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1.
Prev Vet Med ; 226: 106170, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38493570

RESUMO

Antimicrobial resistance within Staphylococcus pseudintermedius poses a significant risk for the treatment of canine pyoderma and as a reservoir for resistance and potential zoonoses, but few studies examine long-term temporal trends of resistance. This study assesses the antimicrobial resistance prevalence and minimum inhibitory concentration (MIC) trends in S. pseudintermedius (n=1804) isolated from canine skin samples at the Cornell University Animal Health Diagnostic Center (AHDC) between 2007 and 2020. Not susceptible (NS) prevalence, Cochran-Armitage tests, logrank tests, MIC50 and MIC90 quantiles, and survival analysis models were used to evaluate resistance prevalence and temporal trends to 23 antimicrobials. We use splines as predictors in accelerated failure time (AFT) models to model non-linear temporal trends in MICs. Multidrug resistance was common among isolates (47%), and isolates had moderate to high NS prevalence to the beta-lactams, chloramphenicol, the fluoroquinolones, gentamicin, the macrolides/lincosamides, the tetracyclines, and trimethoprim-sulfamethoxazole. However, low levels of NS to amikacin, rifampin, and vancomycin were observed. Around one third of isolates (38%) were found to be methicillin resistant S. pseudintermedius (MRSP), and these isolates had a higher prevalence of NS to all tested antimicrobials than methicillin susceptible isolates. Amongst the MRSP isolates, one phenotypically vancomycin resistant isolate (MIC >16 µg/mL) was identified, but genomic sequence data was unavailable. AFT models showed increasing MICs across time to the beta-lactams, chloramphenicol, the fluoroquinolones, gentamicin, and the macrolides/lincosamides, and decreasing temporal resistance (decreasing MICs) to doxycycline was observed amongst isolates. Notably, ATF modeling showed changes in MIC distributions that were not identified using Cochran-Armitage tests on prevalence, MIC quantiles, and logrank tests. Increasing resistance amongst these S. pseudintermedius isolates highlights the need for rational, empirical prescribing practices and increased antimicrobial resistance (AMR) surveillance to maintain the efficacy of current therapeutic agents. AFT models with non-linear predictors may be a useful, breakpoint-independent, surveillance tool alongside other modeling methods and antibiograms.


Assuntos
Anti-Infecciosos , Doenças do Cão , Infecções Estafilocócicas , Staphylococcus , Humanos , Animais , Cães , Vancomicina/uso terapêutico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Anti-Infecciosos/uso terapêutico , Cloranfenicol/uso terapêutico , Lincosamidas/uso terapêutico , Fluoroquinolonas , beta-Lactamas/uso terapêutico , Gentamicinas/uso terapêutico , Macrolídeos/uso terapêutico , Testes de Sensibilidade Microbiana/veterinária , Doenças do Cão/epidemiologia , Doenças do Cão/tratamento farmacológico , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/veterinária , Infecções Estafilocócicas/tratamento farmacológico
2.
Artigo em Inglês | MEDLINE | ID: mdl-38459616

RESUMO

AIMS: This report documents the exposure of passengers and crew of a commercial international flight to the zoonotic pathogen Brucella canis after an infected dog aborted in the passenger cabin of the aircraft. This case demonstrates the challenges associated with brucellosis screening and the risks that airline personnel, airport employees and travellers face when animals with unrecognized zoonotic infections are transported. METHODS/RESULTS: The public health investigation of this case was conducted by the Centers for Disease Control, the Illinois Department of Health and the Illinois Department of Agriculture, in collaboration with a local veterinary clinic and several academic and federal diagnostic laboratories. It included an extensive diagnostic evaluation of the dam and aborted foetuses to confirm a diagnosis of canine brucellosis. Passengers, airline personnel and staff from the veterinary clinic where the dogs were treated underwent risk assessments, and clinic staff also received detailed guidance regarding infection prevention practices. CONCLUSIONS: Animal shelters and breeding programs are recommended to screen dogs routinely for brucellosis, but it is not unusual for domestic or imported animals to have unknown health histories, including the dog's brucellosis status, at the time of purchase, adoption, or re-homing. Testing recommendations and requirements vary by state, making it challenging for state public health and animal health agencies to monitor and respond appropriately. This case highlights the importance of Brucella spp. screening in sexually intact dogs prior to breeding, purchase, or domestic or international transportation of the dogs. The transportation of pregnant dogs may present a previously unrecognized public health threat in addition to contributing to unnecessary stress and health risks for pregnant animals.

