Subinhibitory Concentrations Of Antimicrobials Research Articles

Extended period of selection for antimicrobial resistance due to recirculation of persistent antimicrobials in broilers.

Antimicrobials can select for antimicrobial-resistant bacteria. After treatment the active compound is excreted through urine and faeces. As some antimicrobials are chemically stable, recirculation of subinhibitory concentrations of antimicrobials may occur due to coprophagic behaviour of animals such as chickens. The persistence of three antimicrobials over time and their potential effects on antimicrobial resistance were determined in four groups of broilers. Groups were left untreated (control) or were treated with amoxicillin (unstable), doxycycline or enrofloxacin (stable). Antimicrobials were extracted from the faecal samples and were measured by LC-MS/MS. We determined the resistome genotypically using shotgun metagenomics and phenotypically by using Escherichia coli as indicator microorganism. Up to 37 days after treatment, doxycycline and enrofloxacin had concentrations in faeces equal to or higher than the minimal selective concentration (MSC), in contrast to the amoxicillin treatment. The amoxicillin treatment showed a significant difference (P ≤ 0.01 and P ≤ 0.0001) in the genotypic resistance only directly after treatment. On the other hand, the doxycycline treatment showed approximately 52% increase in phenotypic resistance and a significant difference (P ≤ 0.05 and P ≤ 0.0001) in genotypic resistance throughout the trial. Furthermore, enrofloxacin treatment resulted in a complete non-WT E. coli population but the quantity of resistance genes was similar to the control group, likely because resistance is mediated by point mutations. Based on our findings, we suggest that persistence of antimicrobials should be taken into consideration in the assessment of priority classification of antimicrobials in livestock.

Assessing Helicobacter pylori motility and biofilm formation in subinhibitory concentrations of antimicrobials

Numerous studies have shown that subinhibitory concentrations of antimicrobials can alter bacterial virulence factors. This study evaluates motility and biofilm formation by H. pylori 43504 grown in subinhibitory concentrations of amoxicillin (AMX), clarithromycin (CLA), or tetracycline (TET). For the swimming and swarming motility assays, H. pylori 43504 suspensions were prepared with the strain alone or with the strain in AMX, CLA, or TET at ½ MIC. Next, the media were incubated at 37 ºC, under microaerophilia. To assess biofilm formation in the presence of one of the antimicrobials at subinhibitory antimicrobial concentrations, bacterial suspensions (109 CFU/mL) were prepared in 2.5% FBS containing AMX, CLA, or TET at ½ MIC. After incubation for 10 days, H. pylori 43504 grown in medium containing AMX, CLA, or TET at ½ MIC presented greater swimming motility and lower swarming motility than the non-treated strain. H. pylori 43504 grown in medium containing AMX, CLA, or TET at ½ MIC showed stronger biofilm production than the non-treated strain. Our results showed that AMX, CLA, or TET at subinhibitory concentrations favors H. pylori 43504 swimming motility and biofilm formation after incubation for 3 days. This may have clinical consequences and make the microorganism difficult to eradicate.

Influence of sub-inhibitory concentrations of antimicrobials on micrococcal nuclease and biofilm formation in Staphylococcus aureus

A major contributor to biomaterial associated infection (BAI) is Staphylococcus aureus. This pathogen produces a protective biofilm, making eradication difficult. Biofilms are composed of bacteria encapsulated in a matrix of extracellular polymeric substances (EPS) comprising polysaccharides, proteins and extracellular DNA (eDNA). S. aureus also produces micrococcal nuclease (MN), an endonuclease which contributes to biofilm composition and dispersion, mainly expressed by nuc1. MN expression can be modulated by sub-minimum inhibitory concentrations of antimicrobials. We investigated the relation between the biofilm and MN expression and the impact of the application of antimicrobial pressure on this relation. Planktonic and biofilm cultures of three S. aureus strains, including a nuc1 deficient strain, were cultured under antimicrobial pressure. Results do not confirm earlier findings that MN directly influences total biomass of the biofilm but indicated that nuc1 deletion stimulates the polysaccharide production per CFU in the biofilm in in vitro biofilms. Though antimicrobial pressure of certain antibiotics resulted in significantly increased quantities of polysaccharides per CFU, this did not coincide with significantly reduced MN activity. Erythromycin and resveratrol significantly reduced MN production per CFU but did not affect total biomass or biomass/CFU. Reduction of MN production may assist in the eradication of biofilms by the host immune system in clinical situations.

