sub-MIC Of Antibiotics Research Articles

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

Effects of sub-inhibitory concentration of antibiotic and heat stress on the expression of type II TA system genes in Brucella spp.

ObjectiveBacteria can react to stress conditions using the Toxin-Antitoxin (TA) system. This study investigated the expression of TA system genes under heat and antibiotic stresses in Brucella spp. MethodsTo determine the effects of sub-inhibitory concentration (sub-MIC) of rifampin on bacterial survival and growth, a colony-forming unit was quantitated, and turbidity was assessed following the treatment of Brucella isolates, with ½ minimum inhibitory concentration (MIC) of antibiotic at different time intervals. Also, Brucella isolates were exposed to heat stress (42 °C) compared to the control (37 °C). Finally, the expression of TA system genes in Brucella isolates was evaluated one hour after treatment using the quantitative reverse transcription PCR (qRT-PCR) method. ResultsOur results showed that the growth of the Brucella isolates reduced in the presence of the sub-MIC of antibiotics compared to the control. The results of the qPCR assay showed that, in the presence of rifampicin the expression of the TA system genes increased and, under the heat stress conditions, the expression of the TA system genes increased compared to controls expect brnT / brnA system. ConclusionAlthough the exact role of the TA system in response to various stresses is not yet fully understood, our study provided information on the effectiveness of the type II TA system under heat and antibiotic stress conditions by examining the gene expression of type II systems in Brucella isolates.

Effects of sub-inhibitory concentration of antibiotic and heat stress on the expression of type II TA system genes in Brucella spp.

ObjectiveBacteria can react to stress conditions using the Toxin-Antitoxin (TA) system. This study investigated the expression of TA system genes under heat and antibiotic stresses in Brucella spp. MethodsTo determine the effects of sub-inhibitory concentration (sub-MIC) of rifampin on bacterial survival and growth, a colony-forming unit was quantitated, and turbidity was assessed following the treatment of Brucella isolates, with ½ minimum inhibitory concentration (MIC) of antibiotic at different time intervals. Also, Brucella isolates were exposed to heat stress (42 °C) compared to the control (37 °C). Finally, the expression of TA system genes in Brucella isolates was evaluated one hour after treatment using the quantitative reverse transcription PCR (qRT-PCR) method. ResultsOur results showed that the growth of the Brucella isolates reduced in the presence of the sub-MIC of antibiotics compared to the control. The results of the qPCR assay showed that, in the presence of rifampicin the expression of the TA system genes increased and, under the heat stress conditions, the expression of the TA system genes increased compared to controls expect brnT / brnA system. ConclusionAlthough the exact role of the TA system in response to various stresses is not yet fully understood, our study provided information on the effectiveness of the type II TA system under heat and antibiotic stress conditions by examining the gene expression of type II systems in Brucella isolates.

The issue beyond resistance: Methicillin-resistant Staphylococcus epidermidis biofilm formation is induced by subinhibitory concentrations of cloxacillin, cefazolin, and clindamycin.

Staphylococcus epidermis is one of the most frequent causes of device-associated infections due to biofilm formation. Current reports noted that subinhibitory concentrations of antibiotics induce biofilm production in some bacteria. Accordingly, we evaluated the effect of exposure of different subinhibitory concentrations of cloxacillin, cefazolin, clindamycin, and vancomycin on the biofilm formation of methicillin-resistant S. epidermidis (MRSE). Antimicrobial susceptibility testing and minimum inhibitory/bactericidal concentration of antimicrobial agents were determined. MRSE isolates were selected, and their biofilm formation ability was evaluated. The effect of subinhibitory concentrations of cloxacillin, cefazolin, clindamycin, and vancomycin, antibiotics selected among common choices in the clinic, on MRSE biofilm formation was determined by the microtitre method. Besides, the effect of subinhibitory concentrations of cloxacillin, cefazolin, clindamycin, and vancomycin on the expression of the biofilm-associated genes icaA and atlE was evaluated by Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR). Antimicrobial susceptibility patterns of MRSE strains showed a high level of resistance as follows: 80%, 53.3%, 33.3%, 33.3%, and 26.6%, for erythromycin, trimethoprim-sulfamethoxazole, tetracycline, clindamycin, and gentamicin, respectively. Besides, 73.3% of S. epidermidis strains were Multidrug-resistant (MDR). Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were in the range of 0.5 to512 μg/mL and 1 to1024 μg/mL for cloxacillin, 0.125 to256 μg/mL and 1 to512 μg/mL for cefazolin, 0.125 to64 μg/mL and 4 to&gt;1024 μg/mL for clindamycin, and 2 to32 μg/mL and 4 to32 μg/mL for vancomycin, respectively. The findings showed that subinhibitory concentrations of cloxacillin, cefazolin, and clindamycin induce biofilm production in MRSE strains. In particular, the OD values of strains were in the range of 0.09-0.95, 0.05-0.86, and 0.06-1 toward cloxacillin, cefazolin, and clindamycin, respectively. On the other hand, exposure to subinhibitory vancomycin concentrations did not increase the biofilm formation in MRSE strains. The findings also demonstrated that sub-MIC of antibiotics up-regulated biofilm-associated genes. In particular, atlE and icaA were up-regulated 0.062 to 1.16 and 0.078 to 1.48 folds, respectively, for cloxacillin, 0.11 to 0.8, and 0.1 to 1.3 folds for cefazolin, 0.18 to 0.98, and 0.19 to 1.4 folds, respectively, for clindamycin. In contrast, the results showed that sub-MIC of vancomycin did not increase the biofilm-associated genes. These findings overall show that exposure to sub-MIC of traditional antibiotics can cause biofilm induction in MRSE, thereby increasing the survival and persistence on various surfaces that worsen the condition of comorbid infections.

