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.

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.

Duties of the Psychiatric Committee for Preventive Measures. Present situation and proposed normative changes / Zadania Komisji Psychiatrycznej do Spraw Srodków Zabezpieczajacych. Stan obecny i postulaty zmian normatywnych.

The aim of this paper is to analyze the present legal model of the Psychiatric Committee for Preventive Measures and formulate proposed changes in regulations based on research findings. In 2003 the legislator delegated the qualifying procedures to the Committee, which resulted in lengthening the time until the moment of detaining the convict in a closed facility, which may cause harm to both the convict and the society. It is proposed that the classification be performed by experts, who must be heard by the court anyway before preventive measures are decreed, with the possibility of consulting the Committee in difficult or doubtful cases, if needed. Most tasks of the Committee, however, should be related to exercising control and to do this, it is necessary for the Committee to liaise with the court's penitentiary supervisors.

Effect of prenatal manganese intoxication on [(3)H]glucose uptake in the brain of rats lesioned as neonates with 6-hydroxydopamine.

In the present study we examined the effects of prenatal manganese (Mn) intoxication on [(3)H]glucose uptake in the brain of rats lesioned as neonates with 6-hydroxydopamine (6-OHDA). MnCl(2) . 4H(2)O (10,000 ppm) was added to the drinking water of pregnant Wistar rats for the duration of pregnancy. On the day of parturition, Mn was discontinued as an additive to the drinking water. The control group consisted of rats that consumed water without Mn. Three days after birth, rats in both groups (control and Mn) were pretreated with desipramine hydrochloride (20 mg/kg) and pargyline hydrochloride (50 mg/kg) and injected bilaterally icv with one of three doses of 6-OHDA hydrobromide (15 mug, 30 mug or 67 mug base form in saline on each side) or with saline (control). 6-[(3)H]-D-glucose (500 muCi/kg, ip) was administered to male offspring in adulthood; after 15 min, brain specimens were taken (frontal cortex, hippocampus, striatum, thalamus with hypothalamus, pons and cerebellum) for determination of radioactivity in a liquid scintillation counter. Low dose 6-OHDA (15 mug icv) increased [(3)H]glucose uptake in all brain regions (p < 0.05) in both control and Mn-intoxicated animals. In rats lesioned with a moderate dose of 6-OHDA (30 mug icv), [(3)H]glucose uptake was unaltered in both control and Mn-exposed rats. High dose 6-OHDA (67 mug icv) reduced [(3)H]glucose uptake in all brain regions of Mn-exposed rats (except for cerebellum) compared with the saline group (all, p < 0.05). There was no change in regional brain uptake of [(3)H]glucose in control rats. In conclusion, this study shows that mild neuronal insult (15 mug icv 6-OHDA) increased glucose uptake in the brain while severe damage (concomitant 60 mug icv 6-OHDA and Mn treatment) significantly diminished this process.

Neonatal co-lesion by DSP-4 and 5,7-DHT produces adulthood behavioral sensitization to dopamine D(2) receptor agonists.

To assess the possible modulatory effects of noradrenergic and serotoninergic neurons on dopaminergic neuronal activity, the noradrenergic and serotoninergic neurotoxins DSP-4 N-(2-chlorethyl)-N-ethyl-2-bromobenzylamine (50.0 mg/kg, sc) and 5,7-dihydroxytryptamine (5,7-DHT) (37.5 microg icv, half in each lateral ventricle), respectively, were administered toWistar rats on the first and third days of postnatal ontogeny, and dopamine (DA) agonist-induced behaviors were assessed in adulthood. At eight weeks, using an HPLC/ED technique, DSP-4 treatment was associated with a reduction in NE content of the corpus striatum (> 60%), hippocampus (95%), and frontal cortex (> 85%), while 5,7-DHT was associated with an 80-90% serotonin reduction in the same brain regions. DA content was unaltered in the striatum and the cortex. In the group lesioned with both DSP-4 and 5,7-DHT, quinpirole-induced (DA D(2) agonist) yawning, 7-hydroxy-DPAT-induced (DA D(3) agonist) yawning, and apomorphine-induced (non-selective DA agonist) stereotypies were enhanced. However, SKF 38393-induced (DA D(1) agonist) oral activity was reduced in the DSP-4 + 5,7-DHT group. These findings demonstrate that DA D(2)- and D(3)-agonist-induced behaviors are enhanced while DA D(1)-agonist-induced behaviors are suppressed in adult rats in which brain noradrenergic and serotoninergic innervation of the brain has largely been destroyed. This study indicates that noradrenergic and serotoninergic neurons have a great impact on the development of DA receptor reactivity (sensitivity).

