Subinhibitory Concentrations Of Antibiotics Research Articles

Characterization of Salmonella Phage P1-CTX and the Potential Mechanism Underlying the Acquisition of the blaCTX-M-27 Gene.

The P1 phage has garnered attention as a carrier of antibiotic resistance genes (ARGs) in Enterobacteriaceae. However, the transferability of ARGs by P1-like phages carrying ARGs, in addition to the mechanism underlying ARG acquisition, remain largely unknown. In this study, we elucidated the biological characteristics, the induction and transmission abilities, and the acquisition mechanism of the blaCTX-M-27 gene in the P1 phage. The P1-CTX phage exhibited distinct lytic plaques and possessed a complete head and tail structure. Additionally, the P1-CTX phage was induced successfully under various conditions, including UV exposure, heat treatment at 42 °C, and subinhibitory concentrations (sub-MICs) of antibiotics. Moreover, the P1-CTX phage could mobilize the blaCTX-M-27 gene into three strains of Escherichia coli (E. coli) and the following seven different serotypes of Salmonella: Rissen, Derby, Kentucky, Typhimurium, Cerro, Senftenberg, and Muenster. The mechanism underlying ARG acquisition by the P1-CTX phage involved Tn1721 transposition-mediated movement of blaCTX-M-27 into the ref and mat genes within its genome. To our knowledge, this is the first report documenting the dynamic processes of ARG acquisition by a phage. Furthermore, this study enriches the research on the mechanism underlying the phage acquisition of drug resistance genes and provides a basis for determining the risk of drug resistance during phage transmission.

Impact of sub-inhibitory antibiotic concentrations on biofilm formation among nosocomial isolates of Enterococcus species

Impact of sub-inhibitory antibiotic concentrations on biofilm formation among nosocomial isolates of Enterococcus species

Understanding the effects of sub-inhibitory antibiotic concentrations on the development of β-lactamase resistance based on quantile regression analysis.

Quantile regression is an alternate type of regression analysis that has been shown to have numerous advantages over standard linear regression. Unlike linear regression, which uses the mean to fit a linear model, quantile regression uses a data set's quantiles (or percentiles), which leads to a more comprehensive analysis of the data. However, while relatively common in other scientific fields such as economic and environmental modeling, it is infrequently used to understand biological and microbiological systems. We analyzed a set of bacterial growth rates using quantile regression analysis to better understand the effects of antibiotics on bacterial fitness. Using a bacterial model system containing 16 variant genotypes of the TEM β-lactamase enzyme, we compared our quantile regression analysis to a previously published study that uses the Tukey's range test, or Tukey honestly significantly difference (HSD) test. We find that trends in the distribution of bacterial growth rate data, as viewed through the lens of quantile regression, can distinguish between novel genotypes and ones that have been clinically isolated from patients. Quantile regression also identified certain combinations of genotypes and antibiotics that resulted in bacterial populations growing faster as the antibiotic concentration increased-the opposite of what was expected. These analyses can provide new insights into the relationships between enzymatic efficacy and antibiotic concentration. Quantile regression analysis enhances our understanding of the impacts of sublethal antibiotic concentrations on enzymatic (TEM β-lactamase) efficacy and bacterial fitness. We illustrate that quantile regression analysis can link patterns in growth rates with clinically relevant mutations and provides an understanding of how increasing sub-lethal antibiotic concentrations, like those found in our modern environment, can affect bacterial growth rates, and provide insight into the genetic basis for varied resistance.

