Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Campylobacter sp. à uma importante causa de enterite de origem alimentar com alta incidÃncia na populaÃÃo infantil de paÃses em desenvolvimento. No entanto, o diagnÃstico especÃfico de sua etiologia segue como um desafio, visto que mÃtodos moleculares e imunoenzimÃticos tÃm se mostrado mais sensÃveis. Postulamos que o conhecimento de sua virulÃncia e o diagnÃstico especÃfico possam ajudar na identificaÃÃo e potencial controle da campilobacteriose na infÃncia. Foi determinada a etiologia de diarreia por Campylobacter sp., em um estudo transversal sobre diarreia em crianÃas de 0-36 meses residentes da Ãrea urbana de Fortaleza, CearÃ, Brasil, que necessitaram de atendimento mÃdico de urgÃncia por causa de doenÃa diarreica. ApÃs a aprovaÃÃo Ãtica do estudo, um questionÃrio foi aplicado para qualificar as condiÃÃes clÃnicas apresentadas por cada crianÃa no momento da admissÃo. O DNA foi extraÃdo diretamente de amostras fecais coletadas de 226 crianÃas. Para a detecÃÃo do agente etiolÃgico, utilizamos diagnÃstico molecular (PCR e RT-PCR) e diagnÃstico imunoenzimÃtico (ELISA), alÃm da detecÃÃo de genes associados à virulÃncia de C. jejuni (PCR). Campylobacter sp. foi encontrado em 8,9% (20/225) das amostras, por diagnÃstico microbiolÃgico convencional. C. jejuni e C. coli foram detectados em 19,5% (44/226) e 1,3% (3/226) das amostras diarreicas, respectivamente, por PCR. Os diagnÃsticos por RT-PCR e ELISA alcanÃaram 26,7% (60/225) e 37,9% (58/153), respectivamente. Quando considerada a combinaÃÃo de diagnÃsticos (positividade no diagnÃstico microbiolÃgico ou no imunoenzimÃtico e ao menos em um dos testes moleculares) a prevalÃncia encontrada foi de 16,4% (37/226). A concordÃncia entre os testes para diagnÃstico utilizados foi de moderada a regular, de acordo com o Ãndice de Kappa. Genes associados à virulÃncia foram detectados nas seguintes proporÃÃes de amostras positivas para C. jejuni: flaA, 79,5% (35/44); racR, 97,7% (43/44) e dnaJ, 88,6% (39/44) â relacionados à adesÃo bacteriana e colonizaÃÃo; ciaB, 97,7% (43/44); pldA, 45,4% (20/44) e pVir 0% (0/44) â relacionados à invasÃo; e cdtABC em 95,4% (42/44) das amostras, operon relacionado à produÃÃo da toxina citoletal distensora (CDT). Sinais e sintomas especÃficos, tais como sangue nas fezes, vÃmito, febre e/ou dor abdominal, apesar de bastante frequentes, nÃo foram associados com a detecÃÃo de C. jejuni. O perfil de distribuiÃÃo dos genes de virulÃncia de C. jejuni nÃo apresentou correlaÃÃo com a apresentaÃÃo clÃnica da doenÃa, mesmo quando tal perfil foi categorizado de acordo com a funÃÃo das proteÃnas codificadas pelos genes, o que nos leva a crer que outros fatores, talvez relacionados à susceptibilidade do hospedeiro, possam ser mais importantes do que a variabilidade genÃtica do micro-organismo. ConcluÃmos que Campylobacter sp. foi detectado em percentual relevante da populaÃÃo estudada, principalmente quando os mÃtodos diagnÃsticos foram utilizados de forma combinada. Em geral, os genes de virulÃncia foram detectados em uma alta proporÃÃo das amostras positivas para C. jejuni, embora os genes relacionados à invasÃo tenham sido menos frequentemente encontrados. Corroboramos dados de outros grupos sobre a necessidade de revisÃo do diagnÃstico para Campylobacter sp. em prol da inclusÃo de metodologias mais sensÃveis e espÃcie-especÃficas, alÃm da busca por marcadores para inflamaÃÃo intestinal e fatores preditivos de cultura negativa. / Campylobacter sp. is an important cause of food-borne gastroenteritis with high incidence in children living in developing countries. However, the specific diagnosis of its etiology remains as a challenge, since conventional diagnosis by culture is now challenged by molecular and immunoenzymatic methods, which have greater sensitivity. We postulate that the knowledge of its virulence and specific diagnosis may assist in identifying and potentially controling campylobacteriosis in childhood. We determined the etiology of Campylobacter sp. associated diarrhea, in a cross-sectional study of diarrhea in children aged 0-36 months from the urban area of Fortaleza, CearÃ, Brazil, who required emergency medical care because of diarrheal disease. After ethical approval of the study, a questionnaire was applied to describe the clinical conditions presented by each child at the time of admission. DNA was extracted directly from fecal samples collected from 226 children. For the determination of the etiologic agent we used molecular diagnostics (PCR and RT-PCR) and diagnostic immunoassay (ELISA), besides the detection of virulence associated genes of C. jejuni (PCR). Campylobacter sp. was found in 8.9% (20/225) of the samples by