Journal of Chemotherapy
ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20
Antimicrobial susceptibility and the in vitro postantibiotic effects of vancomycin and ciprofloxacin against Bacillus cereus isolates Karmen Godič Torkar, Branka Bedenić & Vanda Plečko To cite this article: Karmen Godič Torkar, Branka Bedenić & Vanda Plečko (2016) Antimicrobial susceptibility and the in vitro postantibiotic effects of vancomycin and ciprofloxacin against Bacillus cereus isolates, Journal of Chemotherapy, 28:3, 151-158, DOI: 10.1179/1973947815Y.0000000069 To link to this article: http://dx.doi.org/10.1179/1973947815Y.0000000069
Published online: 27 May 2016.
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Date: 07 August 2016, At: 20:39
Antimicrobial Original Research Paper
Antimicrobial susceptibility and the in vitro postantibiotic effects of vancomycin and ciprofloxacin against Bacillus cereus isolates Karmen Godicˇ Torkar1, Branka Bedenic´2, Vanda Plecˇko2 University in Ljubljana, Faculty of Health Sciences, Slovenia, 2Clinical Department of Clinical and Molecular Microbiology, School of Medicine, University Hospital Center, Zagreb, Croatia
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Background: Vancomycin and ciprofloxacin were often used in the therapy of infections associated with Bacillus cereus. Methods: Four B. cereus food and clinical isolates were chosen for determination of time–kill curves and postantibiotic effects (PAE) of ciprofloxacin and vancomycin. Results: According to the minimum inhibition concentration (MIC), breakpoints defined by CLSI for Staphylococcus spp. were all four strains intermediate for vancomycin (MIC 5 4 mg/ml) and sensitive to ciprofloxacin (MIC 5 0.2 mg/ml) except the strain Bc63 resistant to the last antimicrobial (MIC 5 1.6 mg/ml). The lowest CFU values of tested strains were reached after 3–5 hours of exposure to 4| MIC of vancomycin, and after 6–7 hours exposure to 10| MIC of ciprofloxacin. The maximum reduction of the CFU in the presence of vancomycin and ciprofloxacin was about 2.46 log10 and 2.48 log10, respectively. The average duration of the PAE of vancomycin and ciprofloxacin was 0.94 and 1.60 hours, respectively. The statistically significant differences between PAEs induced with 3| MIC, 4| MIC and 8| MIC of vancomycin were observed (P v 0.05). Both antibiotics did not affect the sporulation of tested bacterial strains. Conclusions: The differences in PAE duration were strain and antimicrobial dependent. Keywords: Bacillus cereus, Antibiotic susceptibility, Postantibiotic effect, Time-killing effect, Sporulation
Introduction Bacillus cereus is a Gram-positive rod-shaped bacteria, which is frequently found as a saprophyte in the environment.1,2 Its endospores survive pasteurization and are resistant to various disinfectants.3–5 It grows easily during food storage and may be responsible for food poisoning and related emetic or diarrhoeal syndromes.6 It may also cause ocular and wound infections, bacteraemia, septicaemia, meningitis, endocarditis, endophthalmitis, osteomyelitis, necrotizing fasciitis, salpingitis, myeloaplasia and pneumonia, mainly among immunocompromised subjects.5–8 In general, most B. cereus isolates are resistant to penicillins and cephalosporins as a consequence of beta–lactamase production. It seems that susceptibility to clindamycin, erythromycin, chloramphenicol, ciprofloxacin, vancomycin, aminoglycosides and tetracycline is common. Specifically, vancomycin, ciprofloxacin, clindamycin, imipenem and aminoglycosides have been recommended as treatment options for severe B. cereus diseases.6,9,10
Correspondence to: Karmen Godicˇ Torkar, University in Ljubljana, Faculty of Health Sciences, Ljubljana, Slovenia. Email: [email protected]
ß 201 6 Edizioni Scientifiche per l’Informazione su Farmaci e Terapia DOI 10.1179/1973947815Y.0000000069
Persistent suppression of bacterial growth after short antimicrobial exposure is called a postantibiotic effect (PAE). It is the lag phase or recovery period of bacterial growth after a brief exposure to antibiotics.11 Studies on the pharmacodynamics of different antibiotics against Gram-positive bacteria are focussed mostly on staphylococci, streptococci and enterococci,12 but there are no available bibliographical references on PAE on B. cereus. The aim of this study was to investigate the time– kill kinetic and PAE of vancomycin and ciprofloxacin on B. cereus isolates.