Identification of Antibiotics Susceptibility Patterns at Sub-inhibitory Concentrations in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram negative bacterium that has been recognized as an opportunistic pathogen. It is the most common bacterium associated with nosocomial infections and ventilator-associated pneumonia. It exhibits high innate resistance to various ranges of antibiotics thereby causing high morbidity and mortality rate. This research was aimed to identify the antibiotics susceptibility patterns at sub-inhibitory concentration in Pseudomonas aeruginosa. One hundred and fifty (150) clinical swab specimens were collected from urinary catheters; wound and ear infections of patients and the swabs inoculated using standard microbiology method. The isolates were characterized based on the bacteriological methods such as morphology and biochemical tests. The isolates were further confirmed by species specific by PCR amplification of 16S rRNA and the amplicons were analyzed by gel electrophoresis; and further genomic sequencing was done and blast with NCBI database mining. The antimicrobial susceptibility of the isolates was done by disc diffusion methods. The result of the isolation showed 22(59.46%) from wound infections, 12(32.43%) from ear infections and 3(8.11%) from urinary catheter. The isolates were Gram-negative, produced β-hemolysis on blood agar and the morphology is small pigmented circular. The isolates showed positive results to catalase, oxidase, citrate, nitrate and indole tests. The amplification of the 16S rRNA gene region resulted in the band size of 1500bp PCR product and the BLAST analysis gave 99% similarity. The results of susceptibility analysis showed that the isolates from the urinary catheter, wound and ear infection were 82%, 68% and 47% respectively, resistance to Piperacillin tazobactem, Cefoperazon, Ofloxacin, Tetracycline, Amikacin, Gentamycin, Bacitracin, Clarithromycin, Cefalotin, Levofloxacin and Cefpiroma. Antimicrobial susceptibility tests in P. aeruginosa isolates revealedshowed that they were multi-drug resistant. At sub-inhibitory concentrations of antibiotics within the microbial environment, Pseudomonas aeruginosa becomes more resistant. Perhaps, these antimicrobials could have other signaling activities within the environment. Therefore, there is a need for more research work to develop therapeutics combination to combat the recalcitrant nature of Pseudomonas aeruginosa.

Sub-inhibitory concentrations of colistin and imipenem impact the expression of biofilm-associated genes in Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic pathogen that is responsible for nosocomial infections. Imipenem and colistin are drugs that are commonly used to treat severe infections caused by A. baumannii, such as sepsis, ventilator-associated pneumonia, and bacteremia. However, some strains of A. baumannii have become resistant to these drugs, which is a concern for public health. Biofilms produced by A. baumannii increase their resistance to antibiotics and the cells within the inner layers of biofilm are exposed to sub-inhibitory concentrations (sub-MICs) of antibiotics. There is limited information available regarding how the genes of A. baumannii are linked to biofilm formation when the bacteria are exposed to sub-MICs of imipenem and colistin. Thus, this study's objective was to explore this relationship by examining the genes involved in biofilm formation in A. baumannii when exposed to low levels of imipenem and colistin. The study found that exposing an isolate of A. baumannii to low levels of these drugs caused changes in their drug susceptibility pattern. The relative gene expression profiles of the biofilm-associated genes exhibited a change in their expression profile during short-term and long-term exposure. This study highlights the potential consequences of overuse and misuse of antibiotics, which can help bacteria become resistant to these drugs.

Role of membrane vesicles in the transmission of vancomycin resistance in Enterococcus faecium

Clonal transmission and horizontal gene transfer (HGT) contribute to the spread of vancomycin-resistant enterococci (VRE) in global healthcare. Our study investigated vesiduction, a HGT mechanism via membrane vesicles (MVs), for vanA and vanB genes that determine vancomycin resistance. We isolated MVs for VRE of different sequence types (STs) and analysed them by nanoparticle tracking analysis. Selected MV samples were subjected to DNA sequence analysis. In resistance transfer experiments, vancomycin-susceptible enterococci were exposed to MVs and bacterial supernatants of VRE. Compared to bacteria grown in lysogeny broth (MVs/LB), cultivation under vancomycin stress (MVs/VAN) resulted in increased particle concentrations of up to 139-fold (ST80). As a key finding, we could show that VRE isolates of ST80 and ST117 produced remarkably more vesicles at subinhibitory antibiotic concentrations (approx. 9.2 × 1011 particles/ml for ST80 and 2.4 × 1011 particles/ml for ST117) than enterococci of other STs (range between 1.8 × 1010 and 5.3 × 1010 particles/ml). In those MV samples, the respective resistance genes vanA and vanB were completely verifiable using sequence analysis. Nevertheless, no vancomycin resistance transfer via MVs to vancomycin-susceptible Enterococcus faecium was phenotypically detectable. However, our results outline the potential of future research on ST-specific MV properties, promising new insights into VRE mechanisms.

