The effect of growth media and physical treatments on the adhesion properties of canine probiotics

Aims

The manufacturing processes have been reported to influence the properties of probiotics with potential impact on health properties. The aim was to investigate the effect of different growth media and inactivation methods on the properties of canine‐originated probiotic bacteria alone and in combination mixture.

Methods and Results

Three established dog probiotics, Lactobacillus fermentum VET9A, Lactobacillus plantarum VET14A and Lactobacillus rhamnosus VET16A, and their combination mixture were evaluated for their adhesion to dog mucus. The effect of different growth media, one reflecting laboratory and the other manufacturing conditions, and inactivation methods (95°C, 80°C and UV irradiation) on the mucus adhesion of the probiotic strains was characterized. Evaluation of dog probiotics was supported by cell visualization using transmission electron microscopy (TEM). Higher adhesion percentage was reported for probiotic strains growing in laboratory rather than in manufacturing conditions (P < 0·05). Inactivation by heat (95°C, 80°C) decreased the adhesion properties when strains were cultivated in soy‐based growth media compared with those grown in MRS broth (P < 0·05). TEM observations uncovered differences in cell‐surface components in nonviable forms of probiotic strains as compared with their viable forms.

Conclusions

Manufacturing process conditions such as growth media and pretreatment methods may significantly affect the adhesive ability of the tested strains.

Significance and Impact of the Study

Growth conditions, growth media, pretreatment methods and different probiotic combinations should be carefully considered for quality control of existing probiotics and for identification of new probiotics for dogs. These may also have an impact on health benefits for the host.     

Pneumococcal immune evasion: ZmpC inhibits neutrophil influx

Neutrophil recruitment is essential in clearing pneumococcal infections. The first step in neutrophil extravasation involves the interaction between P‐selectin on activated endothelium and P‐Selectin Glycoprotein 1 (PSGL‐1) on neutrophils. Here, we identify pneumococcal Zinc metalloproteinase C as a potent inhibitor of PSGL‐1. ZmpC degrades the N‐terminal domain of PSGL‐1, thereby disrupting the initial rolling of neutrophils on activated human umbilical vein endothelial cells. Furthermore, mice infected with wild‐type strain in the model of pneumococcal pneumonia showed lower lungs neutrophil infiltration compare to animals infected with ZmpC mutant. In addition, we confirmed the association of zmpC with serotype 8 and 11A and found it to be associated with serotype 33F as well. In conclusion, wereport PSGL‐1 as a novel target for ZmpC and show that ZmpC inhibits neutrophil extravasation during pneumococcal pneumonia.     

Back from the Brink: The Holocene History of the Carpathian Barbel Barbus carpathicus

As a result of specific adaptations and habitat preferences strongly rheophilic fish species may show high levels of endemism. Many temperate rheophilic fish species were subjected to a series of range contractions during the Pleistocene, and then successfully expanded during the Holocene, colonising previously abandoned areas. The Carpathian barbel (Barbus carpathicus Kotlík, Tsigenopoulos, Ráb et Berrebi 2002) occurs in the montane streams in three basins of the main Central European rivers in the northern part of the Carpathian range. We used genetic variation within 3 mitochondrial and 9 microsatellite loci to determine a pattern of postglacial expansion in B. carpathicus. We found that overall genetic variation within the species is relatively low. Estimate of time to the most recent common ancestor (tMRCA) of mitochondrial sequences falls within the Holocene. The highest levels of genetic variation found in upper reaches of the Tisa river in the Danube basin suggest that glacial refugia were located in the south-eastern part of the species range. Our data suggest that the species crossed different watersheds at least six times as three genetically distinct groups (probably established in different expansion episodes) were found in northern part of the species range. Clines of genetic variation were observed in both the Danube and Vistula basins, which probably resulted from subsequent bottlenecks while colonizing successive habitats (south eastern populations) or due to the admixture of genetically diverse individuals to a previously uniform population (Vistula basin). Therefore, B. carpathicus underwent both demographic breakdowns and expansions during the Holocene, showing its distribution and demography are sensitive to environmental change. Our findings are important in the light of the current human-induced habitats alterations.     

Apolipoprotein E,brain injury and neurodevelopmental outcome of children

Apolipoprotein E plays an important role in neurodegenerative processes in adulthood, whereas its neurodevelopmental role is uncertain. We aimed to study the effect of apolipoprotein E on neurodevelopment in a cohort liable to neurodevelopmental changes. The cohort consisted of very preterm (<32 gestational weeks) and/or very low birth weight (<1500 g) children, and the longitudinal follow‐up protocol included sequential cranial ultrasounds during infancy, brain magnetic resonance imaging at term‐equivalent age, neurological and cognitive assessment (Mental Developmental Index) at the corrected age of 2 years and cognitive and neuropsychological assessments (Wechsler Preschool and Primary Scale of Intelligence and Developmental NEuroPSYchological Assessment) at the chronological age of 5 years. Apolipoprotein E genotypes were determined from 322 children. Ultrasound and magnetic resonance imaging data were available for 321 (99.7%) and 151 (46.9%) children, respectively. Neurodevelopmental assessment data were available for 138 (42.9%) to 171 (53.1%) children. Abnormal findings in ultrasounds and magnetic resonance imaging were found in 163 (50.8%) and 64 (42.4%) children, respectively. Mild cognitive delay at the corrected age of 2 years and the chronological age of 5 years was suspected in 21 (12.3%) of 171 and 19 (13.8%) of 138 children, respectively. In the Developmental NEuroPSYchological Assessment, 47 (32.6%) of 144 children had significantly impaired performances in more than one study subtest. No associations between the apolipoprotein E genotypes and imaging findings or measured neurodevelopmental variables were found. Apolipoprotein E genotypes do not appear to have major impact on brain vulnerability or neurodevelopment in children .     

