Horsing around: Escherichia coli ST1250 of equine origin harbouring epidemic IncHI1/ST9 plasmid with bla CTX-M-1 and an operon for short-chain fructooligosaccharides metabolism.

The relatedness of the equine-associated Escherichia coli ST1250 and its single- and double-locus variants (ST1250-SLV/DLV), obtained from horses in Europe, was studied by comparative genome analysis. A total of 54 isolates of E. coli ST1250 and ST1250-SLV/DLV from healthy and hospitalized horses across Europe [Czech Republic (n=23), the Netherlands (n=18), Germany (n=9), Denmark (n=3) and France (n=1)] from 2008-2017 were subjected to whole-genome sequencing. An additional 25 draft genome assemblies of E. coli ST1250 and ST1250-SLV/DLV were obtained from the public databases. The isolates were compared for genomic features, virulence genes, clade structure and plasmid content. The complete nucleotide sequences of eight IncHI1/ST9 and one IncHI1/ST2 plasmids were obtained using long-read sequencing by PacBio or MinION. In the collection of 79 isolates, only 10 were phylogenetically close (<8 SNP). The majority of isolates belonged to phylogroup B1 (73/79, 92.4%) and carried bla CTX-M-1 (58/79, 73.4%). The plasmid content of the isolates was dominated by IncHI1 of ST9 (56/62, 90.3%) and ST2 (6/62, 9.7%), while 84.5% (49/58) bla CTX-M-1 genes were associated with presence of IncHI1 replicon of ST9 and 6.9% (4/58) with IncHI1 replicon of ST2 within the corresponding isolates. The operon for the utilization of short chain fructooligosaccharides (fos operon) was present in 55 (55/79, 69.6%) isolates, and all of these carried IncHI1/ST9 plasmids. The eight complete IncHI1/ST9 plasmid sequences showed the presence of bla CTX-M-1 and the fos operon within the same molecule. Sequences of IncHI1/ST9 plasmids were highly conserved (>98% similarity) regardless of country of origin and varied only in the structure and integration site of MDR region. E. coli ST1250 and ST1250-SLV/DLV are phylogenetically-diverse strains associated with horses. A strong linkage of E. coli ST1250 with epidemic multi-drug resistance plasmid lineage IncHI1/ST9 carrying bla CTX-M-1 and the fos operon was identified.

Broad-Host Dissemination of Plasmids Coharboring the fos Operon for Fructooligosaccharide Metabolism with Antibiotic Resistance Genes.

The fos operon encoding short-chain fructooligosaccharide (scFOS) utilization enables bacteria of the family Enterobacteriaceae to grow and be sustained in environments where they would struggle to survive. Despite several cases of the detection of the fos operon in isolates of avian and equine origins, its global distribution in bacterial genomes remains unknown. The presence of the plasmid-harbored fos operon among resistant bacteria may promote the spread of antibiotic resistance. A collection of 11,538 antimicrobial-resistant Enterobacteriaceae isolates from various sources was screened for the fosT gene encoding the scFOS transporter. Out of 307 fosT-positive isolates, 80% of them originated from sources not previously linked to fosT (humans, wastewater, and animals). The chromosomally harbored fos operon was detected in 163/237 isolates subjected to whole-genome sequencing. In the remaining 74 isolates, the operon was carried by plasmids. Further analyses focusing on the isolates with a plasmid-harbored fos operon showed that the operon was linked to various incompatibility (Inc) groups, including the IncHI1, IncF-type, IncK2, IncI1, and IncY families. Long-read sequencing of representative plasmids showed the colocalization of fos genes with antibiotic resistance genes (ARGs) in IncHI1 (containing a multidrug resistance region), IncK2 (blaTEM-1A), IncI1 [sul2 and tet(A)], and IncY [aadA5, dfrA17, sul2, and tet(A)] plasmids, while IncF-type plasmids had no ARGs but coharbored virulence-associated genes. Despite the differences in the locations and structures of the fos operons, all isolates except one were proven to utilize scFOSs. In this study, we show that the fos operon and its spread are not strictly bound to one group of plasmids, and therefore, it should not be overlooked. IMPORTANCE It was believed that members of the family Enterobacteriaceae are unable to grow under conditions with short-chain fructooligosaccharides as the only source of carbon. Nevertheless, the first Escherichia coli isolate from chicken intestine was able to utilize these sugars owing to the chromosomally harbored fos operon. Studies on E. coli isolates from horses discovered the horizontal transfer of the fos operon on IncHI1 plasmids along with genes for antibiotic resistance. The first plasmid detected was pEQ1, originating from the feces of a hospitalized horse in the Czech Republic. Follow-up studies also revealed the dissemination of the IncHI1 plasmid-harbored fos operon in the Netherlands, Germany, Denmark, and France among healthy horses. Despite several cases of detection of the fos operon, its global distribution in bacterial genomes remains unknown. The fos operon possibly plays a role in the adaptation of plasmids among resistant bacteria and therefore may promote the spread of antibiotic resistance.