Materials and Methods Bacterial isolates Four representative B. cereus strains out of 63 isolates from food and clinical samples were selected for time–kill studies and the determination of PAE: Bc10, Bc16, Bc63 and Bc100. The isolates were obtained and identified from 2006 to 2008. The strains, Bc10 and Bc16, were isolated from clinical specimens (wound and faeces) collected from patients hospitalized in University Medical Centre Ljubljana. The strains, Bc63 and Bc100, were isolated from raw milk and milk product from Slovenian dairy.
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2.22 ^ 0.71 2.13 ^ 0.40 1.58 ^ 0.38
1.21 ^ 0.13 0.74 ^ 0.28
0.43 0.56 0.58 0.28 0.61 0.49 0.49 ^ ^ ^ ^ ^ ^ ^ 4 (4) 4 (4) Bc63 Bc100 Mean values
MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; PAE, postantibiotic effect; SD, standard deviation.
0.48 ^ 0.30 1.84 ^ 0.20 1.56 ^ 0.48 1.6 (1.6) 0.2 (0.2)
1.25 £ 10 £
2.15 1.23 0.61 1.72 2.89 2.22 1.96 2.47 ^ 0.64 1.44 ^ 0.79 10 £ 10 £ 0.2 (0.2) 0.2 (0.2)
0.63 2.97 0.13 0.42 0.42 0.60 0.45 ^ ^ ^ ^ ^ ^ ^ 1.49 3.74 3.85 1.44 0.24 1.08 1.67 4 (4) 4 (4) Bc10 Bc16
4£ 3£ 4£ 8£ 4£ 4£ 4£
Ciprofloxacin Vancomycin PAE (hours) ^ SD MIC (MBC) (mg/ml)
Conc., £ MIC at PAE
According to the results of susceptibility testing, the B. cereus strains Bc10, Bc16, Bc63 and Bc100 were exposed to 16 mg/ml (app. 4| MIC) for vancomycin and 2 mg/ml (10| MIC) for ciprofloxacin, except for the strain Bc63 with MIC 1.6 mg/ml of ciprofloxacin, for which this concentration represented 1.25| MIC (Table 1). The antibiotics were added to the logarithmic-phase broth culture, obtained by the dilution of the overnight culture, containing approximately 107 CFU/ml (1 : 100 dilution).16 Viable counting was performed on samples collected every hour for 8 hours and then at 24 hours. The samples were decimal diluted in phosphate-buffered saline (PBS), and 100 ml of dilutions and the original cultures were seeded on MH agar for viable counting. The growing colonies were counted after 24 hours of incubation at 37uuC. All experiments were performed in triplicate for each of four chosen B. cereus isolates, as described in previous studies.16,17
PAE (hours) ^ SD
Time –kill assay
Conc., £ MIC at PAE
The minimum inhibition concentration (MIC) and the minimum bactericidal concentration (MBC) of vancomycin and ciprofloxacin of four B. cereus strains (Bc10, Bc16, Bc63 and Bc100) were determined with a microdilution method, using Mueller–Hinton broth (MH, Merck, Germany) in accordance with the national standards.15 The concentrations of vancomycin and ciprofloxacin after dilution and the addition of the bacterial suspension ranged from 0.06 to 128 mg/ml and from 0.05 to 100 mg/ml, respectively.