Autoinducer-2 influences tetracycline resistance in Streptococcus suis by regulating the tet(M) gene via transposon Tn916

The concern over increasing resistance to tetracyclines (TCs), such as tetracycline and chlortetracycline, necessitates exploration of new approaches to combating infection in antimicrobial therapy. Given that bacteria use the chemical language of autoinducer 2 (AI-2) signaling molecules in order to communicate and regulate group behaviors, we asked whether the AI-2 signaling influence the tetracyclines antibiotics susceptibility in S. suis. Our present work demonstrated that MIC increased when exogenous AI-2 was added, when compared to the wild type strain. When grown in the presence of sub-MIC of antibiotics, it has been shown that exogenous AI-2 increases growth rate and biofilm formation. These results suggest that the TCs resistance in S. suis could involve a signaling mechanism. Base on the above observations, transcriptomic analyses showed significant differences in the expression of tet(M) of tetracyclines resistance genes, as well as differences in Tn916 transposon related genes transcription, as judged by RT-PCR. Our results provide strong evidence that AI-2 signaling molecules is may involve in TCs antibiotic resistance in S. suis by regulating tet(M) gene via Tn916 transposon. This study may suggest that targeting AI-2 signaling in bacteria could represent an alternative approach in antimicrobial therapy.

LuxS/AI-2 system is involved in fluoroquinolones susceptibility in Streptococcus suis through overexpression of efflux pump SatAB

Increasing resistance to fluoroquinolones (FQs), such as norfloxacin and enrofloxacin, supports the need for the discovery of novel molecules and alternative approaches in antimicrobial therapy. Quorum sensing (QS) is a promising target for next-generation anti-infective agents designed to address the evolving drug resistance in bacterial pathogens. Given that the LuxS/autoinducer-2 (AI-2) quorum-sensing system regulates microbial group behaviors, we hypothesized that this system influences the FQ susceptibility in Streptococcus suis. It was found that a luxS mutant (ΔluxS) of S. suis possesses an increased susceptibility to FQs compared to the wild type strain. When grown in the presence of sub-MIC of antibiotics, the ΔluxS strain showed a significant decrease in growth rate and biofilm formation. These results suggest that the FQ resistance in S. suis could involve a signaling mechanism associated with the LuxS/AI-2 quorum-sensing system. HPLC (High Performance Liquid Chromatography) analyses showed a significant increase in the intracellular accumulation of enrofloxacin in the ΔluxS strain compared to the wild type strain. This increase was less pronounced in the presence of exogenous AI-2. Moreover, the expression of satA and satB genes was decreased in the ΔluxS strain. Exogenous AI-2 reversed the down-regulated gene expression observed in the ΔluxS strain. Our study brought strong evidence that the LuxS/AI-2 system in S. suis is involved in FQ susceptibility by regulating the efflux pump SatAB. LuxS is highly conserved among Gram-positive bacteria and may therefore represent a novel antimicrobial target for an alternative approach in antimicrobial therapy.

