Viability Of Strains Research Articles

Penicillin Treatment Failure in Rabbit Syphilis Due to the Persistence of Treponemes (Treponema paraluisleporidarum Ecovar Cuniculus) in the Focus of Infection.

Rabbit venereal spirochetosis, a disease caused by Treponema paraluisleporidarum ecovar Cuniculus (TPeC), affects both wild and pet rabbits, and is transmitted sexually and via direct contact among animals. Treatment of syphilis in pet rabbits requires administration of antibiotics, including penicillin G, chloramphenicol, or fluoroquinolones. The aim of this work was to elucidate the cause of penicillin treatment failure in rabbit syphilis in a pet rabbit treated in Brno, Czech Republic, and to assess the phylogenetic relatedness of the agent to previously characterized pathogenic treponemes. Following amputation of the infected digits, the second round of penicillin treatment using the same dosage and application route resulted in the disappearance of clinical symptoms within a period of two weeks. The bacterium was successfully isolated from the claws, propagated in three experimental rabbits, and the resulting TPeC strain was designated as Cz-2020. Analysis of four genetic loci revealed that the Cz-2020 strain was similar but also clearly distinct from the only TPeC strain, which had been characterized in detail to date, i.e., the Cuniculi A strain, which was isolated in North America. The strain Cz-2020 represents the first available viable TPeC strain of European origin. DNA sequences encoding five penicillin-binding proteins of the strain Cz-2020 were compared to those of Cuniculi A, which is known to be sensitive to penicillin. The sequences differed in six nucleotides resulting in single amino acid changes in Penicillin-binding protein 1, 2, and 3. Since the second round of treatment was successful, we conclude that the penicillin treatment failure in the first round resulted from the presence of infection foci in claws where treponemes persisted.

Cultivation of the gut bacterium Prevotella copri DSM 18205T using glucose and xylose as carbon sources.

Prevotella copri DSM18205T is a human gut bacterium, suggested as a next‐generation probiotic. To utilize it as such, it is, however, necessary to grow the species in a reproducible manner. Prevotella copri has previously been reported to be highly sensitive to oxygen, and hence difficult to isolate and cultivate. This study presents successful batch cultivation strategies for viable strain inoculations and growth in both serum bottles and a stirred tank bioreactor (STR), without the use of an anaerobic chamber, as long as the cells were kept in the exponential growth phase. A low headspace volume in the STR was important to reach high cell density. P. copri utilized xylose cultivated in Peptone Yeast Xylose medium (PYX medium), resulting in a comparable growth rate and metabolite production as in Peptone Yeast Glucose medium (PYG medium) in batch cultivations at pH 7.2.Up to 5 g/L of the carbon source was consumed, leading to the production of succinic acid, acetic acid, and formic acid, and cell densities (OD620 nm) in the range 6−7.5. The highest yield of produced succinic acid was 0.63 ± 0.05 g/g glucose in PYG medium cultivations and 0.88 ± 0.06 g/g xylose in PYX medium cultivations.

First isolation and genotyping of Toxoplasma gondii strains from domestic animals in Tunisia

The isolation and molecular typing of Toxoplasma gondii strains provide an essential basis for a better understanding of the parasite’s genetic diversity, determinants of its geographical distribution and associated risks to human health. In this study, we isolated and genetically characterized T. gondii strains from domestic animals in Southern and coastal area of Tunisia. Blood, hearts and/or brains were collected from 766 domestic animals (630 sheep and 136 free-range chickens). Strain isolation from these samples was performed using mouse bioassay and genotyping was carried out with a multiplex PCR technique using 15 microsatellite markers. Thirty viable strains of T. gondii were successfully isolated from tissues of sheep (19/142) and chickens (11/33). In addition, 3 strains could be successfully genotyped from animal tissues for which mouse bioassay was unsuccessful. A large predominance of type II strains (n = 29) was found in the sampled regions, followed by type III (n = 3) and, for the first time in Tunisia, a single isolate of Africa 4 lineage from a sheep. Analyses of population genetics showed the presence of a divergent population of type II lineage in Tunisia, supporting limited recent migrations of strains between Tunisia and other countries of the world.

