, with homologous recombination being the likely mechanism of blaSHV copy number expansion. In this study, we investigated the mutation rate of non-ESBL expressing K. pneumoniae isolates to an ESBL-positive status by using the MSS-maximum likelihood method. Our data showed that blaSHV mutation rate of a non-ESBL expressing isolate is lower than the mutation rate of the other single base changes on the chromosome, even with a plasmid-borne blaSHV gene. On the other hand, mutation rate from a low MIC ESBL-positive (≤ 8 µg/mL for cefotaxime) to high MIC ESBL-positive (≥16 µg/mL for cefotaxime) is very high. This is because only gene copy number increase is needed which is probably mediated by homologous recombination that typically takes place at a much higher frequencies than point mutations. Using a subinhibitory concentration of novobiocin, as a homologous recombination inhibitor, revealed that this is the case.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-06-01T14:13:34Z No. of bitstreams: 1 texto completo.pdf: 11919399 bytes, checksum: 7b65e307cd59fdd0169cabc3a06ca449 (MD5) / Made available in DSpace on 2016-06-01T14:13:34Z (GMT). No. of bitstreams: 1 texto completo.pdf: 11919399 bytes, checksum: 7b65e307cd59fdd0169cabc3a06ca449 (MD5) Previous issue date: 1999-08-13 / Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A presença e a distribuição de bactérias e leveduras em frutos de café, desinfestados superficialmente, foram observadas, ulilizando-se técnicas de microscopia com colorações biológicas e fluorocromo, inclusão em parafina e técnicas imunológicas. As preparações a fresco e de inclusão em parafina possibilitaram observar a existência de um gradiente de distribuição de bactérias e leveduras na polpa do café, com predomínio de leveduras em sua porção mais externa. Foram encontradas, também, bactérias nas sementes de café. A aplicação de fluoresceína diacetato, em cortes a fresco, não se mostrou eficiente, dada a dificuldade de visualização dos microrganismos in situ e a inespecificidade da reação. Os anticorpos específicos, para a bactéria Klebsiella oxytoca, foram obtidos; porém, a aplicação de imunofluorescência usando anticorpos como sonda, para detecção das bactérias,em tecidos de frutos de café, necessita de adaptações. / The presence and distribution of bacteria and yeast in coffee beans disinfected superficially were observed microscopically with biological and fluorochromic dyes, pemianent slides and immunological techniques. Fresh preparations and permanent slides permitted observation of the existence of a distribution gradient of bacteria and yeast within the coffee pulp, with a predominance of yeast in the outer portion. Bacteria were also found in the coffee seeds. Application of fluorescein diacetate in fresh cuts was not found efficient due to difficulty in visualizing the microorganisms in situ and the non specificity of the reaction. Specific antibodies for the bacteria Klebsiella oxytoca were obtained but the application of immunofluorescence using antibodies as probes for the detection of bacteria in coffee bean tissues requires further adaptations. / Dissertação antiga escaneada.