Emerging Variants of the Integrative and Conjugant Element ICEMh1 in Livestock Pathogens: Structural Insights, Potential Host Range, and Implications for Bacterial Fitness and Antimicrobial Therapy.

Horizontal gene transfer of integrative and conjugative elements (ICE) in bacterial pathogens of the bovine respiratory disease (BRD) complex has emerged as a significant cause of antimicrobial resistance (AMR) and therapeutic failure and mortalities in cattle. The aim of this study was to assess an AMR ICE occurring in Pasteurella multocida from a case of BRD, designated ICEMh1PM22 for its structure and host genome insertion site, and to identify consequences for host fitness and antimicrobial therapy. The modular structure of ICEMh1-like elements found in several related livestock pathogens was compared to ICEMh1PM22, and the repertoire of cargo genes in variable ICE modules was functionally categorized. AMR genes were identified as frequent additions to the variable modules of ICEMh1-like elements. Random PCR-based mapping of ICEMh1PM22-genome junctions in transconjugants provided evidence that ICEMh1PM22 integrates into the tRNA-leu for the UUG codon, and not into tRNA-leu for other codons. This was separately confirmed in the genomes of ICEMh1-like-harboring livestock pathogens. Bacterial genera harboring receptive tRNA-leuUUG were identified to establish the potential host range of ICEMh1-like elements. ICEMh1PM22-carrying transconjugants in P. multocida and Mannheimia haemolytica were less fit than isogenic strains without the ICE when grown without antimicrobial selection. This fitness cost was abrogated in the presence of subinhibitory concentrations of antimicrobials. Despite this cost, ICEMh1PM22 was retained in transconjugants in extended culture. To identify possible therapeutic efficiencies, antimicrobial combinations were screened for synergistic interactions against AMR ICEMh1PM22-carrying transconjugants. No antimicrobial combination tested exhibited synergistic interactions against AMR P. multocida or M. haemolytica harboring ICEMh1PM22. In conclusion, this study provided information on the structural variation of ICEMh1-like elements, refined the ICE insertion site and potential host range, and demonstrated the risk and consequences for AMR following horizontal transfer of ICE into BRD pathogens.

Resistance gene transfer: induction of transducing phage by sub-inhibitory concentrations of antimicrobials is not correlated to induction of lytic phage.

Objectives: Horizontal gene transfer of antimicrobial resistance (AMR) genes between clinical isolates via transduction is poorly understood. MRSA are opportunistic pathogens resistant to all classes of antimicrobial agents but currently no strains are fully drug resistant. AMR gene transfer between Staphylococcus aureus isolates is predominantly due to generalized transduction via endogenous bacteriophage, and recent studies have suggested transfer is elevated during host colonization. The aim was to investigate whether exposure to sub-MIC concentrations of antimicrobials triggers bacteriophage induction and/or increased efficiency of AMR gene transfer. Methods: Isolates from MRSA carriers were exposed to nine antimicrobials and supernatants were compared for lytic phage particles and ability to transfer an AMR gene. A new technology, droplet digital PCR, was used to measure the concentration of genes in phage particles. Results: All antibiotics tested induced lytic phage and AMR gene transduction, although the ratio of transducing particles to lytic particles differed substantially for each antibiotic. Mupirocin induced the highest ratio of transducing versus lytic particles. Gentamicin and novobiocin reduced UV-induced AMR transduction. The genes carried in phage particles correlated with AMR transfer or lytic particle activity, suggesting antimicrobials influence which DNA sequences are packaged into phage particles. Conclusions: Sub-inhibitory antibiotics induce AMR gene transfer between clinical MRSA, while combination therapy with an inhibiting antibiotic could potentially alter AMR gene packaging into phage particles, reducing AMR transfer. In a continually evolving environment, pathogens have an advantage if they can transfer DNA while lowering the risk of lytic death.