Improvement of Myelopoiesis in Cyclophosphamide-Immunosuppressed Mice by Oral Administration of Viable or Non-Viable Lactobacillus Strains.

Myelosuppression is the major dose-limiting toxicity of cancer chemotherapy. There have been many attempts to find new strategies that reduce myelosuppression. The dietary supplementation with lactic acid bacteria (LAB) improved respiratory innate immune response and the resistance against respiratory pathogens in immunosupressed hosts. Although LAB viability is an important factor in achieving optimal protective effects, non-viable LAB are capable of stimulating immunity. In this work, we studied the ability of oral preventive administration of viable and non-viable Lactobacillus rhamnosus CRL1505 or L. plantarum CRL1506 (Lr05, Lr05NV, Lp06V or Lp06NV, respectively) to minimize myelosuppressive and immunosuppressive effects derived from chemotherapy. Cyclophosphamide (Cy) impaired steady-state myelopoiesis in lactobacilli-treated and untreated control mice. Lr05V, Lr05NV and Lp06V treatments were the most effective to induce the early recovery of bone marrow (BM) tissue architecture, leukocytes, myeloid, pool mitotic and post-mitotic, peroxidase positive, and Gr-1Low/High cells in BM. We selected the CRL1505 strain for being the one capable of maintaining its myelopoiesis-enhancing properties in its non-viable form. Although the CRL1505 treatments do not modify the Cy ability to induce apoptosis, both increased the incorporation of BrdU in BM cells. Consequently, Lr05NV and Lr05V treatments were able to promote early recovery of LSK cells (Lin-Sca-1+c-Kit+ cells), multipotent progenitors (Lin-Sca-1+c-Kit+CD34+ cells), and myeloid cells (Gr-1+Ly6G+Ly6C- cells) with respect to the untreated Cy control. In addition, these treatments were able to increase the frequency of IL17A-producing innate lymphoid cells in the intestinal lamina propria (IL-17A+RORγt+CD4-NKp46+ cells) after Cy injection. These results were correlated with an increase in the IL-17A serum levels, a GM-CSF high expression and a CXCL12 lower expression in BM. Therefore, both Lr05V and Lr05NV treatments are able to activate beneficially the IL-17A/GM-CSF axis and accelerate the recovery of Cy-induced immunosuppression by increasing BM myeloid precursors. We demonstrated for the first time the beneficial effect of CRL1505 strain on myelopoiesis affected by a chemotherapeutic drug. Furthermore, Lr05NV could be a good and safe resource for reducing chemotherapy-induced leukopenia. The results are a starting point for future research and open up broad prospects for future applications of the immunobiotics.

Mycotoxin Removal by Lactobacillus spp. and Their Application in Animal Liquid Feed.

The removal of mycotoxins from contaminated feed using lactic acid bacteria (LAB) has been proposed as an inexpensive, safe, and promising mycotoxin decontamination strategy. In this study, viable and heat-inactivated L. acidophilus CIP 76.13T and L. delbrueckii subsp. bulgaricus CIP 101027T cells were investigated for their ability to remove aflatoxin B1 (AFB1), ochratoxin A (OTA), zearalenone (ZEA), and deoxynivalenol (DON) from MRS medium and PBS buffer over a 24 h period at 37 °C. LAB decontamination activity was also assessed in a ZEA-contaminated liquid feed (LF). Residual mycotoxin concentrations were determined by UHPLC-FLD/DAD analysis. In PBS, viable L. acidophilus CIP 76.13T and L. delbrueckii subsp. bulgaricus CIP 101027T cells removed up to 57% and 30% of ZEA and DON, respectively, while AFB1 and OTA reductions were lower than 15%. In MRS, 28% and 33% of ZEA and AFB1 were removed, respectively; OTA and DON reductions were small (≤15%). Regardless of the medium, heat-inactivated cells produced significantly lower mycotoxin reductions than those obtained with viable cells. An adsorption mechanism was suggested to explain the reductions in AFB1 and OTA, while biodegradation could be responsible for the removal of ZEA and DON. Both viable LAB strains reduced ZEA by 23% in contaminated LF after 48 h of incubation. These findings suggest that LAB strains of L. acidophilus CIP 76.13T and L. delbrueckii subsp. bulgaricus CIP 101027T may be applied in the feed industry to reduce mycotoxin contamination.

