Presence of pathogenic Escherichia coli is correlated with bacterial community diversity and composition on pre-harvest cattle hides

Citation: Chopyk, J., Moore, R. M., DiSpirito, Z., Stromberg, Z. R., Lewis, G. L., Renter, D. G., . . . Wommack, K. E. (2016). Presence of pathogenic Escherichia coli is correlated with bacterial community diversity and composition on pre-harvest cattle hides. Microbiome, 4, 11. doi:10.1186/s40168-016-0155-4Background: Since 1982, specific serotypes of Shiga toxin-producing Escherichia coli (STEC) have been recognized as significant foodborne pathogens acquired from contaminated beef and, more recently, other food products. Cattle are the major reservoir hosts of these organisms, and while there have been advancements in food safety practices and industry standards, STEC still remains prevalent within beef cattle operations with cattle hides implicated as major sources of carcass contamination. To investigate whether the composition of hide-specific microbial communities are associated with STEC prevalence, 16S ribosomal RNA (rRNA) bacterial community profiles were obtained from hide and fecal samples collected from a large commercial feedlot over a 3-month period. These community data were examined amidst an extensive collection of prevalence data on a subgroup of STEC that cause illness in humans, referred to as enterohemorrhagic E. coli (EHEC). Fecal 16S rRNA gene OTUs (operational taxonomic units) were subtracted from the OTUs found within each hide 16S rRNA amplicon library to identify hide-specific bacterial populations. Results: Comparative analysis of alpha diversity revealed a significant correlation between low bacterial diversity and samples positive for the presence of E. coli O157:H7 and/or the non-O157 groups: O26, O111, O103, O121, O45, and O145. This trend occurred regardless of diversity metric or fecal OTU presence. The number of EHEC serogroups present in the samples had a compounding effect on the inverse relationship between pathogen presence and bacterial diversity. Beta diversity data showed differences in bacterial community composition between samples containing O157 and non-O157 populations, with certain OTUs demonstrating significant changes in relative abundance. Conclusions: The cumulative prevalence of the targeted EHEC serogroups was correlated with low bacterial community diversity on pre-harvest cattle hides. Understanding the relationship between indigenous hide bacterial communities and populations may provide strategies to limit EHEC in cattle and provide biomarkers for EHEC risk assessment

Effect of platelet-rich fibrin matrix in complex with artificial material Nubiplant on expression of chondrogenic marker genes and morphogenesis of the nucleus pulposus cells of intervertebral discs in rats

The purpose was to study the barrier and biological properties of platelet-rich fibrin matrix (PRFM), an artificial biopolymer Nubiplant, and a mixture of PRFM / Nubiplant by assessing the viability and morphological characteristics of nucleus pulposus (NP) cells in rats, as well as the expression level of chondrogenic marker genes during cell cultivation in the presence of these matrices.Materials and methods. PRFM was obtained from platelet-rich plasma using a SiO2 coagulation activator. A suspension of nucleus pulposus cells was obtained from the caudal spine of rats. Cultivation was carried out in the presence of one of three matrices — PRFM, Nubiplant, or their mixture for 3, 7, and 14 days under standard culture conditions in an EC-160 incubator (Nüve, Turkey). Observation of the living culture was carried out in the area bordering with the matrix within one field of view using an inverted microscope (Nicon TS100, Japan). The expression of chondrogenic marker genes in the cell culture of the NP was determined by the method of PCR with reverse transcription.Results. The study of the viability and morphological characteristics of NP cells during their cultivation for 3, 7, and 14 days in the presence of PRFM, PRFM / Nubiplant, or Nubiplant showed a decrease in the content of living cells in control samples; in cultures with PRFM and PRFM / Nubiplant, the number of living cells significantly exceeded the control values, aggregation of cells was observed in the area bordering with the matrices from the side of the application. None of the experimental samples showed the outflow of cells to the opposite side of the matrix after 14 days of cultivation; thus, PRFM, Nubiplant, and their mixture can perform barrier functions to keep the cell population in a certain location. Expression of the COL II, ACAN, GPC3, ANXA3, PTN, MGP, and VIM genes by the NP cells during cultivation for 3 and 7 days in the presence of PRFM and PRFM / Nubiplant increased as compared to the control samples.Conclusions. The use of PRFM, Nubiplant, or a mixture of PRFM / Nubiplant during the cultivation of NP cells demonstrated the absence of cell outflow to the opposite side of the studied matrices during the study period (14 days). The use of PRFM, Nubiplant, or a mixture of PRFM / Nubiplant promoted the formation of cell colonies with chondrocyte-like morphology in the zone bordering with the matrices and maintained cell viability throughout the study period. PRFM and PRFM / Nubiplant contributed to the maintenance of the expression of chondrogenic genes in the NP cells in the zone bordering the matrices. The results obtained indicate the positive effect of the matrix based on platelet-rich fibrin on the NP cells and its barrier functions, which is promising for the use of PRMF for preventing the formation of cicatricial adhesion

