Clinicopathological Factors as Predictors for Establishment of Patient Derived Head and Neck Squamous Cell Carcinoma Organoids.

INTRODUCTION: Patient derived organoids (PDOs) are 3D in vitro models and have shown to better reflect patient and tumor heterogeneity than conventional 2D cell lines. To utilize PDOs in clinical settings and trials for biomarker discovery or drug response evaluation, it is valuable to determine the best way to optimize sample selection for maximum PDO establishment. In this study, we assess patient, tumor and tissue sampling factors and correlate them with successful PDO establishment in a well-documented cohort of patients with head and neck squamous cell carcinoma (HNSCC). METHODS: Tumor and non-tumorous adjacent tissue samples were obtained from HNSCC patients during routine biopsy or resection procedures at the University Medical Center Utrecht. The tissue was subsequently processed to establish PDOs. The sample purity was determined as the presence of epithelial cells in the culture on the day of organoid isolation as visualized microscopically by the researcher. PDO establishment was recorded for all samples. Clinical data was obtained from the medical records and was correlated to PDO establishment and presence of epithelial cells. RESULTS: Organoids could be established in 133/250 (53.2%) primary tumor site tissues. HNSCC organoid establishment tended to be more successful if patients were younger than the median age of 68 years (74/123 (60.2%) vs. 59/127 (46.5%), p = 0.03). For a subset of samples, the presence of epithelial cells in the organoid culture on the day of organoid isolation was recorded in 112/149 (75.2%) of these samples. When cultures were selected for presence of epithelial cells, organoid establishment increased to 76.8% (86/112 samples). CONCLUSION: This study found a trend between age and successful organoid outgrowth in patients with HNSCC younger than 68 years and emphasizes the value of efficient sampling regarding PDO establishment.

p-mTOR, p-ERK and PTEN Expression in Tumor Biopsies and Organoids as Predictive Biomarkers for Patients with HPV Negative Head and Neck Cancer.

BACKGROUND: Survival rates of head and neck squamous cell carcinoma (HNSCC) have only marginally improved in the last decades. Hence there is a need for predictive biomarkers for long-time survival that can help to guide treatment decisions and might lead to the development of new therapies. The phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling pathway is the most frequently altered pathway in HNSCC, genes are often mutated, amplificated and overexpressed causing aberrant signaling affecting cell growth and differentiation. Numerous genetic alterations of upstream and downstream factors have currently been clarified. However, their predictive value has yet to be established. Therefore we assess the predictive value of p-mTOR, p-ERK and PTEN expression. METHODS: Tissue microarrays (TMA's) of HPV-negative patients with oropharyngeal (n = 48), hypopharyngeal (n = 16) or laryngeal (n = 13) SCC, treated with primary chemoradiation (cisplatin/carboplatin/cetuximab and radiotherapy), were histologically stained for p-mTOR, PTEN and p-ERK. Expression was correlated to overall survival (OS), disease free survival (DFS) and locoregional control (LRC). Also p-mTOR was histologically stained in a separate cohort of HNSCC organoids (n = 8) and correlated to mTOR-inhibitor everolimus response. RESULTS: High p-mTOR expression correlated significantly with worse OS in multivariate analysis in the whole patient cohort [Hazar Ratio (HR) 1.06, 95%CI 1.01-1.11, p = 0.03] and in the cisplatin/carboplatin group with both worse OS (HR 1.09, 95%CI 1.02-1.16, p = 0.02) and DFS (HR 1.06, 95%CI 1.00-1.12, p = 0,04). p-ERK expression correlated significantly with DFS in univariate analysis in the whole patient cohort (HR 1.03, 95%CI 1.00-1.05, p = 0.04) and cisplatin/carboplatin group (HR 1.03, 95%CI 1.00-1.07, p = 0.04). PTEN-expression did not correlate with OS/DFS/LRC. Better organoid response to everolimus correlated significantly to higher p-mTOR expression (Rs = - 0.731, p = 0.04). CONCLUSIONS: High p-mTOR expression predicts and high p-ERK expression tends to predict worse treatment outcome in HPV negative HNSCC patients treated with chemoradiation, providing additional evidence that these markers are candidate prognostic biomarkers for survival in this patient population. Also this study shows that the use of HNSCC organoids for biomarker research has potential. The role of PTEN expression as prognostic biomarker remains unclear, as consistent evidence on its prognostic and predictive value is lacking.