Effect of digestion and thermal processing on the stability of microbial cell-aflatoxin B1 complex

This study investigates the removal of aflatoxin B1 by viable and heat treated cells of Saccharomyces cerevisiae (PTCC 5052) and Lactobacillus rhamnosus GG (ATCC 53103) in a model system as a function of time and salt concentration. Moreover, the stability of microbial cell-aflatoxin B1 complex was evaluated at elevated temperature, high shear stress as well as under simulated digestive conditions. The rate of aflatoxin B1 adsorption was found to be significantly different for viable and heat treated cells. Heat inactivation of L. rhamnosus GG and S. cerevisiae dramatically increased the rate of toxin adsorption to 97.74% and 92.34%, respectively. Desorption of aflatoxin B1 from L. rhamnosus GG was approximately 3 times greater than viable S. cerevisiae and nearly1.5 times higher than heat treated cells. The maximum desorption of aflatoxin B1 from complexes occurred after 3 h incubation in simulated intestinal fluid. Shear stress and simulated saliva had no impact on the breakage of cell-aflatoxin B1 complex. The complexes formed by heat treated cells exhibited high stability against thermal treatment, whilst those generated by viable strains experienced toxin release during heating, which was much higher for S. cerevisiae (%4.68) than L. rhamnosus GG (%0.99).

First report of macrolide-resistant and -susceptible Mycoplasma pneumoniae clinical strains isolated from a single case

ObjectivesThe aim of this study was to investigate the biological characteristics and effect of antibiotic treatment for different Mycoplasma pneumoniae isolates co-infecting the same patient. MethodsTwo throat swab specimens from a single patient, on the day of admission (Sp01) and discharge (Sp13), were liquid cultured and subcultured on agar medium to obtain M. pneumoniae monoclones. The 23S rRNA gene of 50 monoclones from specimens Sp01 and Sp13 were analysed. Real-time PCR assay was used for detection of mutations and genotyping. Two typical monoclones were isolated for antimicrobial susceptibility testing. ResultsGenotype 1 monoclones accounted for 70.8% (34/48) in Sp01 and 95.7% (44/46) in Sp13. All genotype 1 monoclones were of the 4-5-7-2 multilocus variable-number tandem-repeat analysis (MLVA) type, while all genotype 2 monoclones were 3-5-6-2 MLVA type. The genotype 1 monoclone, which harboured the A2063G mutation in 23S rRNA gene, was resistant to erythromycin and azithromycin in vitro, whilst genotype 2, which did not carry the mutation, was susceptible to macrolides. The proportion of macrolide-resistant M. pneumoniae monoclones in the specimen cultures collected rose from 70.8% to 95.7% at the time of discharge. ConclusionThis is the first report on the isolation of macrolide-resistant and -susceptible strains of M. pneumoniae from the same patient. After treatment, the proportion of macrolide-resistant M. pneumoniae increased, but the patient still carried viable macrolide-susceptible strains, meaning that the macrolide-susceptible strains did not disappear completely.

Functional Differences between E. coli and ESKAPE Pathogen GroES/GroEL.