The hyaluronan metabolism in the UV-irradiated human epidermis and the relevance of in vitro epidermal models.

Exposure to the sun affects the skin and may eventually result in UV-induced skin damage. It is generally known that hyaluronan (HA) is one of the main structural and functional components of the skin. However, UV-related changes in the HA metabolism in the skin have not yet been elucidated. Using qRT-PCR, confocal microscopy and LC-MS/MS we compared the naturally sun-exposed (SE), sun-protected, experimentally repeatedly UVA + UVB-exposed and acutely (once) UVA + UVB irradiated skin of Caucasian women. The epidermis was harvested by means of suction blistering 24 h after the acute irradiation. In addition, the epidermis was compared with a UV-irradiated in vitro reconstituted 3D epidermis (EpiDerm) and an in vitro 2D culture of normal human keratinocytes (NHEK). The amount of HA was found to be statistically significantly enhanced in the acutely irradiated epidermis. The acute UV evinced the upregulation of HA synthases (HAS2 and HAS3), hyaluronidases (HYAL2 and HYAL3), Cluster of differentiation 44 (CD44), and Cell Migration Inducing Proteins (CEMIP and CEMIP2), while only certain changes were recapitulated in the 3D epidermis. For the first time, we demonstrated the enhanced gene and protein expression of CEMIP and CEMIP2 following UV irradiation in the human epidermis. The data suggest that the HA metabolism is affected by UV in the irradiated epidermis and that the response can be modulated by the underlying dermis.

Genomic Analysis of a Strain Collection Containing Multidrug-, Extensively Drug-, Pandrug-, and Carbapenem-Resistant Modern Clinical Isolates of Acinetobacter baumannii.

In this study, we characterize a new collection that comprises multidrug-resistant (MDR), extensively drug-resistant (XDR), pandrug-resistant (PDR), and carbapenem-resistant modern clinical isolates of Acinetobacter baumannii collected from hospitals through national microbiological surveillance in Belgium. Bacterial isolates (n = 43) were subjected to whole-genome sequencing (WGS), combining Illumina (MiSeq) and Nanopore (MinION) technologies, from which high-quality genomes (chromosome and plasmids) were de novo assembled. Antimicrobial susceptibility testing was performed along with genome analyses, which identified intrinsic and acquired resistance determinants along with their genetic environments and vehicles. Furthermore, the bacterial isolates were compared to the most prevalent A. baumannii sequence type 2 (ST2) (Pasteur scheme) genomes available from the BIGSdb database. Of the 43 strains, 40 carried determinants of resistance to carbapenems; blaOXA-23 (n = 29) was the most abundant acquired antimicrobial resistance gene, with 39 isolates encoding at least two different types of OXA enzymes. According to the Pasteur scheme, the majority of the isolates were globally disseminated clones of ST2 (n = 25), while less frequent sequence types included ST636 (n = 6), ST1 (n = 4), ST85 and ST78 (n = 2 each), and ST604, ST215, ST158, and ST10 (n = 1 each). Using the Oxford typing scheme, we identified 22 STs, including two novel types (ST2454 and ST2455). While the majority (26/29) of blaOXA-23 genes were chromosomally carried, all blaOXA-72 genes were plasmid borne. Our results show the presence of high-risk clones of A. baumannii within Belgian health care facilities with frequent occurrences of genes encoding carbapenemases, highlighting the crucial need for constant surveillance.

