Agnieszka Marek

Prevalence and antibiotic resistance of Enterococcus strains isolated from poultry

The aim of this study was to evaluate the frequency of occurrence of bacteria of the genus Enterococcus in poultry, to identify them by means of matrixassisted laser desorption/ionisation time-of-flight mass spectrometry (MALDITOF MS), and to analyse the antimicrobial susceptibility of the isolated strains to the drugs most frequently used in poultry. The material for the bacteriological tests was obtained mainly from the heart (97%) of the birds investigated. Of a total of 2,970 samples tested, 911 (30.7%) tested positive for Enterococcus spp. Enterococci were detected in broilers (88.1%), laying hens (5.3%), turkeys (3.9%), breeding hens (2.2%), and geese (0.4%). The most commonly identified species were Enterococcus (E.) faecalis (74.7%), E. faecium (10.1%), E. gallinarum (5.5%), E. hirae (4.6%), and E. cecorum (4.1%). The most frequent resistance properties were resistance to sulphamethoxazole/trimethoprim (88%), tylosin (71.4%), enrofloxacin (69.4%), doxycycline (67.3%), and lincomycin/spectinomycin (56.1%). Only one vancomycin-resistant Enterococcus, E. cecorum from a broiler, was found.

The effect of feed supplementation with zinc chelate and zinc sulphate on selected humoral and cell-mediated immune parameters and cytokine concentration in broiler chickens.

The ability of poultry to withstand infectious disease caused by bacteria, viruses or protozoa depends upon the integrity of the immune system. Zinc is important for proper functioning of heterophils, mononuclear phagocytes and T lymphocytes. Numerous data indicate that the demand for zinc in poultry is not met in Poland due to its low content in feeds of vegetable origin. The aim of the study was to determine the effect of supplementation of inorganic (ZnSO4 and ZnSO4+ phytase enzyme), and organic forms of zinc (Zn with glycine and Zn with glycine and phytase enzyme) on selected parameters of the cellular and humoral immune response in broiler chickens by evaluating the percentage of CD3+CD4+, CD3+CD8+, CD25+, MHC Class II, and BU-1+ lymphocytes, the phagocytic activity of monocytes and heterophils, and the concentration of IL-2, IL-10 and TNF-α in the peripheral blood. Flow cytometry was used to determine selected cell-mediated immune response parameters. Phagocytic activity in whole blood was performed using the commercial Phagotest kit (ORPEGEN-Pharma, Immuniq, Poland). The results showed that supplementation with zinc chelates causes activation of the cellular and humoral immune response in poultry, helping to maintain the balance between the Th1 and Th2 response and enhancing resistance to infections. In contrast with chelates, the use of zinc in the form of sulphates has no immunomodulatory effect and may contribute to the development of local inflammatory processes in the digestive tract, increasing susceptibility to infection.

Effects of feed supplementation with glycine chelate and iron sulfate on selected parameters of cell-mediated immune response in broiler chickens

Because little is known about the impact of chelated (Fe-Gly, Fe-Gly+F) and inorganic (FeSO4, FeSO4+F) iron products on immune response parameters in broiler chickens, the objective of the study was to determine the effects of inorganic and organic forms of iron on selected parameters of the cell-mediated immune response in broiler chickens by assessing the percentage of CD3(+)CD4(+), CD3(+)CD8(+), CD25(+), and MHC Class II lymphocytes, as well as the CD4(+)/CD8(+) ratio and IL-2 concentration in the peripheral blood. The experiments were conducted using 50day-old Ross 308 roosters. The test material was peripheral blood. Flow cytometry was used to determine selected cell-mediated immune response parameters. The results obtained indicate that the use of iron chelates in the diet of broiler chickens may stimulate cellular defense mechanisms. As a result of the experiment an increase was observed in the percentage of Th1, mainly T CD4(+) and T CD8(+). It was also noted that application of chelated iron can increase production of T CD8(+) cytotoxic cells and IL-2, which promotes the bodys natural response to developing inflammation. There were no changes in T CD4(+), T CD8(+), T CD25(+) or MHC II lymphocyte subpopulations in the chickens following application of the inorganic form of iron.

Characterisation of Staphylococcus aureus and Staphylococcus aureus – like strains isolated from table eggs

Abstract The aim of the study was to provide a detailed phenotypic and genotypic characterisation of Staphylococcus aureus strains and the group of microorganisms with unusual biochemical patterns (called Staphylococcus aureus-like) isolated from table chicken eggs. All of the strains tested exhibited resistance to at least one of the 17 antibiotics tested, and 55.55% of isolates were found to be resistant to five or more of them. PCR used for detection of the methicillin resistance gene (mecA) confirmed the presence of a specific product of 533 bp in the case of two of the isolated S. aureus-like strains. Analysis of the phylogenetic relationship between eight of S. aureus and ten S. aureus-like strains distinguished 18 macrorestriction profiles following digestion with SmaI endonuclease, indicating that there were no identical strains with the same macrorestriction profile. However, the presence of methicillin-resistant strains indicates a serious risk to consumer health.