MIC (MBC) (mg/ml)
Susceptibility testing
Bacterial strains
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Vancomycin and ciprofloxacin (American Pharmocopoeia, Rockville, MD, USA) were used for the study of antibiotic susceptibility, the PAE and the time–kill effect. Each agent was freshly prepared according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) in the appropriate solvent. The stock solutions of vancomycin and ciprofloxacin were prepared by diluting the antibiotic powder with distilled water.15
Ciprofloxacin
Antimicrobial agents
Vancomycin
These four strains were selected based on their resistance to chosen antimicrobials. Colony morphology, cell morphological and physiological characteristics, as well as haemolytic activity were determined using conventional procedures.13 The isolates were identified with the API 50 CHB and API 10S test systems using the API WEB identification programme (bioMerieux, Marcy-I’Etoile, France). The control strain used to validate the antimicrobial susceptibility testing was Staphylococcus aureus ATCC 29213.14
Reduction of log CFU/ml after 2 hours exposure to antibiotics
PAEs of vancomycin and ciprofloxacin against B. cereus
Table 1 Postantibiotic effects (PAEs) of vancomycin induced by concentrations of 16 μg/ml and of ciprofloxacin in concentration 2 μg/ml, on four B. cereus strains. Additionally, the PAE of vancomycin on concentrations of 12 and 32 μg/ml on the strain Bc16 was also tested. The reduction in viable bacterial cells after 2hours exposure to antibiotics was represented as well
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The bactericidal activity and bacteriostatic activity were determined. Bactericidal activity was defined as a 3 log10 decrease in CFU/ml (99.9% kill). Bacteriostatic activity was defined as v99.9% kill of bacterial cells after exposure to an antimicrobial.18
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The effect of antimicrobials to sporulation Vancomycin and ciprofloxacin in concentrations 16 and 2 mg/ml, respectively, were added to the logarithmic-phase broth cultures of four tested strains, prepared as for time–kill assay. The samples were divided into two, and one of them was thermized at 80uuC for 10 min.19 The heat shock procedure killed the vegetative bacteria without affecting the viability of the spore population. Then the samples that were and were not subjected to heat shock were quantitatively cultured on antibiotic-free MH agar. The number of colonies of thermized and unthermized samples was counted after incubation at 30uuC for 72 hours to determine the effect of each regimen on the spore and total B. cereus populations.19,20 The control cultures without antimicrobials were tested using the same procedure. The number of spores and the percentage of sporulation were calculated after recovery of viable spores and bacteria on MH agar from three independent experiments. The colony-forming units per millilitre of unthermized samples represented the number of spores and vegetative cells, while the colony-forming units per millilitre in thermized samples represented the number of spores.21
Determination of PAE
The strains were exposed to 16 mg/ml (app. 4| MIC) for vancomycin and 2 mg/ml (10| MIC for ciprofloxacin for 2 hours. The exception was Bc63, where the concentration of ciprofloxacin used in the experiment represented 1.25| MIC. Additionally, the exposure to 3| MIC (12 mg/ml) and 8| MIC (32 mg/ml) of vancomycin was implemented with the strain Bc16. The lower concentrations with about 4| MIC and 8| MIC of ciprofloxacin in our preliminary studies had no bacteriostatic effect. The antibiotic was added to the logarithmic-phase broth culture, obtained by dilution of the overnight culture, containing approximately 107 CFU/ml. After 2 hours, the antibiotic was removed by centrifugation at 4000 | g/10 minutes and the washing of the pellet twice in 5 ml of saline; the recovery of the tested strains was followed for 8 hours and then at 24 hours. Viable counting was performed in the same manner as for time–kill assay.16,17 The PAE was calculated using the following formula: PAE 5 T { C, where T was the time necessary for the number of viable organisms in the test culture to increase by 1 log10 (10-fold) above the number, observed immediately after removal of
PAEs of vancomycin and ciprofloxacin against B. cereus
antibiotics. C was the time necessary for the number of viable organisms in the untreated culture to increase by 1 log10 above the number observed immediately after the completion of the same procedure used in the test procedure for drug removal (centrifugation).16,22
Statistical analysis The data were log-transformed prior to analysis. The IBS SPSS Statistics 22 Programme was used for statistical analyses. Descriptive statistics for calculating the mean, standard deviation, variance minimum and maximum values were chosen. Comparisons between the groups were done with an analysis of variance (ANOVA) with post hoc tests. Comparisons between the PAE of different antimicrobials and their concentrations were calculated with a Wilcoxon signed rank test. Any P value below 0.05 was considered significant.