A smart sensor for monitoring antimicrobial interventions in wound infections

Effective therapeutic monitoring of wound infections is crucial for wound care. We explore a novel approach using a previously described 3D-printed, colourimetric indicator to non-invasively monitor antimicrobial interventions in wound infection. The indicator contains a pH-sensitive dye which detects carbon dioxide production from wound pathogens by changing colour from blue to yellow. The sensor accurately reported the minimum inhibitory concentration of antibiotic against Pseudomonas aeruginosa in a sealed 96-well plate. Subinhibitory antibiotic concentrations resulted in the sensor changing colour, whilst remaining blue for inhibitory concentrations. The sensor was applied to a porcine skin wound model with Pseudomonas aeruginosa biofilms, testing several antimicrobial treatments: antibiotic monotherapy, phage monotherapy, and a combination of both. After treatment, the sensor, sealed with the wound, enabled visual monitoring of antimicrobial efficacy. Through comparing post-treatment bioburden and sensor response, greater log-fold reductions in P. aeruginosa following antimicrobial treatment correlated with longer sensor response times. Sensors monitoring unsuccessfully treated biofilms changed colour, while those treated at or above the minimum biofilm eradication concentration elicited no colour change, indicating P. aeruginosa eradication. This sensor holds promise for monitoring wound infection treatment efficacy, improving patient outcomes, and promoting undisturbed wound healing as a non-invasive point-of-care tool.

Subinhibitory antibiotic concentrations promote the excision of a genomic island carried by the globally spread carbapenem-resistant Klebsiella pneumoniae sequence type 258.

The ICEKp258.2 genomic island (GI) has been proposed as an important factor for the emergence and success of the globally spread carbapenem-resistant Klebsiella pneumoniae sequence type (ST) 258. However, a characterization of this horizontally acquired element is lacking. Using bioinformatic and experimental approaches, we found that ICEKp258.2 is not confined to ST258 and ST512, but also carried by ST3795 strains and emergent invasive multidrug-resistant pathogens from ST1519. We also identified several ICEKp258.2-like GIs spread among different K. pneumoniae STs, other Klebsiella species and even other pathogen genera, uncovering horizontal gene transfer events between different STs and bacterial genera. Also, the comparative and phylogenetic analyses of the ICEKp258.2-like GIs revealed that the most closely related ICEKp258.2-like GIs were harboured by ST11 strains. Importantly, we found that subinhibitory concentrations of antibiotics used in treating K. pneumoniae infections can induce the excision of this GI and modulate its gene expression. Our findings provide the basis for the study of ICEKp258.2 and its role in the success of K. pneumoniae ST258. They also highlight the potential role of antibiotics in the spread of ICEKp258.2-like GIs among bacterial pathogens.

Sub-inhibitory concentrations of ceftriaxone induce morphological alterations and PIA-independent biofilm formation in Staphylococcus aureus

The exposure of bacteria to sub-inhibitory concentrations of antibiotics is of biological significance since it can occur in vivo under many circumstances, including low-dose treatment, poor adherence to a regimen, poor drug penetration, drug-drug interactions, and antibiotic resistance of the pathogen. In this study, we investigated the effects of subinhibitory concentrations of four antibiotics: ampicillin, ceftriaxone, gentamicin, and norfloxacin, which are commonly used in clinical settings and on cell morphology and biofilm formation in Staphylococcus aureus as one of the leading causes of nosocomial and biofilm-associated infections. Nine clinical S. aureus biofilm-producing isolates and two known biofilm-producing reference strains, S. aureus ATCC 29213 and S. aureus ATCC 6538, were used in this study. Sub-MICs of beta-lactam antibiotics (ampicillin and ceftriaxone) significantly induced biofilm formation in S. aureus ATCC 29213 and S. aureus ATCC 6538 and in six clinical isolates out of the nine selected isolates when compared with the antibiotic-free control group (P < 0.05), with an approximately 2- to 2.5-fold increase. Gentamicin and norfloxacin induced biofilms in S. aureus ATCC 29213 and S. aureus ATCC 6538, while gentamicin and norfloxacin induced biofilms only in three and two of the nine tested isolates, respectively (P < 0.05). The chemical nature of the biofilm matrix produced by half the MIC of ceftriaxone in the six isolates that showed increased biofilm was all non-polysaccharide in composition (PIA-independent). Gene expression of biofilm-encoding genes atl and sarA in biofilms of the two tested strains (S. aureus ATCC 6538) and clinical strain (S. aureus 16) showed a significant upregulation after exposure to half MIC of ceftriaxone. Additionally, the bacterial cell morphological changes in planktonic cells caused by half MIC of ceftriaxone were evaluated by scanning electron microscopy, which demonstrated a significant cell enlargement when compared with the antibiotic-free control (P < 0.05), and some deformed cells were also noticed. In S. aureus clinical isolates, sub-MICs of ampicillin, ceftriaxone, gentamicin, and norfloxacin may stimulate substantial production of biofilm, which could have important clinical significance and make infection treatment challenges. Further, in vivo research is needed to fully comprehend how sub-MIC of antibiotics can affect biofilm formation in clinical settings. Additionally, more research is required to reveal the clinical implications of the morphological alterations in S. aureus brought on by exposure to ceftriaxone at concentrations below its MIC.Graphical

