New biologically active ionic liquids with benzethonium cation-efficient SAR inducers and antimicrobial agents.

BACKGROUND: An urgent need to find new methods for crop protection remains open due to the withdrawal from the market of the most toxic pesticides and increasing consumer awareness. One of the alternatives that can be used in modern agriculture is the use of bifunctional compounds whose actions towards plant protection are wider than those of conventional pesticides. RESULTS: In this study, we present the investigation of the biological efficacy of nine dual-functional salts containing a systemic acquired resistance (SAR)-inducing anion and the benzethonium cation. A significant result of the presented study is the discovery of the SAR induction activity of benzethonium chloride, which was previously reported only as an antimicrobial agent. Moreover, the concept of dual functionality was proven, as the application of presented compounds in a given concentrations resulted both in the control of human and plant bacteria species and induction of SAR. CONCLUSION: The strategy presented in this article shows the capabilities of derivatization of common biologically active compounds into their ionic derivatives to obtain bifunctional salts. This approach may be an example of the design of potential new compounds for modern agriculture. It provides plants with two complementary actions allowing to provide efficient protection to plants, if one mode of action is ineffective. © 2024 Society of Chemical Industry.

Urban wastewater as a conduit for pathogenic Gram-positive bacteria and genes encoding resistance to ß-lactams and glycopeptides.

The emergence and spread of clinical pathogens, antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in the environment pose a direct threat to human and animal health worldwide. In this study, we analyzed qualitatively and quantitatively urban sewage resistome for the occurrence of genes encoding resistance to ß-lactams and glycopeptides in the genomes of culturable bacteria, as well as in the wastewater metagenome of the Central Wastewater Treatment Plant in Kozieglowy (Poland). Moreover, we estimated the presence of pathogenic Gram-positive bacteria in wastewater based on analysis of species-specific virulence genes in the wastewater metagenome. The results show that the final effluent contains alarm pathogens with particularly dangerous mechanisms of antibiotic resistance, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). We also noticed that during the wastewater treatment, there is an increase in the frequency of MRSA and VRE. Furthermore, the results prove the effective removal of vanA, but at the same time show that wastewater treatment increases the relative abundance of mecA and virulence genes (groES and sec), indicating the presence of clinical pathogens E. faecalis and S. aureus in the effluent released to surface waters. We also observed an increase in the relative abundance of mecA and vanA genes already in the aeration tank, which suggests accumulation of contaminants affecting enhanced selection and HGT processes in the activated sludge. Moreover, we found a relation between the taxonomic composition and the copy number of ARGs as well as the presence of pathogens at various stages of wastewater treatment. The presence of clinically relevant pathogens, ARB, including multi-resistant bacteria, and ARGs in the effluent indicates that wastewater treatment plant play a key role in the existence of pathogens and antimicrobial resistance spreading pathway in the environment and human communities, which is a direct threat to public health and environmental protection.

Occurrence of integrons and antibiotic resistance genes in cryoconite and ice of Svalbard, Greenland, and the Caucasus glaciers.

The prevalence of integrons and antibiotic resistance genes (ARGs) is a serious threat for public health in the new millennium. Although commonly detected in sites affected by strong anthropogenic pressure, in remote areas their occurrence, dissemination, and transfer to other ecosystems is poorly recognized. Remote sites are considered as a benchmark for human-induced contamination on Earth. For years glaciers were considered pristine, now they are regarded as reservoirs of contaminants, thus studies on contamination of glaciers, which may be released to other ecosystems, are highly needed. Therefore, in this study we evaluated the occurrence and frequency of clinically relevant ARGs and resistance integrons in the genomes of culturable bacteria and class 1 integron-integrase gene copy number in the metagenome of cryoconite, ice and supraglacial gravel collected on two Arctic (South-West Greenland and Svalbard) and two High Mountain (the Caucasus) glaciers. Altogether, 36 strains with intI1 integron-integrase gene were isolated. Presence of class 1 integron-integrase gene was also recorded in metagenomic DNA from all sampling localities. The mean values of relative abundance of intI1 gene varied among samples and ranged from 0.7% in cryoconite from Adishi Glacier (the Caucasus) to 16.3% in cryoconite from Greenland. Moreover, antibiotic-resistant strains were isolated from all regions. Genes conferring resistance to ß-lactams (blaSHV, blaTEM, blaOXA, blaCMY), fluoroquinolones (qepA, qnrC), and chloramphenicol (cat, cmr) were detected in the genomes of bacterial isolates.