Hyaluronan: A key player or just a bystander in skin photoaging?

Photoaged skin exhibits signs of inflammation, DNA damage and changes in morphology that are visible at the macroscopic and microscopic levels. Photoaging also affects the extracellular matrix (ECM) including hyaluronan (HA), the main polysaccharide component thereof. HA is a structurally simple but biologically complex molecule that serves as a water-retaining component and provides both a scaffold for a number of the proteins of the ECM and the ligand for cellular receptors. The study provides an overview of the literature concerning the changes in HA amount, size and metabolism, and the potential role of HA in photoaging. We also suggest novel HA contributions to photoaging based on our knowledge of the role of HA in other pathological processes, including the senescence and inflammation-triggered ECM reorganization. Moreover, we discuss potential direct or indirect intervention to mitigate photoaging that targets the hyaluronan metabolism, as well as supplementation.

Silver distribution in chronic wounds and the healing dynamics of chronic wounds treated with dressings containing silver and octenidine.

Although silver is an efficient antimicrobial and is a widely used antiseptic in wound healing, previous studies have reported the cytotoxic in vitro effects of silver dressings. Moreover, few studies have addressed the distribution of silver in chronic wounds. The study compares the healing of chronic wounds treated with a standard-of-care silver dressing (Ag-CMC) and a dressing containing antiseptic octenidine (OCT-HA). Biopsies were taken from two wound areas before the commencement of treatment (baseline), after 2 weeks and after 6 weeks (the end of the study). We analyzed the histopathologic wound-healing score, silver distribution, and expression of selected genes. The wound-healing score improved significantly in the wounded area treated with OCT-HA after 2 weeks compared to the baseline and the Ag-CMC. The Ag-CMC wound areas improved after 6 weeks compared to the baseline. Moreover, collagen maturation and decreases in the granulocyte and macrophage counts were faster in the OCT-HA parts. Treatment with OCT-HA resulted in less wound slough. The silver, visualized via autometallography, penetrated approximately 2 mm into the wound tissue and associated around capillaries and ECM fibers, and was detected in phagocytes. The metallothionein gene expression was elevated in the Ag-CMC wound parts. This exploratory study determined the penetration of silver into human chronic wounds and changes in the distribution thereof during treatment. We observed that silver directly affects the cells in the wound and elevates the metallothionein gene expression. Octenidine and hyaluronan dressings provide a suitable alternative to silver and carboxymethyl cellulose dressings without supplying silver to the wound.

Anti-HA antibody does not detect hyaluronan.

It is generally known that hyaluronic acid (HA) is a biocompatible and biodegradable glycosaminoglycan distributed widely throughout epithelial, connective and neural tissues. HA is one of the chief components of the extracellular matrix. Lack of immunogenicity is one of the biggest advantages of the therapeutic use of HA, but it also prevents the production of specific anti-HA antibodies. Contrary to this, there are still several studies performing HA detection by immunohistochemical or immunohistofluorescent method using an anti-HA antibody. Therefore, this short study discusses whether the anti-HA antibody is specific for HA. To verify the specificity of the HA staining the hyaluronidase treatment of histological samples was performed and the ability of anti-HA antibody and biotinylated HA binding protein (bHABP)-based probe to bind to their targets was evaluated. Additionally, the competitive binding assay with short HA oligosaccharides and subsequent histological staining was performed. Both assays showed that the anti-HA antibody is not sufficiently specific for HA and that the bHABP probe is a reliable method for HA detection in histological samples. The conclusion made by previous investigators based on using HA antibodies should be reevaluated and future use of anti-HA antibody should be avoided.

Escherichia coli Sequence Type 457 Is an Emerging Extended-Spectrum-ß-Lactam-Resistant Lineage with Reservoirs in Wildlife and Food-Producing Animals.

Silver gulls carry phylogenetically diverse Escherichia coli, including globally dominant extraintestinal pathogenic E. coli (ExPEC) sequence types and pandemic ExPEC-ST131 clades; however, our large-scale study (504 samples) on silver gulls nesting off the coast of New South Wales identified E. coli ST457 as the most prevalent. A phylogenetic analysis of whole-genome sequences (WGS) of 138 ST457 samples comprising 42 from gulls, 2 from humans (Australia), and 14 from poultry farmed in Paraguay were compared with 80 WGS deposited in public databases from diverse sources and countries. E. coli ST457 strains are phylogenetic group F, carry fimH145, and partition into five main clades in accordance to predominant flagella H-antigen carriage. Although we identified considerable phylogenetic diversity among the 138 ST457 strains, closely related subclades (<100 SNPs) suggested zoonotic or zooanthroponosis transmission between humans, wild birds, and food-producing animals. Australian human clinical and gull strains in two of the clades were closely related (≤80 SNPs). Regarding plasmid content, country, or country/source, specific connections were observed, including I1/ST23, I1/ST314, and I1/ST315 disseminating blaCMY-2 in Australia, I1/ST113 carrying blaCTX-M-8 and mcr-5 in Paraguayan poultry, and F2:A-:B1 plasmids of Dutch origin being detected across multiple ST457 clades. We identified a high prevalence of nearly identical I1/ST23 plasmids carrying blaCMY-2 among Australian gull and clinical human strains. In summary, ST457 is a broad host range, geographically diverse E. coli lineage that can cause human extraintestinal disease, including urinary tract infection, and displays a remarkable ability to capture mobile elements that carry and transmit genes encoding resistance to critically important antibiotics.

Opioid receptors and opioid peptides in the cardiomyogenesis of mouse embryonic stem cells.

The stimulation of myocardium repair is restricted due to the limited understanding of heart regeneration. Interestingly, endogenous opioid peptides such as dynorphins and enkephalins are suggested to support this process. However, the mechanism-whether through the stimulation of the regenerative capacity of cardiac stem cells or through effects on other cell types in the heart-is still not completely understood. Thus, a model of the spontaneous cardiomyogenic differentiation of mouse embryonic stem (mES) cells via the formation of embryoid bodies was used to describe changes in the expression and localization of opioid receptors within cells during the differentiation process and the potential of the selected opioid peptides, dynorphin A and B, and methionin-enkephalins and leucin-enkephalins, to modulate cardiomyogenic differentiation in vitro. The expressions of both κ- and δ-opioid receptors significantly increased during mES cell differentiation. Moreover, their primary colocalization with the nucleus was followed by their growing presence on the cytoplasmic membrane with increasing mES cell differentiation status. Interestingly, dynorphin B enhanced the downregulation gene expression of Oct4 characteristic of the pluripotent phenotype. Further, dynorphin B also increased cardiomyocyte-specific Nkx2.5 gene expression. However, neither dynorphin A nor methionin-enkephalins and leucin-enkephalins exhibited any significant effects on the course of mES cell differentiation. In conclusion, despite the increased expression of opioid receptors and some enhancement of mES cell differentiation by dynorphin B, the overall data do not support the notion that opioid peptides have a significant potential to promote the spontaneous cardiomyogenesis of mES cells in vitro.

Low molecular weight hyaluronan mediated CD44 dependent induction of IL-6 and chemokines in human dermal fibroblasts potentiates innate immune response.

