Comparison of automatic methods MALDI-TOF, VITEK2 and manual methods for the identification of intestinal microbial communities on the example of samples from alpacas (Vicugna pacos).

Introduction: Universally, in microbiological diagnostics the detection of live bacteria is essential. Rapid identification of pathogens enables appropriate remedial measures to be taken. The identification of many bacteria simultaneously facilitates the determination of the characteristics of the accompanying microbiota and/or the microbiological complexity of a given environment. Material and Methods: The effectiveness of the VITEK2 Compact automated microbial identification system and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS), analytical profile index (API) and Remel RapID tests were compared in identification of bacteria isolated from the alpaca gastrointestinal tract. Results: Most isolates were Gram-positive, such as Bacillus cereus, Bacillus flexus, Bacillus licheniformis, Bacillus pumilus and Bacillus subtilis; Enterococcus faecium, Enterococcus gallinarum, Enterococcus hirae and Enterococcus casseliflavus; Staphylococcus aureus, Staphylococcus equorum, Staphylococcus lentus, Staphylococcus pseudintermedius and Staphylococcus sciuri; Paenibacillus amylolyticus; Cellulosimicrobium cellulans; Leuconostoc mesenteroides; Clostridium perfringens; Corynebacterium stationis, Corynebacterium xerosis, and Corynebacterium diphtheriae (the last only isolated manually by API Coryne and the VITEK2 system and Corynebacteria (CBC) card). Corynebacterium diphtheriae was misidentified by MALDI-TOF MS as Candida lipolytica (currently Yarrowia lipolytica). Gram-positive and Gram-variable Micrococcus luteus were also isolated. Gram-negative Enterobacter cloacae, Enterobacter gergoviae, Enterobacter hormaechei and Enterobacter ludwigii; E. coli; Klebsiella pneumoniae subsp. pneumoniae; Citrobacter braakii and Citrobacter freundii; Serratia liquefaciens, Serratia odorifera and Serratia marcescens; Morganella morganii subsp. morganii; Providencia alcalifaciens; Pseudomonas aeruginosa; Stenotrophomonas maltophilia; Moraxella osloensis; and Ochrobactrum intermedium were also found. The yeasts Candida albicans, Candida haemulonii and Candida ciferrii were also present. Conclusion: MALDI-TOF MS enabled the identification of pathogens and opportunistic pathogens from the alpaca gut which may represent a high risk to human and animal health.

Comparison of Phenotype and Genotype Virulence and Antimicrobial Factors of Salmonella Typhimurium Isolated from Human Milk.

Salmonella is a common foodborne infection. Many serovars belonging to Salmonella enterica subsp. enterica are present in the gut of various animal species. They can cause infection in human infants via breast milk or cross-contamination with powdered milk. In the present study, Salmonella BO was isolated from human milk in accordance with ISO 6579-1:2017 standards and sequenced using whole-genome sequencing (WGS), followed by serosequencing and genotyping. The results also allowed its pathogenicity to be predicted. The WGS results were compared with the bacterial phenotype. The isolated strain was found to be Salmonella enterica subsp. enterica serovar Typhimurium 4:i:1,2_69M (S. Typhimurium 69M); it showed a very close similarity to S. enterica subsp. enterica serovar Typhimurium LT2. Bioinformatics sequence analysis detected eleven SPIs (SPI-1, SPI-2, SPI-3, SPI-4, SPI-5, SPI-9, SPI-12, SPI-13, SPI-14, C63PI, CS54_island). Significant changes in gene sequences were noted, causing frameshift mutations in yeiG, rfbP, fumA, yeaL, ybeU (insertion) and lpfD, avrA, ratB, yacH (deletion). The sequences of several proteins were significantly different from those coded in the reference genome; their three-dimensional structure was predicted and compared with reference proteins. Our findings indicate the presence of a number of antimicrobial resistance genes that do not directly imply an antibiotic resistance phenotype.

Detection and Identification of Avian Reovirus in Young Geese (Anser anser domestica) in Poland.