Gestational manganese intoxication and anxiolytic-like effects of diazepam and the 5-HT1A receptor agonist 8-OH-DPAT in male Wistar rats.

In the present study, the effects of prenatal manganese (Mn) intoxication on the anxiolytic-like effects of diazepam and the 5-HT(1A) receptor agonist R-(+)-8-hydroxy-dipropylaminotetralin (8-OH-DPAT) were examined.Wistar dams were exposed to MnCl(2).4H(2)O at 5,000 ppm in the drinking water for the duration of pregnancy. On the day of parturition, Mn was discontinued as an additive in the drinking water. Control rats were derived from dams that consumed tap water and had no exposure to Mn. Male offspring were tested at the age of 12 weeks. The anxiolytic-like effect was assessed in an elevated plus maze device and with the Vogel conflict test. The benzodiazepine anxiolytic diazepam (5 mg/kg, ip) increased the percentage of time spent in open arms in control rats (in comparison to saline treatment) (p < 0.05); no such effect was seen in Mn-exposed rats. Conversely, the serotoninergic 5-HT(1A) agonist 8-OH-DPAT (0.3 mg/kg, ip) increased the percentage of time spent in open arms in both experimental groups. In the Vogel drinking test, an anxiolytic-like effect was also observed in both test groups (in controls this was of borderline significance). In contrast, 8-OH-DPAT did not evoke an anxiolytic-like action in control or in Mn-exposed rats in the anticonflict test. In conclusion, findings indicate that gestational Mn exposure attenuated benzodiazepine-mediated anxiolytic-like effects but not those of the 5-HT(1A) receptor agonist 8-OH-DPAT.

Nitric oxide modulates the amphetamine effect on [3H]glucose uptake in the brain of rats prenatally exposed to lead.

Glucose is the main source of energy for the central nervous system (CNS). In this study, we examined the effects of the psychostimulant amphetamine (AMPH) and the neuronal mediator nitric oxide (NO) on [3H]glucose uptake in the brain of adult rats that had been prenatally exposed to lead. Lead [Pb(CH3COO)2 . 3H2O; 250 ppm] was added to the drinking water of pregnant Wistar rats for the duration of pregnancy. On the day of parturition, lead was discontinued as an additive in the drinking water. Offspring remained ith dams for 21 days. The control group consisted of rats that consumed water without lead. In adulthood, male offspring from both groups (lead-exposed and control) were pretreated with 7-nitroindazole (nNOS blocking agent) (10.0 mg/kg ip) or saline (1.0 ml/kg ip), 30 min before AMPH (1.0 mg/kg ip). After another 30 min, and 15 min before termination, all rats were injected with 6-[3H]-D-glucose (500 muCi/kg ip). Brain specimens were taken (striatum, frontal cortex, hippocampus, and thalamus with hypothalamus, and pons with medulla oblongata) for determination of radioactivity in a liquid scintillation counter. We found that lead did not alter [3H]glucose uptake in brain regions studied (with exception of frontal cortex) but that AMPH increased [3H]glucose uptake in the striatum, frontal cortex and hippocampus, and that the AMPH effect was lessened in the hippocampus of lead-exposed rats. Moreover, the AMPH effect on [3H]glucose uptake in the frontal cortex, hippocampus, thalamus with hypothalamus and pons of control rats was potentiated by 7-NI pretreatment. Similar effect was observed in lead-intoxicated rats (striatum, frontal cortex and hippocampus). These results indicate that NO modulates AMPH-induced [3H]glucose uptake in the brain of rats prenatally exposed to lead.