Effects of sub-minimal inhibitory concentrations of antibiotics on the morphology and surface hydrophobicity of periodontopathic anaerobes

It has been reported that sub-minimal inhibitory concentrations (sub-MICs) of antibiotics are capable of altering bacterial surface properties and phenotype. In this study, the effects of sub-MICs of certain antibiotics on surface hydrophobicity, cell morphology, and protein profile were ascertained using Fusobacterium nucleatum, Porphyromonas gingivalis and Treponema denticola strains, which are pathogenic bacterial species in periodontal diseases. The MICs of antibiotics were determined by culturing bacteria in media supplemented with serially diluted antibiotic solutions, and sub-MIC of antibiotics was used. The effect of sub-MIC of antibiotics on cell morphology was determined by scanning electron microscopy. Microscopic observation of F. nucleatum and P. gingivalis grown at a sub-MIC of amoxicillin revealed cell enlargement. T. denticola grown at a sub-MIC of doxycycline also showed cell elongation. The relative surface hydrophobicity determined by measuring the ability of the bacteria to absorb n-hexadecane revealed an increase in surface hydrophobicity of F. nucleatum grown at sub-MIC of penicillin and amoxicillin, but a decrease with metronidazole; whereas increased hydrophobicity was observed in T. denticola grown at sub-MIC of doxycycline, metronidazole and tetracycline. The surface hydrophobicity of P. gingivalis increased only when grown in sub-MIC of metronidazole. The protein expression profile of the treated bacteria differed from their respective controls. These results confirmed that sub-MIC concentrations of antibiotics can affect the phenotype, surface properties and morphology of periodontal pathogenic anaerobic bacteria.

Sub-inhibitory concentrations of fluoroquinolones increase conjugation frequency

Bacteria are subjected to sub-minimal inhibitory concentrations (sub-MIC) of antibiotics in various niches where the low-dosage treatment plays a key role in antibiotic resistance selection. However, the mechanism of sub-MIC of antibiotics on the resistant gene transfer is largely unknown. Here, we used Escherichia coli SM10λpir in which the RP4 plasmid was chromosomally-integrated as the donor strain, to investigate the effects of sub-MIC of Ciprofloxacin(Cip) or Levofloxacin(Lev) on conjugational transfer of mobilisable plasmid-pUCP24T from SM10λpir to Pseudomonas aeruginosa. The results showed that the transfer frequency was significantly increased by treating E. coli with sub-MIC of Cip or Lev. To investigate the molecular mechanisms, complete transcriptome sequencing was performed. We found that the sub-MIC of Cip or Lev enhanced the expression of several genes on the RP4 plasmid, which was consistent with the conjugation efficiency. Moreover, the expression of genes associated with SOS response in donor SM10λpir was increased, but had no correlation with conjugation efficiency. These findings suggested that sub-MIC of Cip or Lev may promote conjugational transfer by up-regulating the expression of conjugation associated genes via an SOS-independent mechanism.

Toxin production of Clostridium difficile in sub-MIC of vancomycin and clindamycin alone and in combination with ceftazidime.

Toxin production in Clostridium difficile (C.difficile) is a key process for induction of diarrhea. Several factors such as sub-MIC of antibiotics impact on toxin production. The aim of this research is investigation of sub-minimum inhibitory concentration (sub-MIC) of vancomycin (VAN), clindamycin (CLI) separately and in combination with ceftazidime (CAZ) on toxin production in C.difficile. About ∼106 colony forming units (CFU) from 18-h culture of C.difficile ATCC 9689 and clinical isolates A+/B+/CTD-, were cultured anaerobically in the pre-reduced medium with appropriate concentration of separated and in combination antibiotics. After 24 and 48h, 1mL of culture was removed, centrifuged and the supernatant stored at-70°C for later use. The evaluation of toxin production was carried out by the ELISA technique. All antibiotics alone and in combination formats inhibited toxin production over a period of 24h. This effect is also observed in presence of VAN and CLI during a period of 48h. Over a 4h period, CAZ increased toxin production alone and in combination, especially with CLI. The data showed VAN and CLI decrease the level of toxins. In contrast, the CAZ not only increases the level of produced toxin, but also can interfere with VAN and CLI. Based on the results, combination therapy which is performed for treatment or prevention of other infections may cause toxin production and probably the severity of C.difficile AAD to increase.

Identification of Factors Associated with Biofilm Formation Ability in the Clinical Isolates of Helicobacter pylori.