Antibiotic stress selects against cooperation in the pathogenic bacterium Pseudomonas aeruginosa

Cheats are a pervasive threat to public goods production in natural and human communities, as they benefit from the commons without contributing to it. Although ecological antagonisms such as predation, parasitism, competition, and abiotic environmental stress play key roles in shaping population biology, it is unknown how such stresses generally affect the ability of cheats to undermine cooperation. We used theory and experiments to address this question in the pathogenic bacterium, Pseudomonas aeruginosa Although public goods producers were selected against in all populations, our competition experiments showed that antibiotics significantly increased the advantage of nonproducers. Moreover, the dominance of nonproducers in mixed cultures was associated with higher resistance to antibiotics than in either monoculture. Mathematical modeling indicates that accentuated costs to producer phenotypes underlie the observed patterns. Mathematical analysis further shows how these patterns should generalize to other taxa with public goods behaviors. Our findings suggest that explaining the maintenance of cooperative public goods behaviors in certain natural systems will be more challenging than previously thought. Our results also have specific implications for the control of pathogenic bacteria using antibiotics and for understanding natural bacterial ecosystems, where subinhibitory concentrations of antimicrobials frequently occur.

Bacteroides fragilis response to subinhibitory concentrations of antimicrobials includes different morphological, physiological and virulence patterns after in vitro selection

As antimicrobials are introduced into the environment, microorganisms may respond in different ways, sometimes displaying alterations in cellular physiology. Considering the clinical relevance of the Bacteroides fragilis, strains were selected to investigate bacterial response after exposure to subinhibitory concentrations (SIC) of ampicillin (AMP), ampicillin-sulbactam (AMS), clindamycin (CLI), chloramphenicol (CHL), and its relationship to a host model (BALB/c mice) after experimental challenge. Morphological alterations, and biochemical-physiological and genetic profiles were evaluated among drug-selected bacteria. Histopathological evaluation of the liver and spleen, and inflammatory cytokines were determined after bacterial infection in mice. AMP and AMS exposure were related to most significant cellular alterations. Decreased sensitivity to all antimicrobials was observed for all drug-selected bacteria. Down regulation in adherence properties were also observed. Spleen and liver alterations were observed in different patterns. Increased levels of TNF-α, IL-6 and IFN-γ were also observed. Our results show that SICs of AMP, AMS, CLI and CHL may be related to alterations in cell physiology in B. fragilis with implications to the host–bacteria relationship. The data emphasizes the risks of inappropriate chemotherapy, and the concerns regarding ecological consequences lead by SICs of antimicrobials in resident microbiota.

Biofilm formation on biotic and abiotic surfaces in the presence of antimicrobials by Escherichia coli Isolates from cases of bovine mastitis.

Escherichia coli is a highly adaptive microorganism, and its ability to form biofilms under certain conditions can be critical for antimicrobial resistance. The adhesion of four E. coli isolates from bovine mastitis to bovine mammary alveolar (MAC-T) cells, biofilm production on a polystyrene surface, and the expression profiles of the genes fliC, csgA, fimA, and luxS in the presence of enrofloxacin, gentamicin, co-trimoxazole, and ampicillin at half of the MIC were investigated. Increased adhesion of E. coli isolates in the presence of antimicrobials was not observed; however, increased internalization of some isolates was observed by confocal microscopy. All of the antimicrobials induced the formation of biofilms by at least one isolate, whereas enrofloxacin and co-trimoxazole decreased biofilm formation by at least one isolate. Quantitative PCR analysis revealed that all four genes were differentially expressed when bacteria were exposed to subinhibitory concentrations of antimicrobials, with expression altered on the order of 1.5- to 22-fold. However, it was not possible to associate gene expression with induction or reduction of biofilm formation in the presence of the antimicrobials. Taken together, the results demonstrate that antimicrobials could induce biofilm formation by some isolates, in addition to inducing MAC-T cell invasion, a situation that might occur in vivo, potentially resulting in a bacterial reservoir in the udder, which might explain some cases of persistent mastitis in herds.

