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Development of methods to diagnose and predict antibiotic resistance using synthetic biology and computational approachesBriars, Emma Ann 17 March 2022 (has links)
Antibiotic resistance is a quickly emerging public health crisis, accounting for more than 700,000 annual global deaths. Global human antibiotic overuse and misuse has significantly expedited the rate at which bacteria become resistant to antibiotics. A renewed focus on discovering new antibiotics is one approach to addressing this crisis. However, it alone cannot solve the problem: historically, the introduction of a new antibiotic has consistently, and at times rapidly, been followed by the appearance and dissemination of resistant bacteria. It is thus crucial to develop strategies to improve how we select and deploy antibiotics so that we can control and prevent the emergence and transmission of antibiotic resistance.
Current gold-standard antibiotic susceptibility tests measure bacterial growth, which can take up to 72 hours. However, bacteria exhibit more immediate measurable phenotypes of antibiotic susceptibility, including changes in transcription, after brief antibiotic exposure. In this dissertation I develop a framework for building a paper-based cell-free toehold sensor antibiotic susceptibility test that can detect differential mRNA expression. I also explore how long-term lab evolution experiments can be used to prospectively uncover transcriptional signatures of antibiotic susceptibility.
Paper-based cell-free systems provide an opportunity for developing clinically tractable nucleic-acid based diagnostics that are low-cost, rapid, and sensitive. I develop a computational workflow to rapidly and easily design toehold switch sensors, amplification primers, and synthetic RNAs. I develop an experimental workflow, based on existing paper-based cell-free technology, for screening toehold sensors, amplifying bacterial mRNA, and deploying sensors for differential mRNA detection. I combine this work to introduce a paper-based cell-free toehold sensor antibiotic susceptibility test that can detect fluoroquinolone-susceptible E. coli. Next, I describe a methodology for long-term lab evolution and how it can be used to explore the relationship between a phenotype, such as gene expression, and antibiotic resistance acquisition. Using a set of E. coli strains evolved to acquire tetracycline resistance, I explore how each strain's transcriptome changes as resistance increases. Together, this work provides a set of computational and experimental methods that can be used to study the emergence of antibiotic resistance, and improve upon available methods for properly selecting and deploying antibiotics. / 2023-03-17T00:00:00Z
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Algorithms for antibiotic susceptibility testing for pathogens causing sepsisÅhag, Stina January 2017 (has links)
This study is a part of a project at Q-linea that aims to present a rapid diagnostic instrument to speed up the process of identification of pathogens and determination of MIC-values (Minimal Inhibitory Concentration) of antibiotic needed to treat patients with sepsis. Specifically, this report is aimed to describe the development and implementation of algorithms that examine susceptibility profiles ofsepsis related pathogens where the bacteria have been exposed to different antibiotics and by different lapse of concentrations. The developed algorithms are based on a clustering technique that identify inhibited growth and present the lowest concentration needed to slow down the growth of the pathogen. The implemented solution was tested on sepsis related pathogens and the determined MIC values were compared to MIC values generated with a method commonly used in healthcare today. Approximately 90% of instances were correctly classified based on data from six hours long tests which is significantly faster than the reference method which takes 16-24 hours to complete. Furthermore, each result comes with a set of quality measures for validation of the algorithm results. Although, further studies are necessary to increase the performance at the four-hour target time, and more data is needed to validate the developed quality measures.
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Développement de nouvelles méthodes moléculaires pour le typage et l’étude de la sensibilité aux antibiotiques de C. trachomatis / Development of new molecular methods for typing and study of antibiotic susceptibility of Chlamydia trachomatisPeuchant, Olivia 17 November 2011 (has links)
Chlamydia trachomatis est une bactérie à développement intracellulaire obligatoire, divisée en 19 sérovars parmi lesquels les sérovars D-K sont responsables d’infections oculo-génitales et les sérovars L de la lymphogranulomatose vénérienne (LGV). En France, C. trachomatis est le principal agent bactérien responsable d’infections sexuellement transmissibles (IST). Les méthodes moléculaires occupent une place de choix dans le dépistage et l’épidémiologie des infections à C. trachomatis. Grâce à leur utilisation à partir de prélèvements non invasifs, nous disposons de chiffres de prévalence qui s’élèvent à 1,5% dans la population générale, 3,6% chez les femmes âgées de 18 à 24 ans sexuellement actives et 10 à 15% dans les centres à vocation de dépistage des IST. N’ayant aucune donnée chez la femme enceinte, le programme hospitalier de recherche clinique (MATIST) que nous avons mis en place chez les femmes enceintes suivies au CHU de Bordeaux a montré une prévalence de l’infection à C. trachomatis de 2,5%, à M. genitalium de 0,8% et à N. gonorrhoeae de 0%. Chez les femmes de moins de 24 ans, la prévalence était respectivement de 7,9% et 2,4%. La compréhension de l’épidémiologie et de la dissémination des infections à C. trachomatis nécessite la mise au point de techniques de typage performantes d’autant qu’un seul sérovar, le sérovar E, est rencontré dans près de la moitié des cas. Nous avons développé une méthode de typage moléculaire, la MLVA (MultiLocus Variable Number of Tandem Repeat Analysis), qui analyse le polymorphisme associé aux répétitions en tandem et permet un typage intra-sérovar. Cinq VNTRs ont été identifiés. La méthode a été automatisée puis appliquée à 220 souches et échantillons cliniques de C. trachomatis de génovar E, permettant d’identifier 25 types MLVA. Les souches d’origine ano-rectale isolées de patients homosexuels et les souches suédoises appartenant au nouveau variant ont été individualisées au sein de deux types MLVA uniques et distincts, suggérant une origine clonale. L’ensemble des résultats obtenus ont montré que la MLVA est un outil de typage moléculaire performant, plus discriminant que les autres méthodes auxquelles nous l’avons comparée. De plus, dans le cadre de la surveillance épidémiologique de la LGV ano-rectale due au variant L2b qui sévit en Europe depuis 2003 presque exclusivement chez les homosexuels, nous avons identifié le premier cas de LGV ano-rectale chez une femme. Enfin, nous avons développé une technique de PCR en temps réel permettant une