Antibiotic-resistant Mutants Research Articles

Monitoring of Pseudomonas aeruginosa mutational resistome dynamics using an enrichment panel for direct sequencing of clinical samples

Pseudomonas aeruginosa is a major cause of hospital-acquired and chronic infections, characterised by an extraordinary capacity to develop antimicrobial resistance through the selection of chromosomal mutations, leading to treatment failure. Here, we designed and tested a hybridisation-based capture system for the enrichment of genes of interest before sequencing to monitor resistant populations genomics directly from clinical samples. A panel for enrichment before sequencing of close to 200 genes related to P.aeruginosa antimicrobial resistance, multilocus sequence typing, mutability or virulence was designed, synthesised (KAPA HyperCap, Roche) and initially validated invitro using a multidrug-resistant ST175 isolate and representative isolates from major P.aeruginosa clades. Invivo testing included ventilator associated pneumonia by MDR P.aeruginosa in ICU (3-10 sequential samples from 3 patients) and chronic respiratory infection by hypermutable P.aeruginosa in cystic fibrosis (8 sequential samples from a single patient covering a 4-year period). Results from direct sequencing with the enrichment panel were compared with those of whole genome sequencing (WGS) and phenotypic profiling of 10 isolated colonies per sample. Invitro assays confirmed the selectivity of the enrichment panel and the correct identification of the vast mutational resistome of ST175, including specific mutations even when introduced in a 1:100 proportion. Invivo performance was at least equivalent to sequencing 10 colonies per sample, including the accurate identification of the sequence types and the basal and acquired mutational resistome. To note, specific resistance mutations, such as those in ampC leading to resistance to novel β-lactams, could be traced even at frequencies of 1%. Moreover, the coselection of mutator populations and antibiotic resistance mutations, predicted in theoretical and invitro studies, was evidenced invivo. This proof-of-concept study demonstrates that resistance genomics of P.aeruginosa can be analysed directly from clinical samples, determining not only a considerable reduction in turnaround time and cost from a diagnostics perspective, but also an unprecedented potency for accurate monitoring of invivo population dynamics in bacterial infections. Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea-NextGenerationEU.

Investigations of microbial adaptation to singular, binary, and fully formulated quaternary ammonium compounds.

The study was conducted to inform risk assessments concerning microbial exposure to quaternary ammonium biocides (QUATs) by investigating their effects on 10 microbial strains of hygiene relevance. Biocides were divided into three categories: simple aqueous solutions, biocide mixtures, and formulated biocides. Organisms were grown in the presence of biocides for 10 generations and then subsequently for another 10 generations in biocide-free media. Control organisms were passaged 20 times in biocide-free media. Strains were then assessed for biocide and antibiotic susceptibility, changes in growth dynamics, and single nucleotide polymorphisms (SNPs). Biocide mixtures demonstrated greater antimicrobial potency than singular and formulated biocides. Susceptibility changes of under twofold were observed for all biocides tested. Susceptibility decreased significantly for organisms passaged with singular biocides (1.29- to 4.35-fold) and biocide mixtures (1.4- to 1.5-fold), but not for formulated biocides (1.3- to 1.84-fold) compared to controls. Antibiotic susceptibility both increased and decreased in passaged organisms, with heightened susceptibility occurring more frequently in the singular biocide group. Changes in susceptibility and growth dynamics were similar in the passaged and unexposed controls for fitness measures of adapted bacteria; there were no significant differences between biocide groups, but significantly lower generation and doubling times in organisms exposed to singular biocides. Similar frequencies in SNPs occurred for the three biocide groups and unexposed controls. While some adaptations occurred, particularly with singular biocides, the impact on antibiotic resistance and genomic mutations was limited. These findings suggest that the use of formulated QUATs may pose a comparatively lower risk for antimicrobial resistance.IMPORTANCEBiocides are used globally to control microbial growth and effective assessment of the risks and benefits of their use is therefore a high priority. Much of the data used to assess risk has been based on sub-lethal exposure of bacteria to singular biocides in simple aqueous solutions. This work builds on limited prior realism-based studies to demonstrate enhanced potency in biocidal mixtures; the mitigation of resistance selection by formulations and inconsistent cross-resistance effects with both increases and decreases in susceptibility for a wide range of antibiotics. These data can be used to better inform risk assessments of biocide deployment.