3.
Am J Vet Res ; 84(12)2023 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-37972472

RESUMO

OBJECTIVE: This study aims to assess the antimicrobial resistance (AMR) trends among Escherichia coli isolated from cats between 2008 and 2022, utilizing MIC data, within a one-health framework. SAMPLE: The study analyzed MIC results from 1,477 feline E coli isolates that were obtained from samples submitted to the Cornell University Animal Health Diagnostic Center, primarily from the northeastern US. METHODS: MIC values were categorized as susceptible or not susceptible using the Clinical and Laboratory Standards Institute breakpoints. Multidrug resistance (MDR) was analyzed using a Poisson regression model. Additionally, accelerated failure time models were employed to analyze MIC values. RESULTS: Out of the 1,477 E coli isolates examined, 739 (50%) showed susceptibility to all tested antimicrobials. Among the tested antimicrobials, cefazolin (69%) and ampicillin (74% for urinary tract isolates) exhibited the lowest susceptibility. Overall, 15% of isolates were not susceptible to cefovecin. E coli isolates were highly susceptible (> 95%) to antibiotics typically reserved for human use. Almost one-third of the isolates were classified as MDR, with nonurinary isolates more likely to exhibit an MDR pattern. A decrease in MICs for fluoroquinolones and gentamicin in recent years was identified. However, MICs for cephalexin increased from 2016 to 2022 and cefovecin from 2012 to 2019. CLINICAL RELEVANCE: This study highlights the challenge of AMR in feline medicine, emphasizing the importance of responsible antimicrobial use and surveillance to address E coli AMR. The related Currents in One Health by Cazer et al, JAVMA, December 2023, addresses additional feline antimicrobial stewardship topics.


Assuntos
Anti-Infecciosos , Escherichia coli , Gatos , Animais , Humanos , Farmacorresistência Bacteriana , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Testes de Sensibilidade Microbiana/veterinária
4.
J Am Vet Med Assoc ; 261(4): 451-462, 2023 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-36862545

RESUMO

Brucellosis is a highly infectious zoonotic disease of global significance due to its adverse impact on public health, economics, and trade. Despite being one of the most prevalent zoonoses worldwide, attention given to global brucellosis control and prevention has been inadequate. Brucella species of greatest one-health relevance in the US include those infecting dogs (Brucella canis), swine (Brucella suis), and cattle and domestic bison (Brucella abortus). Although not endemic in the US, Brucella melitensis warrants awareness as it poses a risk to international travelers. While brucellosis has been eradicated from domestic livestock in the US, its detection in US companion animals (B canis) and US wildlife reservoirs (B suis and B abortus) and enzootic presence internationally pose a threat to human and animal health, warranting its spotlight on the one-health stage. The challenges of B canis diagnosis in humans and dogs is addressed in more detail in the companion Currents in One Health by Guarino et al, AJVR, April 2023. Human consumption of unpasteurized dairy products and occupational exposure of laboratory diagnosticians, veterinarians, and animal care providers are responsible for human exposures reported to the US CDC. Diagnosis and treatment of brucellosis is challenging due to the limitations of diagnostic assays and the tendency of Brucella spp to produce nonspecific, insidious clinical signs and evade antimicrobial therapy, making prevention essential. This review will focus on zoonotic considerations for Brucella spp found within the US along with their epidemiology, pathophysiology, clinical presentation, treatment, and control strategies.


Assuntos
Bison , Brucelose , Doenças dos Bovinos , Doenças do Cão , Saúde Única , Doenças dos Suínos , Bovinos , Animais , Humanos , Estados Unidos/epidemiologia , Cães , Suínos , Brucelose/epidemiologia , Brucelose/veterinária , Zoonoses/epidemiologia , Brucella abortus , Animais Selvagens
5.
Am J Vet Res ; 84(4)2023 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-36892988

