Acid stress response in campylobacter jejuni

Dissertação de Mestrado Integrado em Medicina VeterináriaConsidering that the acid tolerance is an important virulence factor of foodborne pathogens and the expressive increasing of incidence of Campylobacter jejuni in recent years as etiologic agent of human campylobacteriosis, this work aimed to evaluate the ability of C. jejuni to survive under acid stress. Strains of different origins (reference, turkey isolates) and tolerance to particular stresses (acid and temperature) were tested for survival in liquid defined media with pH values of 5.0 and 7.0 adjusted with 1M HCl. Experimental conditions were performed at 2 different temperatures: 4 ºC and 37 ºC. C. jejuni cells were found to be very sensitive to acid stress at 37 ºC, whereas they were more resistant at 4 ºC. A strain effect was observed at 37 ºC. Temperature of 37 ºC combined with acid stress allowed a rapid decrease in the C. jejuni population, whereas low temperature (4ºC) considerably decreased the effect of acid stress. The survival curves were either described by the Weibull or traditional first-order model and goodness of fit of these models was investigated. Regression coefficients (R2), root mean square error (RMSE) and correlation plots suggestted that Weibull model produced a better fit to the data than the traditional model. Fluorescence Ratio Imaging Microscopy (FRIM) was used to determine intracellular pH (pHi) as an indicator of the physiological state of C. jejuni cells at the single cell level after treatment with hydrochloride acid (1M HCl, pH 4.0) in liquid defined medium. For all the tested strains pHi of healthy cells was found to be above 7.0. After exposure to HCl in liquid medium an immediate decline in pHi to 5.5 (detection limit) was observed in the majority of cells (75%) of one strain within 15 minutes. The FRIM results revealed that the subpopulations with pHi>5.5 increased for this strain, especially subpopulations with 5.5<pHi<6.0 and 6.0<pHi<6.5 following 200 min. of exposure to HCl. This indicates that some strains of C. jejuni may employ certain recovery strategies to extrude protons and to increase pHi.RESUMO - Considerando que a tolerância ao ácido é um importante factor de virulência de agentes patogénicos veiculados por alimentos, condicionando a sobrevivência dos microrganismos, e o expressivo aumento, nos últimos anos, da incidência de Campylobacter jejuni como agente etiológico de campilobacteriose humana, este trabalho teve como objectivo avaliar a capacidade do patogénico C. jejuni sobreviver em condições de stresse ácido. Estirpes de diferentes origens (referência, isolados de peru) sujeitas a diferentes factores de stresse (acidez e temperatura) foram testadas avaliando-se a sua sobrevivência em meio líquido (pH 5.0 e 7.0). As condições experimentais foram efectuadas a duas temperaturas diferentes: 4 ºC e 37 ºC. Células de C. jejuni mostraram-se muitos sensíveis a stresse ácido a 37 ºC, enquanto a 4 ºC foram mais resistentes. O efeito estirpe foi evidenciado a 37 ºC. A temperatura 37ºC em combinação com um pH 5.0 (stresse ácido) causou rápido decréscimo da população de C. jejuni, enquanto que a 4ºC o efeito do stresse ácido diminuiu consideravelmente. As curvas de sobrevivência foram descritas pelos modelos Weibull ou pelo modelo clássico de inactivação de primeira ordem, tendo-se concluído através dos coeficientes de regressão (R2), raízes quadradas do erro médio (RMSE) e gráficos de correlação que o modelo Weibull se adequa melhor aos dados apresentados que o modelo tradicionalmente utilizado. O racio da emissão de fluorescência medida pela técnica de microscopia óptica invertida foi utilizado para determinar o pH intracelular (pHi) como indicador do estado fisiológico das células de C. jejuni, a um nível celular único, após aplicação de um tratamento com ácido clorídrico (1M HCl, pH 4.0) em meio de cultura líquido. Para todas as estirpes testadas, o pHi de células saudáveis encontrava-se acima de 7.0. Após exposição a HCl em meio líquido, foi detectado para uma das estirpes, um decréscimo imediato do pHi para 5.5 (limite de detecção) na maioria das células observadas (75%) ao fim de 15 minutos. Os resultados da medição de fluorescência revelaram um aumento de subpopulações com pHi>5.5 para essa estirpe, nomeadamente subpopulações com 5.5<pHi<6.0 e 6.0<pHi<6.5 em 200 min. de exposição a HCl. Estes resultados indicam que algumas estirpes de C. jejuni poderão utilizar determinadas estratégias de recuperação para exteriorizar protões e elevar o pHi