conventional microbiological diagnosis. C. jejuni and C. coli were detected in 19.5% (44/226) and 1.3% (3/226) of the diarrheic samples, respectively. The diagnostic RT-PCR and ELISA reached 26.7% (60/225) and 37.9% (58/153) of positivity, respectively. When considering the combination of diagnostic (positive in microbiological diagnosis or immunoassay and at least one of the molecular tests) the prevalence was 16.4% (37/226). The agreement between the tests used for diagnosis was moderate to regular, according to Kappa index. The presence of C. jejuniÂs virulence-associated genes that encode proteins related to the pathogenesis of micro-organism were detected in the following proportions of C. jejuni-positive DNA samples: flaA, 79.5% (35/44); racR, 97.7% (43/44) and dnaJ, 88.6% (39/44) â related to bacterial adhesion and colonization; ciaB, 97.7 % (43/44); pldA, 45.4% (20/44) and pVir 0% (0/44) â related to invasion, and cdtABC in 95.4% (42/44) of samples related to citoletal distending toxin (CDT). Specific signs and symptoms such as blood in the stool, vomiting, fever and/or abdominal pain, although quite frequent, were not associated with the detection of C. jejuni. The distribution profile of C. jejuniÂs virulence genes was not correlated with the clinical presentation of the disease, even when this profile was categorized according to the function of the proteins encoded by the genes, which leads us to believe that other factors, perhaps related to host susceptibility, may be more important than genetic variability of the microorganism. We conclude that Campylobacter sp. was detected in a significant percentage of the children 0-36 months with diarrhea, especially when the diagnostic methods were used in combination. In general, the virulence genes were detected in a high proportion of C. jejuni-positive samples, although the invasion-related genes have been found less frequently. Our data corroborates findings from other groups on the need to revise the diagnostic for Campylobacter sp. towards the inclusion of more sensitive and species-specific methods, as well as search for extra markers for intestinal inflammation and predictors of negative culture.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-07-28T13:08:05Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_EDUARDA_versãoFinalDigital.pdf: 2657877 bytes, checksum: 2ce4a0aa738737b2947e45ed5fb9e912 (MD5) / Made available in DSpace on 2016-07-28T13:08:05Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_EDUARDA_versãoFinalDigital.pdf: 2657877 bytes, checksum: 2ce4a0aa738737b2947e45ed5fb9e912 (MD5) Previous issue date: 2016-02-29 / FACEPE / Devido à crescente resistência de isolados clínicos de Staphylococcus spp. a antimicrobianos e a consequente diminuição de opções terapêuticas eficazes, este estudo investigou a influência in vitro de sub-Concentrações Inibitórias Mínimas (sub-CIM) de oxacilina e tigeciclina, isoladamente e em combinação, no crescimento bacteriano e na expressão dos genes mecA, ica e tst em isolados clínicos de Staphylococcus spp.Para realização da primeira etapa deste estudo 45 isolados,identificados quanto a espécie de Staphylococcus spp. e susceptibilidade pelo Vitek2, foram obtidos de dois hospitais públicos de Recife-PE e avaliados pelas técnicas de MALDI-TOF MS e sequenciamento do rDNA 16S para confirmação das espécies bacterianas. A relação clonal dos isolados de Staphylococcus spp. foi determinada pela análise da Região Intergênica 16S-23S para posterior determinação do tipo de SCCmec e do perfil de virulência. O MALDI-TOF MS e sequenciamento do 16S se mostraram mais concordantes na identificação das espécies bacterianas quando comparadas ao Vitek2, sendo identificados isolados de Staphylococcus aureus, S. epidermidis, S.hominis, S. haemolyticus e S. saprophyticus. Trinta e um ribotipos foram determinados, demonstrando grande variabilidade genética. Os ribotipos foram encontrados dispersos nos diferentes setores dos dois hospitais investigados. A maioria dos isolados apresentaram SCCmec tipo IV (51%), seguido pelo tipo III (26%), tipo II (16%) e tipo V (7%). Foi possível encontrar nove perfis de virulência, onde o gene luk foi observado em 80% dos isolados, seguido de 51% para o gene ica, 35,5% para o hlg e 33,3% para o tstratificando a crescente evidência da presença de isolados tradicionalmente comunitários no ambiente hospitalar. Na segunda fase do estudo quatro isolados clínicos com diferentes tipos de SCCmece baixa relação clonal foram analisados in vitro para a determinação da CIM frente a oxacilina e tigeciclina. A influência in vitro destes antimicrobianos no crescimento bacteriano foi avaliada isoladamente e em associação, assim como a expressão