Effect of feed supplementation with zinc glycine chelate and zinc sulfate on cytokine and immunoglobulin gene expression profiles in chicken intestinal tissue

&NA; The aim of the study was to evaluate the effect of inorganic and organic forms of Zn on the expression of cytokines (IL‐2, TNF‐&agr;, IFN‐&ggr;, IL‐12, IL‐17, IL‐4, IL‐10, and TGF‐&bgr;) and immunoglobulins (IgA and IgG) in the tissues of the small intestine (jejunum and ileum) of broiler chickens. In the experiment, 90 broiler chickens were divided into 4 experimental groups and a control group, with 18 birds each. The birds received Zn supplements in inorganic form with and without phytase (ZnSO4 and ZnSO4 + F), and in organic form with glycine, with and without phytase (Zn‐Gly and Zn‐Gly + F). The total rearing period was 42 days. Quantitative real‐time (RT)‐PCR was used to measure the expression of the cytokines and immunoglobulins. The differences between the results obtained for the control and experimental groups, between the groups receiving ZnSO4 and Zn‐Gly, and between groups ZnSO4‐F and Zn‐Gly‐F were analyzed statistically. High relative expression of IL‐2 was observed for the chickens in the groups receiving ZnSO4‐F, Zn‐Gly, and Zn‐Gly‐F on d 42 in comparison to the control group. High relative expression of TNF‐&agr;, IL‐12, and IL‐17 was noted in the group that received ZnSO4 + F. High expression of IgG, IgA, IL‐4, TGF‐&bgr;, and IL‐10 was noted in the groups of chickens that received feed supplemented with Zn‐Gly and Zn‐Gly + F chelates on d 42 of the study in comparison to the control group. In conclusion, supplementation with Zn‐Gly chelates can ensure Th1 and Th2 balance during the immune response in the gut‐associated lymphoid tissue (GALT), and, by increasing IgA and IgG expression, also can stimulate potentiation of the immune response involved in passive protection of the body from infection. In contrast, the use of inorganic forms of Zn, in the form of sulfates, can induce local inflammatory processes in the intestines, which, in the case of long‐term supplementation, lead to the development of infections.

Staphylococcus simulans associated with endocarditis in broiler chickens

ABSTRACT This report suggests a strong association between coagulase-negative Staphylococcus simulans and endocarditis in broiler chickens of a single flock. Clinical signs included increased mortality and lameness, and some dead chickens were found on their backs. Lesions included cauliflower-like, fibrinous vegetative lesions on the left atrioventricular valve; cream-coloured, necrotic foci of varying size in the liver; and necrosis of the femoral head. Histopathological examination of the heart revealed multifocal conglomerates of bacterial colonies attached to the valvular endocardium, threads of fibrin, and inflammatory cells with the presence of heterophils. S. simulans strains were first identified by API ID32, and then confirmed with Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry and by partial sequencing of the rpoB and dnaJ genes. These bacteria were resistant to methicillin but sensitive to vancomycin and characterized by slime production and protease activity.

Identification of strains with phenotypes similar to those of Staphylococcus aureus isolated from table chicken eggs using MALDI-TOF MS and genotyping methods

Abstract The aim of the study was to identify the affinity of 10 Staphylococcus strains isolated from table chicken eggs to specific species. Preliminary analysis performed by API ID32 Staph test identified these strains as S. aureus, but they exhibited a negative reaction in the tube coagulase test. Thus, the analysed strains were initially characterised as Staphylococcus aureus-like (SAL). Further characterisation was performed by genotypic methods, using restriction fragment length polymorphism (RFLP) of the coagulase gene (coa) and sequencing of the gene rpoB. An attempt was also made to identify the isolated Staphylococcus strains by MALDI-TOF mass spectrometry. The results indicated that none of the strains tested belonged to the species S. aureus. The rpoB sequences of five isolates showed the highest sequence similarity to S. haemolyticus, three isolates to S. chromogenes, and one isolate to S. epidermidis. One strain (SAL4) remained unidentified in this analysis. The results obtained using mass spectrometry were comparable to those based on gene sequence analysis. Strain SAL4, which could not be identified by sequencing, was identified by MALDI-TOF as Staphylococcus chromogenes.