Ameloblastomas: clinicopathological features from 70 cases diagnosed in a single Oral Pathology service in an 8-year period

Ameloblastomas are odontogenic tumors that can present some distinct clinicopathological profiles when comparing different populations and studies. Objectives: The aim of the present study was to analyze the clinicopathological features from a series of ameloblastomas diagnosed in a single Oral Pathology service in Brazil in an 8-year period. Study Design: The files were revised and all cases diagnosed as ameloblastomas in the period were retrieved. All hematoxylin and eosin stained histological slides were reviewed and all clinical and radiological information were obtained through a review of the laboratory forms. Data were descriptively analyzed and a comparison was performed with the different ameloblastomas subtypes. Results: Seventy ameloblastomas composed the final sample, including 57 (81%) solid/multicystic, 9 (13%) unicystic, 2 (3%) desmoplastic and 2 (3%) peripheral ameloblastomas. Mean age of the affected patients was in the forth decade of life and there was a slight male predominance. Most tumors presented as multilocular radiolucencies, were located in the posterior mandible and showed the follicular and plexiform histological patterns. There was no difference on the mean age of the patients affected by solid and unicystic ameloblastomas. Conclusions: The present results showed that the clinicopathological features of the ameloblastomas included in this Brazilian sample were similar to the features described in most other worldwide populations

Systematic Review on Global Epidemiology of Methicillin-Resistant Staphylococcus pseudintermedius: Inference of Population Structure from Multilocus Sequence Typing Data

Background and rationale: Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is a major cause of infections in dogs, also posing a zoonotic risk to humans. This systematic review aimed to determine the global epidemiology of MRSP and provide new insights into the population structure of this important veterinary pathogen.Methodology: Web of Science was searched systematically for articles reporting data on multilocus sequence typing (MLST) of S. pseudintermedius isolates from dogs or other animal or human patients and carriers. Data from the eligible studies were then integrated with data from the MLST database for this species. Analysis of MLST data was performed with eBURST and ClonalFrame, and the proportion of MRSP isolates resistant to selected antimicrobial drugs was determined for the most predominant clonal complexes.Results: Fifty-eight studies published over the last 10 years were included in the review. MRSP represented 76% of the 1428 isolates characterized by the current MLST scheme. The population of S. pseudintermedius was highly diverse and included five major MRSP clonal complexes (CCs). CC71, previously described as the epidemic European clone, is now widespread worldwide. In Europe, CC258, which is more frequently susceptible to enrofloxacin and aminoglycosides, and more frequently resistant to sulphonamides/trimethoprim than CC71, is increasingly reported in various countries. CC68, previously described as the epidemic North American clone, is frequently reported in this region but also in Europe, while CC45 (associated with chloramphenicol resistance) and CC112 are prevalent in Asia. It was estimated that clonal diversification in this species is primarily driven by homologous recombination (r/m=7.52).Conclusion: This study provides evidence that S. pseudintermedius has an epidemic population structure, in which five successful MRSP clones with specific traits regarding antimicrobial resistance, genetic diversity and geographical distribution have emerged upon a weakly clonal background through acquisition of SCCmec and other mobile genetic elements

Comparative immunoexpression of ICAM-1, TGF-?1 and ki-67 in periapical and residual cysts

This study compared the immunohistochemical expression of ki-67, transforming growth factor beta 1 (TGF-?1) and intercellular adhesion molecule-1 (ICAM-1) in inflammatory periapical cysts and residual cysts. The study sample was composed by 25 periapical cysts and 25 residual cysts and immunohistochemical reactions were carried out using antibodies directed against ICAM-1, TGF-?1 and ki-67. Clinical, radiological, gross, histological and immunohistochemical data were tabulated for descriptive and comparative analysis using the SPSS software and differences were considered statistically significant when p<0.05%. There were no differences between the expression of ICAM-1 (p=0.239) and TGF-?1 (p=0.258) when comparing both groups. Ki-67 labeling index was higher in residual cysts compared to periapical cysts (p=0.017). Results from the present study suggest that some specific inflammatory stimuli on residual cysts would modulate their mechanisms of etiopathogenesis, growing and repair

DQ High genotypic diversity among methicillin-resistant Staphylococcus pseudintermedius isolated from canine infections in Denmark

