Monika Krüger

Challenging the problem of clostridial identification with matrix-assisted laser desorption and ionization–time-of-flight mass spectrometry (MALDI–TOF MS)

Diverse techniques were applied to effect the identification and classification of isolated clostridial strains. Nevertheless, the correct identification of clostridial strains remains a laborious, time-consuming task which entails a not inconsiderable degree of expertise. In addition to this, traditional methods based on the metabolic properties of the bacteria require rigorously standardized media and growth conditions to assure the attainment of reproducible results. Although DNA-based methods, like the PCR of a species specific gene, are known to yield precise and reproducible results, their degree of effectivity is circumscribed by the fact that even the incidence of a toxin encoding gene is not necessarily linked to nor consequently indicative of the presence of an infectious disease. Moreover, most of these methods postulate an initial assumption concerning the expected bacterial species involved before the choice of PCR primer for use can be made. Consequently, the scope of these methods is restricted to that of targeted analyses. The 16S rDNA sequencing which is assumed to be the gold standard for bacterial classification having the unequivocal advantage of being capable of determining even uncultivable bacteria is nonetheless a time-consuming and costly technique. In the present study we describe the utilization of matrix-assisted laser desorption and ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for whole cell fingerprinting in combination with a dedicated bioinformatic software tool to distinguish between various clostridial species. Total 64 clostridial strains of 31 different species each displayed a mass spectrum unique to the strain involved, to the effect that it was also possible not only to differentiate between the strains examined, but also to establish to which species the individual strains belonged to. Starting with a single colony it was possible to correctly identify a Clostridium species within minutes. It was even possible to identify species which are normally difficult to differentiate by traditional methods, such as C. chauvoei and C. septicum. With the results obtained we were able to assemble a dendrogram of the Clostridium species which showed considerable similarities to dendrograms based upon 16S rDNA sequencing data. To conclude, our findings indicate that, inasmuch as the MALDI-TOF MS technology employed is based on a high-quality reference database, it may serve as an effective tool for the swift and reliable identification and classification of Clostridia.

Curcumin inhibits glyoxalase 1: a possible link to its anti-inflammatory and anti-tumor activity.

Background Glyoxalases (Glo1 and Glo2) are involved in the glycolytic pathway by detoxifying the reactive methylglyoxal (MGO) into D-lactate in a two-step reaction using glutathione (GSH) as cofactor. Inhibitors of glyoxalases are considered as anti-inflammatory and anti-carcinogenic agents. The recent finding that various polyphenols modulate Glo1 activity has prompted us to assess curcumins potency as an Glo1 inhibitor. Methodology/Principal Findings Cultures of whole blood cells and tumor cell lines (PC-3, JIM-1, MDA-MD 231 and 1321N1) were set up to investigate the effect of selected polyphenols, including curcumin, on the LPS-induced cytokine production (cytometric bead-based array), cell proliferation (WST-1 assay), cytosolic Glo1 and Glo2 enzymatic activity, apoptosis/necrosis (annexin V-FITC/propidium iodide staining; flow cytometric analysis) as well as GSH and ATP content. Results of enzyme kinetics revealed that curcumin, compared to the polyphenols quercetin, myricetin, kaempferol, luteolin and rutin, elicited a stronger competitive inhibitory effect on Glo1 (Ki = 5.1±1.4 µM). Applying a whole blood assay, IC50 values of pro-inflammatory cytokine release (TNF-α, IL-6, IL-8, IL-1β) were found to be positively correlated with the Ki-values of the aforementioned polyphenols. Moreover, whereas curcumin was found to hamper the growth of breast cancer (JIMT-1, MDA-MB-231), prostate cancer PC-3 and brain astrocytoma 1321N1 cells, no effect on growth or vitality of human primary hepatocytes was elucidated. Curcumin decreased D-lactate release by tumor cells, another clue for inhibition of intracellular Glo1. Conclusions/Significance The results described herein provide new insights into curcumins biological activities as they indicate that inhibition of Glo1 by curcumin may result in non-tolerable levels of MGO and GSH, which, in turn, modulate various metabolic cellular pathways including depletion of cellular ATP and GSH content. This may account for curcumins potency as an anti-inflammatory and anti-tumor agent. The findings support the use of curcumin as a potential therapeutic agent.

The effect of glyphosate on potential pathogens and beneficial members of poultry microbiota in vitro.

