The Toxin-Antitoxin Systems of the Opportunistic Pathogen Stenotrophomonas maltophilia of Environmental and Clinical Origin.
ABSTRACT: Stenotrophomonas maltophilia is a ubiquitous environmental bacterium that has recently emerged as a multidrug-resistant opportunistic pathogen causing bloodstream, respiratory, and urinary tract infections. The connection between the commensal environmental S. maltophilia and the opportunistic pathogen strains is still under investigation. Bacterial toxin-antitoxin (TA) systems have been previously associated with pathogenic traits, such as biofilm formation and resistance to antibiotics, which are important in clinical settings. The same species of the bacterium can possess various sets of TAs, possibly influencing their overall stress response. While the TA systems of other important opportunistic pathogens have been researched, nothing is known about the TA systems of S. maltophilia. Here, we report the identification and characterization of S. maltophilia type II TA systems and their prevalence in the isolates of clinical and environmental origins. We found 49 putative TA systems by bioinformatic analysis in S. maltophilia genomes. Despite their even spread in sequenced S. maltophilia genomes, we observed that relBE, hicAB, and previously undescribed COG3832-ArsR operons were present solely in clinical S. maltophilia isolates collected in Lithuania, while hipBA was more frequent in the environmental ones. The kill-rescue experiments in Escherichia coli proved higBA, hicAB, and relBE systems to be functional TA modules. Together with different TA profiles, the clinical S. maltophilia isolates exhibited stronger biofilm formation, increased antibiotic, and serum resistance compared to environmental isolates. Such tendencies suggest that certain TA systems could be used as indicators of virulence traits.
Project description:BACKGROUND:Toxin-antitoxin (TA) systems are found on the chromosomes and plasmids of many Bacteria such as Escherichia coli. The roles of TA systems in bacteria are enigmatic. Multiple biological functions of TA systems are proposed including growth modulation, persistence, and biofilm formation. Bio?lms of E. coli are cause of urinary tract infections, as well as bacteraemia. OBJECTIVES:The current study aimed to find the association between biofilm formation and toxin-antitoxin systems in clinical isolates of E. coli. MATERIALS AND METHODS:A total of 150 E. coli isolates were evaluated for biofilm formation by Congo red agar medium (CRA) and microtiter plate assay and the presence of different TA systems including MazEF, RelBE, hipBA, ccdAB and MqsRA. RESULTS:The results of the analysis revealed that 107 E. coli isolates were potent for biofilm formation by CRA. The findings by microtiter plates showed that 102 E. coli isolates were biofilm producers. The results indicated that 80%, 85%, 70%, 91% and 82% of the isolates possessed MazEF, RelBE, hipBA, ccdAB and MqsRA TA loci, respectively. CONCLUSIONS:The analysis recommended that TA genes are prevalent in clinical isolates of E. coli strains. The analysis revealed that hipBA TA system is associated with biofilm formation.
Project description:Toxin-antitoxin (TA) systems are small genetic modules that are widely distributed in the genomes of bacteria and archaea and have been proposed to fulfill numerous functions. Here, we describe the identification and characterization of a type II TA system, comprising the hicAB locus in the human opportunistic pathogen Pseudomonas aeruginosa. The hicAB locus consists of genes hicA and hicB encoding a toxin and its cognate antitoxin, respectively. BLAST analysis revealed that hicAB is prevalent in approximately 36% of P. aeruginosa strains and locates in the same genomic region. RT-PCR demonstrated that hicAB forms a bicistronic operon that is cotranscribed under normal growth conditions. Overproduction of HicA inhibited the growth of Escherichia coli, and this effect could be counteracted by co-expression of HicB. The Escherichia coli kill/rescue assay showed that the effect of HicA is bacteriostatic, rather than bactericidal. Deletion of hicAB had no effect on the biofilm formation and virulence of P. aeruginosa in a mice infection model. Collectively, this study presents the first characterization of the HicAB system in the opportunistic pathogen P. aeruginosa.
