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Antibiotic susceptibility of coagulase-negative staphylococci isolated from very low birth weight babies: comprehensive comparisons of bacteria at different stages of biofilm formation.

Qu Y, Daley AJ, Istivan TS, Garland SM, Deighton MA - Ann. Clin. Microbiol. Antimicrob. (2010)

Bottom Line: Epifluorescence microscopy and confocal laser scanning microscopy in combination with bacterial viability staining and polysaccharide staining were used to confirm the stimulatory effects of antibiotics on biofilms.We conclude that the resistance of coagulase-negative staphylococci to multiple antibiotics initially remain similar when the bacteria shift from a planktonic growth mode into an early attached mode, then increase significantly as the adherent mode further develops.Furthermore, preformed biofilms of some CoNS are enhanced by oxacillin in a dose-dependent manner.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Applied Sciences, RMIT University, Australia.

ABSTRACT

Background: Coagulase-negative staphylococci are major causes of bloodstream infections in very low birth weight babies cared for in Neonatal Intensive Care Units. The virulence of these bacteria is mainly due to their ability to form biofilms on indwelling medical devices. Biofilm-related infections often fail to respond to antibiotic chemotherapy guided by conventional antibiotic susceptibility tests.

Methods: Coagulase-negative staphylococcal blood culture isolates were grown in different phases relevant to biofilm formation: planktonic cells at mid-log phase, planktonic cells at stationary phase, adherent monolayers and mature biofilms and their susceptibilities to conventional antibiotics were assessed. The effects of oxacillin, gentamicin, and vancomycin on preformed biofilms, at the highest achievable serum concentrations were examined. Epifluorescence microscopy and confocal laser scanning microscopy in combination with bacterial viability staining and polysaccharide staining were used to confirm the stimulatory effects of antibiotics on biofilms.

Results: Most coagulase-negative staphylococcal clinical isolates were resistant to penicillin G (100%), gentamicin (83.3%) and oxacillin (91.7%) and susceptible to vancomycin (100%), ciprofloxacin (100%), and rifampicin (79.2%). Bacteria grown as adherent monolayers showed similar susceptibilities to their planktonic counterparts at mid-log phase. Isolates in a biofilm growth mode were more resistant to antibiotics than both planktonic cultures at mid-log phase and adherent monolayers; however they were equally resistant or less resistant than planktonic cells at stationary phase. Moreover, for some cell-wall active antibiotics, concentrations higher than conventional MICs were required to prevent the establishment of planktonic cultures from biofilms. Finally, the biofilm-growth of two S. capitis isolates could be enhanced by oxacillin at the highest achievable serum concentration.

Conclusion: We conclude that the resistance of coagulase-negative staphylococci to multiple antibiotics initially remain similar when the bacteria shift from a planktonic growth mode into an early attached mode, then increase significantly as the adherent mode further develops. Furthermore, preformed biofilms of some CoNS are enhanced by oxacillin in a dose-dependent manner.

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Enhancement of biofilm-mode growth of CoNS cells by oxacillin at the highest serum achievable concentration. Solutions of gentamicin, oxacillin, vancomycin or the three agents in combination in TSB at the highest concentrations achievable in serum, were added to biofilm grown cells of two S. capitis isolates; 15 (biofilm-negative, ica-weak), and 22 (biofilm-negative, ica-positive).  After overnight incubation, bacterial growth was stained with crystal violet and the OD600 was measured.  Error bars represent standard errors of at least 3 individual experiments in triplicate.  *: P = 0.004; ** P < 0.001.
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Figure 1: Enhancement of biofilm-mode growth of CoNS cells by oxacillin at the highest serum achievable concentration. Solutions of gentamicin, oxacillin, vancomycin or the three agents in combination in TSB at the highest concentrations achievable in serum, were added to biofilm grown cells of two S. capitis isolates; 15 (biofilm-negative, ica-weak), and 22 (biofilm-negative, ica-positive). After overnight incubation, bacterial growth was stained with crystal violet and the OD600 was measured. Error bars represent standard errors of at least 3 individual experiments in triplicate. *: P = 0.004; ** P < 0.001.

Mentions: The effects of oxacillin, gentamicin, vancomycin and their combination at the highest achievable serum concentrations on preformed biofilms of 24 CoNS clinical isolates are presented in Table 4. Vancomycin or combinations of vancomycin with oxacillin and gentamicin were the most effective in reducing the density of preformed biofilms (58.3%); followed by oxacillin (29.2%) and gentamicin (8.3%). Oxacillin stimulated biofilm growth of two S. capitis isolates (isolates 15 and 22), converting these biofilm-negative isolates (OD600 < 0.12, when grown with TSB) into weak biofilm producers (0.12 ≤ OD600 < 0.24) or even biofilm-positive phenotypes (OD600 ≥ 0.24) (Figure 1 and Figure 2). S. epidermidis isolate 19 also showed enhancement of biofilm in the presence of oxacillin but the difference did not quite reach clinical significance (p < 0.074). Similar findings were obtained when 1% glucose was added into TSB as the medium for biofilm growth (data not shown). Dose-response studies using isolates 15 and 22 confirmed that oxacillin at concentrations ranging from 0.25 to128 μg/ml enhanced the biofilm-mode growth, with peak enhancement occurring around 8 to 32 μg/ml (data not shown).


