Acquisition Of Antibiotic Resistance Research Articles

The role of Bifidobacterium genus in modulating the neonate microbiota: implications for antibiotic resistance acquisition in early life

ABSTRACT Resistance to antibiotics in newborns is a huge concern as their immune system is still developing, and infections and resistance acquisition in early life have short- and long-term consequences for their health. Bifidobacterium species are important commensals capable of dominating the infant gut microbiome and are known to be less prone to possess antimicrobial resistance genes than other taxa that may colonize infants. We aimed to study the association between Bifidobacterium-dominated infant gut microbiota and the antibiotic resistant gene load in neonates, and to ascertain the perinatal factors that may contribute to the antibiotic resistance acquisition. Two hundred infant fecal samples at 7 days and 1 month of age from the MAMI birth cohort were included in the study and for whom maternal-neonatal clinical records were available. Microbiota profiling was carried out by 16S rRNA amplicon sequencing, and targeted antibiotic resistance genes (ARGs) including tetM, tetW, tetO, blaTEM, blaSHV and ermB were quantified by qPCR. Infant microbiota clustered into two distinct groups according to their Bifidobacterium genus abundance: high and low. The main separation of groups or clusters at each time point was performed with an unsupervised non-linear algorithm of k-means partitioning to cluster data by time points based on Bifidobacterium genus relative abundance. Microbiota composition differed significantly between both groups, and specific bifidobacterial species were enriched in each cluster. Lower abundance of Bifidobacterium in the infant gut was associated with a higher load of antibiotic resistance genes. Our results highlight the relevance of Bifidobacterium genus in the early acquisition and establishment of antibiotic resistance in the gut. Further studies are needed to develop strategies to promote a healthy early colonization and fight against the spread of antibiotic resistances.

Genome analysis of Streptococcus spp. isolates from animals in pre-antibiotic era with respect to antibiotic susceptibility and virulence gene profiles.

Lyophilized Streptococcus spp. isolates(n = 50) from animal samples submitted to the diagnostic laboratory at the University of Connecticut in the 1940s were revivified to investigate the genetic characteristics using whole-genome sequencing (WGS). The Streptococcus spp. isolateswere identified as follows; S. agalactiae (n = 14), S. dysgalactiae subsp. dysgalactiae (n = 10), S. dysgalactiae subsp. equisimils (n = 5), S. uberis (n = 8), S. pyogenes (n = 7), S. equi subsp. zooepidemicus (n = 4), S. oralis (n = 1), and S. pseudoporcinus (n = 1). We identified sequence types (ST) of S. agalactiae, S. dysgalactiae, S. uberis, S. pyogenes, and S. equi subsp. zooepidemicus and reported ten novel sequence types of those species. WGS analysis revealed that none of Streptococcus spp. carried antibiotic resistance genes. However, tetracycline resistance was observed in four out of 15 S. dysgalactiae isolates and in one out of four S. equi subsp. zooepidemicus isolate. This data highlights that antimicrobial resistance is pre-existed in nature before the use of antibiotics. The draft genome sequences of isolates from this study and 426 complete genome sequences of Streptococcus spp. downloaded from BV-BRC and NCBI GenBank database were analyzed for virulence gene profiles and phylogenetic relationships. Different Streptococcus species demonstrated distinct virulence gene profiles, with no time-related variations observed. Phylogenetic analysis revealed high genetic diversity of Streptococcus spp. isolates from the 1940s, and no clear spatio-temporal clustering patterns were observed among Streptococcus spp. analyzed in this study. This study provides an invaluable resource for studying the evolutionary aspects of antibiotic resistance acquisition and virulence in Streptococcus spp.

