Subinhibitory Concentrations Of Antibiotics Research Articles

Effect of subinhibitory concentrations on the spreading of the ampicillin resistance gene blaCMY-2 in an activated sludge microcosm

ABSTRACT As the problem of multi-resistant bacteria grows a better understanding of the spread of antibiotic resistance genes is of utmost importance for society. Wastewater treatment plants contain subinhibitory concentrations of antibiotics and are thought to be hotspots for antibiotic resistance gene propagation. Here we evaluate the influence of sub-minimum inhibitory concentrations of antibiotics on the spread of resistance genes within the bacterial community in activated sludge laboratory-scale sequencing batch reactors. The mixed communities were fed two different ampicillin concentrations (500 and 5000 µg/L) and the reactors were run and monitored for 30 days. During the experiment the β-lactamase resistance gene bla CMY-2 was monitored via qPCR and DNA samples were taken to monitor the effect of ampicillin on the microbial community. The relative copy number of bla CMY-2 in the reactor fed with the sub-minimum inhibitory concentration of 500 µg/L ampicillin was spread out over a wider range of values than the control and 5000 µg/L ampicillin reactors indicating more variability of gene number in the 500 µg/L reactor. This result emphasises the problem of sub-minimum inhibitory concentrations of antibiotics in wastewater. High-throughput sequencing showed that continuous exposure to ampicillin caused a shift from a Bacteroidetes to Proteobacteria in the bacterial community. The combined use of qPCR and high-throughput sequencing showed that ampicillin stimulates the spread of resistance genes and leads to the propagation of microbial populations which are resistant to it.

Molecular and phenotypic characterization of biofilm formation and antimicrobial resistance patterns of uropathogenic staphylococcus haemolyticus isolates in Casablanca, Morocco

This study aimed to establish the correlation between antibiotic resistance and biofilm formation by Staphylococcus haemolyticus and to examine the impact of sub-inhibitory concentrations of antibiotics (sub-MICs) on biofilm formation.Antibiotic susceptibility testing was conducted using the disk diffusion method, and biofilm formation was determined using Congo red agar and microtiter plate methods. Antibiotic resistance and biofilm-associated genes were detected using polymerase chain reaction.The majority of the twenty-one S. haemolyticus isolates were multidrug-resistant, methicillin-resistant (MRSH) and biofilm producers, including 43 % of moderate biofilm producers. A significant correlation was observed between MRSH and MSSH isolates in terms of biofilm production. Vancomycin, gentamicin, and ciprofloxacin at their sub-MICs tended to promote biofilm formation. The eno gene was present in 76.2 % of strains, followed by aap, and atlE.This study revealed a strong correlation between the biofilm-forming ability and antibiotic resistance in S. haemolyticus, which underlines a crucial public health issue.

Identification and Characterization of a Novel Prophage Lysin against Streptococcus dysgalactiae.

Streptococcus dysgalactiae infection can cause bovine mastitis and lead to huge economic losses for the dairy industry. The abuse of antibiotics has resulted in growing drug resistance of S. dysgalactiae, which causes hard-to-treat infections. Bacteriophage lysin, as a novel antibacterial agent, has great potential for application against drug-resistant gram-positive bacteria. However, few studies have been conducted on the prophage lysin of S. dysgalactiae. In this study, we mined a novel prophage lysin, named Lys1644, from a clinical S. dysgalactiae isolate by genome sequencing and bioinformatic analysis. Lys1644 was expressed and purified, and the lytic activity, antibacterial spectrum, optimal pH and temperature, lytic activity in milk in vitro, and synergistic bacteriostasis with antibiotics were assessed. The Lys1644 prophage lysin showed high bacteriolysis activity specifically on S. dysgalactiae, which resulted in CFU 100-fold reduction in milk. Moreover, Lys1644 maintained high activity over a wide pH range (pH 5-10) and a wide temperature range (4-42 °C). Synergistic bacteriostatic experiments showed that the combination of low-dose Lys1644 (50 μg/mL) with a subinhibitory concentration of aminoglycoside antibiotics (kanamycin or spectinomycin) can completely inhibit bacterial growth, suggesting that the combination of Lys1644 and antibiotics could be an effective therapeutic strategy against S. dysgalactiae infection.

Subinhibitory concentrations of glabridin from Glycyrrhiza glabra L. reduce Listeria monocytogenes motility and hemolytic activity but do not exhibit antimicrobial activity.