Efficacy and toxicity of vemurafenib and cobimetinib in relation to plasma concentrations, after administration via feeding tube in patients with BRAF-mutated thyroid cancer: a case series and review of literature.

INTRODUCTION: The combination of vemurafenib, a proto-oncogene B-Raf inhibitor (BRAFi) and cobimetinib, an inhibitor of mitogen-activated protein kinase kinase (MEKi) has shown to improve survival in patients with BRAF V600-mutated melanoma. BRAF mutations are also frequently detected driver mutations in other tumor types, including thyroid carcinoma. Since thyroid carcinoma is not a labeled indication for BRAF/MEKi, a cohort for patients with BRAF V600-mutated thyroid carcinoma was opened within the Drug Rediscovery Protocol (DRUP), a national ongoing pan-cancer multi-drug trial, in which patients receive off-label treatment with approved drugs based on their molecular tumor profile. RESULTS: Here, we present two patients with BRAF-mutated thyroid carcinoma, who were successfully treated with vemurafenib/cobimetinib administered via a feeding tube. Plasma concentrations of vemurafenib and cobimetinib were determined. A partial response was observed in both patients, but they experienced significant toxicity. CONCLUSION: Our cases show that vemurafenib/cobimetinib treatment is effective in BRAF V600-mutated thyroid carcinoma, also when administered via a feeding tube. Although serious side effects occurred in both patients, we hypothesize that this was not attributable to the administration route. Therefore, administration of vemurafenib/cobimetinib by feeding tube is feasible and effective. TRIAL REGISTRATION: Clinical trial identification: NCT02925234.

Validation of skeletal muscle mass assessment at the level of the third cervical vertebra in patients with head and neck cancer.

BACKGROUND: Low skeletal muscle mass (SMM) is associated with adverse outcomes. SMM is often assessed at the third lumbar vertebra (L3) on abdominal imaging. Abdominal imaging is not routinely performed in patients with head and neck cancer (HNC). We aim to validate SMM measurement at the level of the third cervical vertebra (C3) on head and neck imaging. MATERIAL AND METHODS: Patients with pre-treatment whole-body computed tomography (CT) between 2010 and 2018 were included. Cross-sectional muscle area (CSMA) was manually delineated at the level of C3 and L3. Correlation coefficients and intraclass correlation coefficients (ICCs) were calculated. Cohen's kappa was used to assess the reliability of identifying a patient with low SMM. RESULTS: Two hundred patients were included. Correlation between CSMA at the level of C3 and L3 was good (r = 0.75, p < 0.01). Using a multivariate formula to estimate CSMA at L3, including gender, age, and weight, correlation improved (r = 0.82, p < 0.01). The agreement between estimated and actual CSMA at L3 was good (ICC 0.78, p < 0.01). There was moderate agreement in the identification of patients with low SMM based on the estimated lumbar skeletal muscle mass index (LSMI) and actual LSMI (Cohen's κ: 0.57, 95%CI 0.45-0.69). CONCLUSIONS: CSMA at C3 correlates well with CSMA at L3. There is moderate agreement in the identification of patients with low SMM based on the estimated lumbar SMI (based on measurement at C3) and actual LSMI.

Predictive Value of EGFR-PI3K-AKT-mTOR-Pathway Inhibitor Biomarkers for Head and Neck Squamous Cell Carcinoma: A Systematic Review.