A role for the stringent response in ciprofloxacin resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a major cause of nosocomial infections and the leading cause of chronic lung infections in cystic fibrosis and chronic obstructive pulmonary disease patients. Antibiotic treatment remains challenging because P. aeruginosa is resistant to high concentrations of antibiotics and has a remarkable ability to acquire mutations conferring resistance to multiple groups of antimicrobial agents. Here we report that when P. aeruginosa is plated on ciprofloxacin (cipro) plates, the majority of cipro-resistant (ciproR) colonies observed at and after 48 h of incubation carry mutations in genes related to the Stringent Response (SR). Mutations in one of the major SR components, spoT, were present in approximately 40% of the ciproR isolates. Compared to the wild-type strain, most of these isolates had decreased growth rate, longer lag phase and altered intracellular ppGpp content. Also, 75% of all sequenced mutations were insertions and deletions, with short deletions being the most frequently occurring mutation type. We present evidence that most of the observed mutations are induced on the selective plates in a subpopulation of cells that are not instantly killed by cipro. Our results suggests that the SR may be an important contributor to antibiotic resistance acquisition in P. aeruginosa.

Expression of norA, norB and norC efflux pump genes mediating fluoroquinolones resistance in MRSA isolates.

Although fluoroquinolones are used to treat methicillin-resistant Staphylococcus aureus (MRSA)-induced infections, acquisition of antibiotic resistance by bacteria has impaired their clinical relevance. We aimed to evaluate the frequency of norA, norB, and norC efflux pump genes-mediating fluoroquinolones resistance and measure their expression levels in MRSA isolates. 126 S. aureus isolates were collected from different clinical samples of adult hospitalized patients and identified by conventional microbiological methods. MRSA was diagnosed by cefoxitin disc diffusion method and minimum inhibitory concentration (MIC) of ciprofloxacin by broth microdilution method. The expression levels of efflux pump genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR). 80 (63.5%) MRSA isolates were identified and showed high level of resistance to erythromycin (80%), gentamicin (75%), clindamycin (65%) and ciprofloxacin (60 %). norA, norB and norC were detected in 75%, 35% and 55% of the MRSA isolates respectively. norC was the most commonly overexpressed gene measured by qRT-PCR, occurring in 40% of MRSA isolates, followed by norA (35%) and norB (30%). The expression of these genes was significantly higher in ciprofloxacin-resistant than quantitative real-time PCR ciprofloxacin-sensitive MRSA isolates. This study showed high prevalence and overexpression of efflux pump genes among MRSA isolates which indicates the significant role of these genes in the development of multidrug resistance against antibiotics including fluoroquinolones.

Role of RelA-synthesized (p)ppGpp and ROS-induced mutagenesis in de novo acquisition of antibiotic resistance in E. coli

The stringent response of bacteria to starvation and stress also fulfills a role in addressing the threat of antibiotics. Within this stringent response, (p)ppGpp, synthesized by RelA or SpoT, functions as a global alarmone. However, the effect of this (p)ppGpp on resistance development is poorly understood. Here, we show that knockout of relA or rpoS curtails resistance development against bactericidal antibiotics. The emergence of mutated genes associated with starvation and (p)ppGpp, among others, indicates the activation of stringent responses. The growth rate is decreased in ΔrelA-resistant strains due to the reduced ability to synthesize (p)ppGpp and the persistence of deacylated tRNA impeding protein synthesis. Sluggish cellular activity causes decreased production of reactive oxygen species (ROS), thereby reducing oxidative damage, leading to weakened DNA mismatch repair, potentially reducing the generation of mutations. These findings offer new targets for mitigating antibiotic resistance development, potentially achieved through inhibiting (p)ppGpp or ROS synthesis.

Mathematical modelling of antibiotic interaction on evolution of antibiotic resistance: an analytical approach.

The emergence and spread of antibiotic-resistant pathogens have led to the exploration of antibiotic combinations to enhance clinical effectiveness and counter resistance development. Synergistic and antagonistic interactions between antibiotics can intensify or diminish the combined therapy's impact. Moreover, these interactions can evolve as bacteria transition from wildtype to mutant (resistant) strains. Experimental studies have shown that the antagonistically interacting antibiotics against wildtype bacteria slow down the evolution of resistance. Interestingly, other studies have shown that antibiotics that interact antagonistically against mutants accelerate resistance. However, it is unclear if the beneficial effect of antagonism in the wildtype bacteria is more critical than the detrimental effect of antagonism in the mutants. This study aims to illuminate the importance of antibiotic interactions against wildtype bacteria and mutants on the deacceleration of antimicrobial resistance. To address this, we developed and analyzed a mathematical model that explores the population dynamics of wildtype and mutant bacteria under the influence of interacting antibiotics. The model investigates the relationship between synergistic and antagonistic antibiotic interactions with respect to the growth rate of mutant bacteria acquiring resistance. Stability analysis was conducted for equilibrium points representing bacteria-free conditions, all-mutant scenarios, and coexistence of both types. Numerical simulations corroborated the analytical findings, illustrating the temporal dynamics of wildtype and mutant bacteria under different combination therapies. Our analysis provides analytical clarification and numerical validation that antibiotic interactions against wildtype bacteria exert a more significant effect on reducing the rate of resistance development than interactions against mutants. Specifically, our findings highlight the crucial role of antagonistic antibiotic interactions against wildtype bacteria in slowing the growth rate of resistant mutants. In contrast, antagonistic interactions against mutants only marginally affect resistance evolution and may even accelerate it. Our results emphasize the importance of considering the nature of antibiotic interactions against wildtype bacteria rather than mutants when aiming to slow down the acquisition of antibiotic resistance.

Novel mechanisms of diversity generation in Acinetobacter baumannii resistance islands driven by Tn7-like elements.

Mobile genetic elements play an important role in the acquisition of antibiotic and biocide resistance, especially through the formation of resistance islands in bacterial chromosomes. We analyzed the contribution of Tn7-like transposons to island formation and diversification in the nosocomial pathogen Acinetobacter baumannii and identified four separate families that recognize different integration sites. One integration site is within the comM gene and coincides with the previously described Tn6022 elements suggested to account for the AbaR resistance island. We established Tn6022 in a heterologous E. coli host and confirmed basic features of transposition into the comM attachment site and the use of a novel transposition protein. By analyzing population features within Tn6022 elements we identified two potential novel transposon-encoded diversification mechanisms with this dynamic genetic island. The activities of these diversification features were confirmed in E. coli. One was a novel natural gain-of-activity allele that could function to broaden transposition targeting. The second was a transposon-encoded hybrid dif-like site that parasitizes the host dimer chromosome resolution system to function with its own tyrosine recombinase. This work establishes a highly active Tn7-like transposon that harnesses novel features allowing the spread and diversification of genetic islands in pathogenic bacteria.

The Effect of the Stringent Response and Oxidative Stress Response on Fitness Costs of De Novo Acquisition of Antibiotic Resistance.

Resistance evolution during exposure to non-lethal levels of antibiotics is influenced by various stress responses of bacteria which are known to affect growth rate. Here, we aim to disentangle how the interplay between resistance development and associated fitness costs is affected by stress responses. We performed de novo resistance evolution of wild-type strains and single-gene knockout strains in stress response pathways using four different antibiotics. Throughout resistance development, the increase in minimum inhibitory concentration (MIC) is accompanied by a gradual decrease in growth rate, most pronounced in amoxicillin or kanamycin. By measuring biomass yield on glucose and whole-genome sequences at intermediate and final time points, we identified two patterns of how the stress responses affect the correlation between MIC and growth rate. First, single-gene knockout E. coli strains associated with reactive oxygen species (ROS) acquire resistance faster, and mutations related to antibiotic permeability and pumping out occur earlier. This increases the metabolic burden of resistant bacteria. Second, the ΔrelA knockout strain, which has reduced (p)ppGpp synthesis, is restricted in its stringent response, leading to diminished growth rates. The ROS-related mutagenesis and the stringent response increase metabolic burdens during resistance development, causing lower growth rates and higher fitness costs.

Boosting Fitness Costs Associated with Antibiotic Resistance in the Gut: On the Way to Biorestoration of Susceptible Populations.

The acquisition and expression of antibiotic resistance implies changes in bacterial cell physiology, imposing fitness costs. Many human opportunistic pathogenic bacteria, such as those causing urinary tract or bloodstream infections, colonize the gut. In this opinionated review, we will examine the various types of stress that these bacteria might suffer during their intestinal stay. These stresses, and their compensatory responses, probably have a fitness cost, which might be additive to the cost of expressing antibiotic resistance. Such an effect could result in a disadvantage relative to antibiotic susceptible populations that might replace the resistant ones. The opinion proposed in this paper is that the effect of these combinations of fitness costs should be tested in antibiotic resistant bacteria with susceptible ones as controls. This testing might provide opportunities to increase the bacterial gut stress boosting physiological biomolecules or using dietary interventions. This approach to reduce the burden of antibiotic-resistant populations certainly must be answered empirically. In the end, the battle against antibiotic resistance should be won by antibiotic-susceptible organisms. Let us help them prevail.

Rolling the evolutionary dice: Neisseria commensals as proxies for elucidating the underpinnings of antibiotic resistance mechanisms and evolution in human pathogens.

Species within the genus Neisseria are adept at sharing adaptive allelic variation, with commensal species repeatedly transferring resistance to their pathogenic relative Neisseria gonorrhoeae. However, resistance in commensals is infrequently characterized, limiting our ability to predict novel and potentially transferable resistance mechanisms that ultimately may become important clinically. Unique evolutionary starting places of each Neisseria species will have distinct genomic backgrounds, which may ultimately control the fate of evolving populations in response to selection as epistatic and additive interactions coerce lineages along divergent evolutionary trajectories. Alternatively, similar genetic content present across species due to shared ancestry may constrain existing adaptive solutions. Thus, identifying the paths to resistance across commensals may aid in characterizing the Neisseria resistome-or the reservoir of alleles within the genus as well as its depth. Here, we use in vitro evolution of four commensal species to investigate the potential and repeatability of resistance evolution to two antimicrobials, the macrolide azithromycin and the β-lactam penicillin. After 20 days of selection, commensals evolved resistance to penicillin and azithromycin in 11/16 and 12/16 cases, respectively. Almost all cases of resistance emergence converged on mutations within ribosomal components or the mtrRCDE efflux pump for azithromycin-based selection and mtrRCDE, penA, and rpoB for penicillin selection, thus supporting constrained adaptive solutions despite divergent evolutionary starting points across the genus for these particular drugs. Though drug-selected loci were limited, we do identify novel resistance-imparting mutations. Continuing to explore paths to resistance across different experimental conditions and genomic backgrounds, which could shunt evolution down alternative evolutionary trajectories, will ultimately flesh out the full Neisseria resistome.IMPORTANCENeisseria gonorrhoeae is a global threat to public health due to its rapid acquisition of antibiotic resistance to all first-line treatments. Recent work has documented that alleles acquired from close commensal relatives have played a large role in the emergence of resistance to macrolides and beta-lactams within gonococcal populations. However, commensals have been relatively underexplored for the resistance genotypes they may harbor. This leaves a gap in our understanding of resistance that could be rapidly acquired by the gonococcus through a known highway of horizontal gene exchange. Here, we characterize resistance mechanisms that can emerge in commensal Neisseria populations via in vitro selection to multiple antimicrobials and begin to define the number of paths to resistance. This study, and other similar works, may ultimately aid both surveillance efforts and clinical diagnostic development by nominating novel and conserved resistance mechanisms that may be at risk of rapid dissemination to pathogen populations.

Cultivation and Genomic DNA Extraction of Klebsiella pneumoniae.

Klebsiella pneumoniae is a Gram-negative, rod-shaped bacterium of medical significance. It typically exists as part of the normal flora of the human intestine but can cause severe infections in the healthcare setting due to its rapid acquisition of antibiotic resistance. Cultivating and extracting genomic DNA from this bacterium is crucial for downstream characterization and comparative analyses. To provide a standardized approach for growing K. pneumoniae in the laboratory setting, this collection of protocols provides step-by-step procedures for routine culturing, generating growth curves, storing bacteria, and extracting genomic DNA. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Reviving K. pneumoniae from frozen stocks Basic Protocol 2: Cultivating K. pneumoniae in rich growth medium Alternate Protocol: Cultivating in minimal liquid growth medium Basic Protocol 3: Enumerating K. pneumoniae colony forming units Basic Protocol 4: Growth curves Basic Protocol 5: Genomic DNA extraction Basic Protocol 6: Characterizing K. pneumoniae strains based on genomic sequence Basic Protocol 7: Storage of K. pneumoniae frozen stocks in glycerol Basic Protocol 8: Storage of K. pneumoniae in agar stabs.