RESUMO

OBJECTIVE: Brucella canis is a zoonotic bacterial pathogen of dogs that is notoriously difficult to diagnose and treat. Humans can become infected with B canis when an infected pet dog is brought into their home. Our objectives were to describe the clinical presentation and outcomes in dogs treated for B canis and evaluate the performance of the quantitative serologic canine Brucella multiplex (CBM) assay for monitoring treatment response. ANIMALS: Diagnostic records from the Animal Health Diagnostic Center at Cornell University were retrospectively reviewed (2017-2022) for dogs that underwent repeat B canis serologic testing. Medical records were requested to compare the clinical presentations and outcomes for dogs that underwent treatment for B canis. Changes in CBM antibody values were compared between dogs with and without resolution of clinical signs. RESULTS: While treatment protocols varied in the 30 treated dogs meeting the inclusion criteria, poly-antimicrobial therapy was prescribed in 97% (29/30) of cases. Gait abnormalities, spinal pain, and discospondylitis were the most common clinical abnormalities. A difference (P value = .0075) in the percent decrease in CBM assay PO1 antibody values was found in dogs with resolved clinical signs. CLINICAL RELEVANCE: Young dogs presenting with recurring lameness or back pain should be screened for B canis infection. A 40% decline in CBM assay values 2 to 6 months posttreatment can be supportive of response to treatment. Further prospective studies are needed to determine the ideal B canis treatment regimen and the magnitude of public health risks associated with maintaining neutered B canis-infected animals as pets.


Assuntos
Brucella canis , Brucelose , Doenças do Cão , Humanos , Animais , Cães , Brucelose/diagnóstico , Brucelose/tratamento farmacológico , Brucelose/veterinária , Formação de Anticorpos , Estudos Retrospectivos , Doenças do Cão/diagnóstico , Doenças do Cão/tratamento farmacológico , Doenças do Cão/microbiologia , Resultado do Tratamento , Anticorpos Antibacterianos
6.
PLoS One ; 17(1): e0262868, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35061843

RESUMO

A serological COVID-19 Multiplex Assay was developed and validated using serum samples from convalescent patients and those collected prior to the 2020 pandemic. After initial testing of multiple potential antigens, the SARS-CoV-2 nucleocapsid protein (NP) and receptor-binding domain (RBD) of the spike protein were selected for the human COVID-19 Multiplex Assay. A comparison of synthesized and mammalian expressed RBD proteins revealed clear advantages of mammalian expression. Antibodies directed against NP strongly correlated with SARS-CoV-2 virus neutralization assay titers (rsp = 0.726), while anti-RBD correlation was moderate (rsp = 0.436). Pan-Ig, IgG, IgA, and IgM against NP and RBD antigens were evaluated on the validation sample sets. Detection of NP and RBD specific IgG and IgA had outstanding performance (AUC > 0.90) for distinguishing patients from controls, but the dynamic range of the IgG assay was substantially greater. The COVID-19 Multiplex Assay was utilized to identify seroprevalence to SARS-CoV-2 in people living in a low-incidence community in Ithaca, NY. Samples were taken from a cohort of healthy volunteers (n = 332) in early June 2020. Only two volunteers had a positive result on a COVID-19 PCR test performed prior to serum sampling. Serological testing revealed an exposure rate of at least 1.2% (NP) or as high as 5.7% (RBD), higher than the measured incidence rate of 0.16% in the county at that time. This highly sensitive and quantitative assay can be used for monitoring community exposure rates and duration of immune response following both infection and vaccination.


Assuntos
Anticorpos Antivirais/química , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/sangue , COVID-19/epidemiologia , Teste Sorológico para COVID-19/normas , Proteínas do Nucleocapsídeo de Coronavírus/química , Monitoramento Epidemiológico , Feminino , Humanos , Imunoglobulina A/química , Imunoglobulina A/imunologia , Imunoglobulina G/química , Imunoglobulina G/imunologia , Imunoglobulina M/química , Imunoglobulina M/imunologia , Masculino , Pessoa de Meia-Idade , New York/epidemiologia , Fosfoproteínas/química , Fosfoproteínas/imunologia , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , SARS-CoV-2/classificação , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/química
7.
Front Vet Sci ; 8: 743067, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34631864

RESUMO

Cases of cranial nuchal bursitis associated with Borrelia burgdorferi infection have not been thoroughly described. Here, we describe the case of a 17-year-old mare that was presented for low head carriage, dull demeanor, and resistance to haltering. Imaging supported a diagnosis of nuchal bursitis, and bursoscopy with surgical debridement of the nuchal bursa was performed. B. burgdorferi was identified by molecular diagnostics in serial samples of the bursal fluid, with no other organisms identified. Serology revealed significant elevation in antibodies directed against OspA of B. burgdorferi, but not the typical infection markers, OspC and OspF. Intravenous ceftiofur was administered for 80 days, and the nuchal bursa was directly injected with ceftiofur. The mare recovered and was able to return to work with no recrudescence of clinical signs over the following year to date. Infection with B. burgdorferi should be considered as a differential in cases of septic nuchal bursitis.