BACKGROUND: Methicillin-resistant Staphylococcus pseudintermedius (MRSP) has emerged globally in companion animals in the last decade. In Europe, the multidrug-resistant sequence type (ST)71 is widespread, but recently other clones have appeared. The objective of this study was to examine genotypic diversity and antimicrobial resistance of clinical MRSP isolates obtained from dogs, including dogs sampled on multiple occasions, in Denmark over a six-year period. For that purpose a total of 46 clinical MRSP isolates obtained from 36 dogs between 2009 and 2014 were subjected to antimicrobial susceptibility testing, multilocus-sequence typing (MLST) and SCCmec typing. RESULTS: Twenty-three sequence types were identified with ST71, mostly associated with SCCmec II-III, as the most common occurring in 13 dogs. Among the remaining 33 isolates, 19 belonged to clonal complex (CC)258 comprising ST258-SCCmec IV and its single- and double-locus variants. These were susceptible to 4–7 of the 22 antibiotics tested, whereas CC71 isolates were susceptible to only 2–5 antibiotics. Clone-specific differences were especially pronounced for fluoroquinolones and aminoglycosides with most CC71 isolates being resistant and almost all CC258 isolates being susceptible. Sixteen of the 19 CC258 isolates had oxacillin MICs of 0.5 g/L, whereas MICs for CC71 isolates were consistently above 4 g/L. Four of five dogs representing multiple isolates had distinct STs on different sampling events. CONCLUSIONS: The overall genotypic diversity of MRSP is high in Denmark indicating multiple acquisitions of SCCmec into distinct clones, and mutational evolution, which appears to be particularly rapid for certain ancestral clones such as ST258. ST71-SCCmec II-III is the most common MRSP lineage and is typically multidrug-resistant. CC258-SCCmec IV isolates, which emerged in Denmark since 2012, display susceptibility to a wider range of antimicrobials. The isolation of distinct STs in individual dogs over time suggests repeated exposure or short-term genetic evolution of MRSP clones within patients

Clinical and laboratorial profile and histological features on minor salivary glands from patients under investigation for Sjögren's syndrome

Diagnosis of Sjögren's syndrome (SS) is complex and the usefulness of labial minor salivary glands biopsy in this process remains controversial. Objectives: to evaluate the clinical and laboratorial profile and histological features on labial minor salivary glands from patients under investigation of SS. Study D esign: clinical charts from 38 patients under suspicion of SS and submitted to labial minor salivary glands biopsies were reviewed. Clinical and laboratorial data were retrieved from the clinical files and the HE-stained histological slides were reviewed under light microscopy. Results: mean age of the patients was 56.5 years and 97% were females; histological analysis showed that 42% of the cases showed ductal dilatation, lymphocytic foci were found in 52.6% and, from this group, 80% of the cases presented a foci/lobules ratio above 0.8. Acinar/ductal ratio was considered diminished in 39.5% of the samples. Thirty six (95%) and 32 (84%) patients, respectively, complained about xerostomia and xerophthalmia. A study of the time interval of the symptoms that led to SS investigation showed a mean of 116 months. Moreover, sixty-six percent of the patients had already been submitted to immunosuppressive therapy prior to the labial minor salivary gland biopsy. Age of the patients, scintigraphic alterations on salivary function, antinuclear factor (ANF), anti-Ro and anti-La did not show statistical significant association with the histological features. Lobules/foci ratio above 0.8 was the only histological parameter statistically associated with Sjögren's syndrome diagnosis ( p <0.0001). Conclusions: in the studied sample, lymphocytic foci on salivary glands were the only histological parameter as - sociated to the diagnosis of SS. Early indication of labial minor salivary gland biopsy to patients under investiga - tion of SS could limit the effects of immunosuppressive therapy on the histological features associated with the evolution of salivary gland involvement in SS

Clinical and radiological analysis of a series of periapical cysts and periapical granulomas diagnosed in a Brazilian population

Periapical cysts (PC) and periapical granulomas (PG) are the two most common chronic inflammatory periapical diseases, but their clinicoradiological characteristics can vary depending on the methods employed in each study. The aim of the present work was to analyze the clinical and radiological profile of a series of PC and PG diagnosed in a Brazilian population. The files of two Oral Pathology laboratories were reviewed and all cases diagnosed as PG and PC were selected for the study. Clinical and radiological information were retrieved and data were tabulated and descriptively and comparatively analyzed. Final sample was composed by 647 inflammatory periapical lesions, including 244 PG (38%) and 403 PC (62%). The number of women affected by PG was significantly higher than the number of women affected by PC (p=0.037). Anterior region of the maxilla was the most common affected area for both entities (39% of the cases), but the most common anatomical location of PG (anterior maxilla and posterior maxilla) was different from PC (anterior maxilla and posterior mandible) (p<0.0001). Upper lateral incisor was the most affected tooth. The mean radiological size of the PC was larger than the mean radiological size of the PG (p<0.0001) and PC showed well-defined radiological images more frequently than PG (p<0.0001). PC were more common than PG, both showed predilection for adult females, most lesions affected predominantly the anterior maxilla and PC presented larger mean radiological diameter and well-defined images when compared with PG