WILD BIRDS AS A POTENTIAL SOURCE OF KNOWN AND NOVEL MULTILOCUS SEQUENCE TYPES OF ANTIBIOTIC-RESISTANT ENTEROCOCCUS FAECALIS

Abstract:  We assessed the antibiotic resistance and genetic diversity of 27 Enterococcus faecalis isolates from 25 wild bird species in Poland. Resistance to lincomycin (100%) was most common followed by tetracycline (48%), erythromycin (44%), and ciprofloxacin (22%). High-level resistance to streptomycin and kanamycin was observed in 19% and 15% of isolates, respectively. One isolate (4%) exhibited low-level resistance to penicillin and vancomycin, and all isolates were susceptible to gentamicin and chloramphenicol. Antibiotic resistance was linked to the tet(M), tet(L), erm(A), erm(B), msr(A/B), ant(6)-Ia, and aph(3′)-IIIa genes. None of the tested van (vanA, vanB, vanC1, vanC2/C3, vanD, vanE, vanG) genes were found in the vancomycin-resistant isolate. Based on pulsed-field gel electrophoresis and multilocus sequence typing analysis, the E. faecalis population from wild birds revealed high genetic diversity. All isolates were divided into 22 pulsotypes and 18 sequence types (STs), among which seven STs were newly assigned (ST748-ST753 and ST764). The most-prevalent STs were ST290 and ST374 followed by ST287 and ST34. The coexistence of strains assigned to the same STs in wild birds and in nonwildlife populations strongly indicated that many wild bird species could constitute a source of E. faecalis for infections in humans, pets, and farm animals.

A loop-mediated isothermal amplification procedure targeting the sodA gene for rapid and specific identification of Gallibacterium anatis

ABSTRACT This paper reports on the development and validation of a real‐time loop‐mediated isothermal amplification assay (LAMP) for rapid and specific identification of Gallibacterium anatis. To design a set of 6 primers using the LAMP technique, the conserved region of the G. anatis sodA gene was selected as a target. To evaluate primer specificity we used 120 field strains, the reference strain G. anatis ATCC 43329, and 9 non‐G. anatis bacteria. The results confirmed positive reactions for all G. anatis strains tested by LAMP at 63°C for 60 min, with no cross‐reactivity observed for the negative control bacteria, i.e., Haemophilus parainfluenzae (ATCC 51505 and ATCC 33392), Aggregatibacter aphrophilus ATCC 7901, Avibacterium endocarditis, Pasteurella multocida, Actinobacillus pleuropneumoniae, Avibacterium paragallinarum, Ornithobacterium rhinotracheale, and Escherichia coli. The lowest detectable amount of DNA for the LAMP reaction was 0.2561 pg, which was detected in about 34 min, while the highest available concentration of the G. anatis reference strain was detected in about 10 min. The lowest detectable amount of DNA for the real‐time PCR reaction was 21.24 pg, which was detected in about 20 min, while the highest available concentration of the G. anatis reference strain was detected in about 7 min. Moreover, using the real‐time LAMP assay the reaction could be effectively carried out in a volume of just 13 &mgr;L, about half the officially recommended reaction volume (25 &mgr;L). The aim of this study was to develop a highly sensitive and specific G. anatis real‐time LAMP assay that is less time‐consuming and less costly than quantitative PCR.

Association Between the Methicillin Resistance of Staphylococcus aureus Isolated from Slaughter Poultry, Their Toxin Gene Profiles and Prophage Patterns

In this work, 85 strains of Staphylococcus aureus were isolated from samples taken from slaughter poultry in Poland. Attempts were made to determine the prophage profile of the strains and to investigate the presence in their genome of genes responsible for the production of five classical enterotoxins (A–E), toxic shock syndrome toxin (TSST-1), exfoliative toxins (ETA and ETB) and staphylokinase (SAK). For this purpose, multiplex PCR was performed using primer-specific pairs for targeted genes. The presence of the mecA gene was found in 26 strains (30.6%). The genomes of one of the methicillin-resistant S. aureus (MRSA) strains and two methicillin-sensitive S. aureus (MSSA) strains contained the gene responsible for the production of enterotoxin A. Only one MRSA strain and two MSSA strains showed the presence of the toxic shock syndrome toxin (tst) gene. Only one of the MSSA strains had the gene (eta) responsible for the production of exfoliative toxins A. The presence of the staphylokinase gene (sak) was confirmed in 13 MRSA strains and in 5 MSSA strains. The study results indicated a high prevalence of prophages among the test isolates of Staphylococcus aureus. In all, 15 prophage patterns were observed among the isolates. The presence of 77-like prophages incorporated into bacterial genome was especially often demonstrated. Various authors emphasize the special role of these prophages in the spread of virulence factors (staphylokinase, enterotoxin A) not only within strains of the same species but also between species and even types of bacteria.