Complex regulation of the wound healing process involves multiple interactions among stromal tissue cells, inflammatory cells, and the extracellular matrix. Low molecular weight hyaluronan (LMW HA) derived from the degradation of high molecular weight hyaluronan (HMW HA) is suggested to activate cells involved in wound healing through interaction with HA receptors. In particular, receptor CD44 is suggested to mediate cell response to HA of different MW, being the main cell surface HA receptor in stromal tissue and immune cells. However, the response of dermal fibroblasts, the key players in granulation tissue formation within the wound healing process, to LMW HA and their importance for the activation of immune cells is unclear. In this study we show that LMW HA (4.3kDa) induced pro-inflammatory cytokine IL-6 and chemokines IL-8, CXCL1, CXCL2, CXCL6 and CCL8 gene expression in normal human dermal fibroblasts (NHDF) that was further confirmed by increased levels of IL-6 and IL-8 in cell culture supernatants. Conversely, NHDF treated by HMW HA revealed a tendency to decrease the gene expression of these cytokine and chemokines when compared to untreated control. The blockage of CD44 expression by siRNA resulted in the attenuation of IL-6 and chemokines expression in LMW HA treated NHDF suggesting the involvement of CD44 in LMW HA mediated NHDF activation. The importance of pro-inflammatory mediators produced by LMW HA triggered NHDF was evaluated by significant activation of blood leukocytes exhibited as increased production of IL-6 and TNF-α. Conclusively, we demonstrated a pro-inflammatory response of dermal fibroblasts to LMW HA that was transferred to leukocytes indicating the significance of LMW HA in the inflammatory process development during the wound healing process.

Selective in vitro anticancer effect of superparamagnetic iron oxide nanoparticles loaded in hyaluronan polymeric micelles.

Due to its native origin, excellent biocompatibility and biodegradability, hyaluronan (HA) represents an attractive polymer for superparamagnetic iron oxide nanoparticles (SPION) coating. Herein, we report HA polymeric micelles encapsulating oleic acid coated SPIONs, having a hydrodynamic size of about 100 nm and SPION loading capacity of 1-2 wt %. The HA-SPION polymeric micelles were found to be selectively cytotoxic toward a number of human cancer cell lines, mainly those of colon adenocarcinoma (HT-29). The selective inhibition of cell growth was even observed when the SPION loaded HA polymeric micelles were incubated with a mixture of control and cancer cells. The selective in vitro inhibition could not be connected with an enhanced CD44 uptake or radical oxygen species formation and was rather connected with a different way of SPION intracellular release. While aggregated iron particles were visualized in control cells, nonaggregated solubilized iron oxide particles were detected in cancer cells. In vivo SPION accumulation in intramuscular tumor following an intravenous micelle administration was confirmed by magnetic resonance (MR) imaging and histological analysis. Having a suitable hydrodynamic size, high magnetic relaxivity, and being cancer specific and able to accumulate in vivo in tumors, SPION-loaded HA micelles represent a promising platform for theranostic applications.

Changing dynamics of antibiotic resistant Escherichia in Caspian gulls shows the importance of longitudinal environmental studies.

This study is focused on Escherichia spp. isolates resistant to critically important antibiotics (cefotaxime, ciprofloxacin and colistin) among Caspian gull's (Larus cachinnans) chicks nesting in the Nove Mlyny Water Reservoir, Czech Republic. The prevalence of antimicrobial resistance (AMR) in bacteria within wild birds is commonly evaluated using a single sampling event, capturing only a brief and momentary snapshot at a particular location. Therefore, the Caspian gulls in our study were sampled in May 2018 (n = 72) and May 2019 (n = 45), and a water sample was taken from the reservoir (2019). We obtained 197 isolates identified as E. coli by MALDI-TOF MS. A total of 158 representative isolates were whole-genome sequenced, 17 isolates were then reclassified to Escherichia albertii. We observed a higher (86 %; 62/72) occurrence of ESBL/AmpC-producing Escherichia spp. among gulls in 2018 compared to 38 % (17/45) in 2019 (p < 0.00001). The decrease in prevalence was linked to clonal lineage of E. coli ST11893 predominating in 2018 which carried blaCMY-2 and which was not recovered from the gulls in 2019. Oppositely, several Escherichia STs were found in gulls from both years as well as in the water sample including STs commonly recognized as internationally high-risk lineages such as ST10, ST58, ST88, ST117, ST648 or ST744. Phylogenetic analysis of E. coli from EnteroBase from countries where these particular gulls wander revealed that some STs are commonly found in various sources including humans and a portion of them is even closely related (up to 100 SNPs) to our isolates. We demonstrated that the occurrence of AMR in Escherichia can vary greatly in time in synanthropic birds and we detected both, a temporary prevalent lineage and several persistent STs. The close relatedness of isolates from gulls and isolates from EnteroBase highlights the need to further evaluate the risk connected to wandering birds.