A Review of Antibiotic Resistance: Global Reports, Sources, Incidents, Resistance Strategies, and Control Plans

Antimicrobial resistance is a major global issue that is only getting worse. Acquired resistance is defined by the emergence of coding genes for strategies of drug evasion from antimicrobial agents. The Enterobacteriaceae family has been linked to this behavior. Antibiotics like beta-lactams and carbapenems, which are the most used types, are used to treat bacterial infections. From a clinical perspective, research on antibiotic resistance is very important because of the effects it has on human health. Furthermore, one of the rare instances of evolution that can be researched in real time is antibiotic resistance. Therefore, doctors, evolutionary biologists, and ecologists are interested in understanding the general processes involved in the acquisition of antibiotic resistance. Environmental microbes are the source of antibiotic resistance genes that are currently found in human diseases. Therefore, research on both natural and medical environments is necessary to fully comprehend the emergence of antibiotic resistance. Recent findings about the evolutionary processes underlying resistance suggest that viability costs, the founder effect, and ecological connectivity are significant barriers that control the spread of resistance from environmental bacteria to diseases.

A two-component system serves as a central hub for connecting energy metabolism and plasmid dissemination in bacteria.

Mobile genetic elements such as conjugative plasmids play a key role in the acquisition of antibiotic resistance by pathogenic bacteria. Resistance genes on plasmids can be transferred between bacteria using specialized conjugation machinery. Acinetobacter baumannii, the most common bacterium associated with nosocomial infections, harbors a large conjugative plasmid that encodes a type IV secretion system (T4SS). Feng et al. recently found that the A. baumannii T4SS is specialized for plasmid transfer, suggesting that it may be involved in multidrug resistance (Z. Feng, L. Wang, Q. Guan, X. Chu, and Z.-Q. Luo, mBio e02276-23, 2023, https://doi.org/10.1128/mbio.02276-23), T4SS-encoding genes are shown to be controlled by a versatile GacA/S two-component regulatory system. GacA/S is also found to regulate genes involved in central metabolism. The coordinated regulation of metabolism and plasmid conjugation may be a bacterial strategy for adapting to selective pressure from antibiotics.

High oral carriage of multidrug resistant Gram-negative bacilli in adolescents: the SOPKARD-Junior study.

The colonization of the oral cavity by potentially pathogenic antimicrobial-resistant bacteria in adolescents and its consequences is very poorly understood. The present study focused on the occurrence of oral colonization by Gram-negative bacilli (GNB) and their multidrug resistance, including the production of extended-spectrum β-lactamases (ESBLs) and carbapenemases, among healthy adolescents and risk factors associated with GNB colonization. This study was conducted as part of "A program for the early detection of risk factors for lifestyle diseases SOPKARD-Junior" (SOPKARD-Junior). Oral samples were collected from 182 adolescents from four public elementary schools in Sopot, Poland, aged 13-14 years. Bacterial strains were identified by the MALDI-TOF MS method. Screening of antimicrobial resistance was performed using a disk diffusion method. The NG-Test® CARBA-5 was used to detect and differentiate the five most widely distributed carbapenemases. Demographic and clinical data were collected and statistical analysis of risk factors was performed. A total of 68 out of 182 (37.4%) healthy adolescents was documented oral colonization with Gram-negative bacilli, including 50/182 (27.5%) multidrug resistant (MDR-GNB) strains. Over 60% of oral carriage concerned three main genera Enterobacter spp., Pseudomonas spp., and Serratia spp., which were detected in 22.1%, 19.1%, and 19.1% of participants, respectively. Citrobacter spp., Escherichia coli, Klebsiella spp., Hafnia spp., Aeromonas spp., Acinetobacter spp., and Stenotrophomonas spp. were also isolated. The antimicrobial resistance to ampicillin (100%), ceftazidime (69.1%), meropenem (60.3%), gentamycin (60.3%), piperacillin/tazobactam (52.9%), and piperacillin (45.6%) were the most common. Among 73.5% GNB strains multidrug resistance was observed, including all Pseudomonas spp. strains. Among MDR-GNB, 30.4% were resistant to four groups of antibiotics, half of the MDR Pseudomonas spp. strains were resistant to 10 groups of antibiotics. Extended-spectrum β-lactamases were produced by Enterobacter cloacae, Klebsiella spp., and Serratia spp. (7.4%). Colonization by ESBLs-positive GNB strains was significantly associated with recurrent respiratory infections, nasal congestion, and bronchitis (p<0.05). Our study revealed high oral carriage of multi-drug resistant Gram-negative bacilli in healthy adolescents and the association of ESBL-producing strains with respiratory infections. Further studies on oral colonization with GNB are necessary due to the possibility of distinct infections and the acquisition of antibiotic resistance by resident microbiota.