Antibiotic-resistant mutants of lactic acid bacteria: potential food control agents

Background: One of the most pressing challenges facing global agriculture today is the restoration of degraded farmland. Organic fertilisers are increasingly being used as a safer alternative to synthetic forms. The most promising form of environmentally friendly fertiliser is a bioconsortium based on different groups of microorganisms. Lactic acid bacteria (LAB) with high antifungal activity, isolated from Armenian dairy products, can form part of such a bioconsortium and protect plants from phytopathogens. In addition, LAB can be used as a preservative in the food industry due to its GRAS (generally recognised as safe) status. Objective: To obtain antibiotic-resistant pleiotropic lactic acid bacterial mutants with higher antifungal activity and broad-spectrum activity compared to natural strains. Methods: The objects of the study were LAB, whose antifungal activity was studied against phytopathogenic fungi of the genera Penicillium, Aspergillus, Alternaria and Fusarium, which cause crop/food spoilage. To increase antifungal activity, spontaneous pleiotropic mutants resistant to the streptomycin, kanamycin or rifampicin were obtained on the basis of original strains. Results: Among all the LAB tested, Lactobacillus bovis MDC 1061, L. paracasei MDC 10898 and L. buchneri BKM 1599 were selected for their antifungal activity. Based on the latter, 78 antibiotic-resistant mutants with different levels of antifungal activity were obtained. L. bovis MDC 1061 STR-25 and L. buchneri BKM 1599 RIF-7 mutants with up to 98% and 95% inhibition of fungal growth, respectively, were selected as potential food control agents. Conclusion: Antibiotic-resistant mutants L. bovis MDC 1061 STR-25 and L. buchneri BKM 1599 RIF-7 with high antifungal ability and a wide range of its effects were obtained. For the first time, the phenomenon of pleiotropic effect has been used in LAB to improve antifungal activity. After extensive study, these mutants can be used as promising components for creating an effective bioconsortium, as well as a preservative in food production, especially in the case of plant-based functional foods to protect against contamination by fungi. Keywords: lactic acid bacteria, pleiotropic mutants, antifungal activity

Decoding Pseudomonas aeruginosa: Genomic insights into adaptation, antibiotic resistance, and the enigmatic role of T6SS in interbacterial dynamics

Pseudomonas aeruginosa demonstrates a remarkable capacity for adaptation and survival in diverse environments. Furthermore, its clinical importance is underscored by its intrinsic and acquired resistance to a wide range of antimicrobial agents, posing a substantial challenge in healthcare settings. Amidst this complex landscape of resistance, the Type VI Secretion System (T6SS) in P. aeruginosa adds yet another layer of intricacy and allows bacteria to engage in interbacterial competition, potentially influencing their resilience and pathogenicity. Whole genome sequencing (WGS) was conducted on the five isolates under investigation, enabling the identification of antibiotic resistance genes (ARGs) and mutations associated with resistance. All isolates exhibit class C and D β-lactamases, displaying variant differences. The Resistance-nodulation-division (RND) antibiotic efflux pumps, crucial for multidrug resistance, have been encoded chromosomally. When exploring the role of the T6SS in urinary tract infections involving other bacteria, it was noted that P. aeruginosa isolates exhibited reduced counts when co-cultivated with other bacteria. The downregulation of the tssJ gene, associated with the T6SS under bacterial stress, and the exclusion of several cluster genes in this study suggest the hypothesis of a basal state rather than an attack/defence mechanism in the initial contact.

Artificial Intelligence-Driven Analysis of Antimicrobial-Resistant and Biofilm-Forming Pathogens on Biotic and Abiotic Surfaces.

The growing threat of antimicrobial-resistant (AMR) pathogens to human health worldwide emphasizes the need for more effective infection control strategies. Bacterial and fungal biofilms pose a major challenge in treating AMR pathogen infections. Biofilms are formed by pathogenic microbes encased in extracellular polymeric substances to confer protection from antimicrobials and the host immune system. Biofilms also promote the growth of antibiotic-resistant mutants and latent persister cells and thus complicate therapeutic approaches. Biofilms are ubiquitous and cause serious health risks due to their ability to colonize various surfaces, including human tissues, medical devices, and food-processing equipment. Detection and characterization of biofilms are crucial for prompt intervention and infection control. To this end, traditional approaches are often effective, yet they fail to identify the microbial species inside biofilms. Recent advances in artificial intelligence (AI) have provided new avenues to improve biofilm identification. Machine-learning algorithms and image-processing techniques have shown promise for the accurate and efficient detection of biofilm-forming microorganisms on biotic and abiotic surfaces. These advancements have the potential to transform biofilm research and clinical practice by allowing faster diagnosis and more tailored therapy. This comprehensive review focuses on the application of AI techniques for the identification of biofilm-forming pathogens in various industries, including healthcare, food safety, and agriculture. The review discusses the existing approaches, challenges, and potential applications of AI in biofilm research, with a particular focus on the role of AI in improving diagnostic capacities and guiding preventative actions. The synthesis of the current knowledge and future directions, as described in this review, will guide future research and development efforts in combating biofilm-associated infections.

Latest advances in adsorptive decontamination of antibiotics remnants from wastewater by physiochemically fabricated phytowaste materials

Antibiotics, posing a major threat to life on earth and water causing propagation of antibiotic resistant bacteria and antibiotic resistant mutant genes. Traces of these antibiotic toxins are found both in aquatic as well as terrestrial ecosystem. Various techniques, that is, ozonation, membrane filtration, Fenton process, etc., are applied for their removal but have many shortcomings, so adsorption is picked as efficient, simple and relatively safe removal technique. This review article discusses the classification of antibiotics, effect of their unnecessary application and removal methods of their remnants from water sources. Also, compares adsorptive removal of antibiotics remnant with other primitive and advance techniques, suggesting adsorption as one of the effective techniques.

Context-dependent fitness benefits of antibiotic resistance mutations.

Context-dependent fitness benefits of antibiotic resistance mutations.

Molecular Characterization of Antimicrobial Resistance and Virulence Genotyping among Helicobacter pylori-Positive Dyspeptic Patients in North Iran.

Iran has a relatively high prevalence of H. pylori, which correlates with high-risk areas for gastric cancer worldwide. Our study aimed to investigate the underlying genetic mechanisms associated with resistance to metronidazole (frxA, rdxA), clarithromycin (23S rRNA), tetracycline (16S rRNA), and fluoroquinolone (gyrA) in H. pylori-positive dyspeptic patients using PCR and sequencing. We further examined the potential correlation between resistance profiles and various virulence genotypes. The rates of genetic mutations associated with resistance to metronidazole, fluoroquinolone, clarithromycin, and tetracycline were found to be 68%, 32.1%, 28.4%, and 11.1%, respectively. Well-documented multiple antibiotic resistance mutations were detected, such as rdxA and frxA (with missense and frameshift alterations), gyrA (Asp91, Asn87), 23S rRNA (A2142G, A2143G), and 16S rRNA (triple-base-pair substitutions AGA926-928→TTC). The cagA+ and vacA s1/m1 types were the predominant genotypes in our study. With the exception of metronidazole and tetracycline, no significant correlation was observed between the cagA+ and cagL+ genotypes and resistance-associated mutations. The prevalence of antibiotic resistance-associated mutations in H. pylori was remarkably high in this region, particularly to metronidazole, ciprofloxacin, and clarithromycin. By conducting a simultaneous screening of virulence and resistance genotypes, clinicians can make informed decisions regarding the appropriate therapeutic regimen to prevent the escalation of antibiotic resistance against H. pylori infection in this specific geographical location.

Antibiotic resistance alters the ability of Pseudomonas aeruginosa to invade bacteria from the respiratory microbiome.

The emergence and spread of antibiotic resistance in bacterial pathogens is a global health threat. One important unanswered question is how antibiotic resistance influences the ability of a pathogen to invade the host-associated microbiome. Here we investigate how antibiotic resistance impacts the ability of a bacterial pathogen to invade bacteria from the microbiome, using the opportunistic bacterial pathogen Pseudomonas aeruginosa and the respiratory microbiome as our model system. We measure the ability of P. aeruginosa spontaneous antibiotic-resistant mutants to invade pre-established cultures of commensal respiratory microbes in an assay that allows us to link specific resistance mutations with changes in invasion ability. While commensal respiratory microbes tend to provide some degree of resistance to P. aeruginosa invasion, antibiotic resistance is a double-edged sword that can either help or hinder the ability of P. aeruginosa to invade. The directionality of this help or hindrance depends on both P. aeruginosa genotype and respiratory microbe identity. Specific resistance mutations in genes involved in multidrug efflux pump regulation are shown to facilitate the invasion of P. aeruginosa into Staphylococcus lugdunensis, yet impair invasion into Rothia mucilaginosa and Staphylococcus epidermidis. Streptococcus species provide the strongest resistance to P. aeruginosa invasion, and this is maintained regardless of antibiotic resistance genotype. Our study demonstrates how the cost of mutations that provide enhanced antibiotic resistance in P. aeruginosa can crucially depend on community context. We suggest that attempts to manipulate the microbiome should focus on promoting the growth of commensals that can increase the fitness costs associated with antibiotic resistance and provide robust inhibition of both wildtype and antibiotic-resistant pathogen strains.

Identifying Novel Therapeutics for the Resistant Mutant "F533L" in PBP3 of Pseudomonas aeruginosa Using ML Techniques.

Pseudomonas aeruginosa (P. aeruginosa) is a highly infectious and antibiotic-resistant bacterium, which causes acute and chronic nosocomial infections. P. aeruginosa exhibits multidrug resistance due to the emergence of resistant mutants. The bacterium takes advantage of intrinsic and acquired resistance mechanisms to resist almost every antibiotic. To overcome the drug-resistance problem, there is a need to develop effective drugs against antibiotic-resistant mutants. Therefore, in this study, we selected the F533L mutation in PBP3 (penicillin-binding protein 3) because of its important role in β-lactam recognition. To target this mutation, we screened 147 antibacterial compounds from PubChem through a machine-learning model developed based on the decision stump algorithm with 75.75% accuracy and filtered out 55 compounds. Subsequently, out of 55 compounds, 47 compounds were filtered based on their drug-like activity. These 47 compounds were subjected to virtual screening to obtain binding affinity compounds. The binding affinity range of all 47 compounds was -11.3 to -4.6 kcal mol-1. The top 10 compounds were examined according to their binding with the mutation point. A molecular dynamic simulation of the top 8 compounds was conducted to understand the stability of the compounds containing the mutated PBP3. Out of 8 compounds, 3 compounds, namely, macozinone, antibacterial agent 71, and antibacterial agent 123, showed good stability and were validated by RMSD, RMSF, and binding-free analysis. The findings of this study revealed promising antibacterial compounds against the F533L mutant PBP3. Furthermore, developments in these compounds may pave the way for novel therapeutic interventions.

Genomic analysis of Helicobacter pylori in Australia: Antimicrobial resistance, phylogenetic patterns, and virulence factors.

Rates of antimicrobial-resistant Helicobacter pylori infection are rising globally, but little is known about contemporary resistance patterns, virulence factors, and phylogenetic patterns of isolates within Australia. We aimed to characterize antimicrobial resistance and genetic mutations associated with adverse clinical outcomes. Whole genome sequencing, culturing, and antibiotic sensitivity data for refractory H.pylori isolates at Australian centers were collected between 2013 and 2022. Phylogenetic origins, antibiotic resistance mutations, and virulence factors were examined with phenotypic resistance profiles. One hundred thirty-five isolates underwent culture, with 109 of these undergoing whole genome sequencing. Forty-three isolates were isolated from patients in South Australia and 66 from Western Australia. Isolates originated primarily from hpEurope (59.6%), hpEastAsia (25.7%), and hpNEAfrica (6.4%). Antimicrobial resistance to clarithromycin was seen in 85% of isolates, metronidazole in 52%, levofloxacin in 18%, rifampicin in 14%, and amoxicillin in 9%. Most isolates (59%) were multi-drug resistant. Resistance concordance between genetically determined resistance and phenotypic resistance was 92% for clarithromycin and 94% for levofloxacin. Analysis of virulence factors demonstrated cag pathogenicity island (cagPAI) in 67% of isolates and cagA in 61%, correlating with isolate genetic origin. The most virulent s1m1 vacuolating cytotoxin A genotype was present in 26% of isolates. Refractory H.pylori isolates in Australia emanate from multiple global origins. Strong concordance between genetic and phenotypic antibiotic resistance profiles raises the possibility of utilizing genetic profiling in clinical practice. The dynamic landscape of H.pylori in Australia warrants the establishment of a national database to monitor H.pylori resistance and evolving virulence.