Enzymatically stable unsaturated hyaluronan-derived oligosaccharides with selective cytostatic properties.

Hyaluronan, a linear glycosaminoglycan comprising D-N-acetylglucosamine and D-glucuronic acid, is the main component of the extracellular matrix. Its influence on cell proliferation, migration, inflammation, signalling, and other functions, depends heavily on its molecular weight and chemical modification. Unsaturated HA oligosaccharides are available in defined length and purity. Their potential therapeutic utility can be further improved by chemical modification, e. g., reduction. No synthesis of such modified oligosaccharides, either stepwise or by hyaluronan cleavage, has been reported yet. Here we show a three-step synthesis (esterification, depolymerization and reduction) of unsaturated even numbered hyaluronan oligosaccharides with carboxylates and the reducing terminus reduced to an alcohol. Particular oligosaccharides were synthesised. The modified oligosaccharides are not cleaved by mammalian or bacterial hyaluronidase and do not affect the growth of mouse and human fibroblasts. Further, MTT and NRU viability tests showed that they inhibit the growth of human colon carcinoma cells HT-29 by 20-50 % in concentrations 500-1000 µg/mL. Interestingly, this effect takes place regardless of CD44 receptor expression and was not observed with unmodified HA oligosaccharides. These compounds could serve as enzymatically stable building blocks for biologically active substances.

Plasmid-mediated colistin resistance from fresh meat and slaughtered animals in the Czech Republic: nation-wide surveillance 2020-2021.

The aim of this study was to determine the occurrence of plasmid-mediated colistin resistance in domestic and imported meat and slaughter animals in the Czech Republic during 2020-2021 by using selective cultivation and direct PCR testing. A total of 111 colistin-resistant Escherichia coli isolates with mcr-1 gene were obtained from 65 (9.9%, n = 659) samples and subjected to whole-genome sequencing. Isolates with mcr were frequently found in fresh meat from domestic production (14.2%) as well as from import (28.8%). The mcr-1-positive E. coli isolates predominantly originated from meat samples (16.6%), mainly poultry (27.1%), and only minor part of the isolates came from the cecum (1.7%). In contrast to selective cultivation, 205 (31.1%) samples of whole-community DNA were positive for at least one mcr variant, and other genes besides mcr-1 were detected. Analysis of whole-genome data of sequenced E. coli isolates revealed diverse sequence types (STs) including pathogenic lineages and dominance of ST1011 (15.6%) and ST162 (12.8%). Most isolates showed multidrug-resistant profile, and 9% of isolates produced clinically important beta-lactamases. The mcr-1 gene was predominantly located on one of three conjugative plasmids of IncX4 (83.5%), IncI2 (7.3%), and IncHI2 (7.3%) groups. Seventy-two percent isolates of several STs carried ColV plasmids. The study revealed high prevalence of mcr genes in fresh meat of slaughter animals. Our results confirmed previous assumptions that the livestock, especially poultry production, is an important source of colistin-resistant E. coli with the potential of transfer to humans via the food chain. IMPORTANCE We present the first data on nation-wide surveillance of plasmid-mediated colistin resistance in the Czech Republic. High occurrence of plasmid-mediated colistin resistance was found in meat samples, especially in poultry from both domestic production and import, while the presence of mcr genes was lower in the gut of slaughter animals. In contrast to culture-based approach, testing of whole-community DNA showed higher prevalence of mcr and presence of various mcr variants. Our results support the importance of combining cultivation methods with direct culture-independent techniques and highlight the need for harmonized surveillance of plasmid-mediated colistin resistance. Our study confirmed the importance of livestock as a major reservoir of plasmid-mediated colistin resistance and pointed out the risks of poultry meat for the transmission of mcr genes toward humans. We identified several mcr-associated prevalent STs, especially ST1011, which should be monitored further as they represent zoonotic bacteria circulating between different environments.

