Virulence factors of Salmonella spp. isolated from free-living grass snakes Natrix natrix.

Salmonellosis associated with reptiles is a well-researched topic, particularly in China and the United States, but it occurs less frequently in Europe. The growth of the human population and changes in the environment could potentially increase the interaction between humans and free-living reptiles, which are an unidentified source of Salmonella species. In this study, we sought to explore this issue by comparing the microbiota of free-living European grass snakes, scientifically known as Natrix natrix, with that of captive banded water snakes, or Nerodia fasciata. We were able to isolate 27 strains of Salmonella species from cloacal swabs of 59 N. natrix and 3 strains from 10 N. fasciata. Our findings revealed that free-living snakes can carry strains of Salmonella species that are resistant to normal human serum (NHS). In contrast, all the Salmonella species strains isolated from N. fasciata were sensitive to the action of the NHS, further supporting our findings. We identified two serovars from N. natrix: Salmonella enterica subspecies diarizonae and S. enterica subspecies houtenae. Additionally, we identified three different virulotypes (VT) with invA, sipB, prgH, orgA, tolC, iroN, sitC, sifA, sopB, spiA, cdtB and msgA genes, and ß-galactosidase synthesised by 23 serovars. The identification of Salmonella species in terms of their VT is a relatively unknown aspect of their pathology. This can be specific to the serovar and pathovar and could be a result of adaptation to a new host or environment.

Colonization of methicillin-resistant Staphylococcus species in healthy and sick pets: prevalence and risk factors.

BACKGROUND: The characterization of staphylococcal species that colonize pets is important to maintain animal health and to minimize the risk of transmission to owners. Here, the prevalence of Staphylococcus spp. and methicillin resistance was investigated in canine and feline isolates, and risk factors of staphylococcal colonization were determined. Pets were examined and separated into four groups: (1) healthy dogs, (2) healthy cats, and (3) dogs and (4) cats with clinical signs of bacterial infections of skin, mucous membranes, or wounds. Specimens were collected by a veterinary physician from six anatomic sites (external ear canal, conjunctival sacs, nares, mouth, skin [groin], and anus). In total, 274 animals (cats n = 161, dogs n = 113) were enrolled. RESULTS: Staphylococcus species were highly diverse (23 species; 3 coagulase-positive and 20 coagulase-negative species), with the highest variety in healthy cats (19 species). The most frequent feline isolates were S. felis and S. epidermidis, while S. pseudintermedius was the most prevalent isolate in dogs. Risk factors of staphylococcal colonization included the presence of other animals in the same household, medical treatment within the last year, and a medical profession of at least one owner. Methicillin resistance was higher in coagulase-negative (17.86%) compared to coagulase-positive (1.95%) staphylococci. The highest prevalence of methicillin-resistant CoNS colonization was observed in animals kept in homes as the most common (dogs and cats). CONCLUSIONS: The association of methicillin-resistant CoNS colonization with animals most often chosen as pets, represents a high risk of transmission between them and owners. The importance of nosocomial transmission of CoNS was also confirmed. This information could guide clinical decisions during the treatment of veterinary bacterial infections. In conclusion, the epidemiologic characteristics of CoNS and their pathogenicity in pets and humans require further research.

The challenges in the identification of Escherichia coli from environmental samples and their genetic characterization.

Escherichia coli bacteria are an essential indicator in evaluations of environmental pollution, which is why they must be correctly identified. This study aimed to determine the applicability of various methods for identifying E. coli strains in environmental samples. Bacterial strains preliminary selected on mFc and Chromocult media as E. coli were identified using MALDI Biotyper techniques, based on the presence of genes characteristic of E. coli (uidA, uspA, yaiO), as well as by 16S rRNA gene sequencing. The virulence and antibiotic resistance genes pattern of bacterial strains were also analyzed to investigate the prevalence of factors that may indicate adaptation to unsupportive environmental conditions and could have any significance in further identification of E. coli. Of the strains that had been initially identified as E. coli with culture-based methods, 36-81% were classified as E. coli with the use of selected techniques. The value of Cohen's kappa revealed the highest degree of agreement between the results of 16S rRNA gene sequencing, the results obtained in the MALDI Biotyper system, and the results of the analysis based on the presence of the yaiO gene. The results of this study could help in the selection of more accurate and reliable methods which can be used in a preliminary screening and more precise identification of E. coli isolated from environmental samples.

Respiratory microbiota as a good predictor of outcome in humoral immunodeficiency. Methodological and conceptual difficulties in comprehensive microbiome analysis.