Presence of pathogenic \u3ci\u3eEscherichia coli\u3c/i\u3e is correlated with bacterial community diversity and composition on pre-harvest cattle hides

Background: Since 1982, specific serotypes of Shiga toxin-producing Escherichia coli (STEC) have been recognized as significant foodborne pathogens acquired from contaminated beef and, more recently, other food products. Cattle are the major reservoir hosts of these organisms, and while there have been advancements in food safety practices and industry standards, STEC still remains prevalent within beef cattle operations with cattle hides implicated as major sources of carcass contamination. To investigate whether the composition of hide-specific microbial communities are associated with STEC prevalence, 16S ribosomal RNA (rRNA) bacterial community profiles were obtained from hide and fecal samples collected from a large commercial feedlot over a 3-month period. These community data were examined amidst an extensive collection of prevalence data on a subgroup of STEC that cause illness in humans, referred to as enterohemorrhagic E. coli (EHEC). Fecal 16S rRNA gene OTUs (operational taxonomic units) were subtracted from the OTUs found within each hide 16S rRNA amplicon library to identify hide-specific bacterial populations. Results: Comparative analysis of alpha diversity revealed a significant correlation between low bacterial diversity and samples positive for the presence of E. coli O157:H7 and/or the non-O157 groups: O26, O111, O103, O121, O45, and O145. This trend occurred regardless of diversity metric or fecal OTU presence. The number of EHEC serogroups present in the samples had a compounding effect on the inverse relationship between pathogen presence and bacterial diversity. Beta diversity data showed differences in bacterial community composition between samples containing O157 and non-O157 populations, with certain OTUs demonstrating significant changes in relative abundance. Conclusions: The cumulative prevalence of the targeted EHEC serogroups was correlated with low bacterial community diversity on pre-harvest cattle hides. Understanding the relationship between indigenous hide bacterial communities and populations may provide strategies to limit EHEC in cattle and provide biomarkers for EHEC risk assessment

Вплив збагаченого тромбоцитами фібринового матриксу в комплексі зі штучним матеріалом Nubiplant на експресію хондрогенних маркерних генів та морфогенез клітин пульпозного ядра міжхребцевих дисків щурів