BACKGROUND: Understanding molecular pathogenesis of head and neck squamous cell carcinomas (HNSCC) has considerably improved in the last decades. As a result, novel therapeutic strategies have evolved, amongst which are epidermal growth factor receptor (EGFR)-targeted therapies. With the exception of cetuximab, targeted therapies for HNSCC have not yet been introduced into clinical practice. One important aspect of new treatment regimes in clinical practice is presence of robust biomarkers predictive for therapy response. METHODS: We performed a systematic search in PubMed, Embase and the Cochrane library. Articles were included if they investigated a biomarker for targeted therapy in the EGFR-PI3K-AKT-mTOR-pathway. RESULTS: Of 83 included articles, 52 were preclinical and 33 were clinical studies (two studies contained both a preclinical and a clinical part). We classified EGFR pathway inhibitor types and investigated the type of biomarker (biomarker on epigenetic, DNA, mRNA or protein level). CONCLUSION: Several EGFR-PI3K-AKT-mTOR-pathway inhibitor biomarkers have been researched for HNSCC but few of the investigated biomarkers have been adequately confirmed in clinical trials. A more systematic approach is needed to discover proper biomarkers as stratifying patients is essential to prevent unnecessary costs and side effects.

The predictive and prognostic value of low skeletal muscle mass for dose-limiting toxicity and survival in head and neck cancer patients receiving concomitant cetuximab and radiotherapy.

PURPOSE: This study aims to investigate the predictive value of low skeletal muscle mass (SMM) for cetuximab dose-limiting toxicity (DLT) and its prognostic value in head and neck squamous cell carcinoma (HNSCC) patients treated with concomitant cetuximab and radiotherapy. METHODS: Patients diagnosed with HNSCC and treated with primary or adjuvant concomitant cetuximab and radiotherapy were included. Clinical and demographic variables were retrospectively retrieved and SMM was measured at the level of the third cervical vertebra using pre-treatment diagnostic computed tomography or magnetic resonance imaging. An optimal cut-off value for low SMM was determined based on the lowest log-likelihood associated with cetuximab DLT. A multivariate linear regression model was used to determine predictive factors for cetuximab DLT. The prognostic value of low SMM for disease-free and overall survival was analyzed using Kaplan-Meier curves. RESULTS: The optimal cut-off value for low SMM as a predictor of cetuximab DLT was an LSMI ≤ 45.2 cm2/m2. Of the 91 included patients, 74.7% had low SMM and 30.8% experienced cetuximab DLT. At multivariate analysis, low SMM had no predictive value for DLT (OR 0.83; 95% CI 0.27-2.56; p = 0.74). The Kaplan-Meier curve demonstrated that patients with low SMM had significantly lower overall survival (Log Rank χ2 = 5.87; p = 0.02). CONCLUSION: Low SMM is highly prevalent in HNSCC patients treated with concomitant cetuximab and radiotherapy. Low SMM has no predictive value for cetuximab DLT in HNSCC patients. Low SMM is probably not a prognostic factor for overall survival in highly selected HNSCC patients treated with concomitant cetuximab and radiotherapy and unfit for platin-based chemotherapy.

The Drug Rediscovery protocol facilitates the expanded use of existing anticancer drugs.

The large-scale genetic profiling of tumours can identify potentially actionable molecular variants for which approved anticancer drugs are available1-3. However, when patients with such variants are treated with drugs outside of their approved label, successes and failures of targeted therapy are not systematically collected or shared. We therefore initiated the Drug Rediscovery protocol, an adaptive, precision-oncology trial that aims to identify signals of activity in cohorts of patients, with defined tumour types and molecular variants, who are being treated with anticancer drugs outside of their approved label. To be eligible for the trial, patients have to have exhausted or declined standard therapies, and have malignancies with potentially actionable variants for which no approved anticancer drugs are available. Here we show an overall rate of clinical benefit-defined as complete or partial response, or as stable disease beyond 16 weeks-of 34% in 215 treated patients, comprising 136 patients who received targeted therapies and 79 patients who received immunotherapy. The overall median duration of clinical benefit was 9 months (95% confidence interval of 8-11 months), including 26 patients who were experiencing ongoing clinical benefit at data cut-off. The potential of the Drug Rediscovery protocol is illustrated by the identification of a successful cohort of patients with microsatellite instable tumours who received nivolumab (clinical benefit rate of 63%), and a cohort of patients with colorectal cancer with relatively low mutational load who experienced only limited clinical benefit from immunotherapy. The Drug Rediscovery protocol facilitates the defined use of approved drugs beyond their labels in rare subgroups of cancer, identifies early signals of activity in these subgroups, accelerates the clinical translation of new insights into the use of anticancer drugs outside of their approved label, and creates a publicly available repository of knowledge for future decision-making.