Metagenomic analysis of ß-lactamase and carbapenemase genes in the wastewater resistome.

The emergence and spread of resistance to antibiotics among bacteria is the most serious global threat to public health in recent and coming decades. In this study, we characterized qualitatively and quantitatively ß-lactamase and carbapenemase genes in the wastewater resistome of Central Wastewater Treatment Plant in Kozieglowy, Poland. The research concerns determination of the frequency of genes conferring resistance to ß-lactam and carbapenem antibiotics in the genomes of culturable bacteria, as well as in the wastewater metagenome at three stages of treatment: raw sewage, aeration tank, and final effluent. In the final effluent we found bacteria with genes that pose the greatest threat to public health, including genes of extended spectrum ß-lactamases - blaCTX-M, carbapenemases - blaNDM, blaVIM, blaGES, blaOXA-48, and showed that during the wastewater treatment their frequency increased. Moreover, the wastewater treatment process leads to significant increase in the relative abundance of blaTEM and blaGES genes and tend to increase the relative abundance of blaCTX-M, blaSHV and blaOXA-48 genes in the effluent metagenome. The biodiversity of bacterial populations increased during the wastewater treatment and there was a correlation between the change in the composition of bacterial populations and the variation of relative abundance of ß-lactamase and carbapenemase genes. PCR-based quantitative metagenomic analysis combined with analyses based on culture methods provided significant information on the routes of ARBs and ARGs spread through WWTP. The limited effectiveness of wastewater treatment processes in the elimination of antibiotic-resistant bacteria and resistance genes impose the need to develop an effective strategy and implement additional methods of wastewater disinfection, in order to limit the increase and the spread of antibiotic resistance in the environment.

Wolbachia of phylogenetic supergroup E identified in oribatid mite Gustavia microcephala (Acari: Oribatida).

Heritable endosymbionts have been observed in arthropod and nematode hosts. The most-known among them is Wolbachia. Although the bacterium was previously identified in oribatid mites (Acari: Oribatida), it was not assigned to any phylogenetic group. Endosymbionts have a profound influence on their hosts, playing various functions that affect invertebrate's biology such as changing the way of reproduction. Oribatida provide the very unique examples of groups in which even whole families appear to be thelytokous, so we considered that it is worth to investigate the occurrence of endosymbiotic microorganisms in oribatid mites, especially that the knowledge on the symbionts occurrence in this invertebrate group is negligible. We report for the first time Wolbachia in oribatid mite Gustavia microcephala. The sequences of 16S rDNA, gltA, and ftsZ genes of the endosymbiont from the mite showed the highest similarity to Wolbachia found in Collembola. Phylogenetic analysis based on single gene and concatenated alignments of three genes revealed that the bacteria from G. microcephala and Collembola were related and clustered together with supergroup E. Relatively close relationship of Wolbachia from oribatid and collembolan hosts might mean at the evolutionary scale that horizontal transfer of bacteria between these two groups of invertebrates may take place.

In vitro virulence characteristics of rare serovars of Salmonella enterica isolated from sand lizards (Lacerta agilis L.).

