Integrative and Conjugative Elements and Prophage DNA as Carriers of Resistance Genes in Erysipelothrix rhusiopathiae Strains from Domestic Geese in Poland.

Goose erysipelas is a serious problem in waterfowl breeding in Poland. However, knowledge of the characteristics of Erysipelothrix rhusiopathiae strains causing this disease is limited. In this study, the antimicrobial susceptibility and serotypes of four E. rhusiopathiae strains from domestic geese were determined, and their whole-genome sequences (WGSs) were analyzed to detect resistance genes, integrative and conjugative elements (ICEs), and prophage DNA. Sequence type and the presence of resistance genes and transposons were compared with 363 publicly available E. rhusiopathiae strains, as well as 13 strains of other Erysipelothrix species. Four strains tested represented serotypes 2 and 5 and the MLST groups ST 4, 32, 242, and 243. Their assembled circular genomes ranged from 1.8 to 1.9 kb with a GC content of 36-37%; a small plasmid was detected in strain 1023. Strains 1023 and 267 were multidrug-resistant. The resistance genes detected in the genome of strain 1023 were erm47, tetM, and lsaE-lnuB-ant(6)-Ia-spw cluster, while strain 267 contained the tetM and ermB genes. Mutations in the gyrA gene were detected in both strains. The tetM gene was embedded in a Tn916-like transposon, which in strain 1023, together with the other resistance genes, was located on a large integrative and conjugative-like element of 130 kb designated as ICEEr1023. A minor integrative element of 74 kb was identified in strain 1012 (ICEEr1012). This work contributes to knowledge about the characteristics of E. rhusiopathiae bacteria and, for the first time, reveals the occurrence of erm47 and ermB resistance genes in strains of this species. Phage infection appears to be responsible for the introduction of the ermB gene into the genome of strain 267, while ICEs most likely play a key role in the spread of the other resistance genes identified in E. rhusiopathiae.

Case reports involving coinfection with Avibacterium paragallinarum and Ornithobacterium rhinotracheale in broiler chickens and Avibacterium endocarditis in broiler breeding hens in Poland.

ABSTRACTThe study describes three clinical cases of infection with Avibacterium spp.. In case no. 1, respiratory clinical signs and high mortality (0.7-4.2% daily; total 21.2%) in Ross 308 broiler chickens were shown to be caused by coinfection with sequence type 9 of O. rhinotracheale presumptive serotype A and A. paragallinarum presumptive serotype B. The identical (pulsed-field gel electrophoresis) restriction pattern (pulsotype) of seven A. paragallinarum isolates indicated that infectious coryza in broilers was caused by the same clone. In cases 2 and 3, sudden increased deaths in Ross 308 broiler breeders (especially males) with lesions in the endocardium (valvular or mural endocarditis) were shown to be caused by A. endocarditis. Among nine antibiotics tested, florfenicol was the only antibiotic to which all A. paragallinarum and O. rhinotracheale isolates were susceptible. Out of the eight antibiotics tested, 11 A. endocarditis isolates from both clinical cases of infective endocarditis were susceptible to penicillin, amoxicillin, doxycycline and florfenicol. The A. endocarditis isolates tested in both clinical cases had different PFGE patterns (pulsotypes), but identical within a case. The causes of infectious coryza and infective endocarditis in the cases presented have not been determined. In the prevention of infectious diseases in large-scale livestock farming, it is very important to follow the rules of biosecurity.

Pathogenic Potential of Coagulase-Positive Staphylococcus Strains Isolated from Aviary Capercaillies and Free-Living Birds in Southeastern Poland.