détermination objective de la concentration minimale inhibitrice d’un antibiotique donné vis-vis de C. trachomatis. Cette technique a également montré que les antibiotiques étudiés n’avaient qu’une activité bactériostatique sur C. trachomatis. / Chlamydia trachomatis is an obligate intracellular bacterium, divided into 19 serovars, among which serovars D-K are responsible for oculo-genital infections and serovars L of lymphogranuloma venereum (LGV). In France, C. trachomatis is the main bacterial cause of sexually transmitted diseases (STI). Molecular methods are the methods of choice for the C. trachomatis detection and epidemiology. Through their use, it has been shown that the prevalence of C. trachomatis infection rise up to 1.5% in the general population, to 3.6% for sexually experienced women aged 18-24 and to 10-15% in STI medical settings. As no data were available for pregnant women, we conducted a clinical research study (MATIST) in pregnant women at the Bordeaux University hospital. The prevalence of C. trachomatis, M. genitalium and N. gonorrhoeae infections was 2.5%, 0.8% and 0%, respectively. In women under 24 years, the prevalence of C. trachomatis, and M. genitalium infections was 7.9% and 2.4%, respectively. Understanding the epidemiology and the spread of C. trachomatis infection requires the development of efficient typing techniques knowing that a single serovar, serovar E, is found in nearly half the cases. We developed a MLVA (MultiLocus Variable-Number of Tandem Repeat Analysis) method which analyzes the genome polymorphism associated to tandem repeats and allowed intra-serovar subtyping. Five VNTRs were identified. The automated method was applied on 220 C. trachomatis genovar E clinical specimens and isolates, yielding 25 MLVA types. All anorectal isolates from men who have sex with men exhibited the same MLVA type, suggesting clonal spread. In the same way, we confirmed the clonal origin of the Swedish new variant of C. trachomatis. MLVA appears to be a good tool for molecular typing, with a higher discriminatory power than those of other methods used for comparison. Since 2003, a LGV proctitis outbreak caused by the new variant L2b has been reported in Europe in men who have had sex with HIV-positive men. We reported the first case of C. trachomatis L2b proctitis diagnosed in a woman. Finally, we developed a real-time PCR method allowing an objective determination of minimum inhibitory concentration of antibiotics for C. trachomatis. Our results also showed that all antibiotics studied only had bacteriostatic activity on C. trachomatis.
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Molecular epidemiology, virulence potential and antibiotic susceptibility of the major lineages of uropathogenic Escherichia coliAlghoribi, Majed January 2015 (has links)
Uropathogenic E. coli (UPEC) is the most frequent cause of urinary tract infection (UTI), being responsible for up to 85% of community acquired and 40% of nosocomial cases. UPEC strains harbour various virulence factors that contribute to their ability to cause disease. The high prevalence across the globe of multidrug resistant UPEC is a significant threat to therapy. Virulent and resistant UPEC strains have been recognised as belonging to major lineages and we have only recently begun to understand the factors contributing to their successful global dissemination. Work in this thesis was carried out to identify the population structure of E. coli isolates recovered from urosepsis and biliary sepsis, to reveal any differences in genetic background. A total of 100 isolates from the blood and urine of 50 patients presenting with urosepsis and 27 isolates from cases of biliary sepsis were subjected to genotypic and phenotypic analysis, including MLST, virulence gene detection and antibiogram and metabolic profiling. Urosepsis paired isolates showed identical genotypes and antimicrobial resistance profiles. However, several pairs of isolates showed discrepant metabolic activity profiles suggesting niche specific regulation of metabolism. Members of the ST131 clone were significantly associated with antibiotic resistance and ST38 isolates were associated with the highest level of metabolic activity. An in vivo infection model was used to investigate the virulence potential of isolates from the major UPEC lineages. Galleria mellonella larvae inoculated with ST69 and ST127 isolates showed significantly higher mortality rates than those infected with other strains. However, one isolate of ST127 (strain EC18) was avirulent and comparative genomic analyses with a single virulent ST127 strain revealed an IS1 mediated deletion in the O-antigen cluster in strain EC18, which is likely to explain the lack of virulence in the larvae and demonstrates the importance of this cell surface molecule in the model system. Finally, a total of 202 UPEC isolates were recovered from community and hospital urine samples from a tertiary care hospital in Riyadh, Saudi Arabia. Molecular epidemiological investigation of the strains was carried out to examine the overall UPEC population structure, for the first time in any part of Saudi Arabia. The most common lineages were ST131 (17.3%), ST73 (11.4%), ST38 (7.4%), ST69 (7.4%) and ST10 (6.4%). The findings highlight the successful spread of multidrug resistant, CTX-M positive ST38, ST131 and ST405 UPEC in Saudi Arabia. The high proportion (35%) of ESBL producing E. coli isolates is a particular concern and is driving frequent prescription of carbapenem antibiotics. A total of four isolates of ST38 were positive for aggR, which is a virulence marker of enteroaggregative E. coli (EAEC); ST38 strains that cause UTI but have an EAEC genetic background are becoming recognised as novel UPEC and this clonal group warrants further study.
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Epidemiology and antibiotic susceptibility patterns of mycoplasma sp. and ureaplasma urealyticumGovender, Sharlene 12 1900 (has links)
Bibliography / Thesis (PhD (Pathology. Medical Microbiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Overview: Mycoplasmas and ureaplasmas are not routinely diagnosed and
are under researched in South Africa. Prevalence, population shifts especially
concerning genital flora and implications in infection or other conditions are
unknown. Information pertaining to Mycoplasma pneumoniae in respiratory
disease is similarly lacking. There is little information on antimicrobial
susceptibilities and resistance development against Sexually Transmitted
Infections (STI) syndromic management approaches.
Aims: a) Elucidate mycoplasmal and ureaplasmal prevalence and
contributing factors concerning cervical colonisation or preterm delivery in
conjunction with HIV and Chlamydia trachomatis b) Investigate prevalence of
M. pneumoniae in respiratory infections in conjunction with HIV,
Mycobacterium tuberculosis and Pneumocystis jiroveci. c) Determine
antimicrobial susceptibilities of mycoplasmas and ureaplasmas and analyse
resistance genes. d) Assess the inter-generic transfer potential of resistance
gene (tetM) between Ureaplasma spp. and Neisseria gonorrhea.