As the GroES/GroEL chaperonin system is the only bacterial chaperone that is essential under all conditions, we have been interested in the development of GroES/GroEL inhibitors as potential antibiotics. Using Escherichia coli GroES/GroEL as a surrogate, we have discovered several classes of GroES/GroEL inhibitors that show potent antibacterial activity against both Gram-positive and Gram-negative bacteria. However, it remains unknown if E. coli GroES/GroEL is functionally identical to other GroES/GroEL chaperonins and hence if our inhibitors will function against other chaperonins. Herein we report our initial efforts to characterize the GroES/GroEL chaperonins from clinically significant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). We used complementation experiments in GroES/GroEL-deficient and -null E. coli strains to report on exogenous ESKAPE chaperone function. In GroES/GroEL-deficient (but not knocked-out) E. coli, we found that only a subset of the ESKAPE GroES/GroEL chaperone systems could complement to produce a viable organism. Surprisingly, GroES/GroEL chaperone systems from two of the ESKAPE pathogens were found to complement in E. coli, but only in the strict absence of either E. coli GroEL (P. aeruginosa) or both E. coli GroES and GroEL (E. faecium). In addition, GroES/GroEL from S. aureus was unable to complement E. coli GroES/GroEL under all conditions. The resulting viable strains, in which E. coligroESL was replaced with ESKAPE groESL, demonstrated similar growth kinetics to wild-type E. coli, but displayed an elongated phenotype (potentially indicating compromised GroEL function) at some temperatures. These results suggest functional differences between GroES/GroEL chaperonins despite high conservation of amino acid identity.IMPORTANCE The GroES/GroEL chaperonin from E. coli has long served as the model system for other chaperonins. This assumption seemed valid because of the high conservation between the chaperonins. It was, therefore, shocking to discover ESKAPE pathogen GroES/GroEL formed mixed-complex chaperonins in the presence of E. coli GroES/GroEL, leading to loss of organism viability in some cases. Complete replacement of E. coligroESL with ESKAPE groESL restored organism viability, but produced an elongated phenotype, suggesting differences in chaperonin function, including client specificity and/or refolding cycle rates. These data offer important mechanistic insight into these remarkable machines, and the new strains developed allow for the synthesis of homogeneous chaperonins for biochemical studies and to further our efforts to develop chaperonin-targeted antibiotics.

Transmissibility study of two vaccine candidates against bovine tuberculosis

Aims & Objective: We studied the transmissibility in mice of two candidate vaccine M. bovis strains, M. bovis Δmce2 and M. bovis Δmce2ΔphoP, attenuated by gene knock out. The parental strain M. bovis UK (NCTC10772) and a M. bovis wild boar isolate (Mb04-303) were inoculated as controls. Methods: Groups of animals including 3 non-inoculated + 3 subcutaneously inoculated with every strain (1.105 CFU) were placed in the same cage (in duplicate). Necropsy was performed at 60 and 90 days post inoculation (dpi) in search of visible tuberculosis lesions (VTL) in lungs and spleens. Organs were analyzed to determine the bacterial load. Results: None of the animals presented VTL and no M. bovis was detectable in the non-inoculated group at 60 and 90 dpi. M. bovis was isolated from spleen of all the inoculated mice and from lungs of mice inoculated with control strains. The candidate vaccines were not isolated from lungs. Conclusions: This study shows the absence of transmissibility up to 90 days. Recovery of viable control strains from lungs and spleen suggests that the administration route would not alter the capacity of virulent strains to colonize organs. Contrasting, the candidate vaccines were only isolated from spleen. Longer periods should be assayed to evaluate transmissibility of these strains in mice.

Факторы риска распространения возбудителей инфекционных заболеваний в Арктической зоне