The aim of the study was to determine the occurrence and characteristics of coagulase-positive Staphylococcus strains in the carcasses of wild birds and aviary capercaillies in Southeastern Poland. In total, samples taken from 333 birds were examined. The material consisted of swabs from the internal organs of dead birds (heart, liver, and spleen), the tarsal joints, and mucous membranes (conjunctiva and palatine fissure), as well as from unhatched embryos. The isolated Staphylococcus strains were tested for sensitivity to nine antimicrobial agents and the presence of selected virulence genes. An analysis of the similarity of isolates within species was performed using pulsed-field gel electrophoresis (PFGE). The result indicates that coagulase-positive strains accounted for 5.7% and belonged to the species: Staphylococcus aureus, Staphylococcus pseudintermedius, and Staphylococcus delphini. Among isolated strains, 15.8% were multidrug resistant. The most frequently detected virulence genes were hla in 58% of isolates and hlb and hld in 47.4% of isolates. The results of multiplex PCR showed the presence of genes responsible for the production of enterotoxins C, B, E, and J, in single isolates. It can be concluded that coagulase-positive Staphylococcus strains accounted for a small percentage of staphylococci isolated from free-living birds in the study area. The occurrence of multidrug-resistant coagulase-positive Staphylococcus strains in aviary capercaillies suggests that they play a role in the transmission and spread of resistant strains into the environment. Free-living birds may also be a reservoir of enterotoxigenic Staphylococcus strains.

Characterization of Riemerella anatipestifer Strains Isolated from Various Poultry Species in Poland.

Riemerella anatipestifer (R. anatipestifer) is one of the common pathogens found in poultry flocks, resulting in serious economic losses for the poultry industry due to high mortality, reduced growth rate, poor feed conversion, increased condemnations, and high treatment costs. The aim of this study was to phenotypically characterize phylogenetic relationships and assess the presence of resistance gene strains of R. anatipestifer obtained from various poultry species in Poland. A total of 57 isolates of Riemerella were included in this study. A polymerase chain reaction (PCR) and matrix assisted laser desorption ionization mass spectrometry (MALDI-TOF MS) were used for identification of the strains. The phylogenetic relationship of the R. anatipestifer isolates was determined by analysing the rpoB gene sequence. The susceptibility to antibiotics was evaluated by minimum inhibitory concentration (MIC) in liquid media. All of the field strains of R. anatipestifer were grouped into one of two clades resulting from rpoB gene sequencing. High MIC50 and MIC90 values were obtained for gentamycin, amikacin, and colistin. Low MIC50 and MIC90 values were obtained for amoxicillin cefuroxime, cefoperazone, piperacillin, and trimethoprim/sulfamethoxazole. Among the resistance genes, tet(X) and ermF were identified most frequently. This is the first phenotypic characterization of R. anatipestifer strains obtained from poultry flocks in Poland.

Staphylococcal Resistance Patterns, blaZ and SCCmec Cassette Genes in the Nasopharyngeal Microbiota of Pregnant Women.

Antimicrobial resistance in Staphylococcus spp. colonising the nasopharynx can create risk factors of therapeutic treatment failure or prophylaxis in pregnant women. Resistance is mostly encoded on plasmids (e.g., blaZ gene for penicillinase synthesis) or chromosomes (e.g., mecA and mecC for methicillin resistance). The mecA gene is part of the chromosomal mec gene cassette (SCCmec), which is also located on the plasmid. The disc diffusion method for the selected drugs (beta-lactams, fluoroquinolones, streptogramins, aminoglicosides, macrolides, oxasolidinones, tetracyclines and other groups) was used. PCR for blaZ, mecA and mecC genes and SCCmec cassette detection and typing were performed. S. aureus (54.4%) and S. epidermidis (27.9%) were the most prevalent and showed the highest diversity of resistance profiles. The blaZ, mecA and mecC genes were reported in 95.6%, 20.6% and 1.5% of isolates, respectively. The highest resistance was found to beta-lactams, commonly used during pregnancy. Resistance to a variety of antimicrobials, including benzylpenicillin resistance in blaZ-positive isolates, and the existence of a very high diversity of SCCmec cassette structures in all staphylococci selected from the nasopharyngeal microbiota of pregnant women were observed for the first time. Knowledge of the prevalence of antimicrobial-resistant staphylococci in the nasopharynx of pregnant women may be important for the appropriate treatment or prophylaxis of this group of patients.