Genital setting: The prevalence of genital mycoplasmas, ureaplasmas and
Chlamydia on women attending their first prenatal visit, in conjunction with
preterm labour or HIV status was investigated. For preterm labour (2003), 199
women were monitored for preterm delivery (<37 weeks); for colonisation and
HIV (2005), 219 women were screened. Microbial detection was performed on
DNA extracted from endocervical swabs employing PCR techniques.
Colonisation was seen to be highest in the 14-20 year group from 2003. In
women aged ±21 years, co-colonisation was 13% although there was a shift
from co-colonisation with Mycoplasma hominis and Ureaplasma spp. in 2003
to other dual/triple combinations in 2005. Overall major trends from both
collection periods were that the prevalence of Ureaplasma spp. tended to be
higher in women ±26 years, whilst prevalence of C. trachomatis and M.
hominis were lower. No association was evident between colonisation with M.
hominis, U. urealyticum, Ureaplasma parvum and labour outcome. HIV status had no effect on the prevalence/co-colonisation of M. hominis, Ureaplasma
spp. or C. trachomatis.
Respiratory setting: Studies were conducted to determine the prevalence of
community acquired atypical pneumonias in adults (M. pneumoniae and P.
jiroveci) and neonates (mycoplasmas, ureaplasmas and Chlamydia
trachomatis) in order to improve treatment management programmes in the
Port Elizabeth region. Sputum specimens from 102 adult patients presenting
with pneumonia/symptoms of pneumonia admitted to hospitals were assessed
by PCR. Details of patient’s gender, age, HIV and Mycobacterium
tuberculosis status were provided by the hospitals. Women were seen to be at
high risk for community-acquired P. jiroveci colonisation. Overall, prevalence
of P. jiroveci was 52.9% (54/102 patients). P. jiroveci was mainly associated
with HIV (25/74) (P. jiroveci and HIV positive patients in patient sample for
which clinical data and HIV status was available) and co-infection with M.
tuberculosis was observed in 12 HIV cases and one HIV negative patient. No
DHPS (20) or DHFR (17) resistance associated mutations were found in P.
jiroveci. M. pneumoniae was detected in one patient. For prevalence studies
(2007-2008) on atypical pneumonia in neonates, 69 endotracheal aspirates
were obtained. PCR detection of M. hominis, U. urealyticum and C.
trachomatis was performed and U. parvum detected in two specimens.
Antibiotic susceptibilities and resistance genes: The following
investigations on clinical isolates of U. parvum and U. urealyticum were
conducted (i) antibiotic susceptibility profiles, (ii) detection of drug target gene
mutations, or gene acquisitions and (iii) inter-generic resistance gene transfer
potential to Neisseria gonorrhoeae. Culture techniques applied to 132
endocervical specimens provided 66 Ureaplasma cultures (35 U. parvum, 9
U. urealyticum, 22 U. parvum + U. urealyticum). MIC determinations to
ofloxacin, erythromycin, tetracycline, doxycycline, azithromycin and josamycin
were performed. Thirty-seven ureaplasma cultures were fully susceptible to all
antibiotics tested; 21 showed intermediate resistance to erythromycin,
azithromycin and ofloxacin; while seven were resistant to tetracycline, three of
which were also resistant to doxycycline and one also resistant to azithromycin. Concerning ofloxacin resistance directed at quinolone
resistance determining regions, a substitution of Ser83Leu in ParC was
demonstrated in one intermediately-resistant Ureaplasma (MIC 4 µg/ml) while
a triple substitution of Asp112Glu in GyrA along with Ala125Thr and
Ala136Thr in ParC was found in six further intermediately-resistant strains. No
mutations were found in strains with MICs 1 µg/ml. No mutations were
detected in 23S rRNA operons, L4 or L22 proteins. TetM and int-Tn genes
were found in seven tetracycline-resistant strains. On screening 59
tetracycline-susceptible and -intermediate strains, eleven whilst possessing
an int-Tn gene lacked a large region of tetM and 48 only contained small
regions of tetM. The tetM genes of the seven tetracycline-resistant strains
were sequenced and comparisons performed against GenBank sequences of
Neisseria gonorrhoeae, Streptococcus pneumoniae and U. urealyticum. For
five strains tetM was seen to be highly mosaic in structure containing regions
that were similar to those of the GenBank strains and others that were unique.
In the tetM leader region, four hot spot recombination sites were identified that
could certainly influence the formation of the mosaic structures, upstream
insertion sequences/open reading frames and transposon regions that
regulate expression. On characterising the int-Tn genes of the seven
tetracycline-resistant strains, three types were present indicating transposons
from different origins had integrated into ureaplasma genomes. Reciprocal
tetracycline resistance gene transfer between ureaplasmas and N.
gonorrhoeae were unsuccessful. However, low-level tetracycline resistance
(MICs 4-8 µg/ml) was transferred to a U. parvum recipient from one U.
urealyticum and three U. parvum donors that carried tetM with MICs 16-64
µg/ml. On tetM PCR analysis, tetM was not detected in the transformants.
Conclusions: The importance of genital mycoplasmas, ureaplasmas and C.
trachomatis in long term aetiologies requires further investigations, certainly in
relation with syndromic management regimens that fail to reduce colonisation
rates. The high prevalence of P. jiroveci, the presence of M. pneumoniae in
cases of pneumonia and detection of U. parvum in two cases of neonatal
pneumonia investigated emphasises that in the absence of definitive
diagnoses, it is crucial to monitor treatment responses carefully, especially when first line antibiotic preferences are ß-lactams, in order to ensure
adequate and informed delivery of medical care. The finding of transposon
and/or tetM regions in all ureaplasmas investigated with or without full
expression of tetracycline resistance, in conjunction with tetM gene diversity,
certainly places ureaplasmas strongly in the picture for intra- and inter-generic
exchange of antibiotic resistance genes. / AFRIKAANSE OPSOMMING: Oorsig: Mikoplasma en ureaplasma word nie roetineweg gediagnoseer nie
en in Suid Afrika is nog min navorsing daaroor gedoen. Prevalensie,
populasie verskuiwings, veral in genital flora, en die impliksies van infeksie en
ander toestande is onbekend. Inligting rakende Mycoplasma pneumoniae in
respiratoriese siekte is ook gebrekkig. Daar is min inligting beskikbaar
rakende die antimikrobiale vatbaarheid en die ontwikkeling van
weerstandigheid gesien teen die benadering tot sindromiese hantering van
seksueel oordraagbare siektes.