dos genes mecA, ica e tst por RT-qPCR. O ensaioin vitro dos diferentes isolados de Staphylococcus spp. frente a tigeciclina associada à oxacilina resultou em inibição de crescimento bacteriano maior em relação a utilização isolada dos antimicrobianos, além de não influenciar ou diminuir a expressão de todos os genes analisados, dando suporte à ideia de que o uso associado da tigeciclina e oxacilina no tratamento de infecções por isolados MRS portadores dos genes tst e ica possivelmente poderiam promover benefícios na modulação destes genes. / Due to the increasing resistance of clinical isolates of Staphylococcus spp. to antimicrobial and the consequent decrease of effective therapeutic options, this study determined the in vitro influence of sub-minimum inhibitory concentrations (sub-MIC) of oxacillin and tigecycline, alone and in combination, on bacterial growth and expression ofvirulence genes in clinical isolates of Staphylococcus spp. To perform the first stage of this study 45 isolates of Staphylococcus sppwere obtained from two public hospitals of Recife-PE and evaluated by MALDI-TOF MS techniques and 16S sequencing for confirmation of bacterial species. The clonal profile Staphylococcus spp. isolates was determined by PCR-Ribotyping for subsequent determination of the type of SCCmec and virulence profile. The MALDI-TOF MS and sequencing of 16S were more effective in the identification of bacterial species when compared to Vitek2, being identified Staphylococcus aureus, S. epidermidis, S.hominis, S. haemolyticus e S. saprophyticus.Thirty-one ribotypes could be determined among the strains tested, with most harboring the SCCmec type IV (51%), followed by type III (26%), type II (16%) and type V (7%).%). 80% of the isolates presented the luk gene, 51% icaAD, 35.5% hlg and 33.3% tst. These results demonstrate a wide variability and dispersion of the isolates analysed, in different sectors of the hospitals confirming the growing evidence of the presence of traditionally community isolates in the hospital environment. In the second phase of the study, four clinical isolates were selected according to different types of SCCmec were analyzed in vitro to determine the MIC front oxacillin and tigecycline. The antimicrobial effect in vitro tests on bacterial growth was evaluated separately and in combination, as well as the expression of the mecA, ica and tst genes by RT-qPCR. Subjecting the different Staphylococcus spp. isolates in vitro to tigecycline in association with oxacillin resulted in inhibition of bacterial growth that was more potent than subjecting them to the antimicrobials separately, and this also did not influence or decrease the expression of any of the genes analyzed. The present study supports the idea that the associated use of tigecycline and oxacillin for treating infections caused by methicillin-resistant Staphylococcus that is positive for tst and ica could promote possible benefits in modulating these genes.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Porphyromonas gingivalis à um patÃgeno extremamente associado com a etiologia da periodontite crÃnica e agressiva. O objetivo deste estudo foi avaliar atravÃs de reaÃÃo em cadeia da polimerase em tempo real (Real Time-PCR) a presenÃa de Porphyromonas gingivalis (Pg) e dos genÃtipos fimA II e IV em indivÃduos com periodontite agressiva generalizada (PAG). Quarenta indivÃduos com PAG (29,7  8,1 anos) foram analisados clinicamente - Ãndice de Placa (IP), Ãndice Gengival (IG), profundidade de sondagem (PS), nÃvel de inserÃÃo clÃnico (NIC) - e microbiologicamente, atravÃs de Real Time PCR, quanto à presenÃa de Pg e dos genÃtipos fimA II e IV. Amostras de biofilme subgengival foram colhidas do sÃtio proximal com maior PS e maior NIC. MÃdias de PS e NIC desses sÃtios foram respectivamente: 9,5  2,2 mm e 10,2  2,8 mm. P. gingivalis foi observado em 26 (65%) dos indivÃduos. O genÃtipo fimA II foi verificado em 16 (61,53%) enquanto o genÃtipo fimA IV em 7 (26,92%) dos que apresentaram P. gingivalis. Entretanto, nÃo foi observada diferenÃa estatÃstica entre os parÃmetros clÃnicos dos indivÃduos que apresentaram ou nÃo o microrganismo ou seus respectivos genÃtipos. TambÃm nÃo foi verificada associaÃÃo entre a presenÃa dos genÃtipos e idade ou gÃnero dos pacientes. Os dados sugerem uma associaÃÃo entre genÃtipos fimA II de Porphyromonas gingivalis quando da ocorrÃncia deste microrganismo em indivÃduos com periodontite agressiva generalizada. / Porphyromonas gingivalis is a pathogen strongly associated with the etiology of chronic and aggressive periodontitis. The purpose of this study was to evaluate by Real-Time polymerase chain reaction (Real Time-PCR) the presence of Porphyromonas