Oral potentially malignant disorders : clinical-pathological study of 684 cases diagnosed in a Brazilian population

The frequency and distribution of oral potentially malignant disorders (OPMD) may vary among different populations. The aim of the present study was to evaluate the clinical-pathological characteristics of OPMD diagnosed in a Brazilian oral pathology laboratory over a period of 11 years. All cases diagnosed as leukoplakia, speckled leukoplakia, erythroplakia, and actinic cheilitis from 2005 to 2015 were analyzed. Clinical information was obtained from laboratory forms and histological information was obtained from histological slides stained with hematoxylin and eosin. the final sample was comprised of 684 cases, of which 292 were males and 392 were females. The mean age was 58 years. The anatomical site most often involved was the lateral border of the tongue (23%), followed by the lower lip (20%), and by the buccal mucosa/vestibule (18%). Leukoplakia accounted for 82% of the sample (564 cases). The mean size of the leukoplakia and speckled leukoplakia lesions was 13 mm (ranging from 1 to 100 mm) and 15 mm (ranging from 5 to 30 mm), respectively (p=0.460). Males reported smoking and drinking alcohol more frequently than females (p=0.001 and p <0.0001, respectively). In half of the cases, dysplasia was not histologically detected, while slight dysplasia was detected in 28% of the cases. The lesions from patients aged from 41 to 80 years presented moderate and severe dysplasia more often than lesions from patients in other age groups. OPMD were more common in females in their sixties. Females were more frequently affected in all anatomical sites, except for the lips. Leukoplakia lesions were the most common OPMD, followed by actinic cheilitis. The lateral border of the tongue was the most affected anatomical site. OPMD located in the floor of the mouth/sublingual mucosa and lesions from older patients presented severe epithelial dysplasia with greater frequency

Putrescine biosynthesis and export genes are essential for normal growth of avian pathogenic Escherichia coli

[Background] Avian pathogenic Escherichia coli (APEC) is the infectious agent of a wide variety of avian diseases, which causes substantial economic losses to the poultry industry worldwide. Polyamines contribute to the optimal synthesis of nucleic acids and proteins in bacteria. The objectives of this study were to investigate; i) whether APEC E. coli encodes the same systems for biosynthesis and uptake as described for E. coli K12 and ii) the role of polyamines during in vitro growth of an avian pathogenic E. coli strain (WT-ST117- O83:H4T).[Results] Following whole genome sequencing, polyamine biosynthesis and export genes present in E. coli MG1655 (K-12) were found to be identical in WT-ST117. Defined mutants were constructed in putrescine and spermidine biosynthesis pathways (ΔspeB, ΔspeC, ΔspeF, ΔspeB/C and ΔspeD/E), and in polyamines transport systems (ΔpotE, ΔyeeF, ΔpotABCD and ΔpotFGHI). Contrary to what was observed for MG1655, the ΔpotE-ST117 mutant was growth attenuated, regardless of putrescine supplementation. The addition of spermidine or orthinine restored the growth to the level of WT-ST117. Growth attenuation after induction of membrane stress by SDS suggested that PotE is involved in protection against this stress. The ΔspeB/C-ST117 mutant was also growth attenuated in minimal medium. The addition of putrescine or spermidine to the media restored growth rate to the wild type level. The remaining biosynthesis and transport mutants showed a growth similar to that of WT-ST117. Analysis by Ultra-High Performance Liquid Chromatography revealed that the ΔspeB/C mutant was putrescine-deficient, despite that the gene speF, which is also involved in the synthesis of putrescine, was expressed.[Conclusions] Deletion of the putrescine transport system, PotE, or the putrescine biosynthesis pathway genes speB/C affected in vitro growth of APEC (ST117- O83:H4) strain, but not E. coli MG1655, despite the high similarity of the genetic make-up of biosynthesis and transport genes. Therefore, blocking these metabolic reactions may be a suitable way to prevent APEC growth in the host without disturbing the commensal E. coli population.Priscila R. Guerra was supported by a scholarship from CAPES – Brazilian Federal Agency for Support and Evaluation of Graduate Education within the Ministry of Education of Brazil. This study received financial support from Spanish Ministry of Economy, Industry and Competitiveness (AGL2013–45431-R and AGL2016–78708-R), and from the Danish Council for Independent Research (Technology and Production), grant number DFF – 4184-00050. The sponsors had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe