Renata Urban-Chmiel

Identification of Lactobacillus strains of goose origin using MALDI-TOF mass spectrometry and 16S-23S rDNA intergenic spacer PCR analysis.

The objective of our study was to identify Lactobacillus sp. strains of goose origin using MALDI-TOF mass spectrometry, ITS-PCR and ITS-PCR/RFLP. All three techniques proved to be valuable tools for identification of avian lactobacilli and produced comparable classification results. Lactobacillus strains were isolated from 100% of geese aged 3 weeks to 4 years, but from only 25% of chicks aged 1-10 days. Among the 104 strains isolated, we distinguished 14 Lactobacillus species. The dominant species was Lactobacillus salivarius (35.6%), followed by Lactobacillus johnsonii (18.3%), Lactobacillus ingluviei (11.5%) and Lactobacillus agilis (7.7%). The intact-cell MALDI-TOF mass spectrometry enabled rapid species identification of the lactobacilli with minimal pretreatment. However, it produced more than one identification result for 11.5% examined strains (mainly of the species L. johnsonii). ITS-PCR distinguished 12 genotypes among the isolates, but was not able to differentiate closely related strains, i.e. between Lactobacillus amylovorus and Lactobacillus kitasatonis and between Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus zeae. These species were differentiated by ITS-PCR/RFLP using the restriction enzymes TaqI and MseI. The results obtained indicate that ITS-PCR and ITS-PCR/RFLP assays could be used not only for interspecific, but also for intraspecific, typing.

Screening of Lactobacillus strains of domestic goose origin against bacterial poultry pathogens for use as probiotics

Lactobacilli are natural inhabitants of human and animal mucous membranes, including the avian gastrointestinal tract. Recently, increasing attention has been given to their probiotic, health-promoting capacities, among which their antagonistic potential against pathogens plays a key role. A study was conducted to evaluate probiotic properties of Lactobacillus strains isolated from feces or cloacae of domestic geese. Among the 104 examined isolates, previously identified to the species level by whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and analysis of 16S-23S regions of rDNA, dominated Lactobacillus salivarius (35%), followed by Lactobacillus johnsonii (18%) and Lactobacillus ingluviei (11%). All lactobacilli were screened for antimicrobial activity toward Salmonella Enteritidis, Escherichia coli, Clostridium perfringens, Staphylococcus aureus, Pasteurella multocida, and Riemerella anatipestifer using the agar slab method and the well diffusion method. Lactobacillus salivarius and Lactobacillus plantarum exhibited particularly strong antagonism toward all of the indicator strains. In the agar slab method, the highest sensitivity to Lactobacillus was observed in R. anatipestifer and P. multocida, and the lowest in E. coli and S. aureus. The ability to produce H₂O₂was exhibited by 92% of isolates, but there was no correlation between the rate of production of this reactive oxygen species and the antimicrobial activity of Lactobacillus sp. All lactobacilli showed resistance to pH 3.0 and 3.5 and to 2% bile. The data demonstrate that Lactobacillus isolates from geese may have probiotic potential in reducing bacterial infections. The antibacterial activity of the selected lactobacilli is mainly due to lactic acid production by these bacteria. The selected Lactobacillus strains that strongly inhibited the growth of pathogenic bacteria, and were also resistant to low pH and bile salts, can potentially restore the balance of intestinal microflora in geese and could offer an alternative to antibiotic therapy.

Characterization of heat-shock proteins in Escherichia coli strains under thermal stress in vitro.

The aim of this study was to evaluate the effect of heat stress in in vitro conditions on the induction of heat-shock protein (Hsp)70 by Escherichia coli cells, and to determine the localization of Hsps in cell fractions. The material consisted of wild strains of E. coli isolated from the digestive tract of calves, suspended in an exponential-phase culture and subjected to 41.5 °C for 2 h. Individual fractions were analysed by SDS-PAGE and two-dimensional electrophoresis. Western blotting with mouse anti-Hsp70 and anti-Hsp60 mAbs was used to identify the proteins. Electrophoretic analysis of the heat-treated cells detected Hsp70 in all three fractions, cytoplasmic, periplasmic and membrane, which was confirmed by Western blotting. The proteins obtained had diverse localizations in the pH gradient in two-dimensional electrophoresis, which may indicate changes in their conformation and physical properties leading to stabilization and protection of intracellular structures in stress conditions. The presence of these Hsps in different cell fractions indicates a very strong protective adaptation in the bacteria in unfavourable conditions, which is critical for the organism infected by them.