Determination of Anti-phage Antibodies in Calf Sera Following Application of Escherichia Coli and Mannheimia Haemolytica-specific Bacteriophages.

Introduction: The widespread occurrence of drug-resistant bacteria has increased interest in alternatives to antibiotics for combatting bacterial infections, among which bacteriophages play an important role. The ability of phage proteins to induce an anti-phage immune response can significantly limit the effectiveness of treatment, which was the basis for the study described in this article. The aim of the study was to assess the effects of bacteriophages on the induction of an anti-phage humoral response in calves. Material and Methods: The study was conducted using phage components of experimental preparations and sera from calves treated and not treated with phages. Levels of G, M and A immunoglobulins were analysed by ELISA. The assay plates were coated with whole Escherichia coli and Mannheimia haemolytica phages and selected phage proteins obtained in sodium dodecyl sulphate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis. Neutralisation of phages by immunoglobulins was assessed by determining phage titres using double-layer plates. Results: The results confirmed an increased anti-phage response affecting all immunoglobulin classes in the calf sera. The highest significant (P ≤ 0.05) level of antibodies was observed for IgG in the sera of calves receiving phages. The phage neutralisation test showed a significant differences (P ≤ 0.05) in the reduction of phage titres in comparison to untreated calves. Conclusion: Despite the induction of an anti-phage response, no significant negative effect on the antibacterial activity of phages was observed in vitro.

Antimicrobial Resistance and Virulence Genes in Staphylococci Isolated from Aviary Capercaillies and Free-living Birds in South-eastern Poland.

Introduction: The current study characterises Staphylococcus bacteria recovered from dead free-living birds and captive capercaillies kept in south-eastern Poland. The results provide novel information about the antimicrobial resistance phenotype/genotype and the virulence profile of these bacteria. Material and Methods: Samples of internal organs were taken from dead birds. Staphylococcus strains were identified by matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry. Susceptibility to 13 antibiotics was tested using a standard disc diffusion method on Mueller-Hinton agar. All isolates were screened for the presence of antibiotic resistance genes and staphylococcal enterotoxins (A to E), toxic shock syndrome toxin 1, exfoliative toxins A and B and Panton-Valentine leukocidin. Results: A total of 129 bacterial strains belonging to 19 species of the Staphylococcus genus were isolated. A relatively high percentage of them resisted fluoroquinolones, tetracyclines, macrolides and ß-lactams to a significant degree and harboured the tetK, tetM, ermC, mphC and mecA genes. Strains of the coagulase-negative S. sciuri, S. xylosus and S. cohnii were isolated with genes encoding enterotoxin A and toxic shock syndrome toxin. Conclusion: Both coagulase-positive and coagulase-negative staphylococci isolated from aviary capercaillies and free-living birds have significant pathogenic potential, and greater attention must be paid to the coagulase-negative species, which are still often considered mere contaminants. Virulence factors associated with resistance to antimicrobials, this being multiple in some strains, seem most important because they can be easily transferred between animals, especially those living in a given area.

Antibiotic Resistance in Bacteria-A Review.