Phase I and pharmacological study of pazopanib in combination with oral topotecan in patients with advanced solid tumours.

BACKGROUND: This phase I study evaluated the safety, tolerability, maximum tolerated dose (MTD) and pharmacokinetics of two dosing schedules of oral topotecan in combination with pazopanib in patients with advanced solid tumours. METHODS: Stage I of this study was to determine whether there was an impact of pazopanib on topotecan exposure. In stage II, the MTD and safety profile of oral topotecan given weekly on days 1, 8 and 15 in a 28-day cycle; or daily-times-five on days 1-5 in a 21-day cycle, both in combination with daily pazopanib, were explored. RESULTS: In total, 67 patients were enroled. Pazopanib co-administration caused a substantial increase in exposure to total topotecan (1.7-fold) compared with topotecan alone, which is considered clinically relevant. Topotecan had no effect on pazopanib concentrations. Safety findings were consistent with the known profile of both agents. There were three drug-related deaths, liver failure, tumour haemorrhage and myelosuppression. Two patients experienced dose-limiting toxicities (DLTs; hand-foot syndrome, myelosuppression and diarrhoea) on the weekly topotecan schedule and four patients experienced DLTs (myelosuppression) on the daily-times-five topotecan schedule. When combined with pazopanib, 800 mg daily, the recommended doses for oral topotecan are: 8 mg weekly and 2.5 mg daily-times-five. Seven of eight patients with partial response had platinum-resistant ovarian cancer. In addition, 54% of patients had stable disease with 22% stable for 6 months. CONCLUSIONS: Total topotecan exposure is 1.7-fold higher when co-administered with pazopanib. Both schedules of administration were tolerated and would permit further evaluation, especially the weekly schedule.

Eribulin mesylate pharmacokinetics in patients with solid tumors receiving repeated oral ketoconazole.

Purpose To study the influence of repeated oral administration of ketoconazole, a potent CYP3A4 inhibitor, on the plasma pharmacokinetics of eribulin mesylate administered by single-dose intravenous infusion. Eribulin mesylate is a non-taxane microtubule dynamics inhibitor that is currently under development in phase I-III trials for the treatment of solid tumors. Experimental design A randomized, open-label, two treatments, two sequences, crossover phase I study was performed in patients with advanced solid tumors. Treatments were given on day 1 and day 15 and consisted of 1.4 mg/m(2) eribulin mesylate alone or 0.7 mg/m(2) eribulin mesylate plus 200 mg ketoconazole on the day of eribulin mesylate administration and the following day. Pharmacokinetic sampling for determination of eribulin plasma concentration was performed up to 144 h following administration of eribulin mesylate. Also safety and anti-tumor activity were determined. Results Pharmacokinetic sampling and analysis was completed in ten patients. Statistical analysis of dose-normalized log-transformed AUC0-∞ and Cmax indicated that single-dose exposure of eribulin was not statistically different when co-administered with ketoconazole (ratio of geometric least square means: 0.95 (90%CI: 0.80-1.12) and 0.97 (90%CI: 0.83-1.12), respectively) in patients with solid tumors. Ketoconazole had no effect on eribulin clearance and elimination half-life. The most frequently reported treatment related adverse events were fatigue and nausea, each reported in 8/12 patients. Seven patients (58.3 %) achieved stable disease as best overall response. Conclusions The results indicate that eribulin mesylate can be safely co-administered with ketoconazole. Drug-drug interactions are not expected with other CYP3A4 inhibitors.

Pharmacokinetics of eribulin mesylate in patients with solid tumors and hepatic impairment.