The effect of conglutinin on production of reactive oxygen species in bovine granulocytes.

Conglutinin is a high molecular-weight lectin originally detected in bovine serum. It belongs to the family of collectins that bind sugar residues in a Ca2+-dependent manner and are effector molecules in innate immunity. Conglutinin appears to play an important role in immune defense mechanisms, showing antiviral and antibacterial activities when tested in vivo and in vitro. The present study evaluated the effect of conglutinin on the respiratory bursts in bovine peripheral phagocytes. Using nitroblue tetrazolium and hydrogen peroxide assays, we showed that sugar ligand-bound conglutinin stimulated the production of superoxide and H2O2 in granulocytes whereas the non-sugar-bound form of conglutinin inhibited these processes. These results indicate that both forms of conglutinin are able to interact with surface leukocyte receptors but have opposite effects on phagocytic activity. Our findings suggest that conglutinin bound to sugar residues on microbial surfaces can induce oxygen burst in phagocytes, and thereby mediates the elimination of pathogens and prevents the spread of infection.

Assessment of antibiotic susceptibility in Lactobacillus isolates from chickens

BackgroundThe aim of this study was to determine the susceptibility of 88 Lactobacillus isolates derived from chickens to antibiotic substances and to detect drug-resistance genes.ResultsThe minimal inhibitory concentration of 13 antimicrobial substances was determined by the broth microdilution method, and resistance genes were detected by PCR. We recorded a high prevalence of resistance to tiamulin (90% resistant isolates), tetracyclines (74%) and lincomycin (70%), and a moderately high frequency of resistance to enrofloxacin (48%), macrolides (42%), aminoglycosides (12.5–31%), ampicillin (26%) and chloramphenicol (23%). Multi-drug resistance was observed in 79.5% of isolates. The presence of resistance genes was generally correlated with phenotypic resistance, but some molecular determinants were also recorded in susceptible isolates. Among tetracycline resistance genes, the most frequently identified was tetW (45% isolates), followed by tetM (26%) and tetL (24%). The ermB, ermC and lnuA genes, associated with resistance to macrolides and lincosamides, were observed in 39, 12 and 39% of isolates, respectively. Among genes determining resistance to aminoglycoside antibiotics, we identified ant(6)-Ia (10% of isolates), aac(6′)-Ie-aph(2′)-Ia (8%), aph(2″)-Ic (6%) and aadE (4.5%). The cat gene was present in 32 isolates, including 8 of 20 found to be resistant to chloramphenicol. Two genes encoding efflux pumps were identified—the acrA gene was present in all isolates tested, and 10 of 79 lactobacilli determined to be phenotypically resistant to tiamulin contained the lsaE gene. We were unable to explain the resistance mechanism of Lactobacillus isolates to ampicillin, but showed that it did not involve the production of β-lactamases.ConclusionsOur findings indicate that intestinal lactobacilli should be considered a reservoir of resistance genes and that antibiotics must be used prudently in poultry production. The data derived from this study can be used as a basis for reviewing current microbiological breakpoints for categorization of susceptible and resistant strains within the genus Lactobacillus.

Isolation, identification and antibiotic resistance of Campylobacter strains isolated from domestic and free-living pigeons

Abstract 1. The aim of this study was to evaluate the occurrence of Campylobacter spp. in domestic and free-living pigeons and to evaluate the antibiotic resistance profiles. 2. The material consisted of cloacal swabs obtained from 108 homing pigeons and fresh faeces from 72 wild birds from Lublin and its vicinity. The identification of strains isolated on differential/selective media for Campylobacter spp. was carried out by MALDI-TOF and PCR. The susceptibility to antibiotics was evaluated by minimum inhibitory concentration (MIC) in Mueller-Hinton broth. 3. A total of 35 strains of Campylobacter spp. were isolated; 27 were identified as Campylobacter jejuni and 8 as Campylobacter coli. Over half of the isolates were resistant to erythromycin and streptomycin, 40% of strains were resistant to tetracycline and ampicillin and 37% isolates were resistant to amoxicillin. Resistance to two or more antibiotics was observed in all strains tested. 4. The results indicate that both domestic and free-living pigeons are reservoirs for bacteria of the genus Campylobacter, which are characterised by varied and growing resistance to commonly used antibiotics.