Submitted by Marilene Donadel (marilene.donadel@unioeste.br) on 2018-10-04T18:06:39Z No. of bitstreams: 1 Mabel_Arantes_2018.pdf: 2059864 bytes, checksum: 831822ad4d3c7ce18c2fda6d1319af80 (MD5) / Made available in DSpace on 2018-10-04T18:06:39Z (GMT). No. of bitstreams: 1 Mabel_Arantes_2018.pdf: 2059864 bytes, checksum: 831822ad4d3c7ce18c2fda6d1319af80 (MD5) Previous issue date: 2018-09-06 / The biological production of hydrogen (bioH2) through anaerobic fermentation of residual carbon sources is an interesting way to combine the environmental adequation of agro-industrial wastewaters to the generation of value-added products. Wastewaters with fermentable carbon sources have high potential to produce bioH2, being possible using different inoculum and biological reactors. It is the case found in brewery wastewater, generated in large amount at Brazil and around the world, and there are only few studies about it. In this context, this work studied the production of bioH2 by an anaerobic reactor operated in sequencing batch with immobilized biomass, AnSBBR, from brewery wastewater, using two inocula: mixed culture from the natural fermentation of synthetic wastewater based on sucrose and pure culture of Klebsiella pneumoniae, immobilized in expanded clay. Were evaluated the effects of influent concentration (2,8 a 7,2 gTRS L-1), volumetric organic load (2,6 a 12,6 gTRS (L day)-1) and cycle time (12 h e 24 h), in assays taking from 9 to 16 cycles. The inocula gotten through natural fermentation showed potential to produce bioH2 in preliminary experiments (shaken bottles, 0,5 L) but, in the tests on the reactor the production was low, instable and brief (less than 7 days). The pure inocula K. pneumoniae showed a good adaptability to the residue, in activity through the whole experiment. The raise in the volumetric organic load (VOLc) up to 12,6 gTRS (L day)-1 and the reduction of the cycle from 24 h to 12 h had a positive effect in the production and, under those conditions, the best values for the evaluation parameters ahead were obtained: volumetric productivity average of 0,9 LH2 (L day)-1, molar flow rate up to 10,8 mmolH2 h-1, molar yield average de 3,9 molH2 KgTRS-1, with H2 content in biogas between 18 and 42% and TRS and COD removal efficiency of 89% and 23%, respectively, with a predominance of acetic and butyric pathways. Those results indicate a promising production of H2 from brewery wastewater using K. pneumoniae as inocula in AnSBBR reactor, contributing to the development of the bioH2 technology by the proposal of a new inocula to achieve a higher use of this wastewater potential. / A produção biológica de hidrogênio (bioH2) por meio de fermentação anaeróbia de fontes de carbono residuais é uma forma interessante de aliar a adequação ambiental de efluentes agroindustriais à geração de produto de valor agregado. Águas residuárias que possuam fontes de carbono fermentescíveis apresentam alto potencial para a produção de bioH2, o que é possível com diferentes inóculos e reatores biológicos. É o caso da água residuária de cervejaria, gerada em grandes quantidades no Brasil e no mundo e para a qual poucos estudos foram realizados até o momento. Neste contexto, o presente trabalho avaliou a produção de biohidrogênio em reator anaeróbio operado em bateladas sequenciais com biomassa imobilizada, AnSBBR, a partir de água residuária de cervejaria, empregando dois inóculos: cultura mista proveniente da fermentação natural de meio sintético à base de sacarose e cultura pura de Klebsiella pneumoniae, suportados em argila expandida. Foram avaliados os efeitos da concentração afluente (2,8 a 7,2 gART L-1), da carga orgânica volumétrica aplicada (2,6 a 12,6 gART (L dia)-1) e do tempo de ciclo (12 h e 24 h) em ensaios com duração entre 9 e 16 ciclos. O inóculo obtido por fermentação natural apresentou potencial para produção de bioH2 em ensaios preliminares (frascos agitados, 0,5 L) mas na avaliação em reator a produção foi baixa, instável e breve (período inferior a 7 dias). Já o inóculo puro, K. pneumoniae apresentou boa adaptação ao resíduo e atividade durante todo o período avaliado. O aumento da carga orgânica volumétrica aplicada (COVc) até 12,6 gART (L dia)-1 e a redução do tempo ciclo de 24 h para 12 h tiveram um efeito positivo sobre a produção e nestas condições foram obtidos os melhores valores para os parâmetros de avaliação: produtividade volumétrica de H2 média de 0,9 LH2 (L dia)-1, vazão molar de H2 máxima de 10,8 mmolH2 h-1, rendimento de H2 médio de 3,9 molH2 KgART-1, com teor de H2 no biogás entre 18 e 42% e eficiência média de remoção de ART e de DQO de 89% e 23%, respectivamente, com predomínio das rotas metabólicas acética e butírica. Estes resultados indicam uma produção promissora de H2 a partir de água residuária de cervejaria empregando K. pneumoniae como inóculo em reator AnSBBR, contribuindo para o desenvolvimento da tecnologia do bioH2 ao propor um novo inóculo para o aproveitamento do potencial de uso desta água residuária.

