Andrzej Puchalski

Reptiles are considered a potential source of Salmonella transmission to humans. The aim of this research was to determine the incidence of Salmonella in pet reptiles in Poland and to examine Salmonella isolates with regard to their biochemical characteristics, serotype, antimicrobial susceptibility, and pathogenic and zoonotic potential. The research material consisted of 67 reptile faeces samples. The taxonomic affiliation of the Salmonella isolates was determined by MALDI-TOF mass spectrometry, biochemical analyses, and serotyping; whole genome sequencing (WGS) analysis was performed on three isolates whose serotype could not be determined by agglutination. The antimicrobial susceptibility of the Salmonella isolates was determined by the broth dilution method, and in the case of some antimicrobials by the disk diffusion method. The pathogenic and zoonotic potential of the identified serotypes was estimated based on available reports and case studies. The presence of Salmonella was confirmed in 71.6% of faecal samples, with the highest incidence (87.1%) recorded for snakes, followed by lizards (77.8%) and turtles (38.9%). All isolates (n = 51) belonged to the species S. enterica, predominantly to subspecies I (66.7%) and IIIb (25.5%). Among these, 25 serotypes were identified, including 10 that had previously been confirmed to cause reptile-associated salmonellosis (RAS). Salmonella isolates were susceptible to all antimicrobial substances used except streptomycin, to which 9.8% of the strains showed resistance. None of the strains contained corresponding resistance genes. The study demonstrates that pet reptiles kept in Poland are a significant reservoir of Salmonella and contribute to knowledge of the characteristics of reptilian Salmonella strains. Due to the risk of salmonellosis, contact with these animals requires special hygiene rules.

Ligilactobacillus salivarius is an important member of the human and animal gut microbiota, and selected strains are promising probiotics, but knowledge of the characteristics of avian isolates is still limited. In this study, we examined selected phenotypic and genotypic traits of 33 L. salivarius strains from geese, chickens, turkeys and pigeons. The strains varied in terms of cell size, colony morphology, broth growth characteristics, biofilm formation, tolerance to bile, hydrophobicity and phenotypic and genotypic antibiotic resistance profiles. Large variation among strains was noted for the utilization of sorbitol, salicin, trehalose, rhamnose, inulin and N-acetyl-D-glucosamine. The presence of genes related to sugar metabolism, i.e., mipB, tktA, rhaB and LSL_1894, was not always correlated with the biochemical phenotypic profile. Correlations were recorded between the host and utilization of certain sugars as well as tolerance to bile. The repA-type megaplasmid and genes coding for Abp118 bacteriocin were detected in 94% and 51.5% of L. salivarius strains, respectively. Phylogeny based on groEL gene sequences was partly correlated with the origin of the strains and revealed an evolutionary distance between L. salivarius strains from humans and birds. The results of the study contribute to knowledge of the characteristics of the species L. salivarius. Intraspecies variations of L. salivarius strains may affect their ability to colonize specific niches and utilize nutrients and reveal potential strain-dependent effects on host health.

Bacteria of the genus Campylobacter are the most common pathogens causing zoonotic diseases in humans. Therefore, the aim of the study was to isolate Campylobacter bacteria from broiler chickens and evaluate their susceptibility to selected antibiotics by determining minimum inhibitory concentrations (MIC), followed by isolation and characterization of bacteriophages specific for Campylobacter spp. The material for the study consisted of field isolates of Campylobacter spp. obtained from the gut (cecum) of broiler chickens directly after slaughter in slaughterhouses, and bacteriophages specific for these strains. We isolated 48 strains from poultry (140 broiler chickens): 31 strains of Campylobacter jejuni and 17 of Campylobacter coli. Identification of the strains was confirmed by multiplex PCR and MALDI‐TOF mass spectrometry. Over 83% of Campylobacter strains were resistant to ciprofloxacin, and over half the isolates were resistant to erythromycin, gentamicin, and tetracycline. Resistance to three or more antibiotics was observed in 91.6% of all strains. Four bacteriophages were obtained, and on the basis of their morphological structure, they were assigned to two families of the order Caudovirales: Myoviridae and Siphoviridae. A high percentage of the Campylobacter strains were resistant to at least three of the antibiotic groups tested. All of the phages exhibited lytic activity against the Campylobacter spp. isolates, but the antibacterial effect of the phages was not observed for all strains.