The aim of this study was to estimate virulence potential of Salmonella enterica strains colonizing the gut of free-living sand lizards (Lacerta agilis L.). The strains belonged to three Salmonella serovars: Abony, Schleissheim, and Telhashomer. Adhesion and invasion abilities of the strains were determined in quantitative assays using the gentamicin protection method. Induction of apoptosis was assessed using HeLa cell monolayers. PCR assays were used for detection of 26 virulence genes localised within mobile elements: pathogenicity islands, virulence plasmids, and prophage sequences. In vitro studies revealed that all strains had adhesion and invasion abilities to human epithelial cells. The isolates were cytotoxic and induced apoptosis of the cells. The serovars differed in the number of virulence-associated genes: up to 18 genes were present in Salmonella Schleissheim, 17 in Salmonella Abony, whereas as few as six genes were found in Salmonella Telhashomer. Generally, Salmonella Abony and Salmonella Schleissheim did not differ much in gene content connected with the presence SPI-1 to -5. All of the strains lacked genes localised within bacteriophages and plasmids. The presence of virulence-associated genes and in vitro pathogenicity assays suggest that Salmonella sp. strains originating from autochthonous, free-living lizards can potentially infect and cause disease in humans.

Abundance of Class 1 Integron-Integrase and Sulfonamide Resistance Genes in River Water and Sediment Is Affected by Anthropogenic Pressure and Environmental Factors.

In this study, we determined the presence of class 1 integron-integrase gene in culturable heterotrophic bacteria isolated from river water and sediment sampled upstream and downstream of a wastewater treatment plant effluent discharge. Moreover, we quantified intI1 and sulfonamide resistance genes (sul1 and sul2) in the water and sediment using qPCR. There was no correlation between the results from water and sediment samples, which suggests integron-containing bacteria are differentially retained in these two environmental compartments. The discharge of treated wastewater significantly increased the frequency of intI1 among culturable bacteria and the gene copy number in river water, and increased the number of sul1 genes in the sediment. We also observed seasonal differences in the frequency of the class 1 integron-integrase gene among culturable heterotrophs as well as intI1 copy number in water, but not in sediment. The results suggest that the abundance of class 1 integrons in aquatic habitat depends on anthropogenic pressure and environmental factors.

Environmental Isolate of Rahnella aquatilis Harbors Class 1 Integron.

The paper presents first description of class 1 integron in an environmental strain of Rahnella aquatilis, a rarely isolated Gram-negative bacterium of the family Enterobacteriaceae. The strain was isolated from the Warta river water, Poland. Class 1 integrase gene was detected by a PCR assay. Sequencing of the integron's variable region showed the presence of a dfrA1-aadA1 gene cassette array. The integron was located in a 54-kbp plasmid that was transferable to Escherichia coli J-53 recipient strain in a conjugation assay. The integron-bearing R. aquatilis strain was resistant to aminoglycosides, penicillins, trimethoprim, sulfamethoxazole, and trimethoprim/sulfamethoxazole. This paper confirms that water environment play a major role in the spread of integrons and, consequently, antimicrobial resistance, among bacteria of various genera.

Class 1 integrase, sulfonamide and tetracycline resistance genes in wastewater treatment plant and surface water.

Wastewater treatment plants are considered hot spots for multiplication and dissemination of antibiotic-resistant bacteria and resistance genes. In this study, we determined the presence of class 1 integron integrase and genes conferring resistance to tetracyclines and sulfonamides in the genomes of culturable bacteria isolated from a wastewater treatment plant and the river that receives the treated wastewater. Moreover, using PCR-based metagenomic approach, we quantified intI1, tet and sul genes. Wastewater treatment caused the decrease in the total number of culturable heterotrophs and bacteria resistant to tetracycline and sulfonamides, along with the decrease in the number of intI1, sul and tet gene copies per ml, with significant reduction of tet(B). On the other hand, the treatment process increased both the frequency of tetracycline- and sulfonamide-resistant bacteria and intI1-positive strains, and the relative abundance of all quantified antibiotic resistance genes (ARGs) and intI1 gene; in the case of tet(A) and sul2 significantly. The discharge of treated wastewater increased the number of intI1, tet and sul genes in the receiving river water both in terms of copy number per ml and relative abundance. Hence, despite the reduction of the number of ARGs and ARBs, wastewater treatment selects for bacteria with ARGs in effluent.