Doelwitte: a) Om inligting te verskaf oor die prevalensie van mikoplasma en
ureaplasma en bydraende faktore betreffende voortydige kraam tesame met
MIV en Chlamydia trachomatis. b) Ondersoek van die prevalensie van M.
pneumoniae in respiratoriese infeksies tesame met MIV, Mycobacterium
tuberculosis en Pneumocystis jiroveci. c) Bepaling van die antimikrobiale
vatbaarheid van mikoplasma en ureaplasma en analisevan weerstandigheids
gene. d) Bereken die inter-genetiese oordrag potensiaal van
weerstandigheids gene (tetM) tussen Ureaplasma spp. en Naisseria
gonorrhoeae.
Genitale omgewing: Die prevalensie van genitale mikoplasma, ureaplasma
en Chlamydia in vroue tydens hul eerste prenatale besoek, tesame met
vroegtydige kraam en MIV status is ondersoek. In voortydige kraam (2003), is
199 vroue gemonitor vir voortydige kraam (<37 weke); vir kolonisasie en MIV
(2005), is 219 vroue getoets. Mikrobiale toetsing is gedoen deur DNS te win
vanaf endoservikale deppers met PKR tegnieke. Kolonisasie was die hoogste
in die ouderdomsgroep 14.20 jaar, in 2003. In vroue van ±21 jaar was medekolonisasie
13% alhoewel daar en verskuiwing was van mede-kolonisasie met
Mycoplasma hominis en Ureaplasma spp. in 2003 tot ander dubbel/trippel
kombinasies in 2005. Die oorkoepelende tendens in altwee die tydperke van
waarneming was dat die prevalensie van Ureoplasma spp. geneig was om
hoër te wees in vroue ±26 jaar, terwyl prevalensie van C. trachomatis en M.
hominis laer was. Geen assosiasie kon getoon word tussen koloniesasie met M. hominis, U. urealyticum, Ureaplasma parvum en uitkoms van kraam nie.
MIV status het geen effek gehad op die prevalensie/mede-kolonisasie van M.
hominis, Ureaplasma spp. of C. Trachomatis nie.
Respiratories: Studies is gedoen om die prevalensie van gemeenskaps
verworwe atipiese pneumonie in volwassenes (M. pneumoniae en P. jiroveci)
en neonate (mikoplasma, ureaplasma en Chlamydia trachomatis) te bepaal
om behandeling en hantering programme in die Port Elizabeth area te
verbeter. Sputum monsters van 102 volwasse pasiënte wat presenteer het
met pneumonie of simptome van pneumonie en wat tot hospitale toegelaat
was, is ontleed. Besonderhede van die pasiënte se geslag, ouderdom, MIV en
Mycobacterium tuberculosis status is deur die hospitale verskaf. PKR is
gedoen met inleiers gerig teen die volgende gene: P. jiroveci vir die aantoning
van mitokondriale groot subeenheid RNS en vir die analise van mutasies vir
ko-trimoksasool weerstandigheid dihydropteroaat sintetase (DHPS) en
dihydrofolaat reduktase (DHFR); M. pneumoniae vir die aantoning van P1
adhesien en 16S rRNS. Vroue het ‘n hoë risiko vir gemeenskapsverworwe P.
jiroveci kolonisasie gehad. In die algemeen was die prevalensie van P.
jiroveci 52.9% (54/102 pasiënte). P. jiroveci was hoofsaaklik geassosieerd
met MIV (25/74) (P. jiroveci en MIV positiewe pasiënte in die pasiënt monster
waarvoor daar kliniese data en MIV status bekend was) en mede-infeksie met
M. tuberculosis is gesien in 12 MIV gevalle en een MIV negatiewe pasiënt.
Geen DHPS (20) of DHFR (17) weerstandigheids geassosieerde mutasies is
gevind in P. Jiroveci nie. M. pneumoniae was aangetoon in een pasiënt. Vir
prevalensie studies (2007-2008) op atipiese pneumonie in neonate is 69
endotrageale aspirate verkry. PKR toetsing vir M. hominis, U. urealyticum en
C. trachomatis is gedoen met ‘primers’ soos voorheen gepubliseer.
Ureaplasma parvum is aangetoon in twee neonate met PKR met negatiewe
kultuur resultate.
Antibiotika sensitiwiteite en weerstandigheids gene: Die volgende toetse
is gedoen op kliniese isolate van U. parvum en U. urealyticum (i) antibiotika
sensitiwiteits profiele, (ii) aantoning van teiken geen mutasies, of geen
aanwinste en (iii) potensiaal vir inter-generiese weerstandigheids geen oordrag na Neisseria gonorrhoeae. Kultuur tegnieke toegepas op 132
endoservikale monsters het 66 Ureaplasma kulture gelewer (35 U. parvum, 9
U. urealyticum, 22 U. parvum + U. urealyticum). MIK bepaling vir ofloksasien,
eritromisien, tetrasiklien, doksisiklien, azitromisien en josamisien is gedoen.