Antibiotic susceptibility of coagulase-negative staphylococci isolated from very low birth weight babies: comprehensive comparisons of bacteria at different stages of biofilm formation.

Qu Y, Daley AJ, Istivan TS, Garland SM, Deighton MA - Ann. Clin. Microbiol. Antimicrob. (2010)

Enhancement of biofilm-mode growth of CoNS cells by oxacillin at the highest serum achievable concentration. Solutions of gentamicin, oxacillin, vancomycin or the three agents in combination in TSB at the highest concentrations achievable in serum, were added to biofilm grown cells of two S. capitis isolates; 15 (biofilm-negative, ica-weak), and 22 (biofilm-negative, ica-positive).  After overnight incubation, bacterial growth was stained with crystal violet and the OD600 was measured.  Error bars represent standard errors of at least 3 individual experiments in triplicate.  *: P = 0.004; ** P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2902406&req=5

Figure 1: Enhancement of biofilm-mode growth of CoNS cells by oxacillin at the highest serum achievable concentration. Solutions of gentamicin, oxacillin, vancomycin or the three agents in combination in TSB at the highest concentrations achievable in serum, were added to biofilm grown cells of two S. capitis isolates; 15 (biofilm-negative, ica-weak), and 22 (biofilm-negative, ica-positive). After overnight incubation, bacterial growth was stained with crystal violet and the OD600 was measured. Error bars represent standard errors of at least 3 individual experiments in triplicate. *: P = 0.004; ** P < 0.001.
Mentions: The effects of oxacillin, gentamicin, vancomycin and their combination at the highest achievable serum concentrations on preformed biofilms of 24 CoNS clinical isolates are presented in Table 4. Vancomycin or combinations of vancomycin with oxacillin and gentamicin were the most effective in reducing the density of preformed biofilms (58.3%); followed by oxacillin (29.2%) and gentamicin (8.3%). Oxacillin stimulated biofilm growth of two S. capitis isolates (isolates 15 and 22), converting these biofilm-negative isolates (OD600 < 0.12, when grown with TSB) into weak biofilm producers (0.12 ≤ OD600 < 0.24) or even biofilm-positive phenotypes (OD600 ≥ 0.24) (Figure 1 and Figure 2). S. epidermidis isolate 19 also showed enhancement of biofilm in the presence of oxacillin but the difference did not quite reach clinical significance (p < 0.074). Similar findings were obtained when 1% glucose was added into TSB as the medium for biofilm growth (data not shown). Dose-response studies using isolates 15 and 22 confirmed that oxacillin at concentrations ranging from 0.25 to128 μg/ml enhanced the biofilm-mode growth, with peak enhancement occurring around 8 to 32 μg/ml (data not shown).

Bottom Line: Epifluorescence microscopy and confocal laser scanning microscopy in combination with bacterial viability staining and polysaccharide staining were used to confirm the stimulatory effects of antibiotics on biofilms.We conclude that the resistance of coagulase-negative staphylococci to multiple antibiotics initially remain similar when the bacteria shift from a planktonic growth mode into an early attached mode, then increase significantly as the adherent mode further develops.Furthermore, preformed biofilms of some CoNS are enhanced by oxacillin in a dose-dependent manner.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Applied Sciences, RMIT University, Australia.

ABSTRACT

Background: Coagulase-negative staphylococci are major causes of bloodstream infections in very low birth weight babies cared for in Neonatal Intensive Care Units. The virulence of these bacteria is mainly due to their ability to form biofilms on indwelling medical devices. Biofilm-related infections often fail to respond to antibiotic chemotherapy guided by conventional antibiotic susceptibility tests.

Methods: Coagulase-negative staphylococcal blood culture isolates were grown in different phases relevant to biofilm formation: planktonic cells at mid-log phase, planktonic cells at stationary phase, adherent monolayers and mature biofilms and their susceptibilities to conventional antibiotics were assessed. The effects of oxacillin, gentamicin, and vancomycin on preformed biofilms, at the highest achievable serum concentrations were examined. Epifluorescence microscopy and confocal laser scanning microscopy in combination with bacterial viability staining and polysaccharide staining were used to confirm the stimulatory effects of antibiotics on biofilms.

Results: Most coagulase-negative staphylococcal clinical isolates were resistant to penicillin G (100%), gentamicin (83.3%) and oxacillin (91.7%) and susceptible to vancomycin (100%), ciprofloxacin (100%), and rifampicin (79.2%). Bacteria grown as adherent monolayers showed similar susceptibilities to their planktonic counterparts at mid-log phase. Isolates in a biofilm growth mode were more resistant to antibiotics than both planktonic cultures at mid-log phase and adherent monolayers; however they were equally resistant or less resistant than planktonic cells at stationary phase. Moreover, for some cell-wall active antibiotics, concentrations higher than conventional MICs were required to prevent the establishment of planktonic cultures from biofilms. Finally, the biofilm-growth of two S. capitis isolates could be enhanced by oxacillin at the highest achievable serum concentration.

Conclusion: We conclude that the resistance of coagulase-negative staphylococci to multiple antibiotics initially remain similar when the bacteria shift from a planktonic growth mode into an early attached mode, then increase significantly as the adherent mode further develops. Furthermore, preformed biofilms of some CoNS are enhanced by oxacillin in a dose-dependent manner.

Show MeSH
Related in: MedlinePlus