Project description:Type II toxin/antitoxin (TA) systems contribute to the formation of persister cells and biofilm formation for many organisms. Aggregatibacter actinomycetemcomitans thrives in the complex oral microbial community subjected to continual environmental flux. Little is known regarding the presence and function of type II TA systems in this organism or their contribution to adaptation and persistence in the biofilm. We identified 11 TA systems that are conserved across all seven serotypes of A. actinomycetemcomitans and represent the RelBE, MazEF and HipAB families of type II TA systems. The systems selectively responded to various environmental conditions that exist in the oral cavity. Two putative RelBE-like TA systems, D11S_1194-1195 and D11S_1718-1719 were induced in response to low pH and deletion of D11S_1718-1719 significantly reduced metabolic activity of stationary phase A. actinomycetemcomitans cells upon prolonged exposure to acidic conditions. The deletion mutant also exhibited reduced biofilm biomass when cultured under acidic conditions. The D11S_1194 and D11S_1718 toxin proteins inhibited in vitro translation of dihydrofolate reductase (DHFR) and degraded ribosome-associated, but not free, MS2 virus RNA. In contrast, the corresponding antitoxins (D11S_1195 and D11S_1719), or equimolar mixtures of toxin and antitoxin, had no effect on DHFR production or RNA degradation. Together, these results suggest that D11S_1194-1195 and D11S_1718-1719 are RelBE-like type II TA systems that are activated under acidic conditions and may function to cleave ribosome-associated mRNA to inhibit translation in A. actinomycetemcomitans. In vivo, these systems may facilitate A. actinomycetemcomitans adaptation and persistence in acidic local environments in the dental biofilm.
Project description:Type II toxin-antitoxin (TA) systems are highly prevalent in bacterial genomes and have been extensively studied. These modules involve in the formation of persistence cells, the biofilm formation, and stress resistance, which might play key roles in pathogen virulence. SezAT and yefM-yoeB TA modules in Streptococcus suis serotype 2 (S. suis 2) have been studied, although the other TA systems have not been identified. In this study, we investigated nine putative type II TA systems in the genome of S. suis 2 strain SC84 by bioinformatics analysis and identified three of them (two relBE loci and one parDE locus) that function as typical type II TA systems. Interestingly, we found that the introduction of the two RelBE TA systems into Escherichia coli or the induction of the ParE toxin led to cell filamentation. Promoter activity assays indicated that RelB1, RelB2, ParD, and ParDE negatively autoregulated the transcriptions of their respective TA operons, while RelBE2 positively autoregulated its TA operon transcription. Collectively, we identified three TA systems in S. suis 2, and our findings have laid an important foundation for further functional studies on these TA systems.
Project description:BACKGROUND:Toxin-antitoxin (TA) systems are little genetic units generally composed of two genes encoding antitoxin and toxin. These systems are known to be involved in many functions that can lead to growth arrest and cell death. Among the different types of TA systems, the type II gathers together systems where the antitoxin directly binds and inhibits the toxin. Among these type II TA systems, the HicAB module is widely distributed in free-living Bacteria and Archaea and the toxin HicA functions via RNA binding and cleavage. The genome of the symbiotic Sinorhizobium meliloti encodes numerous TA systems and only a few of them are functional. Among the predicted TA systems, there is one homologous to HicAB modules. RESULTS:In this study, we characterize the HicAB toxin-antitoxin module of S. meliloti. The production of the HicA of S. meliloti in Escherichia coli cells abolishes growth and decreases cell viability. We show that expression of the HicB of S. meliloti counteracts HicA toxicity. The results of double hybrid assays and co-purification experiments allow demonstrating the interaction of HicB with the toxin HicA. Purified HicA, but not HicAB complex, is able to degrade ribosomal RNA in vitro. The analysis of separated domains of HicB protein permits us to define the antitoxin activity and the operator-binding domain. CONCLUSIONS:This study points out the first characterization of the HicAB system of the symbiotic S. meliloti whereas HicA is a toxin with ribonuclease activity and HicB has two domains: the COOH-terminal one that binds the operator and the NH2-terminal one that inhibits the toxin.
