Dagmara Stępień-Pyśniak

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.

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.

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.

The effect of feed supplementation with Zakarpacki zeolite (clinoptilolite) on percentages of T and B lymphocytes and cytokine concentrations in poultry

&NA; The available literature lacks information on the effect of Zakarpacki zeolite (clinoptilolite) on the immune system of poultry. Therefore, the aim of this study was to determine the effect of this zeolite on selected indicators of the immune response in poultry by evaluating the expression of cluster of differentiation (CD) surface molecules on T and B lymphocytes and the concentration of IL‐2 and IL‐10 in the blood. Ninety one‐day‐old Ross 308 broiler chicks were used in the study. The birds were divided into 3 groups of 30 each. The same basic diet was used in all groups, but groups II and III received a feed additive in the form of 2% and 3% zeolite. Blood samples were collected from all birds on the 40th day of observations. Weight gain in the birds in both experimental groups was significantly higher, and no clinical symptoms of disease were observed. The percentage of CD4+CD25+ T and B lymphocytes was higher in both groups receiving zeolite, but the percentage of CD8+CD25+ T lymphocytes was higher only in the group receiving 3% zeolite. There were no differences between the groups in the percentage of cells with CD3+ and MHC Class II expression. Higher serum concentrations of IL‐2 and IL‐10 were noted only in group III. The use of zeolites enhances antigen presentation and leads to increased Th1 and Th2 response. Excessive supply of zeolite in the feed leads to a local inflammatory response, which may cause damage to the intestinal barrier.

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.

The effect of dietary supplementation of transcarpathian zeolite on intestinal morphology in female broiler chickens

Abstract Zeolites occur in nature in specific types of rocks, mostly of volcanic origin. Some studies have proved that zeolite added as a dietary supplement in poultry results in weight gain and improves digestion because zeolites slow down the passage rate of digesta through the digestive tract and controls the release of nutrients in the small intestine. The aim of this study was to investigate the effects of different levels of zeolite on intestinal morphometry in female broiler chickens. One‐day‐old Ross 308 broilers were assigned randomly into 3 groups, with 30 birds per treatment. The 3 dietary treatments were: basal diet only (control group), basal diet + 2% zeolite, and basal diet + 3% zeolite. The broiler females were randomly selected from each group, slaughtered samples from duodenum, jejunum /proximal, middle, distal/ and ileum were sampled for histological characteristics at the 40 d of the study. The broilers fed 2% of zeolite were observed to have a tendency towards increased villus height, villus width, villus perimeter, villus section area, and crypt depth throughout the distal regions of small intestine and ileum. Supplementation with 3% of zeolite was associated with a greater villus height, villus width, villus perimeter, villus section area, villus crypts, and mucosa thickness in jejunum and ileum mucosa compared with those same areas in the control. The results suggest that dietary supplementation of zeolite increases intestinal morphology parameters in the gastrointestinal tract of female broiler chickens that improved growth performance.

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.