In the conditions of further development of the Arctic it is especially important to preserve the health of the population permanently or temporarily located in this territory. In recent years significant changes have taken place in the biosphere of the Arctic region under the influence of natural and anthropogenic factors. The population morbidity is accounted for by a number of diseases. However, the infectious component and the factors contributing to its growth remain poorly understood. Objective. To study various biocenoses in the Arctic region as potential risk areas for the spread of infectious diseases among the population. Materials and methods. The material for microbiological studies was selected in 2018–2019 in the archipelagos of Svalbard and Severnaya Zemlya. A total of 139 samples from various samples of natural, anthropogenic and ornithogenic biocenoses were studied. Research methods: classical bacteriological, MALDI-TOF mass spectrometric analysis, statistical data analysis. Results. During the study of Arctic samples were isolated and identified 309 viable bacterial strains and representatives of 117 species of micromycetes. Of all the isolated strains, the most important are Escherichia coli, Yersinia intermedia, representatives of the genera Acinetobacter, Pseudomonas, Stenotrophomonas, Serratia, Enterobacter, Enterococcus, and Staphylococcus. Conclusion. The risk factors for the spread of infectious disease pathogens in the Arctic region were identified: an increase in the number and diversity of isolated microorganisms of medical significance in dynamics; identification of pathogens of infections associated with medical care and saprozoonoses among them. Key words: Arctic, Svalbard and Severnaya Zemlya archipelagos, infectious diseases, pathogens of medical care-related infections and saprozoonoses, polar microorganisms

Oral probiotics to reduce vaginal group B streptococcal colonization in late pregnancy

This study aimed to evaluate the potential of oral probiotics to eradicate vaginal GBS colonization during the third trimester of pregnancy. We screened 1058 women for GBS colonization at 33–37 gestational weeks using a combination of vaginal-to-rectal swab and culture-based methods. Women who tested GBS positive were randomized to either the verum group, receiving a dietary probiotic supplement of four viable strains of Lactobacillus twice-daily for 14 days, or to the placebo group. Women underwent follow-up smears, whereat GBS colonization upon follow-up was considered the primary endpoint. We found that 215 women (20.3%) were positive for GBS upon screening, of which 82 (38.1%) were eligible for study inclusion; 41 (50%) of these were randomized to the verum and placebo groups each. After treatment, 21/33 (63.6%) members of the verum group, and 21/27 (77.8%) of the placebo group were still GBS positive (p = 0.24). Four (9.8%) women in the verum group and one (2.4%) in the placebo group experienced preterm birth (p = 0.20); smokers showed significantly higher rates of preterm birth (p = 0.03). Hence, the findings did not support the hypothesis that oral probiotics can eradicate GBS during pregnancy, although we observed a trend toward reduced GBS persistence after probiotic intake.

The Efficacy of Lactobacillus-Containing Probiotic Supplementation in Hemodialysis Patients: A Randomized, Double-Blind, Placebo-Controlled Trial

This study was performed to determine the effects of probiotic supplementation on cholesterol-triglyceride ratio, an indirect marker of insulin resistance, protein-bound uremic toxins, biomarkers of inflammation, and microbial translocation in end-stage renal disease patients on hemodialysis. Fifty-six patients aged 39-75years were assigned into two groups to receive either probiotic sachets (n=28) or a placebo (n=28) in a randomized double-blinded placebo-controlled clinical trial. The patients in the probiotic group received twice daily sachets that contained a mixture of three viable and freeze-dried strains: Lactococcus lactis subsp. Lactis LL358, Lactobaccillus salivarius LS159, and Lactobaccillus pentosus LPE588 at high dose (100 billion; 1×1011cfu/day) for 6months. A total of 50 patients were available for final analysis. Probiotic supplementation did not have a significant influence on cholesterol-triglyceride ratio. Probiotic supplementation for 6months caused a significant decrease in serum levels of indoxyl sulfate. Compared with the placebo, probiotic supplementation did not result in significant changes in hemoglobin levels, blood urea nitrogen, blood glucose, serum p-cresyl sulfate, inflammatory, and microbial translocation markers. No clinically significant changes in body composition were observed between the two groups during the study period. The probiotic supplementation was well tolerated by all subjects with minimal adverse effects during the 6-month-long study. Our results suggest that high-dose multistrain lactobaccillus probiotic supplementation over 6months as a monotherapy did not significantly decrease markers of insulin resistance, cholesterol-triglyceride ratio, and most of the studied markers, with the exception of levels of indoxyl sulfate in patients on HD.