Extended-spectrum β-lactamases (ESBLs) are plasmid-mediated bacterial enzymes that confer resistance for most β-lactams antibiotics. These enzymes are widespread in microorganisms in hospital settings worldwide. This study estimated the distribution and prevalence of the main ESBLs families among samples of Escherichia coli and Klebsiella spp. in the university hospital of Santa Maria (HUSM), Rio Grande do Sul. During a period of 14 months 90 microorganisms were selected as probable ESBL producers according to the recommendations of Clinical and Laboratory Standards Institute (CLSI). The isolated microorganisms were submitted to phenotypic confirmatory tests for the presence of ESBL. Samples that showed negative results were tested against their susceptibility to cefoxitin. The ESBLs types found in each organism were determined by the research of the genes bla TEM, bla SHV e bla CTX-M by polymerase chain reaction (PCR). Fifty-five (61.1%) samples were confirmed as ESBL positive by the combined disc method and fifty-seven (63.3%) by the double disc method. In the cefoxitin susceptibility test 16 of the 39 samples presented resistance to this agent. Based on PCR, 74 (82,2%) samples harbored TEM-type ESBL gens, 61 (67,8%) SHV-type and 19 (21,1%) CTX-M-type. Only one Escherichia coli isolate appeared harboring genes for the CTX-M family of ESBLs. The distribution of TEM, SHV and CTX-M ESBL families from HUSM presented some similarities and differences compared with ESBLs of other hospital settings. / As β-lactamases de Espectro Estendido (ESBLs) são enzimas bacterianas mediadas por plasmídeos que conferem resistência à maioria dos antibióticos β-lactâmicos. Estas enzimas estão amplamente disseminadas em microrganismos nos ambientes hospitalares do mundo. Este estudo estimou a distribuição e prevalência das principais famílias de ESBLs entre amostras de Escherichia coli e Klebsiella spp. no Hospital Universitário de Santa Maria (HUSM), Rio Grande do Sul. Durante um período de 14 meses, 90 microrganismos foram selecionados como prováveis produtores de ESBLs de acordo com os critérios estabelecidos pelo Clinical and Laboratory Standards Institute (CLSI). Os microrganismos isolados foram submetidos a testes fenotípicos confirmatórios para a presença de ESBL. As amostras que apresentaram resultado negativo nestes testes tiveram sua susceptibilidade testada frente à cefoxitina. Os tipos de ESBLs presentes em cada microrganismo foram determinados pela pesquisa dos genes bla TEM, bla SHV e bla CTX-M através da reação em cadeia da polimerase (PCR). Empregando-se o método do disco combinado, a presença de ESBLs foi confirmada em 55 (61,1%) amostras; quando o método do duplo disco foi utilizado, 57 (63,3%) amostras foram confirmadas. No teste de susceptibilidade à cefoxitina, 16 das 39 amostras testadas apresentaram resistência a este substrato. Com base na PCR, 74 (82,2%) amostras possuíam genes para a família TEM de ESBLs, 61 (67,8%) para a família SHV e 19 (21,1%) para a família CTX-M. Apenas um isolado de Escherichia coli demonstrou possuir genes para a família CTX-M de ESBLs. A distribuição de ESBLs das famílias TEM, SHV e CTX-M no HUSM apresentou semelhanças e diferenças em comparação com ESBLs de outros ambientes hospitalares.

As part of recognizing the interconnected nature of different sectors in relation to health, AMR (antimicrobial resistance) has emerged as an issue of high global importance. E. coli isolates were taken from pig farms in Thailand, which serves as a point of interest in the study of ARGs (antimicrobial resistance genes) in emerging economies. The fecal samples were collected from pigs, humans who came in contact with the pigs, and humans who did not have contact with pigs to be analyzed for ARGS, virulence genes, and plasmids. Data was analyzed with an automated pipeline in the form of ARG-MATEE, the Antimicrobial Resistance Gene Multi-Analysis Tool for Enteric E. coli, a tool designed in this study to be used here and in future investigations. ARG-MATEE regulates and records internal software versions in a produced report which also includes data tables for all non phylogeny results in Boyce–Codd normal form and data visualizations for plasmids, ARGs, virulence genes, and phylogeny. Through the use of ARG-MATEE, the iss virulence gene was seen to be significantly different between testing groups as it is present in only human testing groups, suggesting the loss of function of the iss gene in pigs, showing host specialization.