Sewe-en-dertig kulture was ten volle sensitief vir alle antibiotika wat getoets
is; een-en twintig het intermediere weerstandigheid teenoor eritromisien,
azitromisien en ofloksasien getoon, terwyl sewe weerstandig was vir
tetrasiklien, drie daarvan was ook weerstandig vir doksisiklien. Wat betref
ofloksasien weerstandigheid gemik teen kwinoloon weerstandigheids
bepalende gebiede, is vervanging van Ser83Leu in ParC gedemonstreer in
een intermedier weerstandige Ureaplasma (MIK 4 µml) terwyl en trippel
vervanging van Asp112Glu in GyrA saam met Ala125Thr en Ala136Thr in
ParC gevind is in ses ander intermedier weerstandige stamme. Geen
mutasies is gevind in stamme met MIKs van MICs 1 µg/ml nie. Geeneen van
die ureaplasma was weerstandig vir eritromisien/azitromisien nie en geen
mutasies is gevind in 23S rRNA operons , L4 of L22 proteine nie. TetM en int-
Tn gene is gevind in sewe tetrasiklien weerstandige stamme. 58 Tetrasiklien
sensitiewe en .intermediere stamme is getoets, waarvan elf en int-Tn geen
gekort het sowel as en groot deel van tetM, terwyl 48 slegs klein dele van
TetM bevat het. Die tetM gene van die sewe tetrasiklein-werstandige stamme
se geenvolgorde is bepaal en vergelykings is getref teenoor die GenBank
volgordes van Neisseria gonorrhoeae, Streptococcus pneumoniae en U.
urealyticum. In vyf stamme is gevind dat die tetM geen hoogs mosaiek in
struktuur was met areas wat ooreenstem met die in GenBank stamme, en
ander areas wat uniek is. In die tetM leier area, is vier ehot spot f
herkombinasie areas geidentifiseer wat sekerlik die vorming van die mosaiiek
strukture kon beinvloed, asook transposon areas wat geenuitdrukking bepaal.
Met karakterisering van die int-Tn gene van die sewe tetrasikleinweerstandlige
stamme, was drie tipes teenwoordig waarin transposons vanaf
verskillende oorsprong aangedui was, geintegreerd met die ureaplama
genome. Resiprokale tetrasiklien weerstandigheids geen oordrag tussen
ureaplasma en n. gonorrhoea was nie suksesvol nie. Lae-vlak tetrasiklien
weerstandigheid (MIK fs van 4 . 8 µg/ml) is wel suksesvol oorgedra na en U.
parvum ontvanger vanaf een U. urealyticum en drie U. parvum ontvangers wat tetM gedra het met MIKs van 16-64 µg/ml. Met die analise van tetM met
PKR, kon tetM nie aangetoon word in die transformante nie.
Gevolgtrekkings: Die belang van genitale mykoplasma, ureaplasma en C.
trachomatis in langtermyn etologie benodig verdere ondersoek, veral in die lig
van die sindromiese behandeling regimes wat nie kolonisasie verminder nie.
Die hoe prevalensie van P. jiroveci, die teenwoordigheid van M. pneumoniae
in gevalle van pneumonie en die aantoning van U. parvum in twee gevalle van
neonatale pneumonie benadruk dat, in die afwesigheid van en definitiewe
diagnose, dit noodsaaklik is om respons tot behandeling sorgvuldig te
moniteer, veral indien die eerste lyn antibiotika keuse ß-laktam antimikrobiale
middels of kefalosporiene is, sodat behoorlike en ingeligde gesondheidsorg
gelewer kan word. Die bevinding van transposon en/of tetM gebiede in alle
ureaplasma wat ondersoek is met of sonder volle uitdrukking van tetrasiklien
weerstandigheid, in samehang met tetM diversiteit, plaas verseker
ureaplasma sterk in die prentjie vir intra- en inter-generiese uitruiling van
antibiotika weerstandigheids gene. / Nelson Mandela Metropolitan University / National Research Foundation (NRF Thuthuka) / Medical Research Council
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Growth Dynamics, Antibiotic Susceptibility and the Effect of Sublethal Ciprofloxacin Concentrations in Susceptible and Resistant Escherichia coli in Biofilm / Tillväxtdynamik, Antibiotikakänslighet och Effekten av Subletala Koncentrationer av Ciprofloxacin på Känsliga och Resistenta Escherichia coli i BiofilmFernberg, Jenny January 2019 (has links)
Instead of planktonic growth in nature, many species of bacteria form biofilm to survive in harsh conditions. Although many chronic bacterial infections are caused by bacterial species in a biofilm lifestyle, previous research has focused on studying antibiotic resistance in planktonic growth. Here we used a modified MBEC assay, i.e. biofilm growth on pegs, to determine Escherichia coli biofilm inhibitory concentrations (BIC) of ciprofloxacin, streptomycin and rifampicin and to study the minimal selective concentration (MSC) for ciprofloxacin in E. coli biofilm. We could observe high inhibitory concentrations for all antibiotics in the biofilm pre-formed in media without antibiotics compared to the biofilm formed in antibiotics. We also show preliminary result indicating that sublethal concentrations of ciprofloxacin lead to the selection of ciprofloxacin resistant mutants in biofilm and that the selection level is lower than what was observed in planktonic growing E. coli. With more knowledge in how the biofilm formation precedes in different antibiotic settings, the treatment for chronic biofilm infections used today could be evaluated and changed so that the infections could be eradicated.