The microbiome as a broad term defines the most dynamic ecological system in which, apart from the biocenosis, there is unique niche for its development, equally dynamic, because host-derived, contrary to physical environment in ecology. Although for years, antibodies, was recognized as a key element of the mucosal barrier, there is still no progress in comprehensive linking microbes and humoral elements. Which organism is a pathogen and which is a commensal - there are still no exact answers (and criteria). Also the discrepancy between experimental research, using more and more sophisticated techniques -showing the existence of nearly 900 bacterial species in our mouth was observed. On the contrary, bacterial infections caused by novel species have not been recognised in patients with humoral immunodeficiencies, apart from the capsular cocci and H. influenzae. Research on human virome, including CMV, is also focused on the cellular immunity, but in many situations in the clinic the humoral compartment are the exclusive, still effective, barrier against viral replication. AIM: The aim of study was to perform an integrated microbiome analysis (microbiota and host) for strict linking various microbiota constelation and end-stage clinical manifestation in humoral immunodeficiency. MATERIALS AND METHODS: This cross-study based on own clinical experience reveal crucial gap in the literature. RESULTS: It indicate the natural competition: why the sentinel microorganism in humoral deficiency - S. pneumoniae dominates in some patients, and in others - Viridans Group Streptococci. Crucial role of high specific IgM level in several CVID complication was described. Furthermore, respiratory microbiota translocation with cancer regression and influence of therapeutic regimen on clinical sequel (predominantly infectious or malignant) were presented. The studies indicates numerous methodological and microecological classification difficulties, which are the source of inadequate, often contradictory conclusions from experimental research. CONCLUSIONS: Microecology describe ecosystem that consists of all the organisms and the abiotic physical environment with which they interact: i.e. biocenosis and biotope but clinical immunology -microorganisms in biotic (cellular) niche in host. Unfortunately, these studies are focused on the anecdotal "healthy" biocenosis (healthy microbiome), without analyzing other elements of the niche, for example cancer. Overall, focusing on the gut microbiota clearly restrict our knowledge on the manifestation of humoral deficiencies in respiratory system. The work is a summary of the search for representative clinical models and microbiological methods (diagnostic chain) in clinical immunology practice.

An analysis of the population of Cryptococcus neoformans strains isolated from animals in Poland, in the years 2015-2019.

Fungi belonging to the Cryptococcus neoformans/C. gattii species complex (CNGSC) are pathogens causing severe infections in humans and animals, that for humans may result in a mortality rate ranging up to 70%. The CNGSC is divided into eight major molecular types, that may differ in their virulence and susceptibility. In order to fully understand the epidemiology of cryptococcosis, it is important to study the world distribution and population structure of these pathogens. The present study is the first presenting a population of strains isolated in Poland and one of the few using a multi-species animal group as a source of the specimen. The pathogen was present in 2.375% of the tested animals. The URA5-RFLP and MALDI-TOF MS analyses have revealed that the population consisted exclusively of C. neoformans strains, with a predominance of major molecular type VNIV (C. neoformans var. neoformans). The MALDI-TOF MS was used to perform the CNGSC strains identification on both the species and sub-species level. Despite the fact that the animals providing the specimens were not treated with 5-fluorocytosine, around 10% of the tested population presented MIC values exceeding 64 mg/L, indicating the existence of the 5-fluorocytosine-resistant strains in the environment.

The Phylogenetic Structure of Reptile, Avian and Uropathogenic Escherichia coli with Particular Reference to Extraintestinal Pathotypes.

The impact of the Gram-negative bacterium Escherichia coli (E. coli) on the microbiomic and pathogenic phenomena occurring in humans and other warm-blooded animals is relatively well-recognized. At the same time, there are scant data concerning the role of E. coli strains in the health and disease of cold-blooded animals. It is presently known that reptiles are common asymptomatic carriers of another human pathogen, Salmonella, which, when transferred to humans, may cause a disease referred to as reptile-associated salmonellosis (RAS). We therefore hypothesized that reptiles may also be carriers of specific E. coli strains (reptilian Escherichia coli, RepEC) which may differ in their genetic composition from the human uropathogenic strain (UPEC) and avian pathogenic E. coli (APEC). Therefore, we isolated RepECs (n = 24) from reptile feces and compared isolated strains' pathogenic potentials and phylogenic relations with the aforementioned UPEC (n = 24) and APEC (n = 24) strains. To this end, we conducted an array of molecular analyses, including determination of the phylogenetic groups of E. coli, virulence genotyping, Pulsed-Field Gel Electrophoresis-Restriction Analysis (RA-PFGE) and genetic population structure analysis using Multi-Locus Sequence Typing (MLST). The majority of the tested RepEC strains belonged to nonpathogenic phylogroups, with an important exception of one strain, which belonged to the pathogenic group B2, typical of extraintestinal pathogenic E. coli. This strain was part of the globally disseminated ST131 lineage. Unlike RepEC strains and in line with previous studies, a high percentage of UPEC strains belonged to the phylogroup B2, and the percentage distribution of phylogroups among the tested APEC strains was relatively homogenous, with most coming from the following nonpathogenic groups: C, A and B1. The RA-PFGE displayed a high genetic diversity among all the tested E. coli groups. In the case of RepEC strains, the frequency of occurrence of virulence genes (VGs) was lower than in the UPEC and APEC strains. The presented study is one of the first attempting to compare the phylogenetic structures of E. coli populations isolated from three groups of vertebrates: reptiles, birds and mammals (humans).

Prevalence, Genetic Structure, and Antifungal Susceptibility of the Cryptococcus neoformans/C. gattii Species Complex Strains Collected from the Arboreal Niche in Poland.