Real-time PCR-based quantitation of viable Mycobacterium leprae strain from clinical samples and environmental sources and its genotype in multi-case leprosy families of India.

The potential role of environmental M. leprae in the transmission of leprosy remains unknown. We investigated role of environment as a possible source of viable M. leprae responsible for transmission of leprosy. The samples were collected from 10 multi-case leprosy families comprising, slit skin smear (SSS) from 9 multibacillary (MB), 16 paucibacillary cases (PB), 22 household contacts, and 38 environmental soil samples. The quantum of viable M. leprae was estimated by qRT-PCR using 16S rRNA gene from soil and SSS. Genotypes of M. leprae were determined by gene sequencing. We could observe presence of viable M. leprae in 11 (44%) leprosy cases (M. leprae 16S rRNA gene copies range from 1.78 × 102 to 8.782 × 109) and 4 (18%) household contacts (M. leprae 16S rRNA gene copies range from 2.54 × 103 and 7.47 × 104). Remarkably, presence of viable M. leprae was also noted in 10 (53%) soil samples where in M. leprae 16S rRNA gene copies ranged from 4.36 × 102 to 7.68 × 102. M leprae subtype 1D was noted in most of the leprosy cases their household contacts and in the surrounding soil samples indicating source of infection in household contacts could be from environment or patients. M. leprae 16S rRNA copies were approximately similar in both PB cases and soil samples along with presence of SNP type 1 subtype 1D in both samples indicating source of M. leprae from patients to contacts was either from patients or environment or both.

Underestimated Survival of Campylobacter in Raw Milk Highlighted by Viability Real-Time PCR and Growth Recovery.

Raw milk is a frequent vehicle for transmission of thermophilic Campylobacter, leading to reported outbreaks. Milk is a challenging food matrix for pathogen detection, due to its high protein and lipid content. Limited detection of Campylobacter colony-forming unit (CFU) in raw milk might underestimate the pathogen’s infectious potential. We optimized a viability real-time PCR (qPCR) for application with raw milk. The procedure was robust against variations of milk lots and different Campylobacter strains. Various DNA-intercalating dyes were evaluated for their ability to reduce the PCR signal of dead cells. Only propidium monoazide (PMA) and PMAxx qualified for diagnostic use. Different sedimentation properties of viable and dead Campylobacter jejuni and Campylobacter coli strains in 10-fold diluted milk enhanced viable/dead differentiation. The new method enabled to review survival of Campylobacter spp. in raw milk based on viable cells harboring an intact cell membrane. The data were compared to culturability according to ISO10272-2:2017. A difference of up to 4.5 log10 between viable Campylobacter counts and CFU values became apparent. Relevance of viability qPCR values was corroborated by full recovery of CFU under extremely reduced oxygen concentration in the presence of hydrogen. Recovery of CFU was limited, however, upon prolonged exposure in raw milk. The data confirm that Campylobacter survival in raw milk can be largely underestimated when relying on CFU data only. We conclude that raw milk led to oxidative stress-induced growth arrest in thermophilic Campylobacter, which was reversible by reduction of the oxygen partial pressure in a time-limited way.

Identification of Substrates of Cytoplasmic Peptidyl-Prolyl Cis/Trans Isomerases and Their Collective Essentiality in Escherichia Coli.