Substrate geometry affects population dynamics in a bacterial biofilm

Biofilms inhabit a range of environments, such as dental plaques or soil micropores, often characterized by noneven surfaces. However, the impact of surface irregularities on the population dynamics of biofilms remains elusive, as most experiments are conducted on flat surfaces. Here, we show that the shape of the surface on which a biofilm grows influences genetic drift and selection within the biofilm. We culture Escherichia coli biofilms in microwells with a corrugated bottom surface and observe the emergence of clonal sectors whose size corresponds to that of the corrugations, despite no physical barrier separating different areas of the biofilm. The sectors are remarkably stable and do not invade each other; we attribute this stability to the characteristics of the velocity field within the biofilm, which hinders mixing and clonal expansion. A microscopically detailed computer model fully reproduces these findings and highlights the role of mechanical interactions such as adhesion and friction in microbial evolution. The model also predicts clonal expansion to be limited even for clones with a significant growth advantage-a finding which we confirm experimentally using a mixture of antibiotic-sensitive and antibiotic-resistant mutants in the presence of sublethal concentrations of the antibiotic rifampicin. The strong suppression of selection contrasts sharply with the behavior seen in range expansion experiments in bacterial colonies grown on agar. Our results show that biofilm population dynamics can be affected by patterning the surface and demonstrate how a better understanding of the physics of bacterial growth can be used to control microbial evolution.

Protocol for the selection of Mycobacterium tuberculosis spontaneous resistant mutants to d-cycloserine

Mycobacterium tuberculosis (MTB) is known for its adaptive capability in developing resistance to antibiotics, through the selection of spontaneous mutations that arise during treatment. Generating spontaneous antibiotic-resistant mutants in vitro is challenging but necessary for studying this phenomenon. A protocol was designed and tested to select stable, MTB spontaneous, d-cycloserine (DCS) resistant mutants. Twenty-four colonies resistant to DCS were selected, demonstrating an increase between 1 and 4 times the Minimum Inhibitory Concentration (MIC) set for Mycobacterium tuberculosis H37Rv ATCC 27294 reference strain.

Chance Favors the Prepared Genomes: Horizontal Transfer Shapes the Emergence of Antibiotic Resistance Mutations in Core Genes.

Bacterial lineages acquire novel traits at diverse rates in part because the genetic background impacts the successful acquisition of novel genes by horizontal transfer. Yet, how horizontal transfer affects the subsequent evolution of core genes remains poorly understood. Here, we studied the evolution of resistance to quinolones in Escherichia coli accounting for population structure. We found 60 groups of genes whose gain or loss induced an increase in the probability of subsequently becoming resistant to quinolones by point mutations in the gyrase and topoisomerase genes. These groups include functions known to be associated with direct mitigation of the effect of quinolones, with metal uptake, cell growth inhibition, biofilm formation, and sugar metabolism. Many of them are encoded in phages or plasmids. Although some of the chronologies may reflect epidemiological trends, many of these groups encoded functions providing latent phenotypes of antibiotic low-level resistance, tolerance, or persistence under quinolone treatment. The mutations providing resistance were frequent and accumulated very quickly. Their emergence was found to increase the rate of acquisition of other antibiotic resistances setting the path for multidrug resistance. Hence, our findings show that horizontal gene transfer shapes the subsequent emergence of adaptive mutations in core genes. In turn, these mutations further affect the subsequent evolution of resistance by horizontal gene transfer. Given the substantial gene flow within bacterial genomes, interactions between horizontal transfer and point mutations in core genes may be a key to the success of adaptation processes.

Impact of Microgravity on Virulence, Antibiotic Resistance and Gene Expression in Beneficial and Pathogenic Microorganisms.

During space missions, the impact of the space conditions (both microgravity and radiation) on physiologic and metabolic aspects of the microbiota of astronauts' bodies should be considered. Changes depend on the mission's duration, types of organisms, and ecology. Reported alterations are related to changes in morphology, growth, gene expression, and physiology of cells, resulting in increased virulence, acid, antibiotic resistance, biofilm formation, secondary metabolism, and microbial mutations. Accordingly, recent research indicates the impacts of simulated microgravity on human physiology and bacterial characteristics. This paper has reviewed the aspects of microgravity on changes in microbiota, including virulence, antibiotic resistance, and gene expression. Microgravity can undermine humans and makes influence bacterial pathogenicity. The review of papers shows that some microorganisms showed higher pathogenicity under microgravity conditions. Moreover, sulfamethoxazole had the highest resistance among Gram-positive microorganisms, and gentamicin had the highest resistance in Gram-negative bacteria. All antibiotics reviewed under microgravity conditions were robust in both groups of microorganisms compared to the gravity condition. Furthermore, some gene expression was altered in bacteria under microgravity conditions compared to Earth conditions (standard bacterial growth conditions). Changes in microbial behavior under microgravity directly influence astronauts' health conditions, and a detailed analysis of known facts can provide essential information for the selection of appropriate probiotics for these specific cases during the missions and after the recovery processes. Moreover, the study of microorganisms changes in the absence of gravity will help to understand the mechanisms of causing diseases on Earth and may be applied in clinical practice.