Antibiotics and antibiotic resistance: A bitter fight against evolution

One of the most terrible consequences of Darwinian evolution is arguably the emergence and spread of antibiotic resistance, which is becoming a serious menace to modern societies. While spontaneous mutation, recombination and horizontal gene transfer are recognized as the main causes of this notorious phenomenon; recent research has raised awareness that sub-lethal concentrations of antibiotics can also foster resistance as an undesirable side-effect. They can produce genetic changes by different ways, including a raise of free radicals within the cell, induction of error-prone DNA-polymerases mediated by SOS response, imbalanced nucleotide metabolism or affect directly DNA. In addition to certain environmental conditions, subinhibitory concentrations of antimicrobials may increase, even more, the mutagenic effect of antibiotics. Here, we review the state of knowledge on antibiotics as promoters of antibiotic resistance.

Morphological, biochemical, physiological and molecular aspects of the response of Fusobacterium nucleatum exposed to subinhibitory concentrations of antimicrobials

Subinhibitory concentrations (SICs) of antimicrobials may result in alterations in bacterial biology with implications for its potential aggression. This has considerable importance for the resident microbiota. Our aim was to analyze the effects of SICs of antimicrobials on the morphological, biochemical, physiological and molecular characteristics of the resident anaerobic Fusobacterium nucleatum. Fourteen strains were obtained from F. nucleatum ATCC 25586, selected by culturing on SICs of ampicillin, ampicillin/sulbactam, clindamycin, chloramphenicol, levofloxacin, metronidazole and piperacillin/tazobactam and subsequent culturing in the absence of drugs. Antimicrobial susceptibility, bacterial morphology, biochemical profiles and biofilm formation were evaluated. Genotyping and analysis of protein profiles were also performed. The antimicrobial susceptibility patterns showed that most of the derived strains were less sensitive to the antimicrobials, even after culturing them without drugs. Morphological and cell complexity alterations were observed, mainly in strains grown in SICs of β-lactam; these strains also expressed a reduced ability for biofilm formation. The other strains showed an increase in biofilm formation but no apparent morphological changes. Alterations were observed in the carbohydrate metabolism patterns and in the activity of microbial enzymes. Several proteins were positively or negatively regulated and there was polymorphism in the DNA from all derived strains. Therefore, SICs of antimicrobials induce alterations in F. nucleatum, which directly impact its biology. These results emphasize the risk of inadequate antibioticotherapy, which may have serious implications for clinical microbiology and infectious diseases and also may interfere with the host–bacteria relationship.

Third Prize: Effects of Subinhibitory Concentrations of Ciprofloxacin on Staphylococcus Saprophyticus Adherence and Virulence in Urinary Tract Infections

Staphylococcus saprophyticus is a frequent cause of both uncomplicated and complicated urinary tract infections (UTI) in young females and has recently been established as the most prominent gram-positive uropathogen. Although the effects of subinhibitory concentrations of antimicrobials on numerous other pathogenic bacteria have been studied, little is known regarding how S saprophyticus responds under such conditions. In this study, we investigated the effects of subminimum inhibitory concentrations (MIC) of ciprofloxacin (CIP) on S saprophyticus attachment to glass microscope slides, ureteral stent material, and T24 bladder cells, as well as its effects on S saprophyticus-induced proinflammatory cytokine expression in bladder cells. Adherence to glass microscope slides, ureteral stent material, and bladder cell monolayers were all significantly increased in the presence of sub-MIC levels of CIP. While the S saprophyticus challenge of T24 bladder cell monolayers significantly upregulated both interleukin (IL)-6 and IL-8 expression, sub-MIC CIP abrogated these effects, returning their secretion to control levels. Our results demonstrate that exposure to sub-MIC CIP increases S saprophyticus adherence to both abiotic and biotic surfaces including urinary device material and cultured bladder cells. In addition, low levels of this antimicrobial downregulate S saprophyticus-stimulated proinflammatory cytokine secretion in the bladder. These changes may make S saprophyticus more effective at colonizing the urinary tract and highlights the need for clinicians to consider the impact of subinhibitory concentrations of antimicrobials on bacteria when designing treatment strategies to manage UTI.