Results Susceptibility testing The MIC and MBC of each antimicrobial agent of four tested isolates are shown in Table 1. The interpretive standards for staphylococci14 or S. aureus (vancomycin)23,24 were used as no interpretive standards have been established for B. cereus. Among the 63 strains, four of them were chosen (Bc10, Bc16, Bc63 and Bc100) based on their resistance to vancomycin (MIC54 mg/ml) and the highest MIC values of ciprofloxacin. The only resistant strain to ciprofloxacin was Bc63 (MIC 5 1.6 mg/ml).
Time–kill assay The kill curve profiles following vancomycin and ciprofloxacin exposure are shown in Fig. 1A–D. The untreated control sample showed exponential growth reaching a maximum of 6.7–7.6 log10 CFU/ml 4–6 hours after the start of the experiment. We found statistically significant correlations in the reduction of viable counts between ciprofloxacin and vancomycin with all tested strains. However, the difference in duration of inhibitory activity between the two antibiotics was not statistically significant (P 5 0.064). The lowest CFU value of the cultures treated with antibiotics was reached after approximately 4 hours 4 of exposure to 4| MIC of vancomycin and 6.6 hours of exposure to 10| MIC of ciprofloxacin. These data were the mean values for the strains Bc10, Bc16 and Bc100. The strain Bc63, resistant to ciprofloxacin, exhibited a bacteriostatic effect after 4 hours (vancomycin) to 5 hours (ciprofloxacin), and the lowest CFU values were reached after 6 hours of exposure to each of both antimicrobials. This strain only demonstrated a reduction of viable counts for w3 log10 in the presence of the ciprofloxacin compared with the
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reduction of 3 log10 CFU/ml. The viable count after 6 hours incubation was w3 log10 CFU/ml lower for all strains in comparison with the untreated controls (Fig. 1A–D).
The effect of antimicrobials to sporulation
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The average number of viable cells in all four control unthermized samples (Bc10, Bc16, Bc63 and Bc100) increased to 7.7 log10 CFU/ml over 24 hours. The spore population in the thermized samples slowly decreased to the sixth hour of incubation and then rose by about 2 log10 CFU/ml within 24 hours, when their number reached 4 log10 CFU/ml and represented 0.02% of the total bacterial count. The total number of cells in cultures with added antimicrobials decreased to 3.5 log10 CFU/ml after 6 hours ofincubation and rose to 5.3 log10 CFU/ml in 24 hours. The spores represented only 0.6% (after 6 hours) and 0.01% (after 24 hours) of the total (vegetative and spore) bacterial count. The results did not differ significantly between the cultures exposed to vancomycin and ciprofloxacin (P 5 0.064). After 48 hours incubation, the number of spores and the total number of cells in control samples without antibiotics reached 5.2 log10 CFU/ ml and 7.2 log10 CFU/ml, respectively. The values of the spores and the total bacterial count in samples treated with antibiotics decreased to under 3 log10 CFU/ml (Fig. 2).