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Antibiotico resistenza in S. thermophilus, tratti fenotipici, coniugazione e aggregazione / Antibiotic Resistance in S. Thermophylus, Phenotypic, Traits, Conjugation, AggregationTOSI, LORENZO 15 February 2007 (has links)
Negli ultimi decenni l'utilizzo degli antibiotici a scopo terapeutico o come promotori della crescita nell'allevamento animale ha portato alla comparsa e alla diffusione di microrganismi resistenti. In questo contesto, la presenza di Lattobacilli (LAB) antibiotico resistenti non rappresentano di per sé un rischio clinico. Tuttavia la possibilità che essi ma possono essere veicolo di geni codificanti l'antibiotico-resistenza verso batteri patogeni presenti negli alimenti o nel tratto gastro-intestinale umano (inclusi enterococchi, streptococchi e listeria), costituisce un possibile rischio per la salute umana che deve essere attentamente valutato. Obiettivo di questo lavoro è stato quello di valutare attraverso metodi di indagine fenotipica con le tecniche delle microdiluizioni in brodo, Etest e disc-diffusion, i livelli di antibiotico resistenza per le specie S. thermophilus e L. plantarum verso gli antibiotici tetraciclina, eritromicina, clindamicina, streptomicina, gentamicina, ampicillina. Ceppi atipici appartenenti alla specie S. thermophilus sono stati sottoposti ad analisi genetiche con lo scopo di caratterizzare e localizzare i geni responsabili della resistenza. E' stato inoltre testato il possibile trasferimento orizzontale dei geni di antibiotico resistenza nativi da S. thermophilus verso i batteri Gram-positivi E. faecalis e Listeria monocytogenes. In alcuni ceppi di S. thermophilus resistenti si sono infine osservati e studiati particolari caratteri fenotipici ( fitness ) correlati alla presenza delle determinanti genetiche di antibiotico resistenza nell'ospite batterico. / In the last decades, the use of antibiotics in human therapy or in animal husbandry as growth promoters has induced the development and the diffusion in antibiotic resistant micro-organisms. In this context antibiotic resistant Lactic Acid Bacteria (LAB) do not represent a clinical risk in themselves. However, the possibility that S. thermophilus cultures might transfer antibiotic resistance genes to pathogenic species either present in food or in the gastrointestinal tract (including enterococci, streptococci and listeria) represents a potential clinical risk that needs to be carefully evaluated. The aim of this study was to evaluate by means of phenotypic methods (microdilution, E-test, disc-diffusion) the levels of antibiotic resistance for S. thermophilus and L. plantarum species against the antibiotic tetracycline, erythromycin, clyndamicin, streptomycin, gentamycin and ampicillin. The atypical resistant S. thermophilus strains were subjected to genetic analyses in order to characterise and to localise the antibiotic resistance determinants. Furthermore the ability of the resistant S. thermophilus strains in transferring the antibiotic resistant determinant was assessed in mating experiments using as recipients the Gram-positive bacteria E. faecalis and Listeria monocytogenes. In same resistant S. thermophilus strains, special bacterial fitness related with the presence of the antibiotic resistance determinants in the bacterial hosts were observed and studied.
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Resistência a antibióticos, prevalência dos fatores associados à virulência, tipagem filogenética e perfil filogenético de isolados de Escherichia coli patogênica aviária (APEC)Barbieri, Nicolle Lima January 2010 (has links)
Escherichia coli patogênica aviária (APEC) é a causa de doenças extra-intestinais em aves, que se manifestam na forma de doenças localizadas ou infecções sistêmicas, gerando grandes perdas econômicas para a indústria aviária. O objetivo desse trabalho foi estudar isolados de APEC do sul do Brasil em relação à resistência a agentes antimicrobianos; a prevalência de fatores associados à virulência; a tipagem filogenética e o perfil filogenético. Na avicultura, os agentes antimicrobianos são muito utilizados na prevenção de infecções e na forma de promotores de crescimento. Foram utilizadas 41 isolados de E. coli obtidos de infecções sistêmicas e 144 isolados de celulite, utilizando o método de difusão com discos. Isolados APEC apresentaram baixos níveis de resistência, com excessão da tetraciclina e das sulfonamidas; e para isolados de colissepticemia, também foi observado uma resistência aos antimicrobianos que atuam na parede celular (ampicilina, cefalotina, bacitracina e ceftiofur). Vários fatores de virulência têm sido investigados em cepas APEC, os que têm sido mais frequentemente associados com a patogenicidade são as fímbrias de aderência F1 e Tsh (hemaglutinina sensível à temperatura); o sistema sideróforo aerobactina, a proteína iss (increased serum survival), a cápsula K e a produção de colicina V. Foram realizadas reações da polimerase em cadeia multiplex (PCR), testando um total de 33 fatores nos isolados APEC. Os fatores relacionados a adesão, sideróforos e resistência ao soro foram presentes em todas as amostras. Os fatores que apresentaram maior prevalência nas amostras foram, fimC, ompA, crlA e traT. A tipagem filogenética foi realizada através do método descrito por Clermont et al., (2000), que permite separar os isolados em 4 grupos filogenéticos principais (A, B1, B2 e D). Observamos a maior presença de isolados APEC pertencentes ao grupo D, tanto para celulite quanto para colissepticemia. A análise do perfil filogenético foi realizada pelo método ARDRA, que é baseado na variação da região do espaçamento intergênico (ISR) na região 16S-23S do DNA ribossômico. Os resultados foram analisados formando um dendrograma que mostra a proximidade filogenética dos isolados, indicando que não foi possível separar os clones de celulite dos de colissepticemia e não existem clones endêmicos nas regiões analisadas. Os resultados obtidos no presente estudo, fornecem um panorama geral da resistência aos agentes antimicrobianos, prevalência dos fatores de virulência, tipagem filogenética e perfil filogenético encontrados nos isolados de E. coli aviária do sul do Brasil de infecções de colissepticemia e celulite. / extra-intestinal infections in poultry, called colibacilose, and cause great economic losses to the poultry industry. The aim of this work was to examine APEC strains, isolated from avian cellulitis and colisepticemic chickens from South Brazil, for antibiotic resistance, presence of virulence-associated genes (VAGs), phylogenetic typing and phylogenetic analyses. In poultry, antibiotics are routinely used to prevent infection and promote growth. We analyzed the susceptibility to 15 antibiotics in 144 E. coli isolates collected from cellulitis lesions and in 41 isolates from septicemic chickens. APEC isolates have shown low levels of resistance excepting tetracycline and sulphonamides, and colisepticemic isolates were resistant to antibiotics that act in the cell wall, such as ampicilin, cephalotin, ceftiofur and bacitracin. The virulence factors most frequently associated with APEC pathogenicity are the adhesins F1 and Tsh (Temperature sensitive hemagglutinin), the iron acquisition system aerobactin, the protein Iss (increased serum survival), K1 capsule and production of colicin V. To investigate the presence of VAGs in the isolates, we performed multiplex polymerase chain reactions to test 33 virulence factors. Virulence factors related to adhesion, iron acquisition and serum resistance were present in almost all strains. The factors fimC, ompA, crlA and traT were the most frequent in APEC isolates. The phylogenetic typing were done using the Clermont et al. (2000) method, which classifies E. coli strains into four main phylogenetic groups (A, B1, B2, and D). Our phylogenetic typing has shown that cellulitis and colisseptisemic isolates were more related to group D. Amplified ribossomal restriction analysis (ARDRA) was performed in colissepticemic and celullitis isolates from broiler chickens from Southern Brasil. The similarity among isolates were observed with a dendrogram based on the band pattern. Our results have shown that clones of celullitis and colissepticemic isolates could not be distinguished and there is no endemic clones in regions observed analised. The results of the present work give us a panorama of the susceptibility to antibiotics, virulence factors, phylogenetic typing and phylogenetic analyses currently found in APEC isolates from severe lesions of cellulites and colibacillosis in south Brazil. Besides that, these analyses could be helpful for monitoring and preventing outbreakes of colibacilosis. Controling the first stages of the disease and detecting more prevalent clones in this region may reduce the incidence of the disease.