Increases in the virulence and survival of some pathogens in the presence of subinhibitory concentrations of antibiotics have been reported. However, research on the effects of subinhibitory concentrations of antimicrobial substances derived from traditional Chinese medicine on pathogens is still insufficient. Glabridin is a well-known active isoflavone found in licorice roots that possesses a wide range of biological activities. Therefore, in this study, Listeria monocytogenes (L. monocytogenes) exposed to subinhibitory concentrations of glabridin was used as the research object. The minimum inhibitory concentration (MIC) was determined for L. monocytogenes. We investigated the impacts of subinhibitory concentrations of glabridin on the morphology, motility, biofilm formation, adherence, and survival of L. monocytogenes. The results indicated that the MIC of glabridin for L. monocytogenes was 31.25 μg/mL. At 1/8, 1/4, or 1/2 of the MIC, glabridin did not affect the growth, morphology, flagellar production, or biofilm formation of L. monocytogenes. However, subinhibitory concentrations of glabridin inhibited bacterial swimming and swarming motility and decreased the hemolytic activity of L. monocytogenes. Glabridin reduced the hemolytic activity of L. monocytogenes culture supernatants. The results also showed that subinhibitory concentrations of glabridin had no toxic effect on RAW264.7 cells but decreased the intracellular growth of L. monocytogenes in RAW264.7 cells. Furthermore, subinhibitory concentrations of glabridin triggered ROS production but did not induce MET formation in macrophages. In addition, glabridin did not enhance the capacity of L. monocytogenes to trigger METs or the extracellular killing of macrophages by METs. Thus, we conclude that subinhibitory concentrations of glabridin reduce L. monocytogenes motility and hemolytic activity but do not exhibit antimicrobial activity. Glabridin could be an interesting food additive as a bacteriostatic agent with anti-Listeria activity.

Subminimum Inhibitory Concentrations Tetracycline Antibiotics Induce Biofilm Formation in Minocycline-Resistant Klebsiella pneumonia by Affecting Bacterial Physical and Chemical Properties and Associated Genes Expression.

Biofilm formation of Klebsiella pneumoniae can protect bacteria from antibiotics and is difficult to eradicate. Thus, the influence of subinhibitory concentrations of antibiotics on bacteria is becoming increasingly important. Our study showed that subminimum inhibitory concentrations (sub-MICs) of tetracycline antibiotics can increase biofilm formation in minocycline-resistant Klebsiella pneumoniae clinical strains. However, in the bacterial adhesion and invasion experiments, the adhesion and invasion ability decreased and the survival rate of Galleria mellonella increased. Under sub-MICs of tetracycline antibiotics treatment, abnormal stretching of bacteria was observed by scanning electron microscopy. Treatment with sub-MICs of tetracyclines leads to increased surface hydrophobicity and eDNA content and decreased outer membrane permeability. The expression levels of the fimA, luxS, qseB, and qseC genes decreased, the expression level of mrkA increased, and the expression level of acrA was inconsistent under different tetracycline antibiotics treatments. Together, our results suggested that the increase in Klebsiella pneumoniae biofilm formation caused by sub-MICs of tetracycline antibiotics may occur by affecting bacterial physical and chemical properties and associated genes expression.

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.

Subinhibitory concentrations of meropenem stimulate membrane vesicle production and modulate immune response in Bacteroides fragilis infection

This study explores an adaptation mechanism of Bacteroides fragilis to subinhibitory concentrations of meropenem, characterized by an alteration in the production of membrane vesicles (MVs) and modulation of the host inflammatory response. Using a rat model of infection, we demonstrated a significant increase in the size of MVs accompanied by a nonsignificant increase in their number in the meropenem-treated group compared to the infected control. Both infected groups showed significantly altered hematological parameters and shifts in monocyte on day 8 (average increase of 21.5%). At the same time, significant changes in neutrophils (decrease by 26%) and eosinophils (increase by 3%) were observed only in the infected group but not in the infected meropenem-treated group. On day 16, increased macrophage activation, neovascularization, and fibrosis were observed in the tissues of the antibiotic-treated group. Immunological profile analysis revealed a slight increase in the levels of pro-inflammatory cytokines (IL-5, IL-6, IFN-γ and G-CSF) on day 8 of the experiment, followed by a sharp decrease on day 16 in both infected groups compared to the negative control. At the same time, network analysis of correlations between these immunological factors showed complex changes in response to subinhibitory concentrations of meropenem. The bacterial load did not differ between the infected groups on days 8 and 16, but only in the meropenem-free group a significant decrease in the number of bacteria was observed on day 16 in all samples. These findings suggest that subinhibitory antibiotic concentrations can influence the pathophysiological progression of B. fragilis infection, modulating both the bacterial response and the host immune reaction, potentially leading to a more complex and chronic disease course.

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.