gingivalis (Pg) and fimA genotypes type II and type IV in patients with generalized aggressive periodontitis (GAgP). Forty individuals with aggressive periodontitis (AgP) (29.7  8.1 years) were clinical analyzed through plaque index (PI), gingival index (GI), probing depth (PD), clinical attachment level (CAL) and microbiologically, by Real Time-PCR for the presence of Pg and fimA genotypes type II and type IV. Subgingival biofilm samples were collected from the interproximal periodontal sites (> PD and > CAL). The PD and CAL average of this sites were respectively: 9,5  2,2 mm e 10,2  2,8 mm. P. gingivalis was observed in 26 (65%) of individuals. FimA genotypes type II was detected in 16 (61,53%) while fimA genotypes type IV in 7 (26,92%) of those with P. gingivalis. However, no differences were observed between the clinical parameters of patients who presented or not the organism or its genotypes. There was also no association between the presence of genotypes and age or gender of patients. The data suggest an association between P. gingivalis fimA genotypes upon the occurrence of this microorganism in patients with generalized aggressive periodontitis.

Francisella tularensis and Francisella novicida are closely related facultative intracellular pathogens capable of survival and growth within macrophages. In this work we present evidence to show that F. tularensis uses phase variation to alter lipopolysaccharide (LPS) antigenicity, macrophage nitric oxide (NO) production, and microbial intramacrophage growth. The LPS and lipid A of F. tularensis LVS fail to stimulate production of significant levels of nitric oxide by rat macrophage monolayers. However, spontaneous variants of F. tularensis expressing an antigenically distinct LPS induce rat macrophages to produce increased levels of NO, thereby suppressing intracellular growth. This new form of LPS produced by F. tularensis is also the predominant form of LPS found normally in F. novicida. Rat macrophages infected with F. novicida produce high levels of NO and exhibit suppression of intracellular growth. LPS and lipid A isolated from F. novicida and variants of F. tularensis stimulate increased levels of NO production. In addition, a reverse phase shift can occur which returns the LPS of the F. tularensis variants to the original antigenic form, resulting in reduced macrophage NO production and restoration of intracellular growth. These results suggest that F. tularensis can modulate macrophage NO production through phase variation of its LPS. It was of interest to initiate a study that would ultimately characterize the molecular mechanism of LPS phase variation in Francisella tularensis . To this end, we used shuttle mutagenesis to create a mutant library of F. novicida. We mutagenized a size- restricted plasmid library of F. novicida with the erythromycin- resistant transposon TnMax2. Putative F. novicida LPS mutants created by shuttle mutagenesis were screened visually for aberrant colony phenotypes on agar plates. Of 10464 mutants screened, 5 unique F. novicida LPS mutants were isolated which exhibit three distinct LPS phenotypes as determined by Western immunoblot. A single mutant from each of the three phenotypic groups was further characterized with respect to DNA sequence analysis, intramacrophage growth, and sensitivity to detergent and serum complement. Furthermore, these three loci were shown to hybridize with a corresponding locus in F. tularensis LVS. However, there was no difference in the restriction pattern of the hybridizing bands between LVS and its LPS phase variants, thus indicating that no major genetic rearrangements or insertion/deletion of a large mobile genetic element occurs in these genes during the phase variation process of F. tularensis. The F. novicida valAB locus has previously been cloned, sequenced, and shown to be functionally homologous to the E. coli genes msbA/lpxK. In order to investigate the hypothesis that valAB is involved in transport of LPS to the cell surface, an E. coli strain harboring an NTG-mutagenized temperature sensitive (t.s.) allele of valAB, a nonfunctional copy of msbA/lpxK, and an IPTG-inducible copy of the gene encoding the Chlamydia trachomatis genus-specific LPS epitope (gseA) was constructed. In this study, DNA sequencing was used to locate the temperature sensitive mutations in the valAB locus. Two C to T transitions were found in the valA coding region which result in a S to F change at amino acid 543 and a T to I change at amino acid 458. The ability of E. coli cells harboring this t.s. copy of valAB to transport the Chlamydia LPS epitope across the inner membrane at the permissive and non-permissive temperatures was determined using sucrose density gradient centrifugation and ELISA. It was determined that there was increased association of the LPS epitope with the inner membrane at the non-permissive temperature, thus suggesting that ValA is required for transport of an LPS precursor across the inner membrane. / Graduate