BACKGROUND: A global problem of multi-drug resistance (MDR) among bacteria is the cause of hundreds of thousands of deaths every year. In response to the significant increase of MDR bacteria, legislative measures have widely been taken to limit or eliminate the use of antibiotics, including in the form of feed additives for livestock, but also in metaphylaxis and its treatment, which was the subject of EU Regulation in 2019/6. Numerous studies have documented that bacteria use both phenotypis and gentic strategies enabling a natural defence against antibiotics and the induction of mechanisms in increasing resistance to the used antibacterial chemicals. The mechanisms presented in this review developed by the bacteria have a significant impact on reducing the ability to combat bacterial infections in humans and animals. Moreover, the high prevalence of multi-resistant strains in the environment and the ease of transmission of drug-resistance genes between the different bacterial species including commensal flora and pathogenic like foodborne pathogens (E. coli, Campylobacter spp., Enterococcus spp., Salmonella spp., Listeria spp., Staphylococcus spp.) favor the rapid spread of multi-resistance among bacteria in humans and animals. Given the global threat posed by the widespread phenomenon of multi-drug resistance among bacteria which are dangerous for humans and animals, the subject of this study is the presentation of the mechanisms of resistance in most frequent bacteria called as "foodborne pathoges" isolated from human and animals. In order to present the significance of the global problem related to multi-drug resistance among selected pathogens, especially those danger to humans, the publication also presents statistical data on the percentage range of occurrence of drug resistance among selected bacteria in various regions of the world. In addition to the phenotypic characteristics of pathogen resistance, this review also presents detailed information on the detection of drug resistance genes for specific groups of antibiotics. It should be emphasized that the manuscript also presents the results of own research i.e., Campylobacter spp., E. coli or Enetrococcus spp. This subject and the presentation of data on the risks of drug resistance among bacteria will contribute to initiating research in implementing the prevention of drug resistance and the development of alternatives for antimicrobials methods of controlling bacteria.

Opportunistic Pathogens of Recreational Waters with Emphasis on Antimicrobial Resistance-A Possible Subject of Human Health Concern.

Infections caused by exposure to opportunistic pathogens can cause serious health problems during recreational water use. The problem of diseases caused by microbes transmitted by water is a major public health challenge, especially in developing countries with economic problems and poor hygiene conditions. Moreover, the quality of water in natural reservoirs is often at a very low level in terms of microbiological water purity, which means that their use for recreational purposes, but also as a source of drinking water, may have serious health consequences. Recreational waters pose a threat to human health. Therefore, the quality of recreational waters is closely monitored in many jurisdictions. In this review, we summarize key information on the most common pathogens that can be water-based or waterborne. The issue of antimicrobial resistance among opportunistic pathogens remains equally important. It is important not only to fight pathogens, but also to take action to reduce chemical stressors (especially antibiotics) in the aquatic environment, and to understand the various mechanisms of the spread of antibiotic-resistant genes.

Plasmid-Mediated Fluoroquinolone Resistance Genes in Quinolone-Susceptible Aeromonas spp. Phenotypes Isolated From Recreational Surface Freshwater Reservoir.

Aeromonas spp. are recognized as opportunistic pathogens causing diseases. Infections in humans can result mainly in gastrointestinal and wound diseases with or without progression to septicemia. Although Aeromonas spp. are not known uropathogens and they rarely cause urinary tract infection, we hypothesize that the presence of these bacteria in the water and the contact during, e.g., recreational and bathing activity can create the conditions for the colonization of the human body and may result to diseases in various locations, including the urinary tract. Our study presents the occurrence of aeromonad fluoroquinolone-susceptible phenotypes with the presence of plasmid-mediated fluoroquinolone resistance (PMQR) genes in a natural freshwater reservoir occasionally used for recreational activities. Sixty-nine isolates collected during the bathing period were identified by mass spectrometry and screened for the presence of fluoroquinolone-resistant phenotypes and genotypes. Fluoroquinolone susceptibility was determined as minimal inhibitory concentration values. PMQR qnr genes were detected by PCR. Isolates comprising eight species, namely, mainly Aeromonas veronii (50.7% isolates) and Aeromonas media (24.6% isolates) and rarely Aeromonas eucrenophila, Aeromonas caviae, Aeromonas bestiarum, Aeromonas ichthiosmia, and Aeromonas hydrophila, were selected. All isolates were phenotypically susceptible either to ciprofloxacin or levofloxacin. Unexpectedly, at least one to three of the PMQR genes were detected in 42.0% of the fluoroquinolone-susceptible Aeromonas spp. phenotypes. Mainly the qnrS (34.8% isolates) and qnrA (14.5% isolates) determinants were detected. In conclusion, the freshwater reservoir occasionally used for bathing was tainted with aeromonads, with a high occurrence of opportunistic pathogens such as A. veronii and A. media. MALDI-TOF MS is a powerful technique for aeromonad identification. Our data reveals the mismatch phenomenon between fluoroquinolone-susceptible aeromonad phenotypes and the presence of plasmid-mediated qnr resistance genes. It suggests that phenotypically susceptible bacteria might be a potential source for the storage and transmission of these genes. The exposure during, e.g., a recreational activity may create the potential risk for causing infections, both diagnostically and therapeutically difficult, after expressing the resistance genes and quinolone-resistant strain selection.