Telocytes: new insight into the pathogenesis of gallstone disease

The major mechanisms of gallstone formation include biliary cholesterol hypersecretion, supersaturation and crystallization, mucus hypersecretion, gel formation and bile stasis. Gallbladder hypomotility seems to be a key event that triggers the precipitation of cholesterol microcrystals from supersaturated lithogenic bile. Telocytes, a new type of interstitial cells, have been recently identified in many organs, including gallbladder. Considering telocyte functions, it is presumed that these cells might be involved in the signalling processes. The purpose of this study was to correlate the quantity of telocytes in the gallbladder with the lithogenicity of bile. Gallbladder specimens were collected from 24 patients who underwent elective laparoscopic cholecystectomy for symptomatic gallstone disease. The control group consisted of 25 consecutive patients who received elective treatment for pancreatic head tumours. Telocytes were visualized in paraffin sections of gallbladders with double immunofluorescence using primary antibodies against c‐Kit (anti‐CD117) and anti‐mast cell tryptase. Cholesterol, phospholipid and bile acid levels were measured in gallbladder bile. The number of telocytes in the gallbladder wall was significantly lower in the study group than that in the control group (3.03 ± 1.43 versus 6.34 ± 1.66 cell/field of view in the muscularis propria, P < 0.001) and correlated with a significant increase in the cholesterol saturation index. The glycocholic and taurocholic acid levels were significantly elevated in the control subjects compared with the study group. The results suggest that bile composition may play an important role in the reduction in telocytes density in the gallbladder.     

Superiority of Trans-Oral over Trans-Nasal Sampling in Detecting Streptococcus pneumoniae Colonization in Adults

The human nasopharynx is the main reservoir for Streptococcus pneumoniae. We applied conventional and molecular methods to determine the prevalence of S. pneumoniae nasopharyngeal colonization in adults. Paired trans-orally and trans-nasally obtained nasopharyngeal samples from 268 parents of 24-month-old children were assessed for pneumococcal presence. Parents were classified as colonized when live pneumococci were recovered from either sample cultured on medium selective for S. pneumoniae. Of the 52 (19%) colonized parents 49 (18%) were culture-positive in trans-nasal and 10 (4%) in trans-oral samples. Bacterial growth was harvested from these cultures, DNA isolated and tested by quantitative-PCR (qPCR) targeting lytA and piaA genes specific for S. pneumoniae. A sample was considered positive if signals for both genes were detected. Altogether 105 (39%) individuals were classified as positive for pneumococcus by qPCR including 50 (19%) in trans-nasal and 94 (35%) in trans-oral settings. Although significantly more trans-nasal compared to trans-oral samples were culture-positive for S. pneumoniae at the primary diagnostic step (p<0.001) the opposite was observed in qPCR results (p<0.001). To confirm the presence of live pneumococcus in samples positive by qPCR but negative at the initial diagnostic step, we serially-diluted cell harvests, re-cultured and carefully examined for S. pneumoniae presence. Live pneumococci were recovered from an additional 43 parents including 42 positive in trans-oral and 4 in trans-nasal samples increasing the number of individuals culture- and qPCR-positive to 93 (35%) and positive by either of two methods to 107 (40%). There were significantly more trans-oral than trans-nasal samples positive for pneumococcus by both culture and qPCR (n = 71; 27%; vs. n = 50; 19%; p<0.05). Our data suggest that pneumococcal colonization is more common in adults than previously estimated and point towards the superiority of a trans-oral over a trans-nasal approach when testing adults for colonization with S. pneumoniae.     

Possible Association between Suicide Committed under Influence of Ethanol and a Variant in the AUTS2 Gene

Background

rs6943555 in AUTS2 has been shown to modulate ethanol consumption. We hypothesized that rs6943555 might be associated with completed suicide.

Methods

We genotyped rs6943555 in 625 completed suicides and 3861 controls using real-time TaqMan Allelic Discrimination Assay. All individuals were Polish Caucasians.

Results

We detected an association between suicide and rs6943555 A allele (OR = 1.17, P = 0.018 for allelic comparison, OR = 1.24, P = 0.013 for dominant, and OR = 1.18, P = 0.020 for co-dominant model of inheritance). The association remained significant after adjusting for age and gender (co-dominant: P = 0.002 and dominant model: P = 0.001). After stratifying suicides according to blood ethanol concentration (BAC≤ 20 mg/dl and BAC > 20 mg/dl) the association remained significant only for cases who committed suicide under influence of alcohol (co-dominant: OR  =  1.37, P = 0.004 and dominant model: OR = 1.45, P = 0.006). To validate this finding we genotyped another cohort of 132 cases. We reproduced the association between rs6943555 A allele and suicide under influence of ethanol (allelic comparison: OR = 1.55, P = 0.023; co-dominant : OR = 1.54, P = 0.031; dominant model: OR = 1.84, P = 0.015). Analyzing pooled suicides with BAC >20 mg/dl (N = 300) we found the association of rs6943555 A allele not only vs. controls (allelic OR = 1.41, P = 0.00029) but also vs. cases with BAC ≤ 20 mg/dl (N = 449, allelic OR = 1.33, P = 0.019).

Conclusions

In our study rs6943555 A allele is associated with suicide committed after drinking ethanol shortly before death. The rs6943555 A allele may be linked to adverse emotional reaction to ethanol, which could explain the association with lower consumption in general population as well as the predisposition to suicide under influence of ethanol.