The evolution and adaptation of microorganisms is so rapid it can be seen in the time frame of days. The root cause for their evolution comes from selective environmental pressures that see organisms with beneficial mutations survive otherwise deadly encounters or outperform members of its population who fail to adapt. This does not always result in strict improvement of the individual as in the case of antibiotic resistant bacteria who often display fitness tradeoffs to avoid death (see Reviews [1-3]). For example, when an ampicillin resistance gene (ampC) containing plasmid that is occasionally found in the wild was transformed into S. typhimurium the bacteria had slower growth and impaired invasiveness [4]. In another example, capreomycin use with mycobacteria resulted in lower binding of the drug to the ribosome through mutations in rRNA methylase TlyA 16S rRNA, which decreases the overall fitness of the mycobacteria [5]. The evolutionary interactomes between bacteria and antibiotics do not end there, as antibiotic resistant bacteria often accumulate compensatory mechanisms to regain fitness. These range in effect with some altering individual cellular pathways and others having systemic affects [1]. My work has focused on the intersection of diabetes and related antibiotic resistant bacterial infections. Diabetes is one of the leading health issues in the United States, with over 10% of the adult population and over 26% of the elderly diagnosed (American Diabetes Association) [6]. Herein I further characterize the molecular pathways involved in diabetes, through the study of PAS kinase (PASK) function. PAS kinase is a serine-threonine protein kinase which regulates the pathways disrupted in diabetes, namely triglyceride accumulation, metabolic rate (respiration), adiposity and insulin production and sensitivity [7-9]. In this study I specifically focus on the effects of PAS kinase and its substrate, USF1/Cbf1p, and how their altered metabolic deficiencies can be suppressed using yeast cells. Through this study I further characterized the molecular function of USF1/Cbf1p through the identification of putative co-transcriptional regulators, identify novel genes involved in the regulation of respiration, and uncover a function or a previous uncharacterized protein, Pal1p. Part of the diabetes healthcare challenge results from the wide range of diseases that are associated with diabetes, including obesity [10, 11], renal failure [12, 13], neuropathies and neurodegeneration [14, 15], endocrine dysfunctions [16, 17], and cancers [18]. In addition, diabetes is a leading cause of lower limb amputations, due to poor circulation and the prevalence of ulcers [19-21], many of which are antibiotic resistant [22-25]. Phage therapy, based on the administration of bacterial viruses, is a viable option for the treatment of these diseases, with our lab recently isolating bacteriophages for several clinical cases. In the second half of my thesis, I present the study of the adaptation of bacteriophages to their hosts as well as report contributions of local ecology to their evolution.

No description available.