Low, medium, and high molecular weight hyaluronic acid effects on human dental pulp stem cells in vitro.

Hyaluronic acid (HA), an extracellular biopolymer found throughout the human body, holds promise as a biocompatible and biodegradable scaffold material. High molecular weight (HMW) HA degrades, generating low molecular weight (LMW) fragments with distinct properties. These fragments can influence the behaviour of cells, including human dental pulp stem cells (hDPSCs) incorporated into HA-containing hydrogels or scaffolds. Therefore, a comprehensive examination of the impact of a range of HA molecular weights on hDPSCs is essential before designing HA-based scaffolds for these cells. hDPSC lines were cultured with LMW HA (800 Da, 1600 Da, 15 kDa), medium molecular weight HA (237 kDa), or HMW HA (1500 kDa) over six passages. The various molecular weights had negligible effects on hDPSCs viability, morphology, adhesion, or relative telomere length. Furthermore, the expression of key surface stemness markers (CD29, CD44, CD73, CD90) remained unaltered. HA did not induce osteogenic, chondrogenic, or adipogenic differentiation. Moreover, the potential for chondrogenic and osteogenic differentiation was not adversely affected by LMW or HMW HA. Various molecular weights of HA seem safe, biocompatible and therefore suitable components for hDPSCs-containing scaffolds. These findings affirm that the hDPCSs will not be negatively affected by HA fragments resulting from scaffold degradation.

Plasmid-mediated colistin resistance among human clinical Enterobacterales isolates: national surveillance in the Czech Republic.

The occurrence of colistin resistance has increased rapidly among Enterobacterales around the world. We performed a national survey of plasmid-mediated colistin resistance in human clinical isolates through a retrospective analysis of samples from 2009 to 2017 and a prospective sampling in 2018-2020. The aim of this study was to identify and characterize isolates with mcr genes from various regions of the Czech Republic using whole genome sequencing (WGS). Of all 1932 colistin-resistant isolates analyzed, 73 (3.8%) were positive for mcr genes. Most isolates carried mcr-1 (48/73) and were identified as Escherichia coli (n = 44) and Klebsiella pneumoniae (n = 4) of various sequence types (ST). Twenty-five isolates, including Enterobacter spp. (n = 24) and Citrobacter freundii (n = 1) carrying the mcr-9 gene were detected; three of them (Enterobacter kobei ST54) co-harbored the mcr-4 and mcr-9 genes. Multi-drug resistance phenotype was a common feature of mcr isolates and 14% (10/73) isolates also co-harbored clinically important beta-lactamases, including two isolates with carbapenemases KPC-2 and OXA-48. Phylogenetic analysis of E. coli ST744, the dominant genotype in this study, with the global collection showed Czech isolates belonged to two major clades, one containing isolates from Europe, while the second composed of isolates from diverse geographical areas. The mcr-1 gene was carried by IncX4 (34/73, 47%), IncHI2/ST4 (6/73, 8%) and IncI2 (8/73, 11%) plasmid groups. Small plasmids belonging to the ColE10 group were associated with mcr-4 in three isolates, while mcr-9 was carried by IncHI2/ST1 plasmids (4/73, 5%) or the chromosome (18/73, 25%). We showed an overall low level of occurrence of mcr genes in colistin-resistant bacteria from human clinical samples in the Czech Republic.

Modified hyaluronic acid with enhanced resistance to degradation.

A mild and efficient reduction of negatively charged glucuronate units of hyaluronic acid (HA) into less polar glucose units has not been reported yet. However, this modification could significantly affect physical and chemical properties. Here we show a one-pot procedure where HA is converted into its derivate with carboxyl groups reduced to primary alcohols (HA-Red) without severe polymer degradation. Optimized synthesis aimed at aqueous solutions allowed the preparation of polysaccharides with molecular weights up to 1000 kDa. The chemical structure of HA-Red was proved by 2-dimensional NMR methodologies, FT-IR, LC-MS and SECMALLS. The final materials were exposed to a higher temperature or digested with bovine testicular hyaluronidase (BTH). Obtained data proved higher stability of HA-Red compared to HA, and significant dependence of stability on the degree of modification was observed in most cases. Preliminary in vitro studies showed no negative effects of HA-Red on the growth of 3T3 fibroblasts, which may be promising for applications requiring biodegradable and biocompatible HA derivatives with increased resistance to degradation.