Influence of subinhibitory concentrations of antimicrobials on hydrophobicity, adherence and ultra-structure of Fusobacterium nucleatum

Fusobacterium nucleatum is considered a bridge organism between earlier and later colonizers in dental biofilms and a putative periodontopathogen. In Dentistry, antimicrobial agents are used for treatment and control of infectious diseases associated with dental plaque. Antiseptics have been used in association with antibiotics to reduce infections after oral surgeries. In this study, the influence of subinhibitory concentrations (SC) of chlorhexidine, triclosan, penicillin G and metronidazole, on hydrophobicity, adherence to oral epithelial cells, and ultra-structure of F. nucleatum was examined. All isolates were susceptible to chlorhexidine, triclosan, and metronidazole; however, most of the isolates were susceptible to penicillin G, and all of them were hydrophilic when grown with or without antimicrobials. Adherence was decreased by all antimicrobials. Results suggest that adherence of F. nucleatum was influenced by adhesins because structures such as fimbries or capsule were not observed by transmission electronic microscope.

Safeguarding future antimicrobial options: strategies to minimize resistance

Current antimicrobial therapy for community-acquired respiratory tract infections (RTIs) is empirical and is influenced by local differences in etiology and bacterial susceptibility. As the rates of resistance and cross-resistance to currently available classes of antimicrobial agents increase, their effectiveness becomes compromised. These issues demand improved strategies for antimicrobial usage, and the development of new agents that do not select resistance are essential to safeguard future antimicrobial efficacy. Strategies to minimize antimicrobial resistance among common RTIs include reducing antimicrobial consumption and controlling the development and spread of resistance through appropriate prescribing and the use of short-duration, once-daily treatments to improve patient compliance. Importantly, the ketolides, which are a new family of antimicrobials, have been recently developed specifically for the treatment of community-acquired RTIs. The first member of this new family, telithromycin, has been shown to have potent activity against common and atypical respiratory pathogens, including beta-lactam- and macrolide-resistant strains, and has a low potential to select for or induce cross-resistance. These properties, combined with its good tolerability across patient groups, make telithromycin an attractive option for the first-line empiric treatment of RTIs with the potential to limit the future development of resistance.

Effect of subinhibitory concentrations of antimicrobials on meningococcal adherence.

Meningococci adhere to human pharyngeal cells and agglutinate erythrocytes. These events are dependent upon pili and are reduced by encapsulation. The effect of subinhibitory concentrations of seven antimicrobials on meningococcal adherence, antimicrobials on meningococcal adherence, piliation, hemagglutination (HA), and bacterial proteins was studied to determine their potential for modifying virulence. Piliation was reduced by most antibiotics but was most markedly (greater than 70%) reduced by rifampin, tobramycin, and VCN (vancomycin, colistin, and nystatin). Bacterial proteins as determined by sodium dodecyl sulphate--polyacrylamide gel electrophoresis were altered: tetracycline, sulfamethoxazole, rifampin, and VCN caused loss of a 43-45 K protein and a general decrease in all stainable protein bands, while erythromycin, ampicillin, and tobramycin only caused an increase in a 28 K protein. HA was reduced by ampicillin, tobramycin, erythromycin, and VCN but interstrain variability was present. Epithelial cell adherence was diminished by an average of 45% compared to controls. The meningococcal strains lost HA, piliation, and adherence in the same rank order, however, there was no significant rank correlation of antibiotic inhibitory activities on these parameters. These results indicate that subinhibitory antibiotic concentrations reduce meningococcal piliation and alter other bacterial proteins; these changes are associated with diminished adherence and hemagglutination, alterations which may be markers of meningococcal virulence.