Isolation and Characterization of Lytic Properties of Bacteriophages Specific for M. haemolytica Strains.

Aim of Study The objective of this study was isolation and morphological characterization of temperate bacteriophages obtained from M. haemolytica strains and evaluation of their lytic properties in vitro against M. haemolytica isolated from the respiratory tract of calves. Material and Methods The material for the study consisted of the reference strain M. haemolytica serotype 1 (ATCC®) BAA-410™, reference serotypes A1, A2, A5, A6, A7, A9 and A11, and wild-type isolates of M. haemolytica. Bacteriophages were induced from an overnight bacterial starter culture of all examined M. haemolytica strains treated with mitomycin C. The lytic properties and host ranges were determined by plaque assays. The morphology of the bacteriophages was examined in negative-stained smears with 5% uranyl acetate solution using a transmission electron microscope. The genetic analysis of the bacteriophages was followed by restriction analysis of bacteriophage DNA. This was followed by analysis of genetic material by polymerase chain reaction (PCR). Results Eight bacteriophages were obtained, like typical of the families Myoviridae, Siphoviridae and Podoviridae. Most of the bacteriophages exhibited lytic properties against the M. haemolytica strains. Restriction analysis revealed similarities to the P2-like phage obtained from the strain M. haemolytica BAA-410. The most similar profiles were observed in the case of bacteriophages φA1 and φA5. All of the bacteriophages obtained were characterized by the presence of additional fragments in the restriction profiles with respect to the P2-like reference phage. In the analysis of PCR products for the P2-like reference phage phi-MhaA1-PHL101 (DQ426904) and the phages of the M. haemolytica serotypes, a 734-bp phage PCR product was obtained. The primers were programmed in Primer-Blast software using the structure of the sequence DQ426904 of reference phage PHL101. Conclusions The results obtained indicate the need for further research aimed at isolating and characterizing bacteriophages, including sequence analysis of selected fragments. Moreover, standardization of methods for obtaining them in order to eliminate M. haemolytica bacteria involved in the etiopathogenesis of BRDC is essential.

Purification and electrophoretic characterization of bovine conglutinin.

In this study a two-stage procedure for purification of conglutinin using affinity and ion-exchange chromatography was developed. To isolate conglutinin from bovine serum, its unique ability to bind to complement component iC3b was exploited. Incubation of bovine serum with chromatographic beads (TSK, Toyopearl HW-75 F) at 37 °C allows for iC3b deposition and subsequent binding of conglutinin. A single protein fraction eluted with ethylenediaminetetraacetic acid (EDTA) was then separated on an ion-exchange column in an NaCl gradient. The purification was evaluated by SDS-PAGE and western blotting. Conglutinin analyzed by SDS-PAGE under reducing conditions showed two main bands at 41 and 47 kDa and eight weaker bands. Nonreduced conglutinin appeared as a ladder pattern composed of many fractions ranging from 34 to 630 kDa. The bands at 34, 153, 174, 247, 338 and 387 kDa displayed the highest optical density. In the native conglutinin profile four fractions were observed, and the pI of this protein was below 8.5. The presence of sugar residues in the conglutinin molecule was detected using Schiffs reagent.

Bacteriophage therapy to combat bacterial infections in poultry

Infections in poultry are an economic and health problem in Europe and worldwide. The most common infections are associated with salmonellosis, colibacillosis, campylobacteriosis, and others. The prevalence of Campylobacter-positive poultry flocks in European countries varies from 18% to 90%. In the United States, the prevalence of infected flocks is nearly 90%. A similar percentage of infection has been noted for salmonellosis (about 75–90%) and E. coli (90–95%). The occurence of Clostridium perfringens is a major problem for the poultry industry, with some estimates suggesting colonization of as many as 95% of chickens, resulting in clinical or subclinical infections. In the US, annual economic losses due to Salmonella infections run from

Application of loop-mediated isothermal amplification (LAMP) assays for the detection of bovine herpesvirus 1

1.188 billion to over