Persistent ciprofloxacin exposure induced the transformation of Klebsiella pneumoniae small colony variant into mucous phenotype

IntroductionSmall colony variant (SCV) is a bacterial phenotype closely related to persistent and recurrent infections. SCVs are mutations that occur within bacterial populations, resulting in a change in bacterial morphology and the formation of small colonies. This morphological change may enhance bacterial resistance to antibiotics and contribute to persistent and recurrent infections.MethodsWe isolated Klebsiella pneumoniae (KPN) and its SCV from a child with recurrent respiratory tract infections. KPN and SCV were treated with subinhibitory concentrations of antibiotics. growth curves, serum resistance experiments, macrophage phagocytosis experiments and whole genome sequencing were used to characterize KPN and SCV.ResultsAfter treating KPN and SCV with subinhibitory concentrations of antibiotics, we found that ciprofloxacin induced the SCV transition to the mucoid phenotype. We found that the growth of mucoid Klebsiella pneumoniae was significantly slower than maternal strain and SCV though growth curves. Serum resistance experiments showed that mucoid strains had significantly higher serum resistance compared to maternal strain and SCV. Macrophage phagocytosis experiments revealed that SCV had significantly higher intracellular survival rates compared to maternal strain and mucoid strains. Differential gene analysis of three strains revealed that the mucoid strain contained DNA polymerase V subunit UmuC gene on the plasmid, while the SCV strain had an additional IcmK family IV secretion protein on its plasmid.DiscussionOur study showed the SCV of KPN changed to a mucoid colony when exposed to subinhibitory concentrations of ciprofloxacin. The higher resistance of serum of mucoid colonies was possibly related to the UmuC gene, while the increased intracellular survival of SCV may be related to the IcmK family type IV secretion proteins.

Impact of Environmental Sub-Inhibitory Concentrations of Antibiotics, Heavy Metals, and Biocides on the Emergence of Tolerance and Effects on the Mutant Selection Window in E. coli.

Bacteria's ability to withstand the detrimental effects of antimicrobials could occur as resistance or tolerance with the minimum inhibitory concentration, the mutant prevention concentration, and the mutant selection window as salient concepts. Thus, this study assessed the impact of exposure to extremely high doses of ampicillin on the level of persistence and tolerance development in isolates previously exposed to different concentrations of selected antibiotics, biocides, and heavy metals. These isolates were previously exposed to oxytetracycline (OXYTET), amoxicillin (AMX), copper (Cu), zinc (Zn), benzalkonium chloride (BAC) 10, dimethylammonium chloride (DADMAC) 12 and a combination of all the individual pollutants (ALL). The isolates were exposed to very high concentrations (25 × MIC) of ampicillin, and their tolerance was calculated as the time required to kill 99.9% of the bacterial population (MDK99.9). The MDK99.9 increased by 30 to 50% in test isolates (DADMAC, OXYTET, Zinc = 28 h; BAC, Copper = 30 h; amoxycillin, ALL = 26 h) compared to the untreated control. BAC-exposed isolates decreased from 2.5 × 108 CFU/mL to 2.5 × 104 CFU/mL on the second day, displaying the highest tolerance increase. The tolerance appeared to originate from two sources, i.e., stochastic persistence and genetic-induced persistence, involving multiple genes with diverse mechanisms. The mutant selection window of the isolates to ampicillin, amoxicillin, and oxytetracycline also slightly increased compared to the control, indicating the selective survival of persister cells during the 30-day exposure. These findings indicate that bacterial exposure to sub-inhibitory concentrations of environmental chemical stressors may not always result in the development of antimicrobial resistance but could initiate this process by selecting persisters that could evolve into resistant isolates.

Impact of veterinary antibiotics on plasmid-encoded antibiotic resistance transfer.