PURPOSE: The aim of this study was to determine the plasma pharmacokinetics of eribulin mesylate in patients with solid tumors with mild and moderate hepatic impairment. PATIENTS AND METHODS: A phase I, pharmacokinetic study was performed in patients with advanced solid tumors and normal hepatic function or Child-Pugh A (mild) or Child-Pugh B (moderate) hepatic impairment. Treatments were given on day 1 and 8 of a 21-day cycle and consisted of 1.4, 1.1 and 0.7 mg/m(2) eribulin mesylate, for normal hepatic function, Child-Pugh A and B hepatic impairment, respectively. Also safety and anti-tumor activity were determined. RESULTS: Hepatic impairment increased exposure to eribulin. In patients with Child-Pugh A (N = 7) and Child-Pugh B (N = 5), mean dose-normalized AUC(0-∞) was 1.75-fold (90 % confidence intervals (CI): 1.16-2.65) and 2.48-fold (90 % CI: 1.57-3.92) increased, respectively, compared with patients who have normal function (N = 6). The most frequently reported treatment-related events were alopecia (12/18) and fatigue (7/18) and these were observed across all groups. Nine patients (50 %) had stable disease as best response. CONCLUSIONS: A reduced dose of 1.1 and 0.7 mg/m(2) of eribulin mesylate is recommended for patients with Child-Pugh A or B hepatic impairment, respectively.

Circulating tumor cell detection in advanced non-small cell lung cancer patients by multi-marker QPCR analysis.

BACKGROUND: The aim of this study was to explore circulating tumor cell (CTC) detection in advanced non-small cell lung cancer (NSCLC). CTCs may not only serve as a prognostic marker in selected tumor types, but may also be useful as pharmacodynamic marker in drug development. METHODS: Fourty-six advanced NSCLC patients and fourty-six healthy controls were included in the study and 8.0 ml of peripheral blood was obtained from each of the participants. Immunomagnetic bead enrichment for cells expressing epithelial cell adhesion molecule (EpCAM) was performed, followed by multi-marker quantitative real-time PCR of a panel of marker genes: cytokeratin 7 (CK7), cytokeratin 19 (CK19), human epithelial glycoprotein (EGP) and fibronectin 1 (FN1). Using quadratic discriminant analysis (QDA), expression values were combined into a single score, which indicated CTC-positivity or -negativity. Test characteristics were assessed using receiver operating characteristic (ROC) curve analysis. RESULTS: ROC curve analysis showed capability of discrimination between advanced NSCLC patients and healthy controls (area=0.712; 95% CI 0.606-0.819; P<0.001). A cut-off minimizing overall misclassification for QDA-positivity reached a sensitivity of 46% (95% CI 31-61) and a specificity of 93% (95% CI 82-99). CONCLUSIONS: In this exploratory study, an assay was developed for discriminating CTCs in peripheral blood samples of advanced NSCLC patients from healthy controls. The assay demonstrated an acceptable sensitivity in combination with good specificity. Further validation studies should take place in NSCLC patients and a matched control group.

A multimarker QPCR-based platform for the detection of circulating tumour cells in patients with early-stage breast cancer.

BACKGROUND: The detection of circulating tumour cells (CTCs) has been linked with poor prognosis in advanced breast cancer. Relatively few studies have been undertaken to study the clinical relevance of CTCs in early-stage breast cancer. METHODS: In a prospective study, we evaluated CTCs in the peripheral blood of 82 early-stage breast cancer patients. Control groups consisted of 16 advanced breast cancer patients and 45 healthy volunteers. The CTC detection was performed using ErbB2/EpCAM immunomagnetic tumour cell enrichment followed by multimarker quantitative PCR (QPCR). The CTC status and common clinicopathological factors were correlated to relapse-free, breast cancer-related and overall survival. RESULTS: Circulating tumour cells were detected in 16 of 82 (20%) patients with early-stage breast cancer and in 13 out of 16 (81%) with advanced breast cancer. The specificity was 100%. The median follow-up time was 51 months (range: 17-60). The CTC positivity in early-stage breast cancer patients resulted in significantly poorer relapse-free survival (log rank test: P=0.003) and was an independent predictor of relapse-free survival (multivariate hazard ratio=5.13, P=0.006, 95% CI: 1.62-16.31). CONCLUSION: The detection of CTCs in peripheral blood of early-stage breast cancer patients provided prognostic information for relapse-free survival.

Phenotypic and genetic characterization of resistance against macrolides and lincosamides in Streptococcus gallolyticus strains isolated from pigeons and humans.