Σκοπός της παρούσας ερευνητικής εργασίας ήταν η επιδημιολογική επιτήρηση της φορείας και των λοιμώξεων από Klebsiella pneumoniae που παράγει καρβαπενεμάση KPC (KPC-Kp), ανθεκτικό σε βανκομυκίνη Enterococcus (VRE) και ανθεκτικό σε μεθικιλλίνη Staphylococccus aureus (MRSA) σε ασθενείς που νοσηλεύονται στις Μονάδες Εντατικής Θεραπείας (ΜΕΘ) του Πανεπιστημιακού Γενικού Νοσοκομείου Πατρών (ΜΕΘ Α) και του Νοσοκομείου «Άγιος Ανδρέας» (ΜΕΘ Β) τη χρονική περίοδο Οκτώβριος 2009 έως Φεβρουάριος 2012. H διασπορά της KPC-Kp αποτελεί το σημαντικότερο πρόβλημα στις Ελληνικές ΜΕΘ, με τα ποσοστά της να αυξάνονται στις παθολογικές και χειρουργικές κλινικές. Κατά τη διάρκεια της παρούσας μελέτης, 12.8% των ασθενών που εισήχθηκαν στη ΜΕΘ Α (52 από 405 ασθενείς) ήταν αποικισμένοι από KPC-Kp κατά την εισαγωγή τους με την προηγηθείσα νοσηλεία σε ΜΕΘ, την χρόνια αποφρακτική πνευμονοπάθεια, τη διάρκεια προηγηθείσας νοσηλείας και την προηγηθείσα χορήγηση καρβαπενέμης ή συνδυασμού β-λακτάμης/αναστολέα λακταμάσης να συμβάλλουν στον αποικισμό. Παρατηρήθηκε μία σταδιακή αύξηση των αποικισμένων ασθενών που εισάγονται στη ΜΕΘ με 3.9% (4 από 102 ασθενείς) τους πρώτους 6 μήνες σε σύγκριση με 15.8% (48 από 300 ασθενείς) τους επόμενους 16 μήνες που αντικατοπτρίζει τη σταδιακή διασπορά της KPC-Kp σε κλινικές εκτός ΜΕΘ. Από τους 226 μη αποικισμένους ασθενείς κατά την εισαγωγή στη ΜΕΘ Α, 164 (72.6%) αποικίστηκαν κατά τη διάρκεια της νοσηλείας τους με σημαντικότερους παράγοντες που επηρεάζουν τον αποικισμό να είναι η παρουσία αποικισμένων ασθενών σε διπλανές κλίνες και η νοσηλεία σε κλίνη προηγουμένως αποικισμένου ασθενή, ενώ δε βρέθηκε συσχέτιση ανάμεσα στον αποικισμό και τη θνησιμότητα. Το υψηλό ποσοστό αποικισμού σε συνδυασμό με τους προηγούμενους παράγοντες υποδεικνύει την σημασία της διασποράς της KPC-Kp από ασθενή σε ασθενή μέσω του ιατρονοσηλευτικού προσωπικού και υποδηλώνει τη σημασία πιο αυστηρής εφαρμογής της πολιτικής ελέγχου λοιμώξεων. Συνολικά 53 ασθενείς της ΜΕΘ Α ανέπτυξαν βακτηριαιμία από KPC-Kp με 43.4% θνησιμότητα. Οι σημαντικότεροι παράγοντες που επηρεάζουν τη θνησιμότητα είναι η αντοχή του στελέχους σε κολιστίνη/τιγεκυκλίνη/γενταμικίνη και η σηπτική καταπληξία, ενώ η θεραπεία με συνδυασμό τουλάχιστον δύο δραστικών αντιβιοτικών σχετίζεται με καλύτερη πρόγνωση επιβεβαιώνοντας τα αποτελέσματα προηγούμενων μελετών υπέρ της συνδυαστικής θεραπείας στην καταπολέμηση των λοιμώξεων από KPC-Kp. Η ανάπτυξη αντοχής των στελεχών KPC-Kp έναντι της κολιστίνης ή της τιγεκυκλίνης, οι οποίες αποτελούν τις τελευταίες θεραπευτικές επιλογές για το συγκεκριμένο παθογόνο, είναι ένα ανησυχητικό φαινόμενο. Συνολικά, 24.4% και 17.9% των ασθενών της ΜΕΘ Α αποικίστηκαν από στέλεχος KPC-Kp ανθεκτικό στην κολιστίνη και τιγεκυκλίνη, αντίστοιχα. Όπως αναμενόταν η λήψη των συγκεκριμένων αντιβιοτικών συνέβαλε στον αποικισμό, όμως ο σημαντικότερος παράγοντας για αποικισμό ήταν η παρουσία αποικισμένου ασθενή στις διπλανές κλίνες υποδηλώνοντας τη σημασία της διασποράς των στελεχών και όχι της de novo ανάπτυξη αντοχής. Η σύγκριση των δύο ΜΕΘ, ανέδειξε ότι μεγαλύτερο ποσοστό των ασθενών της ΜΕΘ Α αποικίζονται κατά τη διάρκεια νοσηλείας σε σχέση με τη ΜΕΘ Β (61.8% vs 34.1%) και σε συντομότερο χρονικό διάστημα (10.6 vs 19.9 ημέρες). Τα στοιχεία αυτά μπορούν να ερμηνευτούν από το υψηλότερο ποσοστό εισαγωγών