Pervasive genotype-by-environment interactions shape the fitness effects of antibiotic resistance mutations.

The fitness effects of antibiotic resistance mutations are a major driver of resistance evolution. While the nutrient environment affects bacterial fitness, experimental studies of resistance typically measure fitness of mutants in a single environment only. We explored how the nutrient environment affected the fitness effects of rifampicin-resistant rpoB mutations in Escherichia coli under several conditions critical for the emergence and spread of resistance-the presence of primary or secondary antibiotic, or the absence of any antibiotic. Pervasive genotype-by-environment (GxE) interactions determined fitness in all experimental conditions, with rank order of fitness in the presence and absence of antibiotics being strongly dependent on the nutrient environment. GxE interactions also affected the magnitude and direction of collateral effects of secondary antibiotics, in some cases so drastically that a mutant that was highly sensitive in one nutrient environment exhibited cross-resistance to the same antibiotic in another. It is likely that the mutant-specific impact of rpoB mutations on the global transcriptome underpins the observed GxE interactions. The pervasive, mutant-specific GxE interactions highlight the importance of doing what is rarely done when studying the evolution and spread of resistance in experimental and clinical work: assessing fitness of antibiotic-resistant mutants across a range of relevant environments.

Simultaneous antibiotic removal and mitigation of resistance induction by manganese bio-oxidation process

Microbial degradation to remove residual antibiotics in wastewater is of growing interest. However, biological treatment of antibiotics may cause resistance dissemination by mutations and horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs). In this study, a Mn(Ⅱ)-oxidizing bacterium (MnOB), Pseudomonas aeruginosa MQ2, simultaneously degraded antibiotics, decreased HGT, and mitigated antibiotic resistance mutation. Intracellular Mn(II) levels increased during manganese oxidation, and biogenic manganese oxides (BioMnOx, including Mn(II), Mn(III) and Mn(IV)) tightly coated the cell surface. Mn(II) bio-oxidation mitigated antibiotic resistance acquisition from an E. coli ARG donor and mitigated antibiotic resistance inducement by decreasing conjugative transfer and mutation, respectively. BioMnOx also oxidized ciprofloxacin (1 mg/L) and tetracycline (5 mg/L), respectively removing 93% and 96% within 24 h. Transcriptomic analysis revealed that two new multicopper oxidase and one peroxidase genes are involved in Mn(II) oxidation. Downregulation of SOS response, multidrug resistance and type Ⅳ secretion system related genes explained that Mn(II) and BioMnOx decreased HGT and mitigated resistance mutation by alleviating oxidative stress, which makes recipient cells more vulnerable to ARG acquisition and mutation. A manganese bio-oxidation based reactor was constructed and completely removed tetracycline with environmental concentration within 4-hour hydraulic retention time. Overall, this study suggests that Mn (II) bio-oxidation process could be exploited to control antibiotic contamination and mitigate resistance propagation during water treatment.

Bioaugmentation of a structural extracellular polymeric substances (EPS) producer to improve activated sludge bioflocculation: lessons learned

Abstract Structural extracellular polymeric substances (EPS) contribute to the bioflocculation performance of activated sludge systems. This research investigates the potential of bioaugmentation of a structural EPS producer, Azoarcus communis, as a bioflocculation improvement or remediation approach. An antibiotic-resistant and fluorescent protein-producing mutant was constructed to monitor the survival, persistence, and location of the augmented strain in the membrane bioreactor. Preliminary batch tests against a kaolin clay model system and deflocculated sludge revealed the flocculation potential of this strain. Morphological image analysis and fluorescence microscopy suggest that most of the bacteria augmented in suspension were initially attached to the sludge flocs with, however, only a limited fraction getting incorporated within the activated sludge floc biomass. This limited bioaugmentation prevented assessing its impact on bioflocculation and might be explained by metazoan and protozoan grazing, together with competition with indigenous organisms and sub-optimal growth conditions in the reactor for the engineered strain.