The use of glyphosate modifies the environment which stresses the living microorganisms. The aim of the present study was to determine the real impact of glyphosate on potential pathogens and beneficial members of poultry microbiota in vitro. The presented results evidence that the highly pathogenic bacteria as Salmonella Entritidis, Salmonella Gallinarum, Salmonella Typhimurium, Clostridium perfringens and Clostridium botulinum are highly resistant to glyphosate. However, most of beneficial bacteria as Enterococcus faecalis, Enterococcus faecium, Bacillus badius, Bifidobacterium adolescentis and Lactobacillus spp. were found to be moderate to highly susceptible. Also Campylobacter spp. were found to be susceptible to glyphosate. A reduction of beneficial bacteria in the gastrointestinal tract microbiota by ingestion of glyphosate could disturb the normal gut bacterial community. Also, the toxicity of glyphosate to the most prevalent Enterococcus spp. could be a significant predisposing factor that is associated with the increase in C. botulinum-mediated diseases by suppressing the antagonistic effect of these bacteria on clostridia.

Detection of Glyphosate Residues in Animals and Humans

In the present study glyphosate residues were tested in urine and different organs of dairy cows as well as in urine of hares, rabbits and humans using ELISA and Gas Chromatography-Mass Spectroscopy (GC-MS). The correlation coefficients between ELISA and GC-MS were 0.96, 0.87, 0.97and 0.96 for cattle, human, and rabbit urine and organs, respectively. The recovery rate of glyphosate in spiked meat using ELISA was 91%. Glyphosate excretion in German dairy cows was significantly lower than Danish cows. Cows kept in genetically modified free area had significantly lower glyphosate concentrations in urine than conventional husbandry cows. Also glyphosate was detected in different organs of slaughtered cows as intestine, liver, muscles, spleen and kidney. Fattening rabbits showed significantly higher glyphosate residues in urine than hares. Moreover, glyphosate was significantly higher in urine of humans with conventional feeding. Furthermore, chronically ill humans showed significantly higher glyphosate residues in urine than healthy population. The presence of glyphosate residues in both humans and animals could haul the entire population towards numerous health hazards, studying the impact of glyphosate residues on health is warranted and the global regulations for the use of glyphosate may have to be re-evaluated.

Field Investigations of Glyphosate in Urine of Danish Dairy Cows

In the present study, thirty dairy cows from each of eight Danish dairy farms were investigated for excretion of glyphosate in urine. Blood serum parameters indicative of cytotoxicity as alkaline phosphatase (AP), glutamate dehydrogenase (GLDH), glutamate oxaloacetate transaminase (GOT), creatinine kinase CK), nephrotoxicity, (urea, creatine), cholesterol and the trace elements as manganese (Mn), cobalt (Co), selenium (Se), copper (Cu) and zinc (Zn) were investigated. All cows excreted glyphosate in their urine but in varying concentrations. Increased levels of GLDH, GOT and CK in cows from all farms demonstrate a possible effect of glyphosate on liver and muscle cells. High urea levels in some farms could be due to nephrotoxicity of glyphosate. Also the unexpected very low levels of Mn and Co were observed in all animals which could be explained due to a strong mineral chelating effect of glyphosate. In contrast the mean levels of Cu, Zn and Se were within the normal reference range. In conclusion, this study gives the first documentation to which extent Danish dairy cattle are exposed to Glyphosate and its impact on blood parameters.

Detection of Glyphosate in Malformed Piglets

Glyphosate residues in different organs and tissues as lungs, liver, kidney, brain, gut wall and heart of malformed euthanized one-day-old Danish piglets (N= 38) were tested using ELISA. All organs or tissues had glyphosate in different concentrations. The highest concentrations were seen in the lungs (Range 0.4-80 μg/ml) and hearts (Range 0.15-80 μg/ml). The lowest concentrations were detected in muscles (4.4-6.4 μg/g). The detection of such glyphosate concentrations in these malformed piglets could be an allusion to the cause of these congenital anomalies. Further investigations are urgently needed to prove or exclude the role of glyphosate in malformations in piglets and other animals.

The effects of stress hormones on growth of selected periodontitis related bacteria

The focus of this study was to examine in vitro the effects of stress hormones (catecholamines: epinephrine, norepinephrine, dopamine and hydrocortisone: cortisol) on the growth of four anaerobic species of periodontitis-related bacteria (Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia and Tannerella forsythia) and one facultative anaerobic species (Eikenella corrodens). Bacterial growth was determined by two different methods: fluorescence in situ hybridization (FISH), and the viable count by culture method. To simulate stress, each single strain was grown in a special growth medium with three different concentrations of each hormone, using an anaerobic chamber at 37 °C. Growth of F. nucleatum increased in the presence of all stress hormones. Growth of P. gingivalis was not significantly influenced by any hormone. Growth of P. intermedia and E. corrodens was inhibited by almost all stress hormones tested. Both methods of analysis revealed that the highest concentrations of norepinephrine and cortisol increased the growth of T. forsythia. Different hormones have a different effect on the growth of periodontitis-related bacteria in vitro. It appears that bacterial viability is more strongly influenced than is bacterial metabolic activity. The growth of F. nucleatum particularly and partially of T. forsythia is increased by several stress hormones and may have an additional negative impact on periodontal disease.