Molecular and Serological Characteristics of Avian Pathogenic Escherichia coli Isolated from Various Clinical Cases of Poultry Colibacillosis in Poland.

Escherichia coli infections are a major problem in modern poultry production. Avian pathogenic E. coli (APEC) strains have several mechanisms that enable them to colonize various ecosystems. In this study, 290 E. coli isolates were recovered from clinical cases of colibacillosis in chicken and turkey broilers and from laying and breeding hens. The samples were taken from organs with pathological changes suggesting colibacillosis. The lesions were assigned to three groups depending on their advancement, of which the largest (60% of the isolates) was group 3, with the most extensive changes. The most common serotype was shown to be O78 (14%). The most frequently detected gene among those tested was iss, while papC was the least prevalent. An analysis of the number of genes present per isolate revealed that the presence of four genes was the most common (22%), while only 1% of the strains tested had all eight genes. The most frequently detected genes for each serotype were iss and iucD for O78; irp2 and cvi/cva for O1; irp2, iucD, and iss for O2, and iss and iucD for O8, for which the least frequent was papC. All O18 serotype strains had the iss gene, while none had the vat gene.

Virulence Profiles and Antibiotic Susceptibility of Escherichia coli Strains from Pet Reptiles.

Exotic reptiles are increasingly being bred as pets in many countries around the world, including Poland. However, the close contact between reptiles and their owners provides favourable conditions for the transmission of zoonotic pathogens. In this work, we examined E. coli isolates from 67 captive reptiles regarding their virulence, antibiotic susceptibility, phylogenetic affiliation, and genetic diversity. The incidence of E. coli was highest in snakes (51.6%, 16 isolates/31 samples), and slightly lower in turtles (44.4%, 8/18) and lizards (44.4%, 8/18). Genes encoding virulence factors were confirmed in 50% of isolates and the most common were the traT (37.5%, n = 12), fyuA (21.87%, n = 7), and irp-2 (15.62%, n = 5). The majority (71.87%, n = 23) of E. coli isolates were susceptible to all of the antimicrobial substances used in the study. Streptomycin resistance (21.87%, n = 7) was the most frequent, while resistance to other antimicrobial substances was sporadic. One strain (3.12%) was classified as multidrug-resistant. The presence of resistance genes (aadA, tetA, tetB, tetM, and blaTEM) was confirmed in 12.5% (n = 4) of the isolates. The majority (65.6%, n = 21) of E. coli isolates represented the B1 phylogenetic group. (GTG)5-PCR fingerprinting showed considerable genetic variation in the pool of tested isolates. The frequency of E. coli in reptiles is much lower than in mammals or birds. Due to the presence of virulence genes, characteristic of both intestinal pathogenic E. coli (IPEC) and extraintestinal pathogenic E. coli (ExPEC), reptilian strains of E. coli have pathogenic potential, and therefore people in contact with these animals should follow good hygiene practices.

Antimicrobial resistance and genetic diversity of Enterococcus faecalis from yolk sac infections in broiler chicks.