Resistance genes can be genetically transmitted and exchanged between commensal and pathogenic bacterial species, and in different compartments including the environment, or human and animal guts (One Health concept). The aim of our study was to evaluate whether subdosages of antibiotics administered in veterinary medicine could enhance plasmid transfer and, consequently, resistance gene exchange in gut microbiota. Conjugation frequencies were determined with Escherichia coli strains carrying IncL- (blaOXA-48) or IncI1-type (blaCTX-M-1) plasmids subjected to a series of subinhibitory concentrations of antibiotics used in veterinary medicine, namely amoxicillin, ceftiofur, apramycin, neomycin, enrofloxacin, colistin, erythromycin, florfenicol, lincomycin, oxytetracycline, sulfamethazine, tiamulin and the ionophore narasin. Treatments with subinhibitory dosages were performed with and without supplementation with the antioxidant edaravone, known as a mitigator of the inducibility effect of several antibiotics on plasmid conjugation frequency (PCF). Expression of SOS-response associated genes and fluorescence-based reactive oxygen species (ROS) detection assays were performed to evaluate the stress oxidative response. Increased PCFs were observed for both strains when treating with florfenicol and oxytetracycline. Increased expression of the SOS-associated recA gene also occurred concomitantly, as well as increased ROS production. Addition of edaravone to the treatments reduced their PCF and also showed a decreasing effect on SOS and ROS responses for both plasmid scaffolds. We showed here that some antibiotics used in veterinary medicine may induce transfer of plasmid-encoded resistance and therefore may contribute to the worldwide spread of antibiotic resistance genes.

Multi-omics reveals the increased biofilm formation of Salmonella Typhimurium M3 by the induction of tetracycline at sub-inhibitory concentrations

Exposure to sub-inhibitory concentrations (sub-MICs) of antibiotics could induce the biofilm formation of microorganisms, but its underlying mechanisms still remain elusive. In the present work, biofilm formation by Salmonella Typhimurium M3 was increased when in the presence of tetracycline at sub-MIC, and the highest induction was observed with tetracycline at 1/8 MIC. The integration of RNA-sequencing and untargeted metabolomics was applied in order to further decipher the potential mechanisms for this observation. In total, 439 genes and 144 metabolites of S. Typhimurium M3 were significantly expressed after its exposure to 1/8 MIC of tetracycline. In addition, the co-expression analysis revealed that 6 genes and 8 metabolites play a key role in response to 1/8 MIC of tetracycline. The differential genes and metabolites were represented in 12 KEGG pathways, including five pathways of amino acid metabolism (beta-alanine metabolism, tryptophan metabolism, arginine and proline metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, and glutathione metabolism), three lipid metabolism pathways (biosynthesis of unsaturated fatty acids, fatty acid degradation, and fatty acid biosynthesis), two nucleotide metabolism pathways (purine metabolism, and pyrimidine metabolism), pantothenate and CoA biosynthesis, and ABC transporters. Metabolites (anthranilate, indole, and putrescine) from amino acid metabolism may act as signaling molecules to promote the biofilm formation of S. Typhimurium M3. The results of this work highlight the importance of low antimicrobial concentrations on foodborne pathogens of environmental origin.

Environmental concentrations of antibiotics, biocides, and heavy metals fail to induce phenotypic antimicrobial resistance in Escherichia coli

Most anthropogenically affected environments contain mixtures of pollutants from different sources. The impact of these pollutants is usually the combined effect of the individual polluting constituents. However, how these stressors contribute to the development of antimicrobial resistance in environmental microorganisms is poorly understood. Thus, a 30-day exposure experiment to environmental and sub-inhibitory concentrations of oxytetracycline, amoxicillin, zinc, copper, BAC (benzalkonium chloride) 10 and DADMAC (diallyldimethylammonium chloride) 12, was conducted using fully susceptible E. coli ATCC 25922 to ascertain any development of phenotypic or genotypic resistance. Furthermore, wild-type isolates were collected from the same aquatic environment as the stressors, analysed for phenotypic resistance using the disk diffusion method and genotypically through whole genome sequencing. Exposure to the various concentrations and combinations of the stressors did not trigger phenotypic resistance in the experimental bacteria. Furthermore, genotypic analysis of the WGS on the exposed isolates only found the macrolide resistance mdf(A) gene (also present in the control strain) and the disinfectant resistance gene sitABCD. With further analysis for single nucleotide variants (SNV), mutations were detected for 19 genes that encoded for oxidative stress, DNA repair, membrane proteins efflux systems, growth and persister formations except for the robA, a transcription protein subset of the ArcC/XylS family of proteins, which confer multidrug resistance in E. coli. This indicates that exposure to sub-inhibitory concentrations of antibiotics, heavy metals and biocide residues in the aquatic environmental concentrations of the stressors identified in the current study could not induce phenotypic or genotypic resistance but encoded for genes responsible for the development of persistence and tolerance in bacteria, which could be a precursor to the development of resistance in environmental bacteria.