Fungi belonging to the Cryptococcus neoformans/C. gattii species complex (CNGSC) are etiological agents of serious and not infrequently fatal infections in both humans and animals. Trees are the main ecological niche and source of potential exposition concerning these pathogens. With regard to epidemiology of cryptococcosis, various surveys were performed worldwide, enabling the establishment of a map of distribution and genetic structure of the arboreal population of the CNGSC. However, there are regions, among them Central and Eastern Europe, in which the data are lacking. The present study shows the results of such an environmental study performed in Wroclaw, Poland. The CNGSC strains were detected in 2.2% of the tested trees belonging to four genera. The obtained pathogen population consisted exclusively of C. neoformans, represented by both the major molecular type VNI and VNIV. Within the tested group of isolates, resistance to commonly used antimycotics was not found, except for 5-fluorocytosine, in which about 5% of the strains were classified as a non-wild type.

Cloacal Gram-Negative Microbiota in Free-Living Grass Snake Natrix natrix from Poland.

Reptiles appear to be an important vector for Gram-negative pathogens, therefore, they are epidemiologically relevant. However, the composition of reptilian microbiota has been poorly recognized so far. The majority of studies concern exotic reptiles as asymptomatic carriers of Salmonella serovars. Studies of other intestinal bacteria of reptiles are rare. Only recently, the microbiota of free-living European reptiles have been investigated, however, on the basis of small samples, mainly in protected areas. Here, we aim to investigate cloacal Gram-negative microbiota of free-living Natrix natrix. Snakes (N = 45) used in the study were collected in Kraków (Poland) and its vicinity. Nineteen species of Gram-negative bacteria were isolated. The most common species were: Aeromonas hydrophila, Morganella morganii, Proteus vulgaris, Salmonella spp. The bacteria prevalent in N. natrix cloacal swabs are likely to represent the natural intestinal Gram-negative microbiota of the examined snakes. Importantly, the identified bacteria are pathogenic to humans, which clearly highlights the epidemiological potential of free-living N. natrix. The risk of infection is high for immunocompromised humans, children (under 5 years old), elderly persons, and pregnant women. Our study provides the largest dataset on intestinal Gram-negative microbiota of wild snakes. The presence of multiple human pathogens determined by us calls for the necessity of further studies on reptile-transmitted bacteria in anthropogenic environments.

Molecular Routes to Specific Identification of the Lactobacillus Casei Group at the Species, Subspecies and Strain Level.

The genus Lactobacillus includes, among others, Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus, species that are collectively referred to as the Lactobacillus casei group. Many studies have shown that strains belonging to this group may decrease lactose intolerance, the effects of inflammatory bowel disease, diarrhea, constipation, food allergies and even colon cancer. Moreover, evidences exists of positive effects of these bacteria on mucosal immunity and blood cholesterol level. Because of their beneficial influence on human health, many of them are used as food additives and probiotic pharmaceuticals. It should be stressed that health-promoting properties are not attributed at the species level, but to specific strains. Therefore, procedures are necessary to allow specific identification at each phylogenetic level-genus, species and strain. In this paper we present a practical overview of molecular methods for the identification and differentiation of L. casei bacteria. The research included 30 bacterial strains belonging to three species: L.casei, L. paracasei and L. rhamnosus. Among the tested procedures were genus- and species-specific PCR, multiplex-PCR, Real-Time HRM analysis, RFLP-PCR, rep-PCR, RAPD-PCR, AFLP-PCR, and proteomic methods such as MALDI-TOF MS typing and SDS-PAGE fingerprinting. The obtained results showed that multiplex-PCR and MALDI-TOF MS turned out to be the most useful methods to identify the tested bacteria at the species level. At the strain level, the AFLP-PCR method showed the highest discriminatory power. We hope that the presented results will allow for the easy selection of an appropriate procedure, depending on the experiment conducted and the equipment capabilities of any given laboratory.

Efficacy of MALDI-TOF mass spectrometry as well as genotypic and phenotypic methods in identification of staphylococci other than Staphylococcus aureus isolated from intramammary infections in dairy cows in Poland.

We compared the effectiveness of various methods for the identification of Staphylococcus spp. other than S. aureus isolated from intramammary infections of cows on 3 dairy farms in Lower Silesia, Poland. A total of 131 isolates belonging to 18 Staphylococcus species were identified by sequence analysis of the 16S rRNA and dnaJ genes, as well using a commercial identification system (ID 32 STAPH; bioMérieux) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics). Sequencing of the 16S rRNA gene was found to have low discriminatory value because only 43% of isolates were recognized unequivocally. Much better results were obtained with the dnaJ gene (all isolates were correctly identified at the species level). However, some of these isolates achieved a low similarity level (<97%) and required a confirmatory test (sequencing of the rpoB gene). The performance of ID 32 STAPH was poor. Regardless of the probability level used (80% or 90%), the commercial system obtained identification rates <40%. Using MALDI-TOF MS and the commercial Bruker database, 67% of isolates were identified correctly with scores ≥2.0 (acceptable species-level identification) but this number increased to 97% after the database was expanded. The definitive identification of Staphylococcus spp. other than S. aureus causing intramammary infections in cattle often requires a combination of different procedures, and the existing databases should be updated.