Hospital and community wastewater as a source of multidrug-resistant ESBL-producing Escherichia coli.

Introduction: Hospitals and wastewater are recognized hot spots for the selection and dissemination of antibiotic-resistant bacteria to the environment, but the total participation of hospitals in the spread of nosocomial pathogens to municipal wastewater treatment plants (WWTPs) and adjacent rivers had not previously been revealed. Methods: We used a combination of culturing and whole-genome sequencing to explore the transmission routes of Escherichia coli from hospitalized patients suffering from urinary tract infections (UTI) via wastewater to the environment. Samples were collected in two periods in three locations (A, B, and C) and cultured on selective antibiotic-enhanced plates. Results: In total, 408 E. coli isolates were obtained from patients with UTI (n=81), raw hospital sewage (n=73), WWTPs inflow (n=96)/outflow (n=106), and river upstream (n=21)/downstream (n=31) of WWTPs. The majority of the isolates produced extended-spectrum beta-lactamase (ESBL), mainly CTX-M-15, and showed multidrug resistance (MDR) profiles. Seven carbapenemase-producing isolates with GES-5 or OXA-244 were obtained in two locations from wastewater and river samples. Isolates were assigned to 74 different sequence types (ST), with the predominance of ST131 (n=80) found in all sources including rivers. Extraintestinal pathogenic lineages frequently found in hospital sewage (ST10, ST38, and ST69) were also found in river water. Despite generally high genetic diversity, phylogenetic analysis of ST10, ST295, and ST744 showed highly related isolates (SNP 0-18) from different sources, providing the evidence for the transmission of resistant strains through WWTPs to surface waters. Discussion: Results of this study suggest that 1) UTI share a minor participation in hospitals wastewaters; 2) a high diversity of STs and phylogenetic groups in municipal wastewaters derive from the urban influence rather than hospitals; and 3) pathogenic lineages and bacteria with emerging resistance genotypes associated with hospitals spread into surface waters. Our study highlights the contribution of hospital and municipal wastewater to the transmission of ESBL- and carbapenemase-producing E. coli with MDR profiles to the environment.

Endogenously produced hyaluronan contributes to the regulation of peritoneal adhesion development.

Peritoneal adhesions are postsurgical fibrotic complications connected to peritoneal inflammation. The exact mechanism of development is unknown; however, an important role is attributed to activated mesothelial cells (MCs) overproducing macromolecules of extracellular matrix (ECM), including hyaluronic acid (HA). It was suggested that endogenously-produced HA contributes to the regulation of different fibrosis-related pathologies. However, little is known about the role of altered HA production in peritoneal fibrosis. We focused on the consequences of the increased turnover of HA in the murine model of peritoneal adhesions. Changes of HA metabolism were observed in early phases of peritoneal adhesion development in vivo. To study the mechanism, human MCs MeT-5A and murine MCs isolated from the peritoneum of healthy mice were pro-fibrotically activated by transforming growth factor ß (TGFß), and the production of HA was attenuated by two modulators of carbohydrate metabolism, 4-methylumbelliferone (4-MU) and 2-deoxyglucose (2-DG). The attenuation of HA production was mediated by upregulation of HAS2 and downregulation of HYAL2 and connected to the lower expression of pro-fibrotic markers, including fibronectin and α-smooth muscle actin (αSMA). Moreover, the inclination of MCs to form fibrotic clusters was also downregulated, particularly in 2-DG-treated cells. The effects of 2-DG, but not 4-MU, were connected to changes in cellular metabolism. Importantly, the inhibition of AKT phosphorylation was observed after the use of both HA production inhibitors. In summary, we identified endogenous HA as an important regulator of peritoneal fibrosis, not just a passive player during this pathological process.