Figure 1 (A– D) Representative time–kill plots for B. cereus isolates Bc10 (A), Bc16 (B), Bc63 (C) and Bc100 (D), exposed to 4 3 MIC vancomycin (VAN) and 10 3 MIC ciprofloxacin (CIP). The exception was Bc63, where the concentration of ciprofloxacin used in the experiment represented 1.25 3 MIC. Bc10C, Bc16C, Bc63C and Bc100C were untreated control cultures for each strain.
starting inoculum, whereas this antimicrobial yielded a reduction from 2 log10 CFU/ml but not higher than 3 log10 for the other three strains. Vancomycin yielded a 2 log10 reduction or greater for all four tested strains, but none of them expressed the
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Figure 2 The average values of spore count and vegetative cell count in the cultures of four B. cereus strains (Bc10, Bc16, Bc63 and Bc100) in the absence of the antimicrobials and at the presence of 4 3 MIC of vancomycin and 10 3 MIC (1.25 3 MIC at Bc63) of ciprofloxacin during 48 hours incubation. VAN, total bacterial count (vegetative cells and spores), exposed to vancomycin; VANSP, the number of spores, exposed to vancomycin; CIP, total bacterial count (vegetative cells and spores), exposed to ciprofloxacin; CIPSP, the number of spores, exposed to ciprofloxacin; CON, total bacterial count (vegetative cells and spores), without antimicrobial; CONSP, the number of spores, without antimicrobial.
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PAEs of vancomycin and ciprofloxacin against B. cereus
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Determination of PAEs The average duration of the PAE of 16 mg/ml of vancomycin against three tested B. cereus strains (Bc10, Bc63 and Bc100) ranged between 0.24 and 1.94 hours with a mean value of 0.94 hours. The PAE of 16 mg/ml of ciprofloxacin with a median value of 1.60 hours and range between 0.48 and 2.47 hours was longer than that caused by vancomycin. The extremely long PAE duration of 3.47 hours of vancomycin against the fourth strain, Bc16, was observed. Vancomycin produced a slightly longer PAE (1.67 hours) than ciprofloxacin did (1.56 hours). The PAEs of ciprofloxacin and vancomycin were the shortest with the strain Bc63, while the values of PAE with Bc10 and Bc100 of both antibiotics were quite similar (Table 1). The average reduction values of the bacterial count of all four tested strains after 2 hours exposure to vancomycin and ciprofloxacin were 1.96 log10 and 1.58 log10, respectively. After 6 hours of incubation following antibiotic removal, the average increase in the viable count was 3.08 log10 for vancomycin and 2.65 log10 for ciprofloxacin. The regrowth of the Bc16 culture was significantly longer in comparison to the other strains. The strain, Bc16, differed from other three tested strains in some characteristics. Without added antibiotics, the culture Bc16 grew slightly slower than other strains did. The average number of viable cells after 6 hours incubation increased only by 0.98 log10 CFU/ml in comparison with average 1.40 log10 CFU/ml of the other three untreated cultures (Table 1, Fig. 3B). Bc16 differed significantly in the CFU count during 24 hours growth in comparison to the other three strains (P 5 0.001). Furthermore, it also differed from other strains in the duration of PAE induced by vancomycin (P 5 0.03) and ciprofloxacin (P 5 0.001). As seen in Fig. 3B, the PAE of vancomycin of the strain Bc16 was induced by all three concentrations (3|, 4| or 8| S. aureus MIC). The reduction of CFU after 2 hours exposure to 3| MIC was 1.24 log10. The regrowth of bacterial culture was similar at both 3| MIC and 4| MIC (1.06 and 1.04 log10). At the 8| MIC, there was a significant (P v 0.05) reduction of the inoculum after 2 hours exposure to vancomycin, but the rate of regrowth was high, and the duration of PAE was short. The time of PAE was significantly longer after exposure to 8| MIC in comparison to those induced by lower concentrations (3| MIC and 4| MIC). The duration of PAE of vancomycin was not concentration dependent, while the reduction of the bacterial count was.
Discussion The systemic and wound infections caused by B. cereus are usually treated with antimicrobials,
Figure 3 (A –D) Postantibiotic effect (PAE) of 16 mg/ml of vancomycin (4 3 MIC) (VAN) and 2 mg/ml of ciprofloxacin (10 3 MIC) (CIP) on strains B. cereus Bc10 (A), Bc16 (B), Bc63 (C) and Bc100 (D). The exception was Bc63, where the concentration of ciprofloxacin used in the experiment represented 1.25 3 MIC. Bc10C, Bc16C, Bc63C and Bc100C were untreated control cultures for each strain. The PAE of 12 mg/ml (3 3 MIC, VAN12) and 32 mg/ml (8 3 MIC, VAN32) of vancomycin was additionally tested on the strain Bc16 (B).
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such as aminoglycosides, macrolides, lincosamides, tetracycline and particularly with carbapenems, glycopeptides (vancomycin) and quinolones (ciprofloxacin).25,26 Turnbull et al. (2004)27 also believed that susceptibility to ciprofloxacin was uniform; it has been shown to be highly effective in the treatment of B. cereus wound infections. We evaluated the time–kill curves and postantibiotic (PAE) effect of vancomycin and ciprofloxacin against four B. cereus isolates from food and clinical specimens, which displayed higher MICs for these two antibiotics out of 63 tested. The time–kill analysis was done with an antibiotic concentration corresponding to 4| the MIC of vancomycin and 10| the MIC of ciprofloxacin, according to Athamna et al. (2004).28 These concentrations are in the range of average peak levels in a serum after dosing 500 mg of these antimicrobials, which are used in the routine therapy.29–31 A bactericidal or killing effect, as defined by a 3 log10 reduction in the viable count, occurred with only one strain (Bc63) out of the four strains exposed to ciprofloxacin. In the experiment, we included the ciprofloxacin-resistant Bc63 strain (MIC 1.6 mg/ml, 1.25| MIC) to verify the potential concentrationdependent effect. A bacteriostatic effect, which is defined as 2 log10 reduction in viable count, was observed with other three strains (Bc10, Bc16 and Bc100) in 1–4 hours and 1–5 hours after exposure to vancomycin and ciprofloxacin, respectively. The rapidity of the effectiveness of the killing of both antimicrobials used in the experiment seems low in respect to Gram-negative32 and some other Gram-positive33 bacteria, and similar to that against B. anthracis.21 Some experiments suggest that vancomycin inhibits peptidoglycane biosynthesis by binding to its intermediates (i.e., a substrate-binding antibiotic) and that it has a bacteriostatic effect to B. subtilis.34,35 Berlanga et al. (2004)32 also reported that ciprofloxacin did not act as a bactericide against B. cereus and B. subtilis. Instead, it seems to have a bacteriostatic effect. The observation of low-level activity against Bacillus species may be the consequence of ciprofloxacin’s decreased interaction with the changed target enzymes DNA gyrase and topoisomerase IV rather than its diminished permeability.32,36 Their findings also suggests that ciprofloxacin might not serve as a good substrate for the Bacillus efflux pump, which depends on the proton-motive force.32 The tests carried out by Citron et al. (2006)37 show that doptamycin and ciprofloxacin are both rapidly bactericidal against the B. cereus group, while their killing effect is slower for representative strains of B. subtilis and B. pumilus for which the number of bacterial cells decreased only for 2 log10 during
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24 hours incubation. In our case, there were clearly expressed strain-dependent differences in the inhibitory effects of vancomycin and ciprofloxacin (Fig. 1A–D). The most pronounced killing effect of both antimicrobials was found on strain Bc63, in spite of it having the highest ciprofloxacin MIC. We cannot explain this phenomenon. The number of viable cells after 24 hours incubation was higher than that before the addition of the antibiotic to the culture with the strains, Bc16, Bc63 and Bc100. The strain Bc10 did not show the increase in the number of viable cells after the removal of the antibiotic (Fig. 1A–D). Published studies on the PAE of the antimicrobials against strains B. cereus in the available bibliography are rare. Some of them were performed on pathogen species B. anthracis, which belongs to