Avian reovirus (ARV) is a cause of infections of broiler and turkey flocks, as well as waterfowl birds. This case report describes a reovirus detection in a fattening goose flock. GRV-infected geese suffer from severe arthritis, tenosynovitis, pericarditis, depressed growth, or runting-stunting syndrome (RSS), malabsorption syndrome, and respiratory and enteric diseases. GRV (goose reovirus) caused pathological lesions in various organs and joints, especially in the liver and spleen. GRV infection causes splenic necrosis, which induces immunosuppression, predisposing geese to infection with other pathogens, which could worsen the disease and lead to death. Our results showed that GRV was detected via RT-PCR and isolated in SPF (Specific Pathogen Free) embryos. This is the first report of the involvement of reovirus in arthritis, and the generalized infection of young geese in Poland, resulting in pathological changes in internal organs and sudden death. This study also provides new information about the GRV, a disease that is little known and underestimated.

Multi-Drug Resistance to Salmonella spp. When Isolated from Raw Meat Products.

Salmonella spp. is the most frequent cause of foodborne diseases, and the increasing occurrence of MDR strains is an additional and increasing problem. We collected Salmonella spp. strains isolated from meat (poultry and pork) and analysed their antibiotic susceptibility profiles and the occurrence of resistance genes. To determine the susceptibility profiles and identify MDR strains, we used two MIC methods (MICRONAUT and VITEC2 Compact) and 25 antibiotics. Phenotypic tests showed that 53.84% strains were MDR. Finally, molecular analysis strains revealed the presence of blaSHV, blaPSE-1, blaTEM, but not blaCTX-M genes. Moreover, several genes were associated with resistance to aminoglycosides, cephalosporins, fluorochinolones, sulfonamides, and tetracyclines. This suggests that further research on the prevalence of antibiotic resistance genes (ARGs) in foodborne strains is needed, especially from a One Health perspective.

Wild Duck (Anas platyrhynchos) as a Source of Antibiotic-Resistant Salmonella enterica subsp. diarizonae O58-The First Report in Poland.

The "One Health" approach increasingly demonstrates the global spread of pathogenic microorganisms and their antimicrobial resistance in the environment, both in animals and humans. Salmonella enterica subsp. diarizonae is nowadays very often isolated from cold-blooded reptiles to a lesser extent from sheep, but unfortunately more and more often from humans. However, there are a few studies describing the isolation of Salmonella enterica subsp. diarizonae from migratory wild birds. The mallard duck (Anas platyrhynchos), a wild animal that traverses the continent of Eurasia, can be an excellent indicator of the spread of intestinal microbes as well as their resistance to antibiotics. This is the first report of the Salmonella enterica subsp. diarizonae detection in Poland in a migrating mallard duck. This research presented the identification difficulties associated with the isolation of Salmonella enterica subsp. diarizonae using three different biochemical tests and advanced serology tests. At the same time, we detected very high antimicrobial resistance in the isolated strain. By using the minimum inhibitory concentration (MIC) method, it was found that the isolated strain of S. enterica subsp. diarizonae has high antibiotic resistance against 14 of the 33 tested antimicrobials agents. The resistance genes that have been identified in S. enterica subsp. diarizonae include aadA, strA/strB, and blaTEM.

Citrobacter braakii Yield False-Positive Identification as Salmonella, a Note of Caution.

BACKGROUND: Globally, Salmonella enterica is one of the leading causes of foodborne illness in humans. Food of animal origin is obligatorily tested for the presence of this pathogen. Unfortunately, in meat and meat products, this is often hampered by the presence of background microbiota, which may present as false-positive Salmonella. METHODS: For the identification of Salmonella spp. from meat samples of beef, pork, and poultry, the authorized detection method is PN-EN ISO 6579-1:2017-04 with the White-Kauffmann-Le Minor scheme, two biochemical tests: API 20E and VITEK II, and a real-time PCR-based technique. RESULTS: Out of 42 presumptive strains of Salmonella, 83.3% Salmonella enterica spp. enterica, 14.3% Citrobacter braakii, and 12.4% Proteus mirabilis were detected from 180 meat samples. CONCLUSIONS: Presumptive strains of Salmonella should be identified based on genotypic properties such as DNA-based methods. The aim of this study was the isolation and identification of Salmonella spp. from miscellaneous meat sorts: beef, pork, and poultry.