The purpose was to study the barrier and biological properties of platelet-rich fibrin matrix (PRFM), an artificial biopolymer Nubiplant, and a mixture of PRFM / Nubiplant by assessing the viability and morphological characteristics of nucleus pulposus (NP) cells in rats, as well as the expression level of chondrogenic marker genes during cell cultivation in the presence of these matrices.Materials and methods. PRFM was obtained from platelet-rich plasma using a SiO2 coagulation activator. A suspension of nucleus pulposus cells was obtained from the caudal spine of rats. Cultivation was carried out in the presence of one of three matrices — PRFM, Nubiplant, or their mixture for 3, 7, and 14 days under standard culture conditions in an EC-160 incubator (Nüve, Turkey). Observation of the living culture was carried out in the area bordering with the matrix within one field of view using an inverted microscope (Nicon TS100, Japan). The expression of chondrogenic marker genes in the cell culture of the NP was determined by the method of PCR with reverse transcription.Results. The study of the viability and morphological characteristics of NP cells during their cultivation for 3, 7, and 14 days in the presence of PRFM, PRFM / Nubiplant, or Nubiplant showed a decrease in the content of living cells in control samples; in cultures with PRFM and PRFM / Nubiplant, the number of living cells significantly exceeded the control values, aggregation of cells was observed in the area bordering with the matrices from the side of the application. None of the experimental samples showed the outflow of cells to the opposite side of the matrix after 14 days of cultivation; thus, PRFM, Nubiplant, and their mixture can perform barrier functions to keep the cell population in a certain location. Expression of the COL II, ACAN, GPC3, ANXA3, PTN, MGP, and VIM genes by the NP cells during cultivation for 3 and 7 days in the presence of PRFM and PRFM / Nubiplant increased as compared to the control samples.Conclusions. The use of PRFM, Nubiplant, or a mixture of PRFM / Nubiplant during the cultivation of NP cells demonstrated the absence of cell outflow to the opposite side of the studied matrices during the study period (14 days). The use of PRFM, Nubiplant, or a mixture of PRFM / Nubiplant promoted the formation of cell colonies with chondrocyte-like morphology in the zone bordering with the matrices and maintained cell viability throughout the study period. PRFM and PRFM / Nubiplant contributed to the maintenance of the expression of chondrogenic genes in the NP cells in the zone bordering the matrices. The results obtained indicate the positive effect of the matrix based on platelet-rich fibrin on the NP cells and its barrier functions, which is promising for the use of PRMF for preventing the formation of cicatricial adhesion.Цель: исследовать барьерные и биологические свойства обогащенного тромбоцитами фибринового матрикса (ОТФМ), искусственного биополимера Nubiplant и смеси ОТФМ и Nubiplant путем оценки жизнеспособности и морфологических характеристик клеток пульпозного ядра (ПЯ) межпозвоночных дисков крыс, а также уровня экспрессии хондрогенных маркерных генов при культивировании этих клеток при наличии указанных матриксов.Материалы и методы. ОТФМ получали из обогащенной тромбоцитами плазмы с использованием активатора свертывания SiO2, суспензию клеток ПЯ ‒ из хвостового отдела позвоночника крыс. Культивирование осуществляли при наличии одного из трех матриксов ‒ ОТФМ, Nubiplant или их смеси в течение 3, 7 и 14 суток при стандартных культуральных условиях в инкубаторе ЕС-160 (Nüve, Турция). Наблюдение за живой культурой проводили в пограничной с матриксом зоне в пределах одного поля зрения с использованием инвертированного микроскопа (Nicon TS100, Япония). Экспрессию хондрогенных маркерных генов в культуре клеток ПЯ определяли методом полимеразной цепной реакции с обратной транскрипцией.Результаты. Исследование жизнеспособности и морфологических характеристик клеток ПЯ при их культивировании продемонстрировало снижение содержания живых клеток в контрольных образцах. В культурах с ОТФМ и смесью ОТФМ и Nubiplant количество живых клеток статистически значимо превышало контрольные показатели. Наблюдали агрегацию клеток в пограничной с матриксами зоне со стороны нанесения. Выселения клеток на противоположную сторону матрикса в течение 14 суток ни в одном из экспериментальных образцов не отмечено. Таким образом, ОТФМ, Nubiplant и их смесь могут выполнять барьерные функции по удержанию клеточной популяции в определенном участке. Экспрессия генов COL II, ACAN, GPC3, ANXA3, PTN, MGP и VIM клетками ПЯ при культивировании в течение 3 и 7 суток при наличии ОТФМ и смеси ОТФМ и Nubiplant возрастала по сравнению с контрольными образцами.Выводы. Применение ОТФМ, Nubiplant или смеси ОТФМ и Nubiplant при культивировании клеток ПЯ продемонстрировало отсутствие выселения клеток на противоположную сторону упомянутых матриксов в течение всего периода исследования (14 дней). Использование ОТФМ, Nubiplant или смеси ОТФМ и Nubiplant способствовало формированию колоний клеток хондроцитоподобной морфологии в пограничной с матриксами зоне и поддерживало жизнеспособность клеток в течение всего исследуемого периода. Применение ОТФМ и ОТФМ и Nubiplant позволяло поддерживать экспрессию хондрогенных генов клетками ПЯ в пограничной с матриксами зоне. Полученные результаты свидетельствуют о положительном действии матрикса на основе обогащенного тромбоцитами фибрина на клетки ПЯ и его барьерные функции, что является перспективным для применения ОТФМ с целью предотвращения формирования рубцово-спаечных процессов.Мета: дослідити бар’єрні та біологічні властивості збагаченого тромбоцитами фібринового матриксу (ЗТФМ), штучного біополімеру Nubiplant та їх суміші шляхом оцінки життєздатності та морфологічних характеристик клітин пульпозного ядра (ПЯ) міжхребцевих дисків щурів, а також рівня експресії хондрогенних маркерних генів при культивуванні цих клітин за наявності зазначених матриксів.Матеріали і методи. ЗТФМ отримували зі збагаченої тромбоцитами плазми з використанням активатора згортання SiO2, суспензію клітин ПЯ ‒ із хвостового відділу хребта щурів. Культивування здійснювали за наявності одного з трьох матриксів – ЗТФМ, Nubiplan або їх суміші протягом 3, 7 та 14 діб за стандартних культуральних умов в інкубаторі ЕС-160 (Nüve, Турція). Спостереження за живою культурою проводили в пограничній з матриксами зоні в межах одного поля зору з використанням інвертованого мікроскопа (Nicon TS100, Японія). Експресію хондрогенних маркерних генів у культурі клітин ПЯ визначали методом полімеразної ланцюгової реакції зі зворотною транскрипцією.Результати. Дослідження життєздатності та морфологічних характеристик клітин ПЯ при їх культивуванні продемонструвало зниження вмісту живих клітин у контрольних зразках. У культурах із ЗТФМ та сумішшю ЗТФМ і Nubiplant кількість живих клітин статистично значущо перевищувала контрольні показники. Спостерігали агрегацію клітин у зоні безпосереднього межування з матриксами з боку нанесення. Виселення клітин на протилежний бік матриксу протягом 14 діб у жодному з експериментальних зразків не відзначено. Таким чином, ЗТФМ, Nubiplant та їх суміш можуть виконувати бар’єрні функції щодо утримання клітинної популяції в певній ділянці. Експресія генів COL II, ACAN, GPC3, ANXA3, PTN, MGP та VIM клітинами ПЯ при культивуванні протягом 3 та 7 діб за наявності ЗТФМ і суміші ЗТФМ та Nubiplant зростала порівняно з контрольними зразками.Висновки. Застосування ЗТФМ, Nubiplant або суміші ЗТФМ і Nubiplant при культивуванні клітин ПЯ продемонструвало відсутність виселення клітин на протилежний бік зазначених матриксів протягом усього періоду дослідження (14 діб). Використання ЗТФМ, Nubiplant або суміші ЗТФМ і Nubiplant сприяло формуванню колоній клітин хондроцитоподібної морфології у зоні межування з матриксами та підтримувало життєздатність клітин протягом усього досліджуваного періоду. Застосування ЗТФМ і суміші ЗТФМ та Nubiplant давало змогу підтримувати експресію хондрогенних генів клітинами ПЯ в пограничній з матриксами зоні. Отримані результати свідчать про позитивну дію матриксів на основі збагаченого тромбоцитами фібрину на клітини ПЯ та його бар’єрні функції, що є перспективним для застосування ЗТФМ з метою запобігання формуванню рубцево-спайкових процесів