Background A few reports confirm the ability of Helicobacter pylori to form biofilm. However, conclusive data do not exist concerning the factors that favor this ability. Objectives Evaluation of the factors associated with the biofilm formation ability of H. pylori including bacterial, physical and chemical, and environmental factors was the research’s aim. Materials and Methods H. pylori isolates from gastric biopsy specimens of patients infected chronically were screened for biofilm formation ability. Association of bacterial properties such as motility, auto-aggregation, cell hydrophobicity, and extracellular polymeric substances (EPS) with in vitro biofilm formation ability of H. pylori was evaluated. The effects of environmental factors such as growth-medium, temperature, oxygen-tension, pH, β-cyclodextrin, gastric secreted mucins, and sub-inhibitory concentration of amoxicillin were also evaluated. Results Ability of clinical H. pylori isolates to form biofilm in was quantitatively compared. The coccoid shape H. pylori cells were observed by scanning electron microscopy, the images were illustrative of the attachment of cells to form microcolony. The levels of hydrophobicity, motility and auto aggregation of two isolates with highest and lowest biofilm formation ability were the same. However, the signifi cant role of mucins (P < 0.05) in elevating the biofilm formation was observed. Other factors influencing biofilm formation were: pH, atmosphere and sub-MIC of antibiotics. Conclusion Mucins have a signifi cant role in elevating the biofilm formation, also pH, atmosphere and sub-MIC of antibiotics influence biofilm formation.

Sub-MIC of antibiotics induced biofilm formation of Pseudomonas aeruginosa in the presence of chlorhexidine.

Public health is facing a new challenge due to the alarming increase in bacterial resistance to most of the conventional antibacterial agents. It has been found that only minor cell damage is caused when exposed to sub-lethal levels of antimicrobial. Biofilms can play an important role in producing resistance, which is developed to reservoirs of pathogens in the hospital and cannot be easily removed. The aim of this study was to test whether the sub-lethal dose of antibiotics can induce biofilm formation of P. aeruginosa following incubating in the presence and absence of chlorhexidine. Standard antibiotic-micro broth 96-flat well plates were used for determination of MIC and biofilm assay. The adherence degree of biofilm was determined by estimation of OD 630 nm values using ELISA reader. The mean 22 isolates of P. aeruginosa growing in culture with presence and absence of chlorhexidine, could exhibited the significant (p < 0.001) proportion of adherence followed incubation in sub minimal inhibitory concentrations (Sub-MIC) of cefotaxim, amoxicillin, and azithromycin in comparison with control (antibiotic-free broth), while the sub-MIC of ciprofloxacin revealed significant inhibition of biofilm. Conclusion: Incubating the isolates of P. aeruginosa to sub-MIC of antibiotics exhibited induction of biofilm in the presence of chlorhexidine.

Effect of Sub-Minimal Inhibitory Concentration Antibiotics on Morphology of Periodontal Pathogens

Minimal inhibitory concentration (MIC) is the lowest concentration of antibiotics that inhibits the visible growth of a microorganism. It has been reported that sub-MIC of antibiotics may result in morphological alterations along with biochemical and physiological changes in bacteria. The purpose of this study was to examine morphological changes of periodontal pathogens after treatment with sub-MIC antibiotics. Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, and Porphyromonas gingivalis were used in this study. The MIC for amoxicillin, doxycycline, metronidazole, penicillin, and tetracycline were determined by broth dilution method. The bacterial morphology was observed with bright field microscope after incubating with sub-MIC antibiotics. The length of A. actinomycetemcomitans and F. nucleatum were increased after incubation with metronidazole; penicillin and amoxicillin. P. gingivalis were increased after incubating with metronidazole and penicillin. However, F. nucleatum showed decreased length after incubation with doxycycline and tetracycline. In this study, we observed that sub-MIC antibiotics can affect the morphology of periodontal pathogens.

Binding of complement C3 to Klebsiella pneumoniae treated with sub-MIC cefodizime and chemiluminescence response of human polymorphonuclear leukocytes.

The binding of complement C3 to the cell surface of Klebsiella pneumoniae exposed to human serum complement after treatment with or without sub-MIC of antibiotics was examined by double diffusion immunoprecipitation against anti-human complement C3, and the production of oxygen-derived radicals by human polymorphonuclear leukocytes stimulated by complement-opsonized K. pneumoniae after treatment with or without sub-MIC of antibiotics was measured using the chemiluminescence (CL) assay. Complement C3 bound to the cell surface of K. pneumoniae treated with cefodizime was detected after exposure to human serum complement. The CL response induced by complement-opsonized bacteria after treatment with cefodizime was much higher than the response induced by nontreated bacteria or complement-opsonized bacteria after treatment with other antibiotics. These findings indicate that treatment with sub-MIC cefodizime make K. pneumoniae more susceptible to opsonization by complement and promotes the specific phagocytosis mediated by complement receptors.