The macrolide and lincosamide (ML) resistance phenotype of 65 pigeon and 30 human Streptococcus gallolyticus strains was determined by the disk diffusion method. Constitutive resistance against the tested antibiotics was seen in 13 human and 28 pigeon strains. Simultaneous screening for the presence of erm(B) and mef(A) genes using PCR revealed that the erm(B) gene was present in 40 out of these 41 phenotypically resistant S. gallolyticus strains while the mef(A) gene was detected in only one resistant and one susceptible human-derived strain. The erm(B) genes of 10 human and 10 pigeon S. gallolyticus strains were sequenced and compared. Four human and seven pigeon strains possessed exactly the same sequence for the erm(B) gene. The sequence of the erm(B) gene of the remaining strains differed in one to five nucleotides. These findings could indicate a possible exchange of resistance genes between human and pigeon strains.

Macrolide and lincosamide resistance in the gram-positive nasal and tonsillar flora of pigs.

Macrolide and lincosamide resistance phenotypes and the presence of the erm(A), erm(B), erm(C), and mef(A) genes were determined in 344 bacterial strains belonging to 34 species and nine genera, isolated from the tonsils and nasal cavities of 2-week- and 6-week-old piglets, derived from four different farms. These piglets had never before been treated with macrolides or lincosamides. Macrolide and lincosamide resistance was most frequently present in Streptococcus and Enterococcus strains, of which over two-thirds were resistant. These genera were followed in decreasing order of resistance frequency by Lactobacillus, Rothia, Staphylococcus, Arcanobacterium, Actinomyces, Pediococcus strains. Only five infrequently occurring species did not show resistance. This high frequency of resistance in nontreated piglets indicates that resistant strains circulate in the herds. In streptococci, enterococci, and Lactobacillus strains, resistance was most often encoded by the erm(B) gene and in staphylococci by erm(A) or erm(C). The erm(B) gene was sporadically detected in other bacterial genera (Actinomyces, Rothia, Aerococcus, Pediococcus). The sequence of the erm(B) gene of 29 strains of 11 pigs originating from the four different farms was determined. This sequence was identical in 12 strains and only differed by 1-6 nucleotides in the other strains, indicating that exchanges of resistance genes might occur between bacterial species and genera belonging to the nasal or tonsillar flora of piglets.

Presence of macrolide resistance genes in streptococci and enterococci isolated from pigs and pork carcasses.

Macrolide and lincosamide (ML)-resistant streptococci and enterococci from tonsillar and colon swabs from 33 pigs and 99 pork carcasses swabs from animals originating from different farms in Belgium were isolated, and their ML resistance phenotypes and genotypes were determined by disk diffusion test and PCR assay, amplifying the ermB gene and the mefA gene. From each of the 33 pigs and 88 of the 99 carcasses' swabs, at least one resistant strain was isolated. The predominant phenotype was the constitutively expressed macrolides, lincosamides and streptogramin B (MLS(B)) phenotype. This phenotype was most often encoded by the ermB gene. A minority of the strains showed the M phenotype encoded by the mefA gene in streptococci, or the L or ML phenotype.

Prevalence and mechanism of resistance against macrolides and lincosamides in Streptococcus suis isolates.

Eighty-seven Streptococcus suis isolates recovered in 1999-2000 from diseased pigs, all from different farms, were screened for resistance against macrolide and lincosamide antibiotics by the disk diffusion and agar dilution test and a PCR assay, amplifying the ermB gene and the mefA/E gene. Seventy-one percent of the isolates showed constitutive resistance to macrolide and lincosamide antibiotics (MLS(B)-phenotype). All these isolates were positive for the ermB gene in the PCR, but negative for the mefA/E gene. For all strains minimum inhibitory concentrations (MIC) against five other antimicrobial agents were determined. All strains were susceptible to penicillin. Ninety-nine percent of the isolates were susceptible to enrofloxacin and tiamulin. Eighty-five percent of the strains were resistant to doxycycline. A 540bp fragment of the ermB genes of eight S. suis strains was sequenced and compared with ermB genes of five S. pneumoniae and five S. pyogenes strains of human origin. A 100% homology was found between these fragments in seven S. suis, one S. pneumoniae and three of the S. pyogenes isolates. This study demonstrates that resistance against macrolides, lincosamides and streptogramin B is widespread in S. suis and mediated by ribosome methylation, encoded by the ermB gene.