8.
J Virol ; 95(11)2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33692203

RESUMO

The origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing the global coronavirus disease 19 (COVID-19) pandemic, remains a mystery. Current evidence suggests a likely spillover into humans from an animal reservoir. Understanding the host range and identifying animal species that are susceptible to SARS-CoV-2 infection may help to elucidate the origin of the virus and the mechanisms underlying cross-species transmission to humans. Here we demonstrated that white-tailed deer (Odocoileus virginianus), an animal species in which the angiotensin converting enzyme 2 (ACE2) - the SARS-CoV-2 receptor - shares a high degree of similarity to humans, are highly susceptible to infection. Intranasal inoculation of deer fawns with SARS-CoV-2 resulted in established subclinical viral infection and shedding of infectious virus in nasal secretions. Notably, infected animals transmitted the virus to non-inoculated contact deer. Viral RNA was detected in multiple tissues 21 days post-inoculation (pi). All inoculated and indirect contact animals seroconverted and developed neutralizing antibodies as early as day 7 pi. The work provides important insights into the animal host range of SARS-CoV-2 and identifies white-tailed deer as a susceptible wild animal species to the virus.IMPORTANCEGiven the presumed zoonotic origin of SARS-CoV-2, the human-animal-environment interface of COVID-19 pandemic is an area of great scientific and public- and animal-health interest. Identification of animal species that are susceptible to infection by SARS-CoV-2 may help to elucidate the potential origin of the virus, identify potential reservoirs or intermediate hosts, and define the mechanisms underlying cross-species transmission to humans. Additionally, it may also provide information and help to prevent potential reverse zoonosis that could lead to the establishment of a new wildlife hosts. Our data show that upon intranasal inoculation, white-tailed deer became subclinically infected and shed infectious SARS-CoV-2 in nasal secretions and feces. Importantly, indirect contact animals were infected and shed infectious virus, indicating efficient SARS-CoV-2 transmission from inoculated animals. These findings support the inclusion of wild cervid species in investigations conducted to assess potential reservoirs or sources of SARS-CoV-2 of infection.

9.
Animals (Basel) ; 10(10)2020 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-33023217

RESUMO

Recent removal and relocation of feral donkeys from vast public lands to more concentrated holding pens, training facilities, and offsite adoption locations raises several health and welfare concerns. Very little is known regarding the common equid pathogens that are circulating within the feral donkey population in and around Death Valley National Park, California, USA. The aim of this study was to utilize serologic assays to assess previous exposure of these donkeys to equine herpesvirus 1 (EHV-1), equine influenza (EIV), West Nile virus (WNV), and Borrelia burgdorferi (the causative agent of Lyme disease). The results of this study indicate that this feral equid population is mostly naïve and likely susceptible to these common equid pathogens upon removal from the wild.

10.
Vaccine ; 35(40): 5373-5380, 2017 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-28866291

RESUMO

The influenza A virus undergoes genetic drift and shift, leaving the general population susceptible to emerging pandemic strains, despite seasonal flu vaccination. Here we describe a single dose influenza vaccine derived from recombinant outer membrane vesicles (rOMVs) that display an antigen-mapped heterospecies tandem sequence of the M2 protein from the influenza A virus, released over 30days from poly(lactic-co-glycolide) (PLGA) microparticles. Four weeks post vaccination, BALB/c mice developed high anti-M2e IgG titers that were equivalent to those generated at 8weeks in a typical prime/boost vaccine regimen. Challenge of mice with a lethal dose of mouse adapted influenza virus PR8 (H1N1) 10weeks post vaccination resulted in 100% survival for both rOMV single-dose microparticle and prime/boost vaccinated mice. Anti-M2e IgG1 and IgG2a antibody titers were weighted toward IgG1, but splenocytes isolated from rOMV single-dose microparticle vaccinated mice produced high levels of IFNγ relative to IL-4 in response to stimulation with M2e peptides, supporting a more Th1 biased immune response. The protective immune response was long lasting, eliciting sustained antibody titers and 100% survival of mice challenged with a lethal dose of PR8 six months post initial vaccination. Together, these data support the potential of controlled release rOMVs as an effective single dose, long lasting and rapidly effective vaccine to protect against influenza.


Assuntos
Vacinas contra Influenza/uso terapêutico , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Micropartículas Derivadas de Células/imunologia , Preparações de Ação Retardada , Feminino , Humanos , Imunoglobulina G/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Interleucina-4/metabolismo , Ácido Láctico/química , Camundongos , Camundongos Endogâmicos BALB C , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico
11.
Vaccine ; 35(33): 4140-4147, 2017 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-28668566

RESUMO

Borrelia burgdorferi can induce Lyme disease. Approved Lyme vaccines for horses are currently not available. In an effort to protect horses, veterinarians are using Lyme vaccines licensed for dogs. However, data to assess the response of horses to, or determine the efficacy of this off-label vaccine use are missing. Here, antibodies against outer surface protein A (OspA), OspC, and OspF were quantified in diagnostic serum submissions from horses with a history of vaccination with canine Lyme vaccines. The results suggested that many horses respond with low and often short-lasting antibody responses. Subsequently, four experimental vaccination trials were performed. First, we investigated antibody responses to three canine vaccines in B. burgdorferi-naïve horses. One killed bacterin vaccine induced antibodies against OspC. OspA antibodies were low for all three vaccines and lasted less than 16weeks. The second trial tested the impact of the vaccine dose using the OspA/OspC inducing bacterin vaccine in horses. A 2mL dose produced higher OspA and OspC antibody values than a 1mL dose. However, the antibody response again quickly declined, independent of dose. Third, the horses were vaccinated with 2 doses of a recombinant OspA vaccine. Previous vaccination and/or environmental exposure enhanced the magnitude and longevity of the OspA antibody response to about 20weeks. Last, the influence of intramuscular versus subcutaneous vaccine administration was investigated for the recombinant OspA vaccine. OspA antibody responses were not influenced by injection route. The current work highlights that commercial Lyme vaccines for dogs induce only transient antibody responses in horses which can also be of low magnitude. Protection from infection with B. burgdorferi should not be automatically assumed after vaccinating horses with Lyme vaccines for dogs.


Assuntos
Anticorpos Antibacterianos/sangue , Formação de Anticorpos , Borrelia burgdorferi/imunologia , Doenças dos Cavalos/prevenção & controle , Vacinas contra Doença de Lyme/administração & dosagem , Doença de Lyme/veterinária , Animais , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Cães , Cavalos , Injeções Intramusculares , Doença de Lyme/prevenção & controle , Fatores de Tempo , Vacinação/métodos , Medicina Veterinária/métodos
12.
Vaccine ; 34(10): 1252-8, 2016 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-26827663

RESUMO

Currently approved influenza vaccines predominantly protect through antibodies directed against the highly variable glycoprotein hemagglutinin (HA), necessitating annual redesign and formulation based on epidemiological prediction of predominant circulating strains. More conserved influenza protein sequences, such as the ectodomain of the influenza M2 protein, or M2e, show promise as a component of a universal influenza A vaccine, but require a Th1-biased immune response for activity. Recently, recombinant, bacterially derived outer membrane vesicles (OMVs) demonstrated potential as a platform to promote a Th1-biased immune response to subunit antigens. Here, we engineer three M2e-OMV vaccines and show that all elicit strong IgG titers, with high IgG2a:IgG1 ratios, in BALB/c mice. Additionally, the administration of one M2e-OMV construct containing tandem heterologous M2e peptides (M2e4xHet-OMV) resulted in 100% survival against lethal doses of the mouse-adapted H1N1 influenza strain PR8. Passive transfer of antibodies from M2e4xHet-OMV vaccinated mice to unvaccinated mice also resulted in 100% survival to challenge, indicating that protection is driven largely via antibody-mediated immunity. The potential mechanism through which M2e-OMVs initiated the immune response was explored and it was found that the constructs triggered TLR1/2, TLR4, and TLR5. Our data indicate that OMVs have potential as a platform for influenza A vaccine development due to their unique adjuvant profile and intrinsic pathogen-mimetic nature.


Assuntos
Vesículas Extracelulares/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Proteínas da Matriz Viral/imunologia , Animais , Anticorpos Antivirais/sangue , Escherichia coli/metabolismo , Feminino , Imunidade Inata , Imunoglobulina G/sangue , Vírus da Influenza A Subtipo H1N1 , Camundongos Endogâmicos BALB C , Nanopartículas , Receptores Toll-Like/agonistas , Vacinas Sintéticas/imunologia
13.
Sci Rep ; 5: 15237, 2015 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-26482295

RESUMO

The central enzyme in the Campylobacter jejuni asparagine-linked glycosylation pathway is the oligosaccharyltransferase (OST), PglB, which transfers preassembled glycans to specific asparagine residues in target proteins. While C. jejuni PglB (CjPglB) can transfer many diverse glycan structures, the acceptor sites that it recognizes are restricted predominantly to those having a negatively charged residue in the -2 position relative to the asparagine. Here, we investigated the acceptor-site preferences for 23 homologs with natural sequence variation compared to CjPglB. Using an ectopic trans-complementation assay for CjPglB function in glycosylation-competent Escherichia coli, we demonstrated in vivo activity for 16 of the candidate OSTs. Interestingly, the OSTs from Campylobacter coli, Campylobacter upsaliensis, Desulfovibrio desulfuricans, Desulfovibrio gigas, and Desulfovibrio vulgaris, exhibited significantly relaxed specificity towards the -2 position compared to CjPglB. These enzymes glycosylated minimal N-X-T motifs in multiple targets and each followed unique, as yet unknown, rules governing acceptor-site preferences. One notable example is D. gigas PglB, which was the only bacterial OST to glycosylate the Fc domain of human immunoglobulin G at its native 'QYNST' sequon. Overall, we find that a subset of bacterial OSTs follow their own rules for acceptor-site specificity, thereby expanding the glycoengineering toolbox with previously unavailable biocatalytic diversity.


Assuntos
Edulcorantes/química , Edulcorantes/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biologia Computacional , Mineração de Dados , Genoma Bacteriano , Genômica , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação , Hexosiltransferases/química , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Conformação Molecular , Filogenia , Polissacarídeos , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/metabolismo , Especificidade por Substrato
14.
Biotechnol J ; 8(12): 1445-51, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23894044

RESUMO

Predicting the structural consequences of site-specific glycosylation remains a major challenge due in part to the lack of convenient experimental tools for rapidly determining how glycosylation influences protein folding. To address this shortcoming, we developed a genetic selection that directly links the in vivo folding of asparagine-linked (N-linked) glycoproteins with antibiotic resistance. Using this assay, we identified three known or putative glycoproteins from Campylobacter jejuni (Peb3, CjaA, and Cj0610c) whose folding was significantly affected by N-glycosylation. We also used the genetic selection to isolate a glycoengineered variant of the Escherichia coli colicin E7 immunity protein (Im7) whose intracellular folding and stability were enhanced as a result of N-glycosylation. In addition to monitoring the effect of glycan attachment on protein folding in living cells, this strategy could easily be extended for optimizing protein folding in vivo and engineering glycosylation enzymes, pathways, and hosts for optimal performance.


Assuntos
Asparagina/metabolismo , Engenharia Genética/métodos , Glicoproteínas , Dobramento de Proteína , Estabilidade Proteica , Asparagina/química , Asparagina/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação
15.
Glycobiology ; 22(5): 596-601, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22068020

RESUMO

Asparagine-linked (N-linked) protein glycosylation has been observed in all domains of life, including most recently in bacteria and is now widely considered a universal post-translational modification. However, cell-based production of homogeneous glycoproteins for laboratory and preparative purposes remains a significant challenge due in part to the complexity of this process in vivo. To address this issue, an easily available and highly controllable Escherichia coli-based cell-free system for the production of N-linked glycoproteins was developed. The method was created by coupling existing in vitro translation systems with an N-linked glycosylation pathway reconstituted from defined components. The translation/glycosylation system yielded efficiently glycosylated target proteins at a rate of hundreds of micrograms/milliliters in half a day. This is the first time a prokaryote-based cell-free protein synthesis system has generated N-linked glycoproteins.


Assuntos
Glicoproteínas/biossíntese , Células Procarióticas/metabolismo , Biossíntese de Proteínas , Sistema Livre de Células , Escherichia coli/metabolismo , Glicosilação
16.
Appl Environ Microbiol ; 77(3): 871-81, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21131519

RESUMO

The Campylobacter jejuni pgl gene cluster encodes a complete N-linked protein glycosylation pathway that can be functionally transferred into Escherichia coli. In this system, we analyzed the interplay between N-linked glycosylation, membrane translocation and folding of acceptor proteins in bacteria. We developed a recombinant N-glycan acceptor peptide tag that permits N-linked glycosylation of diverse recombinant proteins expressed in the periplasm of glycosylation-competent E. coli cells. With this "glycosylation tag," a clear difference was observed in the glycosylation patterns found on periplasmic proteins depending on their mode of inner membrane translocation (i.e., Sec, signal recognition particle [SRP], or twin-arginine translocation [Tat] export), indicating that the mode of protein export can influence N-glycosylation efficiency. We also established that engineered substrate proteins targeted to environments beyond the periplasm, such as the outer membrane, the membrane vesicles, and the extracellular medium, could serve as substrates for N-linked glycosylation. Taken together, our results demonstrate that the C. jejuni N-glycosylation machinery is compatible with distinct secretory mechanisms in E. coli, effectively expanding the N-linked glycome of recombinant E. coli. Moreover, this simple glycosylation tag strategy expands the glycoengineering toolbox and opens the door to bacterial synthesis of a wide array of recombinant glycoprotein conjugates.


Assuntos
Escherichia coli/metabolismo , Glicoproteínas/biossíntese , Glicoproteínas/metabolismo , Polissacarídeos/metabolismo , Engenharia de Proteínas/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Meios de Cultura , Escherichia coli/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glicoproteínas/química , Glicoproteínas/genética , Glicosilação , Periplasma/metabolismo , Transporte Proteico , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo
17.
Protein Sci ; 19(10): 2006-13, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20669235

RESUMO

We have developed a filamentous phage display system for the detection of asparagine-linked glycoproteins in Escherichia coli that carry a plasmid encoding the protein glycosylation locus (pgl) from Campylobacter jejuni. In our assay, fusion of target glycoproteins to the minor phage coat protein g3p results in the display of glycans on phage. The glyco-epitope displayed on phage is the product of biosynthetic enzymes encoded by the C. jejuni pgl pathway and minimally requires three essential factors: a pathway for oligosaccharide biosynthesis, a functional oligosaccharyltransferase, and an acceptor protein with a D/E-X(1)-N-X(2)-S/T motif. Glycosylated phages could be recovered by lectin chromatography with enrichment factors as high as 2 × 10(5) per round of panning and these enriched phages retained their infectivity after panning. Using this assay, we show that desired glyco-phenotypes can be reliably selected by panning phage-displayed glycoprotein libraries on lectins that are specific for the glycan. For instance, we used our phage selection to identify permissible residues in the -2 position of the bacterial consensus acceptor site sequence. Taken together, our results demonstrate that a genotype-phenotype link can be established between the phage-associated glyco-epitope and the phagemid-encoded genes for any of the three essential components of the glycosylation process. Thus, we anticipate that our phage display system can be used to isolate interesting variants in any step of the glycosylation process, thereby making it an invaluable tool for genetic analysis of protein glycosylation and for glycoengineering in E. coli cells.


Assuntos
Glicoproteínas/genética , Inovirus/genética , Biblioteca de Peptídeos , Proteínas Virais/genética , Asparagina/genética , Asparagina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Inovirus/metabolismo , Mutação , Oligossacarídeos/metabolismo , Plasmídeos/genética , Polissacarídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Virais/metabolismo
18.
J Gen Intern Med ; 21(1): 7-12, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16423117

RESUMO

OBJECTIVE: To determine the prevalence and influence of specific attending teaching practices on student evaluations of the quality of attendings' teaching in the inpatient component of Internal Medicine clerkships. DESIGN: Nationwide survey using a simple random sample. SETTING: One hundred and twenty-one allopathic 4-year medical schools in the United States. PARTICIPANTS: A total of 2,250 fourth-year medical students. MEASUREMENTS AND MAIN RESULTS: In the spring of 2002, student satisfaction with the overall quality of teaching by attendings in the inpatient component of Internal Medicine clerkships was measured on a 5-point scale from very satisfied to very dissatisfied (survey response rate, 68.3%). Logistic regression was used to determine the association of specific teaching practices with student evaluations of the quality of their attendings' teaching. Attending physicians' teaching practices such as engaging students in substantive discussions (odds ratio (OR)=3.0), giving spontaneous talks and prepared presentations (OR=1.6 and 1.8), and seeing new patients with the team (OR=1.2) were strongly associated with higher student satisfaction, whereas seeming rushed and eager to finish rounds was associated with lower satisfaction (OR=0.6). CONCLUSION: Findings suggest that student satisfaction with attendings' teaching is high overall but there is room for improvement. Specific teaching behaviors used by attendings affect student satisfaction. These specific behaviors could be taught and modified for use by attendings and clerkship directors to enhance student experiences during clerkships.