The aim of study was to isolate, characterize and analyse the protein profiles of Myoviridae-like bacteriophages obtained from M. haemolytica using MALDI TOF mass spectrometry. The material consisted of the M. haemolytica reference strain ATCC® BAA410, reference serotypes A1, A2, A5, A6, A7, A9, and A11, and wild-type isolates of serotype A1. Bacteriophage morphology was examined with a transmission electron microscope. The proteins were separated in SDS-PAGE and two-dimensional electrophoresis and characterized by MALDI-TOF. Among the phages obtained, seven were specific for strains A1, A2, A5, A6, A7 and 25, and PHL-1 was specific for the BAA410 strain. The protein profiles for the phages were very similar to one another, but differed from the reference phage in that they lacked protein fractions with molecular weights of 22.9, 56.3 and 73.1 kDa. 2D electrophoresis revealed significant differences in the size of proteins and their localization in the pH gradient. The most similar profiles were observed in phages specific for strains BAA-410 and A6. In all profiles two main spots were observed in the molecular weight range from 44 to 70 kDa at pH < 4. The results indicate that 2D electrophoresis is a very useful tool for characterization of phage protein profiles. An important objective was to determine the molecular differences between morphologically similar phages belonging to one family and to find similarities to phages specific for other pathogens. The study also assessed the suitability of the methods used to characterize phages.

Bovine respiratory disease (BRD) in cattle, due to the participation of numerous viral and bacterial etiological agents, predisposition factors, and environmental stresses, is a serious health and economic problem in herds of beef cattle. Given the broad scope of the problem of respiratory syndrome and the limited immunoprophylaxis, the aim of this study was to estimate the additive effects of flunixin with florfenicol followed with vitamin E or/and C in reducing the development of oxidative stress and inflammation in the first weeks in the feedlot. The study was conducted on young Simmental cattle (n=50) weighing approx. 160 kg. The animals were divided into 5 groups (n=10 in each group). Group I was the control. The cattle received florfenicol and flunixin in combination with vitamins E or C (Group II and III), florfenicol and flunixin followed with vitamin E and C (Group IV), or florfenicol and flunixin without vitamins (group V). Blood for sera was collected on the day the drugs were administered and on days 3, 7, 14, 21 and 28 of the experiment. The level of oxidative stress was analysed on the basis of the concentration of nitrogen ions (NO) in the sera and of lipid peroxidation end products reacting with thiobarbituric acid (TBARS). Concentrations of haptoglobin (Hp) and serum amyloid A (SAA) in the sera were determined using EIA enzyme immunoassays. The treatment used in the study, involving the application of florfenicol and flunixin with vitamin E or C, substantially reduced the level of oxidative stress, expressed as a reduced concentration of TBARS and NO ions, particularly in groups II and III. It was also confirmed that the treatment inhibited the concentration of acute phase proteins (Hp and SAA). .

The objective of our study is to evaluate the potential use of Amplified 16S Ribosomal DNA Restriction Analysis (16S-ARDRA) and MALDI-TOF mass spectrometry (MS) as methods for species identification of Lactobacillus strains in poultry. A total of 80 Lactobacillus strains isolated from the cloaca of chicken, geese and turkeys were identified to the species level by MALDI-TOF MS (on-plate extraction method) and 16S-ARDRA. The two techniques produced comparable classification results, some of which were additionally confirmed by sequencing of 16S rDNA. MALDI-TOF MS enabled rapid species identification but produced more than one reliable identification result for 16.25 % of examined strains (mainly of the species L. johnsonii). For 30 % of isolates intermediate log(scores) of 1.70–1.99 were obtained, indicating correct genus identification but only presumptive species identification. The 16S-ARDRA protocol was based on digestion of 16S rDNA with the restriction enzymes MseI, HinfI, MboI and AluI. This technique was able to distinguish 17 of the 19 Lactobacillus reference species tested and enabled identification of all 80 wild isolates. L. salivarius dominated among the 15 recognized species, followed by L. johnsonii and L. ingluviei. The MALDI-TOF MS and 16S-ARDRA assays are valuable tools for the identification of avian lactobacilli to the species level. MALDI-TOF MS is a fast, simple and cost-effective technique, and despite generating a high percentage of results with a log(score) <2.00, the on-plate extraction method is characterized by high-performance. For samples for which Biotyper produces more than one reliable result, MALDI-TOF MS must be used in combination with genotypic techniques to achieve unambiguous results. 16S-ARDRA is simple, repetitive method with high power of discrimination, whose sole limitation is its inability to discriminate between species with very high 16S rDNA sequence homology, such as L. casei and L. zeae. The assays can be used for discrimination of Lactobacillus bacteria from different habitats.

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.