A tissue-engineered urinary conduit in a porcine urinary diversion model.

The use of an ileal segment is a standard method for urinary diversion after radical cystectomy. Unfortunately, utilization of this method can lead to numerous surgical and metabolic complications. This study aimed to assess the tissue-engineered artificial conduit for urinary diversion in a porcine model. Tissue-engineered tubular polypropylene mesh scaffolds were used for the right ureter incontinent urostomy model. Eighteen male pigs were divided into three equal groups: Group 1 (control ureterocutaneostomy), Group 2 (the right ureter-artificial conduit-skin anastomoses), and Group 3 (4 weeks before urostomy reconstruction, the artificial conduit was implanted between abdomen muscles). Follow-up was 6 months. Computed tomography, ultrasound examination, and pyelogram were used to confirm the patency of created diversions. Morphological and histological analyses were used to evaluate the tissue-engineered urinary diversion. All animals survived the experimental procedures and follow-up. The longest average patency was observed in the 3rd Group (15.8 weeks) compared to the 2nd Group (10 weeks) and the 1st Group (5.8 weeks). The implant's remnants created a retroperitoneal post-inflammation tunnel confirmed by computed tomography and histological evaluation, which constitutes urostomy. The simultaneous urinary diversion using a tissue-engineered scaffold connected directly with the skin is inappropriate for clinical application.

PCR melting profile as a tool for outbreak studies of Salmonella enterica in chickens.

BACKGROUND: Salmonellosis is of great economic concern in all phases of the poultry industry, from production to marketing, leading to severe economic losses. Monitoring the source of the bacterial contamination has fundamental importance in the spreading of salmonellosis. RESULTS: We applied a ligation-mediated PCR method, PCR MP (PCR melting profile), to type S. enterica ssp. enterica ser. Enteritidis (56 strains) and 43 control strains classified to other serovars isolated from poultry. We demonstrated the PCR MP potential for salmonellosis spreading monitoring. Our rapid test presents higher discriminatory power (0.939 vs. 0.608) compared to current molecular subtyping tool such as pulsed-field gel electrophoresis (PFGE), which ineffectiveness underlies the high degree of clonality of S. Enteritidis. CONCLUSIONS: PCR MP was found to be a highly discriminating, sensitive and specific method that could be a valuable molecular tool, particularly for analyzing epidemiological links of limited number of S. enterica ser. Enteritidis strains.

Effects of inclusion level and source of dietary sodium on performance and meat characteristics of broiler chickens.

The effect of different concentrations of dietary Na from three Na salts (NaCl, NaHCO3 and Na2SO4) was assessed in two experiments carried out on broiler chickens aged from 1 to 35 days. In Exp. 1, diets were supplemented with 0.15, 0.20 and 0.25% Na, which increased the average Na content of the diets to 0.19, 0.24 and 0.30% respectively. In Exp. 2, the amounts of selected Na salts (NaCI and Na2SO4) were reduced and the estimated Na contents of experimental diets amounted to 0.10, 0.13, 0.15 and 0.19%. In view of the risk factors for the development of foot pad dermatitis, our aim was to find an optimum source of Na and to keep dietary Na intake at the minimum level sufficient to support normal growth and acceptable slaughter quality. The present results suggest that the amount of Na required for the undisturbed growth of broilers and adequate feed conversion is not less than 0.15% of additional Na in the starter period (1-14 d), and not less than 0.11% in the grower period (until day 35). Higher dietary Na levels did not lead to further production advantages, and were found to increase the moisture content of droppings. Dry matter concentration of excreta was also affected by Na source. In comparison with NaHCO3, Na2SO4 seemed to be a better alternative for NaCl. Na2SO4 also tended to surpass NaHCO3 as a dietary alternative for NaCl in terms of feed utilisation during the starter period. The applied additional Na levels (0.25 and 0.15%) and Na sources had no effect on the sensory profile and composition of breast meat.