αποικισμένων ασθενών (11.4% vs 1.8%), τη μικρότερη αναλογία νοσηλευτών/ασθενών καθώς και την αυξημένη κατανάλωση καρβαπενεμών στη ΜΕΘ Α. Συνολικά, 305 και 100 στελέχη K. pneumoniae που απομονώθηκαν από τη ΜΕΘ Α και Β, αντίστοιχα, ήταν θετικά για την παρουσία του γονιδίου blaKPC ενώ πέντε στελέχη της ΜΕΘ Α ήταν θετικά και για το γονίδιο blaVIM. Και στις δύο ΜΕΘ τα στελέχη ήταν ανθεκτικά σε πενικιλλίνες, στις κεφαλοσπορίνες, στην αζτρεονάμη, στην τριμεθοπρίμη-σουλφαμεθοξαζόλη (30% των στελεχών της ΜΕΘ Β ήταν ευαίσθητα), στην αμικασίνη, στην τομπραμυκίνη και στις κινολόνες. Η αντοχή στις καρβαπενέμες (67.9% vs 60%), στην κολιστίνη (35.1% vs 18%), στη γενταμικίνη (50.8% vs 24%) και στην τιγεκυκλίνη (17% vs 18%) στα στελέχη των δύο ΜΕΘ κυμαινόταν στα ίδια επίπεδα. Πενήντα επτά και 20 στελέχη της ΜΕΘ Α και Β, αντίστοιχα, ταυτοποιήθηκαν με PFGE, η οποία ανέδειξε την παρουσία δύο τύπων στη ΜΕΘ Α, με τον τύπο Α να απαρτίζεται από το 65.5% των στελεχών, ενώ στη ΜΕΘ Β όλα τα στελέχη ανήκαν στον τύπο Α. Τα ποσοστά αποικισμού από VRE στις δύο ΜΕΘ είναι χαμηλότερα σε σχέση με αυτά της KPC-Kp. Αποικισμός κατά την εισαγωγή στη ΜΕΘ παρατηρήθηκε σε 14.3% (71 από 497 ασθενείς), ενώ κατά τη διάρκεια νοσηλείας ήταν 14.4% (36 από 250 ασθενείς). Ο σημαντικότερος παράγοντας για αποικισμό από VRE κατά τη διάρκεια νοσηλείας είναι η νοσηλεία αποικισμένων ασθενών σε διπλανές κλίνες υποδεικνύοντας ότι η μη τήρηση των μέτρων υγιεινής των χεριών ίσως διαδραματίζει το σημαντικότερο ρόλο στη διασπορά του VRE. Συνολικά 107 στελέχη VRE απομονώθηκαν (100 E. faecium και 7 E. faecalis). Ογδόντα τέσσερα στελέχη έφεραν το γονίδιο vanA και ήταν ανθεκτικά στη βανκομυκίνη και στην τεϊκοπλανίνη, ενώ τα υπόλοιπα 23 έφεραν το γονίδιο vanB και χαρακτηρίζονταν από χαμηλού επιπέδου αντοχή στη βανκομυκίνη (12 στελέχη ήταν ευαίσθητα) και ευαίσθητα στην τεϊκοπλανίνη. Όλα τα στελέχη ήταν ευαίσθητα στη λινεζολίδη, στη δαπτομυκίνη και στην τιγεκυκλίνη. Η MLST αποκάλυψε ότι τα στελέχη E. faecium ανήκουν σε έξι διαφορετικούς κλώνους (STs: ST117, ST17, ST203, ST226, ST786, ST125) με το 90% των E. faecium, ανήκουν στο Κλωνικό Σύμπλεγμα 17 (Clonal Complex CC17). Τα στελέχη E. faecalis ταξινομήθηκαν σε τέσσερις κλώνους (STs: ST6, ST41, ST19, ST28). Τα ποσοστά αποικισμού από MRSA κατά την εισαγωγή και κατά τη διάρκεια νοσηλείας είναι χαμηλά (5.3% και 3.7%, αντίστοιχα) με το σημαντικότερο παράγοντα που σχετίζεται με τον αποικισμό να είναι ο εντερικός αποικισμός με vanA-θετικό στέλεχος Enterococcus. Ο έλεγχος φορείας για MRSA ανέδειξε 28 mecA-θετικά στελέχη S. aureus, με την πλειονότητα (ν=19) να είναι PVL-θετικά, να ανήκουν στον κλώνο ST80 και να είναι ανθεκτικά σε καναμυκίνη, τετρακυκλίνη και φουσιδικό, ενώ τα υπόλοιπα ταξινομήθηκαν σε τέσσερις κλώνους με MLST (6 στον ST239 και από ένα σε ST225, ST72 και ST30). Το στέλεχος που ανήκε στον ST30 ήταν tst-θετικό. Η σύγκριση των στελεχών φορείας S. aureus που απομονώθηκαν από αθενείς (ν=67) και προσωπικό (ν=23) των ΜΕΘ (Ομάδα Α) με τα στελέχη φορείας (ν=53) και βακτηριαιμιών (ν=75) μη νοσηλευόμενων σε ΜΕΘ (Ομάδα Β), ανέδειξε υψηλότερο ποσοστό MRSA (46.9% vs 31.1%) και PVL-θετικών στελεχών (39.8% vs 25.6%) στην Ομάδα Β, ενώ η Ομάδα Α χαρακτηρίζεται από υψηλότερο ποσοστό tst-θετικών στελεχών (21.1% vs 2.3%) υποδεικνύοντας τη σιωπηρή τους διασπορά στους