>Magister Scientiae - MSc / The aim of this study was to investigate the presence of Salmonella in the tea processing environment, to identify and detect potential virulence genes isolated from Salmonella in the tea, and to determine the antibiotic resistance levels of Salmonella isolated from fermented Rooibos. / National Research Foundation (NRF)

Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium that is also a major opportunistic human pathogen in nosocomial infections and cystic fibrosis chronic lung infections. These P. aeruginosa infections can be extremely difficult to treat due to the high intrinsic antibiotic resistance and broad repertoire of virulence factors, both of which are highly regulated. It was demonstrated here that the psrA gene, encoding a transcriptional regulator, was up-regulated in response to sub-inhibitory concentrations of antimicrobial peptides. Compared to wild-type and the complemented mutant, a P. aeruginosa PAO1 psrA / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Most eukaryotes harbor a diverse community of parasitic, mutualistic, and commensal microbial symbionts. Although the diversity of these microbial symbiotic communities has recently drawn considerable attention, theory regarding the evolution of interactions among symbionts and with the host is still in its nascent stages. Here we evaluate the role of interactions among coinfecting symbionts in the evolution of symbiont virulence toward the host. To do so, we place the virulence-transmission trade-off into a community context and model the evolution of symbiont trophic modes along the continuum from parasitism (virulence) to mutualism (negative virulence). We establish a framework for studying multiple infections of a host by the same symbiont species and coinfection by multiple species, using a concept of shared costs, wherein the negative consequences of virulence (or harm) toward the host are shared among symbionts. Our results show that mutualism can be maintained under infection by multiple symbionts when shared costs are sufficiently low, while greater virulence and parasitism toward the host are more likely when shared costs are high. Last, for coinfection by more than one species, we show that if the presence of a mutualist ameliorates some of the costs of pathogen virulence, then the symbiotic community may more often evolve to a more commensal state and maintain mutualisms.

The genetic requirements for Influenza virus to infect and adapt to new species is largely unknown. To understand the evolutionary steps required by a virus to become virulent, a human virus (A/HK/1/68) (HK), avirulent in mice, was subjected to 20 and 21 serial lung-to-lung passages in mouse. Sequence analysis revealed the emergence of eleven mutations within the NS1 gene of the new virulent strains, many of which occurred in binding sites for transcriptional and translational cellular factors. In the present study we have rescued viruses containing each of the NS1 mouse adapted mutations onto A/PR/8/34 (PR8) backbone. We found 9 of 16 NS1 mutants were adaptive by inducing mortality, body weight loss in BALB/c mice and enhanced virus replication in MDCK cells with properties of host cell interferon transcription inhibition. Sequence comparisons with the highly pathogenic A/Hong Kong/156/1997 (H5N1) and the most severe pandemic A/Brevig Mission/1/1918 (H1N1) NS1 genes showed convergent evolution with some of the mouse adapted viruses for F103L plus M106I and V226I plus R227K mutations respectively. The F103L and M106I mutations in the HK NS1 gene were shown to be adaptive by assessment with respect to replication, early viral protein synthesis, interferon-β antagonism and tropism in the mouse lung. We extended the study and proved increased virulence associated with F103L+M106I mutations in their respective H5N1 NS1 gene on the PR8 and HK backbones, as well as the PR8 NS1 gene and the H9N2 (A/Ck/Bj/1/95) gene in the PR8 and A/WSN/33 backbones respectively. However the V226I and R227K mutations in their respective HK and 1918 NS1 genes slightly enhanced virulence and viral growth at later stages of infection. This study demonstrates that NS1 is a virulence factor; involved in multiple viral processes including interferon antagonism and viral protein synthesis. Furthermore, NS1 mutations acquired during mouse adaptation are proven to be adaptive in human, mouse and avian NS1 genes.

