Subinhibitory Concentrations Of Gentamicin Research Articles

Gentamicin at sub-inhibitory concentrations selects for antibiotic resistance in the environment

Antibiotics released into the environment at low (sub-inhibitory) concentrations could select for antibiotic resistance that might disseminate to the human microbiome. In this case, low-level anthropogenic sources of antibiotics would have a significant impact on human health risk. In order to provide data necessary for the evaluation of this risk, we implemented river water microcosms at both sub-inhibitory and inhibitory concentrations of gentamicin as determined previously based on bacterial growth in enriched media. Using metagenomic sequencing and qPCR/RT-qPCR, we assessed the effects of gentamicin on water bacterial communities and their resistome. A change in the composition of total and active communities, as well as a gentamicin resistance gene selection identified via mobile genetic elements, was observed during a two-day exposure. We demonstrated the effects of sub-inhibitory concentrations of gentamicin on bacterial communities and their associated resistome in microcosms (simulating in situ conditions). In addition, we established relationships between antibiotic dose and the magnitude of the community response in the environment. The scope of resistance selection under sub-inhibitory concentrations of antibiotics and the mechanisms underlying this process might provide the basis for understanding resistance dispersion and associated risks in relatively low impacted ecosystems.

Sub-inhibitory concentrations of gentamicin triggers the expression of aac(6′)Ie-aph(2″)Ia, chaperons and biofilm related genes in Lactobacillus plantarum MCC 3011

The study aimed to analyze the effects of sub-inhibitory concentrations of gentamicin on the expressions of high level aminoglycoside resistant (HLAR) bifunctional aac(6′)Ie-aph(2″)Ia, biofilm and chaperone genes in Lactobacillus plantarum. The analysis of the biofilm formation in five isolates obtained from chicken sausages indicated their role in exhibiting phenotypic resistance based on the varied MIC values despite carrying the bifunctional gene. The biofilm formation significantly increased when L. plantarum MCC 3011 was grown in sub-inhibitory concentrations of gentamicin (4 μg/ml), kanamycin (8 μg/ml) and streptomycin (2 μg/ml). Thirty day gentamicin selection increased minimum inhibitory concentration (MIC) values from 4 to 64 and 2 to 256 fold for gentamicin and kanamycin, respectively when compared to the parental cultures. Expression studies revealed that constant exposure to gentamicin had induced chaperon [groEL] and the bifunctional gene, aac(6′)Ie-aph(2″)Ia upto nine fold. Induction of groEL, groES and lamC genes in gentamicin (4 μg/ml) preincubated MCC 3011 indicated their significant role in aminoglycoside mediated response. Our study indicates that constant exposure to sub inhibitory concentrations of gentamicin allows L. plantarum to adapt against higher doses of aminoglycosides. This highlights the risks and food safety issues associated with the use of aminoglycosides in livestock and consumption of farm oriented fermented food products.

Bactericidal Activity and Synergy Studies of Peptide AP-CECT7121 Against Multi-resistant Bacteria Isolated from Human and Animal Soft Tissue Infections.

AP-CECT7121 is an antimicrobial peptide, produced by Enterococcus faecalis CECT7121, with bactericidal activity against Gram-positive bacteria. The aim of this study was to evaluate the bactericidal activity of AP-CECT7121, alone and with gentamicin, against multi-resistant bacteria isolated from human and animals with soft tissue infections. During the period 2014-2015, bacterial strains producing human and animal soft tissue infections were studied. Samples from patients attended at a general hospital and cattle from four dairies in the Province of Buenos Aires (Argentina) were included. Twenty-two methicillin-resistant Staphylococcus aureus (11, human blood samples; 11, cow milk) and five vancomycin-resistant Ent. faecium strains isolated from four mastitic dairy cows were tested. AP-CECT7121 (12mg/L) potency was assessed by time-kill curves alone or with sub-inhibitory concentrations of gentamicin. All staphylococcal strains were susceptible to gentamicin; enterococci did not show high-level gentamicin resistance. Colony counts were carried out at 0, 2, 4, 8, and 24h of incubation. AP-CECT7121 showed bactericidal activity against all the enterococcal strains. In addition, AP-CECT7121 had a bactericidal effect on most staphylococci (16/22). Early AP-CECT7121/gentamicin synergy (4-8h) for all staphylococci was detected. At 24h, synergy (19/22) and indifference (3/22) were observed. Synergy with gentamicin was detected for staphylococci. AP-CECT7121 constitutes an attractive candidate for its use as a natural therapeutic tool for the treatment of infections produced by multi-resistant Staph. aureus and vancomycin-resistant Ent. faecium isolated from humans and animals.