Class 1 integrons and antibiotic resistance of clinical Acinetobacter calcoaceticus-baumannii complex in Poznan, Poland.

Sixty-three clinical isolates of Acinetobacter calcoaceticus-baumannii complex were analyzed for the presence of integrons and antimicrobial resistance. Class 1 integrons were detected in 40 (63.5 %) isolates. None of them had class 2 or class 3 integrons. The majority of the integrons contained aacC1-orfA-orfB-aadA1 gene cassette array. The presence of integrons was associated with the increased frequency of resistance to 12 of 15 antimicrobials tested, multi-drug resistance phenotype, and the overall resistance ranges of the strains.

Association between the presence of class 1 integrons, virulence genes, and phylogenetic groups of Escherichia coli isolates from river water.

Ninety-six class 1 integron-positive and 96 integron-negative Escherichia coli isolates cultured from the water of the Warta River, Poland, were characterized for their phylogenetic group affiliation and for the presence of genes associated with virulence. Most strains belonged to phylogenetic group A, but phylogenetic group affiliation was not related with the presence of integrons. The occurrence of heat-stable toxin gene of enterotoxigenic E. coli, S fimbriae subunit gene sfaS, and siderophore receptor genes, fyuA and iutA, was associated with the presence of class 1 integrons. Moreover, virulence factor score (the total number of virulence-associated genes) was associated with the presence of integrons in groups. The results bring new insight into relations between the presence of integrons in E. coli, virulence traits, as well as phylogenetic group affiliation.

The Yersinia high-pathogenicity island in Escherichia coli and Klebsiella pneumoniae isolated from polymicrobial infections.

We examined 12 pairs of strains of Escherichia coli and Klebsiella pneumoniae isolated from mixed infections in human for the presence of the Yersinia high-pathogenicity island (HPI). In one case both isolates carried the HPI, whereas in 11 cases one strain of the pair was HPI-positive. Although there were differences in the organization of the Yersinia HPI, all HPI-positive isolates were able to produce yersiniabactin. The presence of the Yersinia HPI may enhance the capability of strains involved in mixed infections to replicate in iron-deprived conditions in the host.

Transferable integrons of Gram-negative bacteria isolated from the gut of a wild boar in the buffer zone of a national park.

The aim of this study was to determine the presence of integron-bearing Gram-negative bacteria in the gut of a wild boar (Sus scrofa L.) shot in the buffer zone of a national park. Five Gram-negative strains of Escherichia coli, Serratia odorifera, Hafnia alvei and Pseudomonas sp. were isolated. Four of these strains had class 2 integrase (intI2), and one harbored class 1 integrase (intI1). The integron-positive strains were multiresistant, i.e., resistant to at least three unrelated antibiotics. All of the integrons were transferred to E. coli J-53 (Rif(R)) in a conjugation assay. The results showed that a number of multiresistant, integron-containing bacterial strains of different genera may inhabit a single individual of a wild animal, allowing the possibility of transfer of antimicrobial resistance genes.

Multiresistant Enterobacteriaceae with class 1 and class 2 integrons in a municipal wastewater treatment plant.

In this study, 1832 strains of the family Enterobacteriaceae were isolated from different stages of a municipal wastewater treatment plant, of which 221 (12.1%) were intI-positive. Among them 61.5% originated from raw sewage, 12.7% from aeration tank and 25.8% from the final effluent. All of the intI-positive strains were multiresistant, i.e. resistant to at least three unrelated antimicrobials. Although there were no significant differences in resistance range, defined as the number of antimicrobial classes to which an isolate was resistant, between strains isolated from different stages of wastewater treatment, for five ß-lactams the percentage of resistant isolates was the highest in final effluent, which may reflect a selective pressure the bacteria are exposed to, and the possible route of dissemination of ß-lactam resistant strains to the corresponding river. The sizes of the variable part of integrons ranged from 0.18 to 3.0 kbp and contained up to four incorporated gene cassettes. Sequence analysis identified over 30 different gene cassettes, including 24 conferring resistance to antibiotics. The highest number of different gene cassettes was found in bacteria isolated from the final effluent. The gene cassettes were arranged in 26 different resistance cassette arrays; the most often were dfrA1-aadA1, aadA1, dfrA17-aadA5 and dfrA12-orfF-aadA2. Regarding the diversity of resistance genes and the number of multiresistant bacteria in the final effluent, we concluded that municipal sewage may serve as a reservoir of integron-embedded antibiotic resistance genes.