Protein folding often requires molecular chaperones and folding catalysts, such as peptidyl-prolyl cis/trans isomerases (PPIs). The Escherichia coli cytoplasm contains six well-known PPIs, although a requirement of their PPIase activity, the identity of their substrates and relative enzymatic contribution is unknown. Thus, strains lacking all periplasmic and one of the cytoplasmic PPIs were constructed. Measurement of their PPIase activity revealed that PpiB is the major source of PPIase activity in the cytoplasm. Furthermore, viable Δ6ppi strains could be constructed only on minimal medium in the temperature range of 30–37 °C, but not on rich medium. To address the molecular basis of essentiality of PPIs, proteins that aggregate in their absence were identified. Next, wild-type and putative active site variants of FkpB, FklB, PpiB and PpiC were purified and in pull-down experiments substrates specific to each of these PPIs identified, revealing an overlap of some substrates. Substrates of PpiC were validated by immunoprecipitations using extracts from wild-type and PpiC-H81A strains carrying a 3xFLAG-tag appended to the C-terminal end of the ppiC gene on the chromosome. Using isothermal titration calorimetry, RpoE, RseA, S2, and AhpC were established as FkpB substrates and PpiC’s PPIase activity was shown to be required for interaction with AhpC.

Formaldehyde effects on kanamycin resistance gene of inactivated recombinant Escherichia coli vaccines

Earlier studies have demonstrated the use of inactivated recombinant E. coli (bacterins), to protect against Clostridium spp. in vaccinated animals. These bacterins have a simpler, safer, and faster production process. However, these bacterins carry expression plasmids, containing antibiotic resistance gene, which could be assimilate accidentally by environmental microorganisms. Considering this, we aimed to impair this plasmids using formaldehyde at different concentrations. This compound inactivated the highest density of cells in 24 h. KanR cassette amplification was found to be impaired with 0.8% for 24 h or 0.4% for 72 h. Upon electroporation, E. coli DH5α ultracompetent cells were unable to acquire the plasmids extracted from the bacterins after inactivation procedure. Formaldehyde-treated bacterins were incubated with other viable strains of E. coli, leading to no detectable gene transfer. We found that this compound is effective as an inactivation agent. Here we demonstrate the biosafety involving antibiotic resistance gene of recombinant E. coli vaccines allowing to industrial production and animal application.

Study of the antibiotic residues in poultry meat in some of the EU countries and selection of the best compositions of lactic acid bacteria and essential oils against Salmonella enterica

In this study, the presence of antibiotics (ANB) residues was evaluated in poultry meat purchased from German and Lithuanian markets. In addition, the antimicrobial activity of 13 lactic acid bacteria (LAB) strains, 2 essential oils (EO) (Thymus vulgaris and Origanum vulgare L.), and their compositions were tested for the purpose of inhibiting antibiotic-resistant Salmonella spp. ANB residues were found in 3 out of the 20 analyzed poultry meat samples: sample no. 8 contained enrofloxacin (0.46 μg/kg), sample no. 14 contained both enrofloxacin and doxycycline (0.05 and 16.8 μg/kg, respectively), and sample no. 18 contained enrofloxacin (2.06 μg/kg). The maximum residue limits (MRLs) for the sum of enrofloxacin and ciprofloxacin and for doxycycline in the poultry muscle are 100 μg/kg. Finally, none of the tested poultry meat samples exceeded the suggested MRLs; however, the issue of ANB residues still requires monitoring of the poultry industry in Germany, Poland, and Lithuania, despite the currently established low ANB concentrations. These findings can be explained by the increased use of alternatives to ANB in the poultry industry. Our results showed that an effective alternative to ANB, which can help to reduce the occurrence of antibiotic-resistant salmonella, is a composition containing 1.0% of thyme EO and the following LAB strains: Lactobacillus plantrum LUHS122, Enteroccocus pseudoavium LUHS242, Lactobacillus casei LUHS210, Lactobacillus paracasei LUHS244, Lactobacillus plantarum LUHS135, Lactobacillus coryniformins LUHS71, and Lactobacillus uvarum LUHS245, which can be recommended for poultry industry as components of feed or for the treatment of surfaces, to control the contamination with Salmonella strains. However, it should be mentioned that most of the tested LAB strains were inhibited by thyme EO at the concentrations of 0.5 and 1.0%, except for LUHS122, LUHS210, and LUHS245. Finally, it can be noted that the agents responsible for the inhibitory effect on Salmonella are not the viable LAB strains but rather their metabolites, and further studies are needed to identify which metabolites are the most important.