Staphylococcus aureus Small-Colony Variants from Airways of Adult Cystic Fibrosis Patients as Precursors of Adaptive Antibiotic-Resistant Mutations.

Prototypic Staphylococcus aureus and their small-colony variants (SCVs) are predominant in cystic fibrosis (CF), but the interdependence of these phenotypes is poorly understood. We characterized S. aureus isolates from adult CF patients over several years. Of 18 S. aureus-positive patients (58%), 13 (72%) were positive for SCVs. Characterization included genotyping, SCCmec types, auxotrophy, biofilm production, antibiotic susceptibilities and tolerance, and resistance acquisition rates. Whole-genome sequencing revealed that several patients were colonized with prototypical and SCV-related clones. Some clonal pairs showed acquisition of aminoglycoside resistance that was not explained by aminoglycoside-modifying enzymes, suggesting a mutation-based process. The characteristics of SCVs that could play a role in resistance acquisition were thus investigated further. For instance, SCV isolates produced more biofilm (p < 0.05) and showed a higher survival rate upon exposure to ciprofloxacin and vancomycin compared to their prototypic associated clones. SCVs also developed spontaneous rifampicin resistance mutations at a higher frequency. Accordingly, a laboratory-derived SCV (ΔhemB) acquired resistance to ciprofloxacin and gentamicin faster than its parent counterpart after serial passages in the presence of sub-inhibitory concentrations of antibiotics. These results suggest a role for SCVs in the establishment of persistent antibiotic-resistant clones in adult CF patients.

Heterogeneity among Clinical Intestinal Escherichia coli Isolates upon Acquired Streptomycin Resistance.

Escherichia coli isolates from inflammatory bowel disease (IBD) patients are often multidrug resistant, including to streptomycin. Streptomycin resistance (StrR) mutations can alter bacterial behavior, which may influence intestinal disease. We generated a spontaneous StrR strain of the intestinal adherent-invasive E. coli (AIEC) strain NC101. Whole-genome sequencing revealed a single missense mutation in rpsL that commonly confers StrR, rpsL-K43N. StrR NC101 exhibited a striking loss of aggregation and significantly increased motility, behaviors that can impact host-microbe interactions. Behavioral changes were associated with reduced transcription of csgA, encoding the biofilm component curli, and increased transcription of fliC, encoding flagellin. Scanning electron microscopy (SEM) detailed morphologic changes consistent with the observed alterations in multicellular behavior. Because intestinal E. coli isolates exhibit remarkable strain-specific differences, we generated spontaneous StrR mutants of 10 clinical E. coli phylotype B2 strains from patients with IBD, colorectal cancer, and urinary tract infection. Out of these 10 StrR clinical strains, two had altered colony morphology on Congo red agar (suggesting changes in extracellular products), and three had significant changes in motility. These changes were not associated with a particular rpsL mutation nor with the presence of virulence genes encoding the inflammation-associated E. coli metabolites yersiniabactin or colibactin. We conclude that common mutations in rpsL, which confer StrR, can differentially alter disease-associated phenotypes across intestinal E. coli strains. These findings highlight the heterogeneity among seemingly similar intestinal E. coli strains and reveal the need to carefully study the strain-specific effects of antibiotic resistance mutations, particularly when using these mutations during strain selection studies. IMPORTANCE We demonstrate that StrR, commonly acquired through a single point mutation in rpsL (a gene encoding part of the 30S bacterial ribosome), strikingly alters the morphology and behavior of a key intestinal AIEC strain, NC101. These changes include remarkably diminished aggregation and significantly increased motility, traits that are linked to AIEC-defining features and disease development. Phenotypic changes were heterogeneous among other StrR clinical E. coli strains, underscoring the need to evaluate the strain-specific effects of commonly acquired antibiotic resistance mutations. This is important, as the results of studies using mutant StrR Enterobacteriaceae strains (e.g., for cloning or in vivo selection) may be confounded beyond our demonstrated effects. Long term, these findings can help researchers better distinguish the contribution of specific E. coli traits to functional changes in the microbiota. Evaluating these strain-level differences could provide insight into the diversity of IBD symptoms and lead to improved therapies for microbiota-driven intestinal disorders.