ασθενείς και στο προσωπικό των ΜΕΘ. Προϊόν της παρούσας ερευνητικής εργασίας ήταν η ανεύρεση των παραγόντων κινδύνου για αποικισμό ή λοίμωξη από KPC-Kp, VRE και MRSA με στόχο την καθοδήγηση των μελλοντικών προσπαθειών περιορισμού της διασποράς τους στις δύο ΜΕΘ καθώς και στα ελληνικά νοσοκομεία, τα οποία στο σύνολο τους μαστίζονται από τα συγκεκριμένα παθογόνα. / The purpose of this study was to investigate the colonization and infections caused by KPC-producing Klebsiella pneumoniae (KPC-Kp), vancomycin-resistant Enterococcus (VRE) and methicillin-resistant Staphylococcus aureus in patients hospitalized in the Intensive Care Units of the University Hospital of Patras (ICU A) and the General Hospital “Saint Andrew” during October 2009 and February 2012. The dissemination of KPC-Kp constitutes the most important issue in Greek ICUs, with its percentage rising in medical and surgical wards. During the duration of this study, 12.8% of patients admitted in the ICU A (52 from 405 patients) were colonized upon admission and previous ICU stay, chronic obstructive pulmonary disease, duration of previous hospitalization and previous usage of carbapenem or combination of beta-lactamic/lactamase were found to influence colonization. A gradual increase of the percentage of colonized patients admitted at the ICU from 3.9% (4 from 102 patients) during the first 6 months to 15.8% (48 from 300 patients) the next 16 months that reflects the dissemination of KPC-Kp in non-ICU wards. Among the 226 non-colonized upon ICU A admission patients, 164 (72.6%) became colonized during their stay with the presence of colonized patients in nearby beds and the previous colonized occupant in the same bed were associated with colonization, which did not influence mortality. The high percentage of colonization in combination with the aforementioned factors indicates the importance of the dissemination of KPC-Kp among patients via the personnel and signifies the value of a strict implementation of infection control protocols. In total, 53 patients developed KPC-Kp bloodstream infection during ICU A stay with 43.4% mortality. The most important factors that influence mortality were the resistance of the strain to gentamicin/colistin/tigecycline and septic shock, while the treatment with two active antibiotics was associated with better survival confirming the results of previous studies favoring combination therapy for the treatment of KPC-Kp infection. The development of resistance against colistin or tigecycline, which are considered the last frontier in the treatment of KPC-Kp infections, is an alarming phenomenon. In total, 24.4% and 17.9% of ICU A patients became colonized by KPC-Kp resistant to colictin or tigecycline, respectively. As expected, the administration of colistin or tigecycline influenced colonization, while the most important factor favoring colonization was the presence of colonized patients in nearby patients, indicating the importance of dissemination of these strains against de novo resistance development. The comparison of the two ICUs, found a higher percentage of patients colonized during ICU A stay (61.8% vs 34.1%) and in a shorter period (10.6 vs 19.9 days). These results may be explained by the higher percentage of patients colonized upon admission (11.4% vs 1.8%), the lower nurse/patient ration and the higher carbapenem administration. In total, 305 and 100 strains of K. pneumoniae isolated from patients hospitalized in ICU A and B, respectively, were positive for the presence of blaKPC gene while five strains in ICU A were positive for the blaVIM gene also. All strains were resistant to penicillins, cephalosporins, aztreonam, trimethoprim sulfamethoxazole (30% of ICU B strains were sensitive), amikacin, tombramycin and quinolones. The resistance rates to carbapenems (67.9% vs 60%), colisitn (35.1% vs 18%), gentamicin (50.8% vs 24%) and tigecycline (17% vs 18%) among the ICUs strains were comparable. PFGE of 57 and 20 isolates from ICU A and B, respectively, revealed that ICU A strains belonged in two types, with type A comprising 65.5% of the isolates, while all ICU B isolates belonged in type A. The percentage of VRE colonization in both ICUs were lower in comparison with those of KPC-Kp. During ICU admission 14.3% (71 from 497 patients) was already colonized, while 14.4% (36 from 250 patients) became colonized during stay. The most important factor influencing colonization was the presence of colonized patients in nearby beds, indicating that non adherence with hand hygiene may play a predominate role in VRE dissemination. In total 107 VRE strains were isolated (100 E. faecium and 7 E. faecalis). Eighty four were positive for the vanA gene and resistant to vancomycin and teicoplanin, while the rest were vanB positive and were characterized by low level resistance to vancomycin (12 were in susceptibility range) and susceptible to teicoplanin. All strains were susceptible to linezolid, daptomycin and tigecycline. As MLST revealed, E. faecium strains belonged in six different Sequencing Types (ST117, ST17, ST203, ST226, ST786, ST125) with 90% among them belonging to the Clonal Complex CC17. E. faecalis strains were categorized in four STs (ST6, ST41, ST19, ST28). The proportion of colonized patients by MRSA upon admission and during ICU stay was very low (5.3% and 3.7%, respectively). The most important factor associated with colonization was enteric carriage of vanA-positive Enterococcus. Surveillance cultures revealed 28 mecA-positive S. aureus strains, with the majority (n=19) being PVL-positive, belonging to ST80 and resistant only to kanamycin, tetracycline and fucidic acid, while the remaining were categorized in four STs (6 strains in ST239 and one at ST225, ST72 and ST30). The ST30 strain was tst-positive. The comparison of colonization strains from patients (n=67) and personnel (n=23) of the ICUs (Group A) with the strains of colonization (n=53) and bloodstream infections (n=75) isolated from non-ICU patients (Group B), revealed a higher percentage of MRSA and PVL-positive strains in Group B, while Group A was characterized by higher percentage of tst-positive strains indicating their silent dissemination between ICU patients and personnel. The present study has identified the risk factors for colonization of infection by KPC-Kp, VRE and MRSA, in order to guide the future efforts towards containing their dissemination in the two ICUs, as well as, to the Greek hospitals, which in total are plagued by the aforementioned pathogens.

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