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Análise epidemiológica de cepas APEC e análise do regulador FNR na modulação da virulência de ExPECBarbieri, Nicolle Lima January 2014 (has links)
Escherichia coli é um bacilo Gram-negativo, anaeróbico facultativo e de distribuição cosmopolita. E. coli coloniza o intestino de humanos e outros animais endotérmicos logo após o nascimento, estabelecendo-se como um importante membro da microbiota intestinal. Algumas cepas de E. coli podem adquirir fatores de virulência, assumindo assim, uma natureza patogênica, como é o caso das E. coli patogênicas extraintestinais (ExPEC). As cepas ExPEC apresentam a capacidade de colonizar e se disseminar em diversos nichos no hospedeiro, e são divididas em UPEC (E. coli uropatogênica), NMEC (E. coli causadora de meningite neonatal) e APEC (E. coli patogênica para aves). UPEC, NMEC e APEC compartilham fatores associados à virulência. Para serem aptas a causar doença, cepas ExPEC devem apresentar pelo menos um fator associado à adesão, um fator para captação de ferro (sideróforo) e um fator de resistência ao soro, podendo, também, apresentar genes que codificam toxinas e invasinas. Embora sejam conhecidos muitos fatores de virulência associados à patogenicidade de cepas ExPEC, a regulação da expressão de tais fatores ainda não foi elucidada. Fumarato-nitrato-redutase (FNR) é uma proteína que atua como regulador global, agindo como um sensor da presença de oxigênio em bactérias gramnegativas. Já foi demonstrado que FNR está relacionada à regulação da virulência de bactérias patogênicas como Shigella flexneri e Salmonella enterica serovar Typhimurium. Este trabalho teve como objetivo a análise epidemiológica e caracterização de cepas APEC, bem como a investigação do controle da expressão de fatores associados à virulência de ExPEC pelo regulador global FNR. Os resultados da análise epidemiológica das cepas APEC mostram o perfil de resistência aos agentes antimicrobianos, a prevalência dos fatores de virulência e dos grupos filogenéticos (de acordo com a classificação EcoR) e a relação filogenética dos isolados, fornecendo um panorama da caracterização de E. coli patogênicas aviárias de lesões severas de celulite e de infecção sistêmica oriundas da região sul do Brasil. Em relação ao FNR, este estudo mostrou a influência deste regulador sobre importantes fatores associados à virulência, estando envolvido no controle de várias etapas do estabelecimento da infecção por cepas ExPEC. A deleção de fnr na cepa UPEC CFT 073 reduziu a motilidade, a expressão das fimbrias tipo I e tipo P, reduziu a expressão da hemolisina e controlou a expressão da ilha de patogenicidade do α- cetoglutarato. Além disso, a deleção de fnr fez tornou as bactérias incapazes de invadir células dos rins e da bexiga e de causar doença in vivo em camundongos de 6 semanas. FNR também foi capaz de controlar as etapas da infecção por NMEC 56. Uma vez deletado, as bactérias perderam a capacidade de causar bacteremia, de crescer no fluido cerebrospinal e de causar doença in vivo em ratos de 5 dias de idade. A deleção de fnr em APEC O1 resultou na diminuição da expressão da proteína OmpT plasmidial, da fímbria do tipo I e do auto-transportador AatA. A principal contribuição deste trabalho foi demonstrar que FNR atua na regulação da expressão de importantes fatores associados à virulência de cepas ExPEC (UPEC, NMEC e APEC), sendo importante para o estabelecimento da infecção por essas cepas. Neste trabalho, verificamos que, além da função já conhecida de regular os genes envolvidos na manutenção de um meio anaeróbico, FNR também atua no controle de genes associados à virulência de cepas ExPEC, refletindo na capacidade de causar doença que tais cepas apresentam. / Escherichia coli is a Gram-negative bacillus, facultative anaerobic and has cosmopolitan distribution. E. coli colonizes the intestine of humans and other endothermic animals immediately after birth, establishing as an important member of the intestinal microbiota. Some strains of E. coli can acquire virulence factors thereby assuming a pathogenic nature, as in the case of extraintestinal pathogenic E. coli (ExPEC). ExPEC strains have the ability to colonize and spread out in different niches of the host, and are divided into UPEC (uropathogenic E. coli), NMEC (newborn meningitis E. coli) and APEC (avian pathogenic E. coli). UPEC, NMEC and APEC share virulence factors. To be able to cause disease, ExPEC strains must produce virulence factors required for adherence, for iron uptake (siderophore) and for resistance to serum and may also contain genes encoding toxins and invasins. Although many virulence factors associated with the pathogenicity of ExPEC strains are known, the regulation of the expression of these factors has not yet been fully elucidated. Fumarate nitrate reductase (FNR) is a global regulatory protein, acting as a sensor of oxygen in Gram- negative bacteria. It has been shown that FNR relates virulence of pathogenic bacteria such as Shigella flexneri and Salmonella enterica serovar Typhimurium. The aim of this study was to do an epidemiological analysis and characterization of APEC strains as well as the investigation of regulation of ExPEC’s virulence factors by the global FNR regulator. The results of epidemiological analysis of APEC strains showed the profile of antimicrobial resistance , the prevalence of virulence factors and phylogenetic groups (according to the EcoR group) and the phylogenetic relationship of the isolates, providing an overview of the characterization of avian pathogenic E. coli causing severe cellulitis lesions and systemic infection originating from southern Brazil. In relation to FNR, this study showed the influence of this important regulator of virulence factors that is involved in controlling various stages of establishment of infection by ExPEC strains. Deletion of fnr in UPEC strain CFT 073 reduced motility and expression of type I and type P fimbriae, reduced the expression of hemolysin and control the expression of the pathogenicity island of α -ketoglutarate. Furthermore, fnr mutant strains were unable to invade cells of kidney and bladder, and to colonize the urinary tract of 6 weeks-old mice. FNR was also able to control the stages of infection of NMEC 56. The fnr mutant lost its ability to cause bacteremia, grow in cerebrospinal fluid, cause disease in 5 days old rats. Deletion of fnr in APEC O1 resulted in decreased expression of genes corresponding to the plasmid encoded OmpT protein, type I fimbriae and autotransporter AatA. The main contribution of this work was to demonstrate that FNR regulates expression of important virulence factors of ExPEC strains (UPEC, NMEC and APEC), which is important for the establishment of infection by these strains. In this work, we found that, besides the already known function in regulating genes involved in maintaining an anaerobic environment, FNR also acts in the control of virulenceassociated genes of ExPEC strains, reflecting the ability of these strains to cause disease.