Despite restrictions on the use of antibiotics in poultry, the percentage of multidrug resistant bacteria, isolated from both adult birds and chicks, remains high. These bacteria can spread between countries via hatching eggs or chicks. Antibiotic resistant bacteria can also pose a threat to hatchery and farm workers or to consumers of poultry. The aim of the study was to perform a phenotypic and genotypic analysis of the drug resistance of E. faecalis isolates from yolk sac infections in broiler chicks from Poland and the Netherlands and to determine their genetic diversity. The tests revealed resistance to antibiotics from category D, that is, tetracycline (69.7%); category C - lincomycin (98.7%), erythromycin (51.3%), aminoglycosides (high-level streptomycin and kanamycin resistance - 10.5% and 3.95%, respectively), and chloramphenicol (7.9%); and category B - ciprofloxacin (25% with resistance or intermediate resistance). No resistance to penicillin, ampicillin, high-level gentamicin, tigecycline, or linezolid was noted. Various combinations of the erm(B), tet(M), tet(L), tet(O), ant(6)-Ia, aph(3')-IIIa, ant(4')-Ia, cat, and msr(A/B) genes were detected in all isolates (irrespective of the drug-resistance phenotype). Among isolates that carried the tet(M) and/or the tet(L) gene, 28% also had the Int-Tn gene, in contrast with isolates possessing tet(O). There were 28 sequence types and 43 PFGE restriction patterns. About 60% of isolates were of sequences types ST59, ST16, ST116, ST282, ST36, and ST82. Nine new sequence types were shown (ST836-ST844). In conclusion, broiler chicks can be a source of drug-resistant sequence types of E. faecalis that are potentially hazardous for people and animals. Restrictive programs for antibiotic use in broiler breeding flocks should be developed to decrease drug resistance in day-old chicks and reduce economic losses during rearing.

Characterization and Comparison of Enterococcus spp. Isolates from Feces of Healthy Dogs and Urine of Dogs with UTIs.

Enterococcus spp. are opportunistic pathogens of both humans and animals characterized by high resistance to antimicrobials. Dogs could be intestinal carriers or suffer from Enterococcus infections, mainly urinary tract infections (UTIs). This study aimed to analyze and compare Enterococcus spp. isolated from healthy dog stools and sick dog urine. Overall, 51 isolates (29 from stools and 22 from UTI) were characterized at species level and tested for antimicrobial resistance, biofilm production and presence of resistance and virulence genes. E. faecium and E. faecalis resulted as equally distributed in stools samples, while E. faecalis predominated among UTI isolates. HLAR phenotype was detected in 47.1% isolates; 64.7% isolates were resistant to ampicillin (47.1% with a MIC ≥ 64 µg/mL). High levels of resistance were recorded for fluoroquinolones (enrofloxacin 74.5%, ciprofloxacin 66.7%), clindamycin (84.3%), tetracycline (78.4%) and quinupristin-dalfopristin (78.4%). No vancomycin resistant strains were detected. All but one isolate were multidrug-resistant. Most detected resistance genes were tetM (70.5%), pbp4 (52.9%) and aph(3')-IIIa (39.2%). All isolates were able to produce biofilm, but isolates from UTIs and belonging to E. faecalis more frequently resulted in strong biofilm producers. Most detected virulence genes were asa1 (52.9%), gelE (41.2%), cylA (37.3%) and esp (35.3%); all of them resulted as more frequently associated to E. faecalis. No particular differences emerged between isolates from feces and UTI, considering all evaluated aspects. Our results confirm pet dogs as carriers of multidrug-resistant enterococci; stool microflora could be considered as the most probable source of enterococcal UTI and E. faecalis carried by dogs seems to be more virulent than E. faecium, justifying its more frequent involvement in urinary tract infections.

The presence of pathogens and heavy metals in urban peregrine falcons (Falco peregrinus).