Subinhibitory Concentrations of Azithromycin Decrease Nontypeable Haemophilus influenzae Biofilm Formation and Diminish Established Biofilms

Nontypeable Haemophilus influenzae (NTHi) commonly causes otitis media, chronic bronchitis in emphysema, and early airway infections in cystic fibrosis. Long-term, low-dose azithromycin has been shown to improve clinical outcomes in chronic lung diseases, although the mechanism of action remains unclear. The inhibition of bacterial biofilms by azithromycin has been postulated to be one mechanism mediating these effects. We hypothesized that subinhibitory concentrations of azithromycin would affect NTHi biofilm formation. Laboratory strains of NTHi expressing green fluorescent protein and azithromycin-resistant clinical isolates were grown in flow-cell and static-culture biofilm models. Using a range of concentrations of azithromycin and gentamicin, we measured the degree to which these antibiotics inhibited biofilm formation and persistence. Large biofilms formed over 2 to 4 days in a flow cell, displaying complex structures, including towers and channels. Subinhibitory concentrations of azithromycin significantly decreased biomass and maximal thickness in both forming and established NTHi biofilms. In contrast, subinhibitory concentrations of gentamicin had no effect on biofilm formation. Furthermore, established NTHi biofilms became resistant to gentamicin at concentrations far above the MIC. Biofilm formation of highly resistant clinical NTHi isolates (azithromycin MIC of > 64 microg/ml) was similarly decreased at subinhibitory azithromycin concentrations. Clinically obtainable azithromycin concentrations inhibited biofilms in all but the most highly resistant isolates. These data show that subinhibitory concentrations of azithromycin have antibiofilm properties, provide mechanistic insights, and supply an additional rationale for the use of azithromycin in chronic biofilm infections involving H. influenzae.

The Lon protease of Pseudomonas aeruginosa is induced by aminoglycosides and is involved in biofilm formation and motility

Pseudomonas aeruginosa is an important nosocomial opportunistic human pathogen and a major cause of chronic lung infections in individuals with cystic fibrosis. Serious infections by this organism are often treated with a combination of aminoglycosides and semi-synthetic penicillins. Subinhibitory concentrations of antibiotics are now being recognized for their role in microbial persistence and the development of antimicrobial resistance, two very important clinical phenomena. An extensive screen of a P. aeruginosa PAO1 luciferase gene fusion library was performed to identify genes that were differentially regulated during exposure to subinhibitory gentamicin. It was demonstrated that subinhibitory concentrations of gentamicin and tobramycin induced a set of genes that are likely to affect the interaction of P. aeruginosa with host cells, including the gene encoding Lon protease, which is known to play a major role in protein quality control. Studies with a lon mutant compared to its parent and a complemented strain indicated that this protein was essential for biofilm formation and motility in P. aeruginosa.

Bactericidal activity and synergy studies of BAL9141, a novel pyrrolidinone-3-ylidenemethyl cephem, tested against streptococci, enterococci and methicillin-resistant staphylococci

BAL9141 has been reported to have inhibitory activity against methicillin-resistant Staphylococcus aureus (MRSA), many enterococci, and streptococci with various resistant patterns. BAL9141 potency was assessed by time-kill curves alone or with subinhibitory concentrations of gentamicin (MIC/4). BAL9141 exhibited bactericidal activity alone against all the streptococci and staphylococci. Among ampicillin-susceptible enterococci, BAL9141 was bactericidal against some strains, but no BAL9141 inhibition was observed of ampicillin-resistant Enterococcus faecium. The activity of BAL9141 with gentamicin was slightly enhanced (not synergy) or indifferent against staphylococci. BAL9141 demonstrated bactericidal action alone against Enterococcus faecalis and some E. faecium strains (-4.8 to -6.0 log10 CFU/mL), but static effects were also noted. Drug interactions with gentamicin showed early synergy (4-8 h) for all enterococci, and indifference or synergy at 24 h (no antagonism). BAL9141 (< or = 8 mg/L) showed promising bactericidal activity alone and synergy with gentamicin against some of the vancomycin-resistant enterococci tested.

Subinhibitory concentrations of gentamicin reduce production of listeriolysin, the main virulence factor of Listeria monocytogenes

The facultatively intracellular Gram-positive rod Listeria monocytogenes is the causative agent of severe infections in humans and animals, e.g. sepsis and meningoencephalitis. The treatment of choice is ampicillin in combination with gentamicin [1Jones EM MacGowan AP Antimicrobial chemotherapy of human infection due to Listeria monocytogenes.Eur J Clin Microbiol Infect Dis. 1995; 14: 165-175Crossref PubMed Scopus (99) Google Scholar]. Because of poor penetration through the blood-brain barrier and into the cytoplasm of cells where the listeriae multiply, these antibiotics are likely to reach concentrations below the levels obtained in the blood at the site of infection. Certain antibiotics are able to retard growth of bacteria at concentrations below the minimal inhibitory concentration assessed visually. In addition, some antibiotics are capable of selectively inhibiting the production of virulence factors by pathogenic bacteria [2Doss SA Tillotson GS Amyes SG Effect of sub-inhibitory concentrations of antibiotics on the virulence of Staphylococcus aureus.J Appl Bacteriol. 1993; 75: 123-128Crossref PubMed Scopus (31) Google Scholar]. Ampicillin has recently been shown to reduce the production of listeriolysin [3Nichterlein T Domann E Kretschmar M et al.Sub-inhibitory concentrations of β-lactams and other cell-wall antibiotics inhibit listeriolysin production by Listeria monocytogenes.Int J Antimicrob Agents. 1996; 7: 75-81Abstract Full Text PDF PubMed Scopus (17) Google Scholar]. Listeriolysin is an essential virulence factor of L. monocytogenes because it enables the bacterium to reach the cytoplasm of infected cells, and to start multiplication [4Portnoy DA Chakraborty T Goebel W Cossart P Molecular determinants of Listeria monocytogenes pathogenesis.Infect Immun. 1992; 60: 1263-1270Crossref PubMed Google Scholar]. Inhibition of the production of listeriolysin could also contribute to the therapeutic action of gentamicin on human listeriosis. Therefore, the effect of gentamicin in subinhibitory concentrations on the expression of this virulence factor was determined. This was done as previously described [3Nichterlein T Domann E Kretschmar M et al.Sub-inhibitory concentrations of β-lactams and other cell-wall antibiotics inhibit listeriolysin production by Listeria monocytogenes.Int J Antimicrob Agents. 1996; 7: 75-81Abstract Full Text PDF PubMed Scopus (17) Google Scholar], using an assay measuring the hemolytic activity in the culture supernatants of L. monocytogenes EGD with and without the plasmids containing the β-galactosidase fusions and measuring the activity of the listeriolysin promoter contained on a plasmid fused to the lacZ gene of Escherichia coli. This allowed the measurement of the activity of the listeriolysin promoter as β-galactosidase activity in the transformed strain. The expression of β-galactosidase from this promoter was compared with that of a plasmid contained in L. monocytogenes EGD harboring the promoter of a streptococcal protease fused at the same EcoRI site to the lacZ gene. Subinhibitory concentrations of gentamicin were able to suppress the expression of β-galactosidase from the listeriolysin promoter in concentrations that did not affect growth or the expression of β-galactosidase from the streptococcal promoter (Figure 1). Therefore, gentamicin is able to selectively suppress the production of this virulence factor. Because gentamicin inhibits bacterial protein synthesis by binding to the ribosome, this action might be due to a selective suppression of the translation of mRNAs deriving from the virulence gene itself or from the regulatory gene prfA, whose product regulates the production of listeriolysin and several other virulence factors of L. monocytogenes [4Portnoy DA Chakraborty T Goebel W Cossart P Molecular determinants of Listeria monocytogenes pathogenesis.Infect Immun. 1992; 60: 1263-1270Crossref PubMed Google Scholar]. In contrast to the β-galactosidase assay, the reduction of the hemolytic activity of culture supernatants of the wild-type strains and the transformed strains was not significant. This may be explained by the fact that in contrast to β-galactosidase, listeriolysin is rapidly inactivated at the incubation temperature of 37 °C ([5Geoffroy C Gaillard J-L Alouf JE Berche P Production of thiol-dependent haemolysins by Listeria monocytogenes and related species.J Gen Microbiol. 1989; 135: 481-487PubMed Google Scholar] and own unpublished data), which may have led to greater standard deviations in this assay that make it difficult to demonstrate minor effects on listeriolysin production. In conclusion, subinhibitory concentrations of gentamicin that do not affect growth are able to reduce the production of an essential virulence factor of L. monocytogenes. In combination with a similar effect of ampicillin [3Nichterlein T Domann E Kretschmar M et al.Sub-inhibitory concentrations of β-lactams and other cell-wall antibiotics inhibit listeriolysin production by Listeria monocytogenes.Int J Antimicrob Agents. 1996; 7: 75-81Abstract Full Text PDF PubMed Scopus (17) Google Scholar], this might contribute to the therapeutic action of the two antibiotics in human listeriosis.

Bactericidal Activity of Tigemonam, Alone and in Combination with Gentamicin.

The in vitro bactericidal activity of tigemonam, alone and in combination with gentamicin, was evaluated against various strains of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella enteritidis, Enterobacter cloacae, Proteus mirabilis, and Proteus vulgaris. Minimum inhibitory and bactericidal concentrations were determined on three consecutive days with an inoculum of 105 colony-forming units (CFU)/mL or a culture in the logarithmic growth phase. The results found tigemonam to be rapidly bactericidal against most Enterobacteriaceae isolated. In addition, the killing curves indicated a synergism between tigemonam and subinhibitory concentrations of gentamicin.

Tolerant Staphylococcus aureus Causing Vertebral Osteomyelitis

Vertebral osteomyelitis due to Staphylococcus aureus was suppressed by not cured by optimal therapy with nafcillin sodium; cure eventually was achieved by treatment with cefazolin sodium and gentamicin sulfate. This is vivo result correlated with in vitro observations that showed that the infecting organism was inhibited but not killed by prolonged incubation with nafcillin or cefazolin; killing was readily achieved in vitro by adding subinhibitory concentrations of gentamicin. Bacterial tolerance in this case appeared to be responsible for the failure of vertebral osteomyelitis to be cured by accepted therapy with beta-lactam antibiotics.