The influence of exposure to various concentrations of five antimicrobial agents on intracellular cytotoxin B production in &lt;i&gt;Clostridioides difficile&lt;/i&gt;

Background: Clostridioides difficile is an important cause of healthcare-associated diarrhea. Several anti- microbial agents are known to promote C. difficile infection (CDI). The impact of various concentrations of ampicillin (AMP), cefotaxime (CTX), clindamycin (CC), metronidazole (MTZ) and vancomycin (VAN) on intra-cellular cytotoxin B production was investigated in this study.&#x0D; Methodology: Six clinical strains of C. difficile were grown at minimum inhibitory concentration (MIC) and sub-MIC concentrations of these antibiotics. Inoculum standardization was performed by Miles and Misra method. Intracellular toxin B production was detected using Vero cell cytotoxicity assay in sonicated cultures on days 1, 2, 3, 4, 5 and 7 days of incubation.&#x0D; Results: There was a heterogeneous relationship between antibiotic exposure and the intra-cellular toxin production by the toxigenic strains. Clinical strains of C. difficile when exposed to MIC and sub-inhibitory concentrations of certain antibiotics produced high cytotoxin levels. All toxigenic isolates produced increased levels of cell-bound cytotoxin after exposure to antibiotics but there was no consistent pattern and the response to different doses varied considerably. Metronidazole was the most potent inducer of cell-bound cytotoxin followed by cefotaxime and clindamycin. Vancomycin induced the least amount of cytotoxin activity.&#x0D; Conclusion: The effects of sub-inhibitory concentration of antibiotic that predispose to C. difficile infection may partially suppress the normal gut flora, allowing colonization and growth of C. difficile, and may affect the level of toxin produced.

Involvement of Acquired Tobramycin Resistance in the Shift to the Viable but Non-Culturable State in Pseudomonas aeruginosa.

Persistent and viable but non-culturable (VBNC) Pseudomonas aeruginosa cells are mainly responsible for the recurrence and non-responsiveness to antibiotics of cystic fibrosis (CF) lung infections. The sub-inhibitory antibiotic concentrations found in the CF lung in between successive therapeutic cycles can trigger the entry into the VBNC state, albeit with a strain-specific pattern. Here, we analyzed the VBNC cell induction in the biofilms of two CF P. aeruginosa isolates, exposed to starvation with/without antibiotics, and investigated the putative genetic determinants involved. Total viable bacterial cells were quantified by the validated ecfX-targeting qPCR protocol and the VBNC cells were estimated as the difference between qPCR and cultural counts. The isolates were both subjected to whole genome sequencing, with attention focused on their carriage of aminoglycoside resistance genes and on identifying mutated toxin-antitoxin and quorum sensing systems. The obtained results suggest the variable contribution of different antibiotic resistance mechanisms to VBNC cell abundance, identifying a major contribution from tobramycin efflux, mediated by MexXY efflux pump overexpression. The genome analysis evidenced putative mutation hotspots, which deserve further investigation. Therefore, drug efflux could represent a crucial mechanism through which the VBNC state is entered and a potential target for anti-persistence strategies.

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.

The Fitness of Beta-Lactamase Mutants Depends Nonlinearly on Resistance Level at Sublethal Antibiotic Concentrations.