BACKGROUND AND AIM: Wild birds raised in urban environments may be exposed to many negative factors, including biological and chemical toxic elements. The aim of the study was to assess the occurrence of bacteria and parasites in wild birds, based on the example of the peregrine falcon (Falco peregrinus) as a potential indicator of bacterial drug resistance genes. Toxicological contamination was also analyzed to determine the impact of urbanized areas on this predatory species, in terms of its health, welfare, and survival in urban environments. MATERIALS AND METHODS: The samples consisted of down feathers and fresh feces obtained from seven falcon chicks (during obligatory veterinary examination) reared in two nests located in the Lublin region (Lublin and Pulawy). Bacteria and parasites were isolated directly from feces by classical microbiological methods, polymerase chain reaction, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS). The down feathers and feces of birds were used for toxicological testing by plasma inductively coupled plasma MS to assess the concentrations of selected heavy metals (cadmium [Cd], lead [Pb], arsenic [As], zinc [Zn], and copper [Cu]). RESULTS: The study revealed the presence of a diverse microbiome in the falcon chicks, among which Escherichia coli, Enterococcus spp., and Staphylococcus spp. bacteria and parasites of the genus Caryospora were dominant. The presence of drug resistance genes was also confirmed among the pathogens. The toxicological analysis found high concentrations of toxic heavy metals, including Cd, Pb, As, and Zn, in the downy feathers and feces of peregrine chicks. CONCLUSION: Predatory free-living birds living in urban environments not only can be infected with various pathogens but may also show contamination with heavy metals, which could influence their natural resistance, condition, and welfare.

Antibiotic Resistance and Virulence Profiles of Escherichia coli Strains Isolated from Wild Birds in Poland.

Wild animals are increasingly reported as carriers of antibiotic-resistant and pathogenic bacteria including Enterobacteriaceae. However, the role of free-living birds as reservoirs for potentially dangerous microbes is not yet thoroughly understood. In our work, we examined Escherichia coli strains from wild birds in Poland in relation to their antimicrobial agents susceptibility, virulence and phylogenetic affiliation. Identification of E. coli was performed using MALDI-TOF mass spectrometry. The antibiotic susceptibility of the isolates was determined by the broth microdilution method, and resistance and virulence genes were detected by PCR. E. coli bacteria were isolated from 32 of 34 samples. The strains were most often classified into phylogenetic groups B1 (50%) and A (25%). Resistance to tetracycline (50%), ciprofloxacin (46.8%), gentamicin (34.3%) and ampicillin (28.1%) was most frequently reported, and as many as 31.2% of E. coli isolates exhibited a multidrug resistance phenotype. Among resistance genes, sul2 (31.2% of isolates) and blaTEM (28.1%) were identified most frequently, while irp-2 (31.2%) and ompT (28.1%) were the most common virulence-associated genes. Five strains were included in the APEC group. The study indicates that wild birds can be carriers of potentially dangerous E. coli strains and vectors for the spread of resistant bacteria and resistance determinants in the environment.

Biodiversity of Ligilactobacillus salivarius Strains from Poultry and Domestic Pigeons.

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.

Biofilm-Formation Ability and the Presence of Adhesion Genes in Coagulase-Negative Staphylococci Isolates from Chicken Broilers.

The aim of the study was to analyze the biofilm-production capacity of 87 coagulase-negative Staphylococcus strains (CoNS) isolated from broiler chickens and to determine the occurrence of biofilm-associated genes. The biofilm production capacity of staphylococci was assessed using the microtiter plate method (MTP), and the frequency of genes was determined by PCR. The ability to form a biofilm in vitro was shown in 79.3% of examined strains. Strong biofilm capacity was demonstrated in 26.4% of strains, moderate capacity in 25.3%, weak capacity in 27.6%, and a complete lack of biofilm production capacity in 20.7% of strains. The icaAB gene responsible for the production of extracellular polysaccharide adhesins was detected in 6.9% of strains. The other four genes, i.e., bap (encoding biofilm-associated protein), atlE (encoding cell surface protein exhibiting vitronectin-binding activity), fbe (encoding fibrinogen-binding protein), and eno (encoding laminin-binding protein) were detected in 5.7%, 19.5%, 8%, and 70.1% of strains, respectively. Demonstration of genes that play a role in bacterial biofilm formation may serve as a genetic basis to distinguish between symbiotic and potentially invasive coagulase-negative staphylococcal strains.