Project description:Stenotrophomonas maltophilia is a non-fermenting Gram-negative bacterium that is ubiquitous in the environment. In humans, this opportunistic multi-drug-resistant pathogen is responsible for a plethora of healthcare-associated infections. Here, we utilized a whole genome sequencing (WGS)-based phylogenomic core single nucleotide polymorphism (SNP) approach to characterize S. maltophilia subgroups, their potential association with human infection, and to detect any possible transmission events. In total, 89 isolates (67 clinical and 22 environmental) from Germany were sequenced. Fully finished genomes of five strains were included in the dataset for the core SNP phylogenomic analysis. WGS data were compared with conventional genotyping results as well as with underlying disease, biofilm formation, protease activity, lipopolysaccharide (LPS) SDS-PAGE profiles, and serological specificity of an antibody raised against the surface-exposed O-antigen of strain S. maltophilia K279a. The WGS-based phylogenies grouped the strains into 12 clades, out of which 6 contained exclusively human and 3 exclusively environmental isolates. Biofilm formation and proteolytic activity did correlate neither with the phylogenetic tree, nor with the origin of isolates. In contrast, the genomic classification correlated well with the reactivity of the strains against the K279a O-specific antibody, as well as in part with the LPS profiles. Three clusters of clinical strains had a maximum distance of 25 distinct SNP positions, pointing to possible transmission events or acquisition from the same source. In conclusion, these findings indicate the presence of specific subgroups of S. maltophilia strains adapted to the human host.
Project description:Iron has been shown to regulate biofilm formation, oxidative stress response and several pathogenic mechanisms in Stenotrophomonas maltophilia. Thus, the present study is aimed at identifying various iron acquisition systems and iron sources utilized during iron starvation in S. maltophilia. The annotations of the complete genome of strains K279a, R551-3, D457 and JV3 through Rapid Annotations using Subsystems Technology (RAST) revealed two putative subsystems to be involved in iron acquisition: the iron siderophore sensor and receptor system and the heme, hemin uptake and utilization systems/hemin transport system. Screening for these acquisition systems in S. maltophilia showed the presence of all tested functional genes in clinical isolates, but only a few in environmental isolates. NanoString nCounter Elements technology, applied to determine the expression pattern of the genes under iron-depleted condition, showed significant expression for FeSR (6.15-fold), HmuT (12.21-fold), Hup (5.46-fold), ETFb (2.28-fold), TonB (2.03-fold) and Fur (3.30-fold). The isolates, when further screened for the production and chemical nature of siderophores using CAS agar diffusion (CASAD) and Arnows's colorimetric assay, revealed S. maltophilia to produce catechol-type siderophore. Siderophore production was also tested through liquid CAS assay and was found to be greater in the clinical isolate (30.8%) compared to environmental isolates (4%). Both clinical and environmental isolates utilized hemoglobin, hemin, transferrin and lactoferrin as iron sources. All data put together indicates that S. maltophilia utilizes siderophore-mediated and heme-mediated systems for iron acquisition during iron starvation. These data need to be further confirmed through several knockout studies.
Project description:The percentage of bacterial infections refractory to standard antibiotic treatments is steadily increasing. Among the most problematic hospital and community-acquired pathogens are methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PA). One novel strategy proposed for treating infections of multidrug-resistant bacteria is the activation of latent toxins of toxin-antitoxin (TA) protein complexes residing within bacteria; however, the prevalence and identity of TA systems in clinical isolates of MRSA and PA has not been defined. We isolated DNA from 78 MRSA and 42 PA clinical isolates and used PCR to probe for the presence of various TA loci. Our results showed that the genes for homologs of the mazEF TA system in MRSA and the relBE and higBA TA systems in PA were present in 100% of the respective strains. Additionally, reverse transcriptase PCR analysis revealed that these transcripts are produced in the clinical isolates. These results indicate that TA genes are prevalent and transcribed within MRSA and PA and suggest that activation of the toxin proteins could be an effective antibacterial strategy for these pathogens.