Enterococcus haemoperoxidus sp. nov. and Enterococcus moraviensis sp. nov., isolated from water.

A polyphasic taxonomic approach was used to study atypical enterococci isolated from surface waters. All strains were characterized by physiological and biochemical tests as well as by genotyping. The results of biochemical tests and tRNA intergenic length polymorphism analysis (tDNA-PCR) divided all studied strains uniformly into two groups. Because these groups were clearly separated from all enterococcal species described to date, 16S rDNA sequence analysis, DNA base composition analysis and DNA-DNA hybridization of representative strains were done to elucidate the taxonomic position of the analysed groups. On the basis of the results obtained, the names Enterococcus haemoperoxidus (type strain CCM 4851T = LMG 19487T) and Enterococcus moraviensis (type strain CCM 4856T = LMG 19486T) are proposed for the two hitherto undescribed species. The type strains and reference cultures have been deposited in the Czech Collection of Microorganisms (CCM), Masaryk University, Brno, Czech Republic, and in the BCCM/LMG Culture Collection, Ghent University, Belgium.

Enterococcus villorum sp. nov., an enteroadherent bacterium associated with diarrhoea in piglets.

The taxonomic positions of five enteroadherent bacterial pig isolates, showing phenotypic characteristics most similar to those of Enterococcus durans and Enterococcus hirae, were investigated in a polyphasic study that included 16S rDNA sequence analysis, DNA-DNA hybridizations, DNA base-ratio determinations, whole-cell protein fingerprinting, D11344-primed PCR typing and an extensive examination of phenotypic properties. The results demonstrated that the organisms represent a new species in the Enterococcus faecium species group, for which the name Enterococcus villorum sp. nov. is proposed. The type strain is LMG 12287T (= CCM 4887T).

Intracellular survival and multiplication of virulent and less virulent strains of Streptococcus bovis in pigeon macrophages.

The intracellular fate of pigeon S. bovis strains ingested by macrophages was studied in vivo and in vitro. During in vivo experiments, histological and electron microscopical examinations demonstrated numerous cocci, which appeared to be actively multiplying, within splenic macrophages of pigeons experimentally inoculated with a highly virulent S. bovis serotype 1 strain. In pigeons inoculated with a low virulence serotype 3 strain, intracellular cocci were only occasionally observed. For in vitro experiments, pigeon peritoneal macrophages were inoculated with a S. bovis serotype 1 or serotype 3 strain and incubated. Following an initial decrease, an increase in the number of intracellular bacteria was observed in tests performed with the S. bovis serotype 1 strain, demonstrating intracellular multiplication. Macrophages in these experiments had all died after 7 h of incubation, possibly indicating that the intracellular replication of S. bovis resulted in the release of substances toxic for macrophages. In experiments performed with the S. bovis serotype 3 strain, the number of intracellular bacteria continuously decreased, reflecting killing of organisms. Significant changes in the number of adhering macrophages in S. bovis serotype 3 inoculated cultures were not observed. These results indicate S. bovis in pigeons is a facultative intracellular bacterium and intracellular multiplication may be involved in virulence.

Influence of the oestrous cycle on experimental intrauterine E. coli infection in the sow.

The influence of the oestrous cycle on the onset of endometritis in the sow was studied. Ten pubertal, unmated gilts of the Belgian Negative Landrace were used. Nine gilts were inoculated into the uterus by laparotomy with a suspension of an E. coli strain isolated from the uterus of a discharging sow from a herd having many problems with vaginal discharge and a lowered fertility. One gilt was as a control inoculated with 2 ml of a PBS-solution. All sows inoculated during dioestrus developed clinical symptoms, but only 1 of the 5 gilts inoculated at standing oestrus developed a vaginal discharge. These data confirm the hypothesis that the stage of the oestrous cycle has an important influence on the onset of endometritis. The resistance to E. coli infections was higher when the gilts were inoculated during oestrus.