Screening strategy targeting the presence of food enzyme-producing fungi in food enzyme preparations

Food enzymes (FE) are often produced through microbial fermentation, for which fungal strains are commonly used. Verifying the absence of viable strains and associated DNA of producer organisms in FE's is important for both the safety evaluation of the product and the control of the food chain quality.For this purpose, a first line generic screening strategy, targeting FE producing fungal species, is proposed, based on a PCR amplification of the internal transcribed spacer (ITS) region followed by sequencing and sequence analysis for genus identification. To simplify the sequence analysis, an in-house constructed database was assembled, containing ITS reference sequences from the FE producing fungal species available from public databases and our own generated amplicons. Finally, the performance of the proposed strategy was assessed in terms of specificity and sensitivity. Some experiments were also conducted in order to evaluate the applicability of the method and to verify the feasibility of its implementation in control laboratories.

Fibroin gene expression and antioxidant enzymes are elevated in Bombyx mori when reared on preferred host plants

Silk fibroin filaments are natural proteins produced by silkworm, Bombyx mori, which is bounded by sericin polymers in silk thread used in the cocoon. Fibroin gene expression and antioxidant enzymes were studied in the midgut of Bombyx mori larvae when reared on tropical mulberry plant varieties (Jorhat, TR10, BC2-59, and Hmute). In this study, the B. mori strain FC2 x FC1 showed a high expression of fibroin at both mRNA and protein levels when reared on four plant varieties. B. mori strains FC1 x FC2 (reared on Local, Jorhat and BC2-59) and CSR4 x CSR2 (reared on Jorhat, TR10 and BC2-59) had a higher level of expression of fibroin protein. The strain FC2 X FC1 showed relatively higher SOD and moderate catalase activities. This study reveals the differential expression of fibroin and tolerance to stress in B. mori strains which might justify the rationality of using the most suitable host plants and economically viable strains (FC2 X FC1) for producing commercially the best quality silk threads.

Use of serological and molecular techniques for detection of Leptospira sp. carrier sheep under semiarid conditions and the importance of genital transmission route

It is possible that there are peculiarities in the epidemiology of leptospirosis in regions with a semiarid climate, where the environment is often adverse, allowing the occurrence of alternative transmission routes. The objective of the work was to generate contributions to the diagnosis and epidemiology of Leptospira sp. infection in sheep reared in semiarid conditions, using serological, molecular and microbiological techniques for diagnosis in dry and rainy seasons. Samples of blood, vaginal fluid, urine, bladder, kidney, uterus, uterine tube, ovary and placenta were collected from 104 sheep (52 animals per season – dry and rainy) slaughtered in the Brazilian semiarid. Diagnostic tests performed were microscopic agglutination test (MAT), polymerase chain reaction (PCR) and bacterial isolation. Anti-Leptospira sp. antibodies were found in 26 (25%) of the animals analyzed by MAT at 1:50 dilution (cut-off 50), while 69 (66.3%) animals had at least one organ/fluid with the presence of Leptospira sp. DNA. Overall, PCR was performed on 758 fragments of organs/fluids from the genital and urinary tracts, and 519 (68.5%) samples tested positive. PCR-positivity was statistically different in dry (46.2%) and rainy (11.5%) seasons for vaginal fluid. It was possible to perform the DNA sequencing in nine samples with 99% similarity to L. interrogans and recovery of viable strains in three samples of vaginal fluid. Regardless of the biological material used in PCR to detect carrier animals and the season, the highest MAT sensitivity values were obtained with cut-off 50 compared to 100. The results obtained indicate that, even in the adverse environmental conditions of the semiarid region, leptospires may survive and propagate by alternative routes of transmission, such as sexual, and the presence of PCR-positive genital tracts in ewes suggests that sexual transmission may play an important role in the epidemiology of the disease in sheep in Brazilian semiarid. In addition, it is suggested the use of titer 50 as cut-off point at serology in semiarid conditions.