Amphetamine and mCPP effects on dopamine and serotonin striatal in vivo microdialysates in an animal model of hyperactivity.

In the neonatally 6-hydroxydopamine (6-OHDA)-lesioned rat hyperlocomotor activity, first described in the 1970s, was subsequently found to be increased by an additional lesion with 5,7-dihydroxytryptamine (5,7-DHT) (i.c.v.) in adulthood. The latter animal model (i.e., 134 microg 6-OHDA at 3 d postbirth plus 71 microg 5,7-DHT at 10 weeks; desipramine pretreatments) was used in this study, in an attempt to attribute hyperlocomotor attenuation by D,L-amphetamine sulfate (AMPH) and m-chlorophenylpiperazine di HCl (mCPP), to specific changes in extraneuronal (i.e., in vivo microdialysate) levels of dopamine (DA) and/or serotonin (5-HT). Despite the 98-99% reduction in striatal tissue content of DA, the baseline striatal microdialysate level of DA was reduced by 50% or less at 14 weeks, versus the intact control group. When challenged with AMPH (0.5 mg/kg), the microdialysate level of DA went either unchanged or was slightly reduced over the next 180 min (i.e., 20 min sampling), while in the vehicle group and 5,7-DHT (alone) lesioned group, the microdialysate level was maximally elevated by approximately 225% and approximately 450%, respectively--and over a span of nearly 2 h. Acute challenge with mCPP (1 mg/kg salt form) had little effect on microdialysate levels of DA, DOPAC and 5-HT. Moreover, there was no consistent change in the microdialysate levels of DA, DOPAC, and 5-HT between intact, 5-HT-lesioned rats, and DA-lesioned rats which might reasonably account for an attenuation of hyperlocomotor activity. These findings indicate that there are other important neurochemical changes produced by AMPH- and mCPP-attenuated hyperlocomotor activity, or perhaps a different brain region or multiple brain regional effects are involved in AMPH and mCPP behavioral actions.

Influence of paraquat on dopaminergic transporter in the rat brain.

Selective toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a parkinsonism inducing compound, is well known to be related to an uptake of its active metabolite MPP+ into dopaminergic neurons by dopamine transporter (DAT). The aim of the present study was to examine whether paraquat, a commonly used herbicide, which is an 1-methyl-4-phenyl-pyridinium ion (MPP+) analogue, affects DAT in vivo in rats. Paraquat administered at a dose of 10 mg/kg ip decreased the binding of [3H]GBR 12,935 to DAT measured by quantitative autoradiography in the dorsal and ventral caudate-putamen, but not in the substantia nigra pars compacta. Moreover, this compound increased the level of 3-methoxytyramine (3-MT) and 3-MT/dopamine ratio in the anterior and posterior caudate-putamen measured by HPLC with electrochemical detection. No other alterations in the levels of dopamine and its metabolites were found in the caudate-putamen and substantia nigra. The present study seems to suggest that systemic paraquat administration affects striatal DAT and dopamine metabolism in the nigrostriatal neurons in rats which may be crucial for its neurotoxic effects on dopaminergic neurons.

Central effects of nafadotride, a dopamine D3 receptor antagonist, in rats. Comparison with haloperidol and clozapine.

The aim of this study was to examine behavioral and biochemical effects of nafadotride, the new dopamine D3 receptor antagonist, and to compare it with haloperidol (dopamine D2 receptor antagonist) and clozapine (predominate dopamine D4 receptor antagonist). Each drug was injected to adult male Wistar rats intraperitoneally, each at a single dose and for 14 consecutive days. Thirty minutes after single or last injection of the examined drugs, the following behavioral parameters were recorded: yawning, oral activity, locomotion, exploratory activity, catalepsy and coordination ability. By HPLC/ED methods, we determined the effects of the examined antagonists on the levels of biogenic amines in striatum and hippocampus: dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxytyramine (3-MT), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and noradrenaline (NA). Additionally, DA and 5-HT synthesis rate was determined in striatum and 5-HT in hippocampus. The results of the study indicate that nafadotride, the dopamine D3 receptor antagonist, has a behavioral and biochemical profile of action different from that of haloperidol but partially similar to that of clozapine.