Assuntos
Atitude do Pessoal de Saúde , Estágio Clínico , Comportamento do Consumidor , Medicina Interna/educação , Corpo Clínico Hospitalar , Ensino/métodos , Adulto , Feminino , Humanos , Modelos Logísticos , Masculino , Análise Multivariada , Estudantes de Medicina/psicologia , Inquéritos e Questionários , Estados Unidos
19.
Ann Surg ; 241(2): 373-80, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15650650

RESUMO

OBJECTIVE: To identify factors that predict fourth- and fifth-year surgical resident satisfaction of attending teaching quality. SUMMARY BACKGROUND DATA: With the training of surgical residents undergoing major changes, a key issue facing surgical educators is whether high-quality surgeons can still be produced. Innovative techniques (eg, computer simulation surgery) are being developed to substitute partially for conventional teaching methods. However, an aspect of training that cannot be so easily replaced is the faculty-resident interaction. This study investigates resident perceptions of attending teaching quality and the factors associated with this faculty-resident interaction to identify predictors of resident educational satisfaction. METHODS: A national survey of clinical fourth- and fifth-year surgery residents in 125 academically affiliated general surgery training programs was performed. The survey contained 67 questions and addressed demographics, hospital, and service characteristics, as well as surgery, education, and clinical care-related factors. Univariate analyses were performed to describe the characteristics of the sample; multivariate analyses were performed to evaluate the factors associated with resident educational satisfaction. RESULTS: The response rate was 61.5% (n = 756). Average age was 32 years; most were male (79%), white (72%), and married (69%); 42% had children. Ninety-five percent of respondents graduated from U.S. medical schools, and the average debt was $80,307. Of 20 potentially mutable factors, 6 variables had positive associations with resident education satisfaction and 7 had negative associations. Positive factors included the resident being the operating surgeon in major surgeries, substantial citing of evidence-based literature by the attending, attending physicians giving spontaneous or unplanned presentations, increasing the continuity of care, clinical teaching aimed at the chief resident level, and having clinical decisions made together by both the attending and resident. There were 7 negative factors such as overly supervising in surgery, being interrupted so much that teaching was ineffective, and attending physicians being rushed and/or eager to finish rounds. CONCLUSION: This study identifies several factors that were associated with resident educational satisfaction. It offers the perspective of the learners (ie, residents) and, importantly, highlights mutable factors that surgery faculty (and departments) may consider changing to improve surgery resident education and satisfaction. Improving such satisfaction may help to produce a better product.


Assuntos
Cirurgia Geral/educação , Internato e Residência , Corpo Clínico Hospitalar , Ensino , Adulto , Feminino , Humanos , Satisfação no Emprego , Masculino , Pessoa de Meia-Idade , Estados Unidos
20.
Surgery ; 136(3): 567-72, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15349103

RESUMO

BACKGROUND: The number of general surgery (GS) residency applicants had been decreasing before 2003. This national survey of fourth-year medical students elucidates factors related to the basic surgery clerkship that are associated with the decision to enter a GS residency. METHODS: A national sample of 2250 fourth-year medical students from all 4-year allopathic US medical schools was surveyed in spring 2002. Multivariate analyses were performed to identify mutable predictors for students entering GS. RESULTS: Data from 1531 fourth-year medical students from 121 different medical schools (response rate=68%) showed that 5.6% planned to enter GS. In multivariate analyses, the strongest predictor of entering GS was satisfaction with the quality of attending teaching (odds ratio 2.14, P <.01) in surgery clerkships. Several clerkship factors, such as frequency of call nights and total hours worked., were not as strongly associated with entering GS residency, Subsequent analyses showed that predictors of satisfaction with the quality of attending teaching included intraoperative activities (ie, suturing, cutting, and stapling), having attending-led rounds, and performing a history and physical with an attending. Significant negative predictors of satisfaction included observing or retracting only in surgery. CONCLUSIONS: In this national survey, factors are identified that are significantly associated with students entering a GS residency. Some of these mutable factors may increase the pool of GS residency applicants.


Assuntos
Escolha da Profissão , Educação Médica/normas , Cirurgia Geral/educação , Internato e Residência , Estudantes de Medicina/psicologia , Adulto , Coleta de Dados , Educação Médica/estatística & dados numéricos , Feminino , Humanos , Internato e Residência/estatística & dados numéricos , Satisfação no Emprego , Masculino , Corpo Clínico Hospitalar/educação , Corpo Clínico Hospitalar/normas , Ensino/normas
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