Presence of pathogenic Escherichia coli is correlated with bacterial community diversity and composition on pre-harvest cattle hides

Publisher's PDFBackground: Since 1982, specific serotypes of Shiga toxin-producing Escherichia coli (STEC) have been recognized as significant foodborne pathogens acquired from contaminated beef and, more recently, other food products. Cattle are the major reservoir hosts of these organisms, and while there have been advancements in food safety practices and industry standards, STEC still remains prevalent within beef cattle operations with cattle hides implicated as major sources of carcass contamination. To investigate whether the composition of hide-specific microbial communities are associated with STEC prevalence, 16S ribosomal RNA (rRNA) bacterial community profiles were obtained from hide and fecal samples collected from a large commercial feedlot over a 3-month period. These community data were examined amidst an extensive collection of prevalence data on a subgroup of STEC that cause illness in humans, referred to as enterohemorrhagic E. coli (EHEC). Fecal 16S rRNA gene OTUs (operational taxonomic units) were subtracted from the OTUs found within each hide 16S rRNA amplicon library to identify hide-specific bacterial populations. Results: Comparative analysis of alpha diversity revealed a significant correlation between low bacterial diversity and samples positive for the presence of E. coli O157:H7 and/or the non-O157 groups: O26, O111, O103, O121, O45, and O145. This trend occurred regardless of diversity metric or fecal OTU presence. The number of EHEC serogroups present in the samples had a compounding effect on the inverse relationship between pathogen presence and bacterial diversity. Beta diversity data showed differences in bacterial community composition between samples containing O157 and non-O157 populations, with certain OTUs demonstrating significant changes in relative abundance. Conclusions: The cumulative prevalence of the targeted EHEC serogroups was correlated with low bacterial community diversity on pre-harvest cattle hides. Understanding the relationship between indigenous hide bacterial communities and populations may provide strategies to limit EHEC in cattle and provide biomarkers for EHEC risk assessment.University of Delaware, Delaware Biotechnology Institut