Phenotypic and genotypic characterization of Enterococcus spp. from yolk sac infections in broiler chicks with a focus on virulence factors.

Bacterial infections of yolk sacs contribute to increased mortality of chicks, chronic infections during their rearing, or increased selection in the flock, which in turn leads to high economic losses in poultry production worldwide. The aim of this study was a phenotypic and genotypic characterization of enterococci isolated from yolk sac infections (YSI) of broiler chickens from Poland and the Netherlands. Biochemical, matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) MS, and rpoA gene sequencing identification was performed. Moreover, phenotypic and genotypic characterization of virulence factors and analysis of the clonal relationship of isolates by MALDI-TOF MS and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) were performed. The biochemical test identified 70 isolates as Enterococcus faecalis and 6 as Enterococcus mundtii. The results of MALDI-TOF MS were 100% concordant with those obtained by rpoA gene sequencing, and all 76 isolates were identified as E. faecalis. Differences were noted in the ß-glucuronidase, ß-glucosidase, α-galactosidase, phosphatase, melibiose, lactose, and raffinose tests that is going about the results of biochemical identification. None of the isolates were beta-hemolytic on blood agar in aerobic conditions, but all but one were gelatinase positive. Among biofilm-forming isolates (30/76; 39.5%), as many as 66.7% (20/30) were Polish E. faecalis strains. Most of the isolates carried virulence genes, that is gelE, ace, asa1, efaAfs, fsrA, fsrB, fsrC, cob, cpd, and ccf, but none had the hyl gene. Some isolates harbored cyl operon genes. One Polish strain (ST16) had all of the tested cyl genes and the esp gene, considered clinically important, and showed the highest biofilm-forming ability. Nearly 50% of the isolates showed close genetic relatedness in ERIC typing. In contrast with MALDI-TOF MS cluster analysis, ERIC-PCR results did not show a relationship with the origin of the strains. Using MALDI-TOF MS, 7 peaks were found in Polish and Dutch isolates, which may type them as species-specific biomarkers in E. faecalis from YSI.

The in vitro efficacy of eye drops containing a bacteriophage solution specific for Staphylococcus spp. isolated from dogs with bacterial conjunctivitis.

BACKGROUND: The purpose of the study was to evaluate the in vitro antibacterial effect of experimental eye drops with bacteriophages in elimination of Staphylococcus spp. isolated from dogs with bacterial conjunctivitis.. The bacterial material was collected from dogs with independent clinical signs of bacterial conjunctivitis. Staphylococcus spp. were identified by phenotypic and genotypic methods (MALDI-TOF MS mass spectrometry). Antibiotic resistance was determined by the disc-diffusion method. Phage activity (Plaque forming units, PFU) was determined on double-layer agar plates. Phages with lytic titres > 108 PFU were used to prepare eye drops. The stability of the antibacterial titre was evaluated for preparations stored in sealed bottles as well as after opening and reclosing. RESULTS: The tests confirmed the occurrence of Staphylococcus spp. strains as etiological agents of bacterial conjunctivitis in dogs. A high percentage of strains were resistant to more than three antibiotics. The experimental phage eye drops used in the study exhibited 100% efficacy in vitro against the tested Staphylococcus isolates. Particularly noteworthy is the long duration of activity and constant antibacterial lytic titre of ≥108 PFU/mL of two eye drop solutions, nos. 7 and 12, after the bottle had been opened (21 days) and after hermetically sealed packaging (28 days) at 4-8 °C. CONCLUSIONS: The results represent the first stage of research and require continuation in vivo. If positive effects are obtained in animals, the results can be used in applied research in humans and animals.