Aeromonas spp.-mediated cell-contact cytotoxicity is associated with the presence of type III secretion system.

In the study we examined the production of cytotonic and cytotoxic toxins and the presence of a type III secretion system (TTSS) in 64 Aeromonas spp. strains isolated from fecal specimens of patients with gastroenteritis. We observed that contact of the bacteria with host epithelial cells is a prerequisite for their cytotoxicity at 3 h incubation. Cell-contact cytotoxic activity of the strains was strongly associated with the presence of the TTSS. Culture supernatants of the strains induced low cytotoxicity effects at the same time of incubation. Cell-free supernatants of 61 (95%) isolates expressed cytotoxic activity which caused the destruction of HEp-2 cells at 24 h. Moreover, 44% strains were cytotonic towards CHO cells and 46% of strains invaded epithelial cells.

Antimicrobial resistance of integron-harboring Escherichia coli isolates from clinical samples, wastewater treatment plant and river water.

The presence and persistence of antibiotic resistant bacteria in the environment is thought to be a growing threat to public health. The route of the spread of multiresistant bacteria from human communities to aquatic environment may lead through wastewater treatment plants that release treated wastewater to a water reservoir. In this study we used multiplex PCR assay to determine the frequency of integron presence in Escherichia coli isolates cultured from wastewater treatment plant (WWTP) (integrons were detected in 11% of E. coli isolates), river water upstream (6%) and downstream (14%) the discharge of WWTP, and clinical specimens (56%). Antimicrobial resistance of the integron-positive isolates, determined by disk diffusion method, varied between E. coli of different origin. Isolates from the downstream river, compared to those cultured from upstream river, were more frequently resistant to kanamycin, cephalotin, co-trimoxazole, trimethoprim, and fluoroquinolones. Moreover, they displayed broader resistance ranges, expressed as the number of classes of antimicrobials to which they were resistant. The results may suggest that WWTP effluent contributes to increased frequency of integron-positive E. coli isolates in the river downstream the WWTP and to their elevated resistance level.

Resistance patterns and integron cassette arrays of Enterobacter cloacae complex strains of human origin.

The aim of this research was to analyse the resistance patterns and characterize the distribution and genetic content of resistance integrons within Enterobacter cloacae complex strains originating from hospitalized patients. The strains were included in the E. cloacae complex study following sequence analysis of the hsp60 gene. The determination of resistance towards eight classes of antimicrobials was followed by PCR detection of integrons and analyses of the size and sequences of their variable parts. The majority of 69 clinical strains of the E. cloacae complex were identified as Enterobacter hormaechei. They were isolated from a variety of samples, including urine, wounds, blood and stools. The remaining isolates belonged to E. cloacae clusters III and IV, E. cloacae subsp. cloacae and Enterobacter kobei. Fifty-two isolates (75.4 %) were resistant to more than three unrelated antibiotics. The resistance for each antibiotic, except imipenem, was significantly associated with the presence of integrons. Class 1 integrons were detected in 55 % of isolates: 63.3 % of 'E. hormaechei subsp. steigerwaltii', 50 % of E. cloacae cluster III, 40 % of 'E. hormaechei subsp. oharae', 33 % belonging to E. cloacae cluster IV and 20 % of 'E. hormaechei subsp. hormaechei' were intI1-positive. All of the integrons were located on transferable genetic elements. The transferred resistance primarily included that to aminoglycosides, ticarcillin, piperacillin, sulfamethoxazole, trimethoprim and tetracycline. Sequence analysis of the variable regions of integrons identified two groups of genes: those encoding aminoglycoside adenylotransferases responsible for resistance to aminoglycosides, and dfr cassettes conferring resistance to trimethoprim. Integrons of the E. cloacae complex showed limited variability of genes encoding resistance to therapeutics and were stable in structure with the following cassette arrays: dfrA12-orfF-aadA2, aadB-aadA2, dfrA1-aadA1 and aacA4-aadA1. Hospital-dependent differences in type and arrays of gene cassettes were observed, which seemed to be conserved and not liable to changes.