Whole-transcriptome analysis after the acquisition of antibiotic resistance of Cronobacter sakazakii: Mechanisms of antibiotic resistance and virulence changes

The emergence of antibiotic-resistant bacteria led to the misuse of antibiotics, resulting in the emergence of more resistant bacteria and continuous improvement in their resistance ability. Cronobacter sakazakii (C. sakazakii) has been considered a pathogen that harms infants. Incidents of C. sakazakii contamination have continued globally, several studies have indicated that C. sakazakii is increasingly resistant to antibiotics. A few studies have explored the mechanism of antibiotic resistance in C. sakazakii, and some have examined the antibiotic resistance and changes in virulence levels. We aimed to investigate the antibiotic resistance mechanism and virulence differences in C. sakazakii. The level of virulence factors of C. sakazakii was modified after induction by antibiotics compared with the antibiotic-sensitive strains, and the XS001-Ofl group had the strongest capacity to produce enterotoxin (85.18 pg/mL) and hemolysin (1.47 ng/mL). The biofilm formation capacity after induction significantly improved. The number of bases and mapped reads in all groups accounted for more than 55 % and 70 %, as detected by transcriptomic analysis. The resistance mechanism of different antibiotics was more common in efflux pumps, cationic antimicrobial peptides, and biofilm formation pathways. The level of antibiotic resistance mainly affected the expression of virulence genes associated with flagella assembly and synthesis.

RecA levels modulate biofilm development in Acinetobacter baumannii.

Infections caused by Acinetobacter baumannii, a Gram-negative opportunistic pathogen, are difficult to eradicate due to the bacterium's propensity to quickly gain antibiotic resistances and form biofilms, a protective bacterial multicellular community. The A. baumannii DNA damage response (DDR) mediates the antibiotic resistance acquisition and regulates RecA in an atypical fashion; both RecALow and RecAHigh cell types are formed in response to DNA damage. The findings of this study demonstrate that the levels of RecA can influence formation and dispersal of biofilms. RecA loss results in surface attachment and prominent biofilms, while elevated RecA leads to diminished attachment and dispersal. These findings suggest that the challenge to treat A. baumannii infections may be explained by the induction of the DDR, common during infection, as well as the delicate balance between maintaining biofilms in low RecA cells and promoting mutagenesis and dispersal in high RecA cells. This study underscores the importance of understanding the fundamental biology of bacteria to develop more effective treatments for infections.

Within-Host Resistance and Virulence Evolution of a Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae ST11 Under Antibiotic Pressure.

Hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) has recently aroused an extremely severe health challenge and public concern. However, the underlying mechanisms of fitness costs that accompany antibiotic resistance acquisition remain largely unexplored. Here, we report a hv-CRKP-associated fatal infection and reveal a reduction in virulence due to the acquisition of aminoglycoside resistance. The bacterial identification, antimicrobial susceptibility, hypermucoviscosity, virulence factors, MLST and serotypes were profiled.The clonal homology and plasmid acquisition among hv-CRKP strains were detected by XbaI and S1-PFGE. The virulence potential of the strains was evaluated using Galleria mellonella larvae infection model, serum resistance assay, capsular polysaccharide quantification, and biofilm formation assay. Genomic variations were identified using whole-genome sequencing (WGS). Four K. pneumoniae carbapenemase (KPC)-producing CRKP strains were consecutively isolated from an 86-year-old patient with severe pneumonia. Whole-genome sequencing (WGS) showed that all four hv-CRKP strains belonged to the ST11-KL64 clone. PFGE analysis revealed that the four ST11-KL64 hv-CRKP strains could be grouped into the same PFGE type. Under the pressure of antibiotics, the antimicrobial resistance of the strains increased and the virulence potential decreased. Further sequencing, using the Nanopore platform, was performed on three representative isolates (WYKP586, WYKP589, and WYKP594). Genomic analysis showed that the plasmids of these three strains underwent a large number of breaks and recombination events under antibiotic pressure. We found that as aminoglycoside resistance emerged via acquisition of the rmtB gene, the hypermucoviscosity and virulence of the strains decreased because of internal mutations in the rmpA and rmpA2 genes. This study shows that ST11-KL64 hv-CRKP can further evolve to acquire aminoglycoside resistance accompanied by decreased virulence to adapt to antibiotic pressure in the host.

Reactive oxygen species accelerate de novo acquisition of antibiotic resistance in E. coli

Reactive oxygen species (ROS) produced as a secondary effect of bactericidal antibiotics are hypothesized to play a role in killing bacteria. If correct, ROS may play a role in development of de novo resistance. Here we report that single-gene knockout strains with reduced ROS scavenging exhibited enhanced ROS accumulation and more rapid acquisition of resistance when exposed to sublethal levels of bactericidal antibiotics. Consistent with this observation, the ROS scavenger thiourea in the medium decelerated resistance development. Thiourea downregulated the transcriptional level of error-prone DNA polymerase and DNA glycosylase MutM, which counters the incorporation and accumulation of 8-hydroxy-2'-deoxyguanosine (8-HOdG) in the genome. The level of 8-HOdG significantly increased following incubation with bactericidal antibiotics but decreased after treatment with the ROS scavenger thiourea. These observations suggest that in E.coli sublethal levels of ROS stimulate de novo development of resistance, providing a mechanistic basis for hormetic responses induced by antibiotics.

Multiplex System: Identification of Vancomycin (Vana) And Methicillin (Meca) Resistance Genes In Staphylococcus Aureus

Introduction: Emergence of Methicillin resistant Staphylococcus aureus (MRSA) and Vancomycin resistant Staphylococcus aureus (VRSA) strain from different regions of the world poses a grave concern to human health. Antibiotic resistance in S. aureus is mainly because of the genetic factors which modify or disrupt their target site on bacteria. Methods: This study is focused to identify the vancomycin and methicillin resistance gene in antibiotic sensitive and resistant S. aureus. The pure cultures of S. aureus were isolated, subjected to morphological and biochemical characterization. Antibiotic susceptibility testing was done to check the resistance pattern. DNA isolation was followed by genotyping of the antibiotic resistance genes (VanA and MecA) and housekeeping gene (AroE) was done through multiplex PCR method. All the strains showed the colonial, microscopic and biochemical characteristics (catalase and coagulase positive) specific for S. aureus. Results: Majority of the strains were resistant to cefixime (80 %) and least resistance was observed with fusidic acid (0%), while resistance frequency of the remaining antibiotics falls between them. All the strains showed the presence of housekeeping AroE gene with frequency of VanA is 2% and MecA is 24% which coincides with the findings of antibiotic resistance testing. For VanA, there might be other resistance genes of vancomycin cassette which confer the resistance against it. Conclusion: The study will help to discriminate the vancomycin and methicilin sensitive and resistance strains of S. aureus based on their respective genetic factors and help to validate the underlying mechanism in the acquisition of antibiotic resistance.

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.