ABSTRACT
Background & objectives: Bacterial biofilms a multi-layered defence, comprise extracellular DNA (eDNA) and proteins, protect bacteria from harmful environment and nutrient limitation and utilize the mutual benefits within a community. Bacterial biofilms also defend bacteria from harsh environments such as antibiotic treatment. This leads to poor antibiotic penetration, slow growth, adaptive stress responses, and formation of persister cells. This study was done to determine the relation of antibiotic resistance deciphered by the biofilms in Lactobacillus plantarum, a lactic acid bacteria (LAB) with probiotic significance. Methods: The gentamicin-resistant L. plantarum isolates were allowed to form biofilms and subjected to DNase I and proteinase K treatment. The optical density (OD) values were recorded for the biofilm assay and the cell count for the number of viable cells was taken for the control and the test samples. Percentage reduction was calculated based on the difference between the initial and final OD for both the parameters. Results: The biofilm assay revealed that the native L. plantarum isolates which were phenotypically susceptible, possessed the ability to form biofilms. The OD values were significantly decreased in comparison to the biofilm-forming control culture when these were treated with DNase I and proteinase K. Interpretation & conclusions: The study revealed that the biofilms formed by L. plantarum comprised of eDNA and proteins which was evidenced by the reduction in OD values and percentage in comparison to the control upon DNase I and proteinase K treatment. This indicates that the eDNA and biofilm matrix proteins are vital constituents of biofilms and may carry significant risk when coupled with antibiotic resistance.
ABSTRACT
En este trabajo se evaluó el efecto de las bacterias persistentes presentes en un inóculo de alta densidad de una cepa autóctona de Escherichia coli sobre la eficacia de enrofloxacina y ciprofloxacina mediante ensayos in vitro de curvas de muerte bacteriana y de determinación de la concentración preventiva de mutantes. En las curvas de muerte realizadas sobre inóculos de alta densidad, ningún antibiótico presentó actividad bactericida y solo permitieron la sobrevida de bacterias persistentes. En el ensayo para determinar la concentración preventiva de mutantes, sobre la superficie del agar de las placas con elevadas concentraciones de enrofloxacina y ciprofloxacina, las bacterias persistentes permanecieron viables sin desarrollar colonias y adoptando morfología filamentosa como una forma de adaptación y supervivencia. Se discute la utilidad clínica de las concentraciones preventivas de mutantes de enrofloxacina y ciprofloxacina sobre E. coli ya que, estas elevadas concentraciones permitirían la sobrevida de una sub-población de bacterias persistentes originando un reservorio biológico que podría dar origen a infecciones crónicas y a favorecer la emergencia de mutantes resistentes.
This work evaluated the effect of persister cells present in a high inocula size of a wild strain of Escherichia coli on the efficacy of enrofloxacin and ciprofloxacin by in vitro time-kill curve assays and mutant prevention concentration testing. In time-kill curves performed with high inocula size, no antibiotics showed bactericidal activity, but only allowed the survival of persister cells. In the assay to determine the mutant prevention concentration, on the surface of agar plates containing high enrofloxacin and ciprofloxacin concentrations, persister cells remained viable and without bacterial colonies development and adopting filamentous morphology as a form of adaptation and survival. The clinical usefulness of mutant prevention concentrations of enrofloxacin and ciproflocxacin against Escherichia coli is discussed, as these high concentrations would allow the survival of a sub-population of persister cells originating a biological reservoir that could give rise to chronic infections and favor the emergence of resistant mutants.
ABSTRACT
En este trabajo se evaluó in vitro: (i) el efecto del pH sobre la actividad bactericida de ciprofloxacina (CFX) frente a una cepa autóctona de Escherichia coli y (ii) el efecto de las bacterias persistentes sobre el modo de acción concentración dependiente de CFX. La actividad antibacteriana de CFX disminuyó a causa del descenso del pH, por lo que los valores de concentración inhibitoria mínima (CIM), concentración bactericida mínima (CBM) y concentración de erradicación bacteriana mínima (CEBM) se incrementaron cuando el pH del medio de cultivo descendió de 7,4 a valores de 6,5 y 5,5. La cinética de eliminación bacteriana de CFX fue bifásica a causa de la selección de una sub-población de bacterias persistentes que presentaron una velocidad de eliminación más lenta. Por lo tanto la actividad bactericida de CFX fue definida por su concentración en relación a la CIM y el tiempo durante el cual se mantuvo la exposición de las bacterias a ésta.
In this in vitro assay was evaluated: (i) the effect of pH on the bactericidal activity of ciprofloxacin (CFX) against a native strain of Escherichia coli, (ii) the effect of persister bacteria on the concentration-dependent mode of action of CFX. The antibacterial activity of CFX decreased with reductions of pH, so the values of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum eradication bacterial concentration (MEBC) were increased when the pH of the culture medium decreased from 7.4 to 6.5 and 5.5. The kinetics of bacterial elimination of CFX presented a biphasic pattern because of the selection of a sub-population of persistent bacteria which presented a slower elimination rate. Therefore, the antibacterial activity of CFX was determined by its concentration in reference to MIC values and the time during which the exposure of the bacteria was maintained.
ABSTRACT
Background & objectives: Available literature shows paucity of reports describing antibiotic and metal resistance profile of biofilm forming clinical isolates of Acinetobacter haemolyticus. The present study was undertaken to evaluate the antibiotic and metal resistance profile of Indian clinical isolate of A. haemolyticus MMC 8 isolated from human pus sample in planktonic and biofilm form. Methods: Antibiotic susceptibility and minimum inhibitory concentration were determined employing broth and agar dilution techniques. Biofilm formation was evaluated quantitatively by microtiter plate method and variation in complex architecture was determined by scanning electron microscopy. Minimum biofilm inhibiting concentration was checked by Calgary biofilm device. Results: Planktonic A. haemolyticus MMC 8 was sensitive to 14 antibiotics, AgNO3 and HgC12 resistant to streptomycin and intermediately resistant to netilmycin and kanamycin. MMC 8 exhibited temporal variation in amount and structure of biofilm. There was 32 – 4000 and 4 – 256 fold increase in antibiotic and metal salt concentration, respectively to inhibit biofilm over a period of 72 h as against susceptible planktonic counterparts. Total viable count in the range of 105 -106cfu / ml was observed on plating minimum biofilm inhibiting concentration on Muller-Hinton Agar plate without antimicrobial agents. Biofilm forming cells were several folds more resistant to antibiotics and metal salts in comparison to planktonic cells. Presence of unaffected residual cell population indicated presence of persister cells. Interpretation & conclusions: The results indicate that biofilm formation causes enhanced resistance against antibiotics and metal salts in otherwise susceptible planktonic A. haemolyticus MMC 8.
ABSTRACT
Persister cells are dormant or slowly grown variations in microbial populations .They are highly tolerant to antibiot-ics but the tolerance are not inherited as the genetic resistance , when persisters are inoculated to fresh medium , most bacteria are still susceptive to antibiotic , while only a small fractiont become persisters again .Persisters are believed to be closely related to clinic bio-film forming and the recurrence and recalcitrance of chronic infections .Different persisters have been found in Escherichia coli , Pseud-omonas aeruginosa , Staphyococcus aureus , Mycobacterium tuberculosis , Candida albicans and so on , and a few mechanisms about per-sisters formation have been studied .This article reviews the current progresses of research methods and achievements on persisters from different levels .