Project description:<h4>Background</h4><i>Stenotrophomonas</i> species are multi-resistant bacteria with ability to cause opportunistic infections.<h4>Objective</h4>We isolated 45 <i>Stenotrophomonas</i> species from soil, sewage and the clinic with the aim of investigating their susceptibility to commonly used antimicrobial agents.<h4>Methodology</h4>The identities of isolates were confirmed with 16S rRNA gene sequence and MALDI-TOF analysis. Anti-microbial resistance, biofilm production and clonal diversity were also evaluated. The minimum inhibitory concentration technique as described by Clinical & Laboratory Standards Institute: CLSI Guidelines (CLSI) was employed for the evaluation of isolate susceptibility to antibiotics.<h4>Result</h4>Forty-five <i>Stenotrophomonas</i> species which include 36 environmental strains and 9 clinical strains of <i>S. maltophilia</i> were considered in this study. 32 (88.9 %) environmental strains were identified to be <i>S. maltophilia</i>, 2 (5.6 %) were <i>Stenotrophomonas nitritireducens</i>, and 2 (5.6 %) cluster as <i>Stenotrophomonas</i> spp. <i>Stenotrophomonas</i> isolates were resistant to at least six of the antibiotics tested, including <i>Trimethoprim</i>/<i>Sulfamethoxazole</i> (<i>SXT</i>).<h4>Conclusion</h4>Environmental isolates from this study were resistant to SXT which is commonly used for the treatment of <i>S. maltophilia</i> infections. This informs the need for good public hygiene as the environment could be a reservoir of multi-resistant bacteria. It also buttresses the importance of surveillance study in the management of bacterial resistance.
Project description:Background Stenotrophomonas maltophilia (S. maltophilia) is an important opportunistic pathogen that can be isolated in hospitals. With the abuse of broad spectrum antibiotics and invasive surgical devices, the rate of S. maltophilia infection is increasing every year. This study was an epidemiological analysis of the clinical and molecular characteristics of S. maltophilia infection in a Chinese teaching hospital. The goal was to obtain a comprehensive understanding of the status of S. maltophilia infection to provide strong epidemiological data for the prevention and treatment of S. maltophilia infection. Results A total of 93 isolates from Renji Hospital affiliated with the Shanghai Jiaotong University School of Medicine were included, in which 62 isolates were from male patients. In addition, 81 isolates were isolated from sputum samples. A total of 86 patients had underlying diseases. All patients received antibiotics. Multilocus sequence typing (MLST) analysis indicated that 61 different sequence types (STs) were found (including 45 novel STs), and MLST did not show significantly dominant STs. Pulsed field gel electrophoresis (PFGE) results showed that 93 isolates could be divided into 73 clusters, and they also showed weak genetic linkages between isolates. The resistant rates to trimethoprim/sulfamethoxazole (TMP/SMX) and levofloxacin were 9.7 and 4.3%, respectively, and all isolates were susceptible to minocycline. Four virulence gene’s loci Stmpr1, Stmpr2, Smf-1, and Smlt3773 were positive in 79.6, 91.4, 94.6, and 52.7% of the isolates, respectively. Three biofilm genes rmlA, spgM, and rpfF were positive in 82.8, 92.5, and 64.5% of the isolates, respectively. Mean biofilm forming level of OD492 was 0.54?±?0.49. We did not find any significant difference between different genders and different age-groups. We retrospectively analyzed data from patients in the intensive care unit (ICU) and the control group. The independent risk factors of those who were infected in the ICU included immunosuppression and the increased antibiotic usage. Conclusions Most of the patients had prior medical usage histories and baseline diseases. The positive rate of virulence genes was high, the drug resistance rate of S. maltophilia was low, and the biofilm formation ability was strong. The increased use of antibiotics was an independent risk factor for S. maltophilia infection, which should receive more attention. No obvious clonal transmissions were found in the same departments.