Adaptive evolutionary processes are constrained by the availability of mutations which cause a fitness benefit and together make up the fitness landscape, which maps genotype space onto fitness under specified conditions. Experimentally derived fitness landscapes have demonstrated a predictability to evolution by identifying limited "mutational routes" that evolution by natural selection may take between low and high-fitness genotypes. However, such studies often utilize indirect measures to determine fitness. We estimated the competitive fitness of mutants relative to all single-mutation neighbors to describe the fitness landscape of three mutations in a β-lactamase enzyme. Fitness assays were performed at sublethal concentrations of the antibiotic cefotaxime in a structured and unstructured environment. In the unstructured environment, the antibiotic selected for higher-resistance types-but with an equivalent fitness for a subset of mutants, despite substantial variation in resistance-resulting in a stratified fitness landscape. In contrast, in a structured environment with a low antibiotic concentration, antibiotic-susceptible genotypes had a relative fitness advantage, which was associated with antibiotic-induced filamentation. These results cast doubt that highly resistant genotypes have a unique selective advantage in environments with subinhibitory concentrations of antibiotics and demonstrate that direct fitness measures are required for meaningful predictions of the accessibility of evolutionary routes. IMPORTANCE The evolution of antibiotic-resistant bacterial populations underpins the ongoing antibiotic resistance crisis. We aim to understand how antibiotic-degrading enzymes can evolve to cause increased resistance, how this process is constrained, and whether it can be predictable. To this end, competition experiments were performed with a combinatorially complete set of mutants of a β-lactamase gene subject to subinhibitory concentrations of the antibiotic cefotaxime. While some mutations confer on their hosts high resistance to cefotaxime, in competition these mutations do not always confer a selective advantage. Specifically, high-resistance mutants had equivalent fitnesses despite different resistance levels and even had selective disadvantages under conditions involving spatial structure. Together, our findings suggest that the relationship between resistance level and fitness at subinhibitory concentrations is complex; predicting the evolution of antibiotic resistance requires knowledge of the conditions that select for resistant genotypes and the selective advantage evolved types have over their predecessors.

Effect of sub-inhibitory concentrations of antibiotic on the production and N-acetylglucosamine scale of methicillin- resistant Staphylococcus aureus biofilm

The ability of Staphylococcus spp. to produce biofilm is one of the virulence factors that facilitate the adhesion and colonization on a different surface. In this study, the effects of sub minimum inhibitory concentration (sub-MIC) of some antibiotics were evaluated on induction of the biofilm producing ability and free N-acetylglucoseamine scale in 29 isolates of methicillin resistance staphylococcus aureus. To this end, the antibiogram and biofilm producing functions of the studied isolates were assessed by Kirby-Bauer and tissue culture plate method, respectively. Vancomycin, trimethoprim-sulfamethoxazole and clindamycin were used in antibiogram. The free N-acetylglucoseamine scale in the inducted biofilm after treatment with antibiotic was evaluated by TLC method. Based on the attained results, all the isolates were susceptible to vancomycin and were capable of producing biofilm in weak (40%), moderate (56.6%) and strong (3.33%) levels. Also, biofilm production was induced in 36.66% of isolates (11/30) from moderate to strong level by sub-MIC vancomycin. An invisible change in free N-acetyl glucoseamine scale was demonstrated in the exopolysaccharide (EPS) structure of the studied isolates biofilm.By comparing of results and literature reviews, free N-acetyl glucoseamine scale in all studied strains was lower than 5µg in before and after inducted biofilm or maybe is not exist. Certainly, for studied of structural N-acetyl glucoseamine scale, using more exact methods of extraction and measurement are need.

Contribution of different class 2 integron elements to fitness costs in multi-drug resistant Escherichia coli and evaluation of their adaptability in “farm-to-table” environments

Integrons play a pivotal role in the dissemination of antimicrobial resistance, because they can capture and express exogenous antimicrobial resistance genes. This study aimed to elucidate the structure and contribution of different elements of class 2 integrons to fitness costs in their host bacteria and evaluate their adaptability to the “farm-to-table” process. We mapped 27 typical class 2 integrons of Escherichia coli isolated from aquatic foods and pork products, each harboring an inactive truncated class 2 integrase gene and the gene cassette (GC) array dfrA1-sat2-aadA1 with strong Pc2A/Pc2B promoters. Notably, the fitness costs associated with class 2 integrons depended on the Pc promoter strength and quantity and content of GCs in the array. Additionally, the costs of integrases were activity-dependent, and a balance was identified between GC capture ability and integron stability, which could explain the inactive truncated integrase identified. Although typical class 2 integrons exhibited low-cost structures in E. coli, the bacteria incurred biological costs, including decreasing growth rates and biofilm formation, in farm-to-table environments, especially under low-nutrient conditions. Nevertheless, sub-inhibitory antibiotic concentrations led to the selection of class 2 integron-carrying bacteria. This study provides important insights into how integrons may travel from preharvest to consumer goods.