Efficacy of early treatment with toltrazuril in prevention of coccidiosis and necrotic enteritis in chickens

In the present study, efficacy of the toltrazuril treatment for prevention of coccidiosis and necrotic enteritis was tested. Ninety-six 14-day-old commercial broiler chickens were caged and divided into eight groups (n=12), designated groups 1 to 8. Chickens of groups 1 to 6 were inoculated orally at 18 days of age with 25,000 oocysts of Eimeria tenella and 75,000 oocysts of Eimeria brunetti. At 22 days of age, chickens of groups 1 to 6 were infected with 109 colony-forming unit Clostridium perfringens. Chickens of group 1 were treated with 75 parts/106 toltrazuril in drinking water for 8 h on two consecutive days up to 12 h before Eimeria infection, while chickens of groups 2 to 5 were treated with the same dose of toltrazuril at 12 h, 36 h, 60 h and 84 h after Eimeria infection, respectively. The non-treated group 6 served as a positive control. Chickens in group 7 were treated with toltrazuril at 17 and 18 days of age, and those of group 8 remained uninfected and non-treated as a negative control. The feed conversion ratio was higher in the positive control compared with other groups. The mortality rates were 16.8% and 41.7% in the late toltrazuril-treated (at 84 h) and infected non-treated chickens, respectively. Lesions scores of necrotic enteritis or coccidiosis in infected, non-treated chickens were significantly more severe compared with negative controls (P<0.01) and late toltrazuril-treated (at 84 h) chickens (P<0.05). In conclusion, application of toltrazuril before Eimeria challenge protected chickens from coccidiosis and indirectly from successive necrotic enteritis caused by C. perfringens infection.

Development of the genital microflora in stallions used for artificial insemination throughout the breeding season

An important factor influencing stallion fertility is the microbial contamination of semen. Aims of this study were to investigate changes in the microbiological population of the genital mucosa and semen in artificial insemination stallions (n=16) from before to after one breeding season (February-August). MALDI-TOF-MS (matrix assisted laser desorption/ionization time-of-flight mass spectrometry) was used for identification of microbial agents. For bacteriology, swabs from the urethral opening, urethral fossa and penile sheath as well as semen were collected at 4-week-intervals. For semen motility and percentage of morphologically normal spermatozoa, changes over time (P<0.001) occurred. In 14.3% of genital swabs and 25.0% of ejaculates no microbial growth was found. Intensity of total microbial growth increased throughout the breeding season (P<0.001). From the penile sheath, between 1.4±0.1 microbial species in February and 3.3±0.4 in August were identified. From semen, 1.1±0.3 microbial species in February and 2.9±0.6 in August were obtained. The number of microbial species isolated from the sheath of the penis (2.0±0.1) and urethral fossa (2.0±0.1) was greater (P<0.01) compared with the urethral opening (1.6±0.1) and semen (1.5±0.1). The microbial flora consisted of aerobic and anaerobic bacteria, dominated by coagulase-negative staphylococci, alpha-haemolytic streptococci and coryneforms. Only occasionally potentially pathogenic agents (E. coli, Ps. aeruginosa) were found. The microbial flora was not related to seminal characteristics.

Antagonistic effect of different bacteria on Clostridium botulinum types A, B, D and E in vitro

Clostridium botulinum is an anaerobic, Gram-positive, spore-forming rod that produces a potent neurotoxin. Seven types (A, B, C1+2, D, E, F and G) of C botulinum are recognised, based on the antigenic specificity of the botulinum neurotoxin (BoNT) produced by each strain. Types A, B, E and F cause human botulism, while types B, C and D cause disease in farm animals (Goonetilleke and Harris 2004, Simpson 2004, Radostits and others 2007, Popoff and Bouvet 2009). Animals most commonly affected are wild fowl and poultry, cattle, horses and some species of fish. There are three major recognised disease entities in humans, food-borne botulism, infant botulism and wound botulism . Recently, Rodloff and Kruger (2012) suggested that a new form of chronic, visceral botulism may exist that affects both human beings and animals. The antagonism between C botulinum and bacterial members of the microecosystem …