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Resistência a antibióticos, prevalência dos fatores associados à virulência, tipagem filogenética e perfil filogenético de isolados de Escherichia coli patogênica aviária (APEC)Barbieri, Nicolle Lima January 2010 (has links)
Escherichia coli patogênica aviária (APEC) é a causa de doenças extra-intestinais em aves, que se manifestam na forma de doenças localizadas ou infecções sistêmicas, gerando grandes perdas econômicas para a indústria aviária. O objetivo desse trabalho foi estudar isolados de APEC do sul do Brasil em relação à resistência a agentes antimicrobianos; a prevalência de fatores associados à virulência; a tipagem filogenética e o perfil filogenético. Na avicultura, os agentes antimicrobianos são muito utilizados na prevenção de infecções e na forma de promotores de crescimento. Foram utilizadas 41 isolados de E. coli obtidos de infecções sistêmicas e 144 isolados de celulite, utilizando o método de difusão com discos. Isolados APEC apresentaram baixos níveis de resistência, com excessão da tetraciclina e das sulfonamidas; e para isolados de colissepticemia, também foi observado uma resistência aos antimicrobianos que atuam na parede celular (ampicilina, cefalotina, bacitracina e ceftiofur). Vários fatores de virulência têm sido investigados em cepas APEC, os que têm sido mais frequentemente associados com a patogenicidade são as fímbrias de aderência F1 e Tsh (hemaglutinina sensível à temperatura); o sistema sideróforo aerobactina, a proteína iss (increased serum survival), a cápsula K e a produção de colicina V. Foram realizadas reações da polimerase em cadeia multiplex (PCR), testando um total de 33 fatores nos isolados APEC. Os fatores relacionados a adesão, sideróforos e resistência ao soro foram presentes em todas as amostras. Os fatores que apresentaram maior prevalência nas amostras foram, fimC, ompA, crlA e traT. A tipagem filogenética foi realizada através do método descrito por Clermont et al., (2000), que permite separar os isolados em 4 grupos filogenéticos principais (A, B1, B2 e D). Observamos a maior presença de isolados APEC pertencentes ao grupo D, tanto para celulite quanto para colissepticemia. A análise do perfil filogenético foi realizada pelo método ARDRA, que é baseado na variação da região do espaçamento intergênico (ISR) na região 16S-23S do DNA ribossômico. Os resultados foram analisados formando um dendrograma que mostra a proximidade filogenética dos isolados, indicando que não foi possível separar os clones de celulite dos de colissepticemia e não existem clones endêmicos nas regiões analisadas. Os resultados obtidos no presente estudo, fornecem um panorama geral da resistência aos agentes antimicrobianos, prevalência dos fatores de virulência, tipagem filogenética e perfil filogenético encontrados nos isolados de E. coli aviária do sul do Brasil de infecções de colissepticemia e celulite. / extra-intestinal infections in poultry, called colibacilose, and cause great economic losses to the poultry industry. The aim of this work was to examine APEC strains, isolated from avian cellulitis and colisepticemic chickens from South Brazil, for antibiotic resistance, presence of virulence-associated genes (VAGs), phylogenetic typing and phylogenetic analyses. In poultry, antibiotics are routinely used to prevent infection and promote growth. We analyzed the susceptibility to 15 antibiotics in 144 E. coli isolates collected from cellulitis lesions and in 41 isolates from septicemic chickens. APEC isolates have shown low levels of resistance excepting tetracycline and sulphonamides, and colisepticemic isolates were resistant to antibiotics that act in the cell wall, such as ampicilin, cephalotin, ceftiofur and bacitracin. The virulence factors most frequently associated with APEC pathogenicity are the adhesins F1 and Tsh (Temperature sensitive hemagglutinin), the iron acquisition system aerobactin, the protein Iss (increased serum survival), K1 capsule and production of colicin V. To investigate the presence of VAGs in the isolates, we performed multiplex polymerase chain reactions to test 33 virulence factors. Virulence factors related to adhesion, iron acquisition and serum resistance were present in almost all strains. The factors fimC, ompA, crlA and traT were the most frequent in APEC isolates. The phylogenetic typing were done using the Clermont et al. (2000) method, which classifies E. coli strains into four main phylogenetic groups (A, B1, B2, and D). Our phylogenetic typing has shown that cellulitis and colisseptisemic isolates were more related to group D. Amplified ribossomal restriction analysis (ARDRA) was performed in colissepticemic and celullitis isolates from broiler chickens from Southern Brasil. The similarity among isolates were observed with a dendrogram based on the band pattern. Our results have shown that clones of celullitis and colissepticemic isolates could not be distinguished and there is no endemic clones in regions observed analised. The results of the present work give us a panorama of the susceptibility to antibiotics, virulence factors, phylogenetic typing and phylogenetic analyses currently found in APEC isolates from severe lesions of cellulites and colibacillosis in south Brazil. Besides that, these analyses could be helpful for monitoring and preventing outbreakes of colibacilosis. Controling the first stages of the disease and detecting more prevalent clones in this region may reduce the incidence of the disease.
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