Urinary tract infections are generally preceded by transfer of organisms from the distal urethra to the bladder (20, 148). However, although urinary infections are predominantly due to pure cultures of Escherichia coli, the distal urethra contains a mixed flora in which E. coli is relatively uncommon and anaerobes predominate (73, 103). This discrepancy between the bladder and distal urethral flora may be due to differential adhesion or differential growth rates. In this dissertation I have tested the hypothesis that differential growth rates of urethral organisms in urine explains the predominance of E. coli as a pathogen. These experiments showed that the balance between bacterial growth and washout may have a pivotal role in the pathogenesis of infection and perhaps therefore in treatment. A model of the human bladder used for the pathogenesis studies was then used to study the activity of mecillinam and ampicillin under conditions simulating human urinary infection. The model proved realistic especially for synergy studies where shortcomings in conventional in vitro methods are a cause for concern. The following topics were studied. 1. Urine was chosen as a test medium for definitive experiments because growth rates of organisms other than E. coli were different in broth and in urine. A method for sterilizing urine in bulk was developed which did not affect growth supporting properties. 2. E. coli was shown to grow faster and to have a shorter lag period than almost all other organisms when studied in shake culture. 3. A continuous culture model of the human urinary bladder was employed for differential growth studies of organisms in sterilized human urine. This model reproduced many of the characteristics of the human lower urinary tract and enabled study of the balance between bacterial growth and the tendency of urine to wash organisms out of the tract. 4. Mixed cultures of approximately equal numbers of E. coli and a second potential urinary' pathogen were introduced into the bladder model and quantitative cultures performed at intervals up to 24 h. In 15 experiments E. coli eventually dominated the second pathogen which was sometimes undetectable at 24 h. Similar changes in bacterial populations seen in infected patients indicate that differential growth rates may be an important determinant of the pathogenicity of E. coli. 5. The use of the bladder model was then extended to investigations of antibiotic activity under realistic conditions. The value of the model for synergy studies with ampicillin and mecillinam was assessed by parallel conventional in vitro tests and an animal infection protection test*. The bladder model gave similar results to mammalian studies and appeared to be far superior to conventional methods. This model may be valuable in the initial assessment of new urinary antibiotics. 6. A representative array of organisms for the above study was selected following a survey of resistance patterns of 2000 clinical isolates of Enterobacteriaceae. An incidental by-product of this survey was the establishment of a breakpoint for mecillinam susceptibility in the Kirby-Bauer antibiotic disk test. 7. Work on the effect of mecillinam and/or ampicillin upon bacterial viability was extended to investigations of the relative contribution of permeability barriers and 3-lactamases to antibiotic susceptibility. Unlike ampicillin, mecillinam resistance of 77 clinical isolates of bacteria appeared to be independent of intracellular 3-lactamase levels, suggesting that the barrier effect may be more pronounced in bacterial resistance to mecillinam than to ampicillin. Kinetic studies using urine as a growth medium, and in particular the use of a bladder model have provided a unifying explanation of many features of both the pathogenesis and treatment of urinary infections. * Carried out by Dr. R.C. Cleeland. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Rhizoctonia zeae is a common pathogen of tall fescue and other turfgrasses in various regions of the United States. Disease caused by R. zeae typically occurs during hot, humid summer months when conditions are ideal for growth of the fungus but less than ideal for growth of tall fescue. While R. zeae has been reported on turfgrasses in several Mid-Atlantic states, there are no records of this pathogen being present in Virginia. Rhizoctonia isolates were obtained from samples of various turfgrasses exhibiting typical Rhizoctonia disease symptoms in Virginia. Additional isolates were obtained from several other states. All were characterized with respect to species characteristics as well as pathogenicity and virulence on two common cultivars of tall fescue. All isolates were pathogenic on tall fescue but there was some variability in virulence. There was consistently slightly less disease present on Crossfire II than on Kentucky 31. Experiments were also conducted to determine the impact of prior exposure to high air temperatures on the severity of disease. Preliminary data showed that one week of exposure to higher air temperatures caused an initial increase in overall turf quality, but as length of exposure increased the quality of turfgrass declined. Tall fescue plants were subjected to 0, 7, and 35 days of heat stress prior to inoculation with several isolates of R. zeae. No relationship was found between predisposing heat stress and disease severity. / Master of Science