Phylogenetic groups, virulence genes and quinolone resistance of integron-bearing Escherichia coli strains isolated from a wastewater treatment plant.

We investigated phylogenetic affiliation, occurrence of virulence genes and quinolone resistance in 109 integron-containing strains of Escherichia coli isolated from a wastewater treatment plant. Selection for integron-bearing strains caused a shift toward phylogroup D, which was most numerous, followed by A, B1 and B2. Phylogroups D and B2, both of which are reported to include virulent extraintestinal pathotypes, made up 50.5% of all isolates and were present in every stage of wastewater treatment, including final effluent. Diarrheagenic pathotypes made up 21% of the strains. The average virulence factor genes score was low (1.40) and the range was from 0 to 5. Quinolone and fluoroquinolone resistance was observed in 56.0% and 50.4% of the strains, respectively; however, it was not associated with virulence factor score. Although the average virulence factor score was low, 17.4% of strains had three and more virulence genes. They were isolated mostly from raw sewage, but 30% of them were cultured from final effluent. Release of multiresistant integron-bearing E. coli strains with virulence traits into the environment may create potential threat and be of public health concern.

Virulence properties and integron-associated antibiotic resistance of Klebsiella mobilis strains isolated from clinical specimens.

This study examined Klebsiella mobilis isolates cultured from clinical specimens for virulence-associated properties and antibiotic resistance. The strains produced a number of siderophores, including enterobactin, aerobactin and yersiniabactin. All isolates were able to adhere to and invade epithelial cells. They had cytotoxic activity, which caused destruction of human laryngeal epithelial HEp-2 cells and evoked lysis of murine macrophage J774 cells. Analyses of HEp-2 and J774 cellular morphology and DNA fragmentation in the cells showed features typical of cells undergoing apoptosis. Some K. mobilis strains harboured class 1 integrons carrying the aadA1 gene encoding an aminoglycoside adenyltransferase.

Isolates of the Enterobacter cloacae complex induce apoptosis of human intestinal epithelial cells.

Strains of the Enterobacter cloacae complex are becoming increasingly important human pathogen. The aim of the study was to identify, by sequencing the hsp60 gene, the species of clinical isolates phenotypically identified as E. cloacae and to examine them for virulence-associated properties: the ability of adhesion, invasion to HEp-2 cells and the induced apoptosis of infected epithelial cells. The majority of the strains were identified as Enterobacter hormaechei with E. hormaechei subsp. steigerwaltii being the most frequent subspecies. Other strains belonged to E. hormaechei subsp. oharae, E. cloacae cluster III, and E. cloacae cluster IV. The strains were examined for virulence-associated properties: the ability to adhesion and invasion to HEp-2 cells and the apoptosis induction of infected epithelial cells. All strains revealed adherence ability and most of them (71%) were invasive to epithelial cells. Analyses of cellular morphology and DNA fragmentation in the HEp-2 cells exhibited typical features of cells undergoing apoptosis. We observed morphological changes, including condensation of nuclear chromatin, formation of apoptotic bodies and blebbing of cell membrane. The lowest apoptotic index did not exceed 6%, whereas the highest reached 49% at 24h and 98% at 48 h after infection. Forty strains (73%) induced fragmentation of nuclear DNA and characteristic intranucleosomal pattern with the size of about 180-200 bp in DNA extracted from infected cells at 48 h after infection. The results indicated that the bacteria of the E. cloacae complex may adhere to and penetrate into epithelial cells and induce apoptosis, which could be an important mechanism contributing to the development diseases.