the group B. cereus sensu lato as well as B. cereus.38 According to Deziel et al. (2005),39 the exposure to subinhibitory concentrations of antimicrobials, such as quinolones, might promote sporulation instead of the killing the bacterial cells. This might be the case in our study: the regrowth occurred in the presence of ciprofloxacin with all four tested strains. This antimicrobial is not a protein synthesis inhibitor,40 so it does not inhibit the production of the molecules and enzymes that are needed for vegetative bacteria to form spores. Louie et al.21 reported that ciprofloxacin in high concentrations rapidly killed vegetative B. anthracis so that the total population consisted primarily of spores. In his study, the heat-shock studies demonstrated that there were spores in 10% of the population in 24-hour bacterial culture prior to the start of the antibiotic treatments, while in our tested cultures in log-phase the spores were almost absent (only 0.04%), whereas after 48 hours the number of spores was 2 log10 CFU/ml lower than the total bacterial count (1%). In all four B. cereus strains treated with antimicrobials, we detected the difference between the total (primarily vegetative bacteria) and the spore population after the first 6 hours, even though the total population was decreasing. At this point, the vegetative B. cereus represented 99.4% of the bacterial population (Fig. 2). There was no difference in the ratio between the spores and vegetative cells of the untreated culture and the two cultures with antibiotics, or between the cultures with vancomycin and ciprofloxacin (P 5 0.064). We examined the PAE effect induced by 10| MIC of ciprofloxacin and at 4| MIC of vancomycin. The duration of PAE of vancomycin was 1.67 hours, which is only slightly longer than that of ciprofloxacin (1.56 hours). Vancomycin induced longer PAE than ciprofloxacin did but only with the strain Bc16, which grew slower and weaker in
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the culture even without antimicrobials. Against other three tested strains, ciprofloxacin was more effective, and its PAEs took longer than those of vancomycin. The reason for such results was probably a higher concentration of ciprofloxacin used in the study (Table 1, Fig. 3A–D). Our preliminary experiments confirmed that the concentrations of 4| MIC and even 8| MIC of ciprofloxacin did not significantly reduce the number of viable bacteria of the tested B. cereus strains (data are not shown in this article). These results are comparable with the study by Athamna et al. (2004)28 in which PAEs of the 10| MIC ciprofloxacin and vancomycin against B. anthracis strains were 2–3 and 1–2 hours, respectively. The differences in PAEs of both antimicrobials were statistically significant (P 5 0.006). Moreover, the duration of PAE was strain dependent. The duration of PAE of ciprofloxacin and vancomycin was the shortest for the strain Bc63, due to its resistance to higher concentrations of ciprofloxacin. The PAEs of vancomycin in different concentrations of Bc16 were compared. Its activity in PAE might be characteristic of a concentration-dependent drug, because increasing drug concentration above 4| MIC had significant beneficial effects on bacterial counts and their re-growth (P v 0.05).
Conclusions This study confirms that some B. cereus isolates have developed resistance to vancomycin and ciprofloxacin, which are often used in the therapy of infections associated with this bacteria. The bactericidal effect was determined in only one strain exposed to ciprofloxacin, although the antimicrobial concentrations of even 10| MIC were used in the experiment. The bacteriostatic effect was observed after 1–5 hours, depending on the strain; the regrowth was significant for all tested strains. Vancomycin and ciprofloxacin did not have any significant effect on the sporulation on tested B. cereus strains. The differences in PAE were dependent upon the B. cereus strain, the antibiotic tested, and the antibiotic concentration. Organisms differ in their growth curves, and the rate of the resumption of more rapid growth and different drugs can cause different effects. This must be taken into consideration when comparisons of growth rates are made after antimicrobial treatment.41 Postantibiotic effect was found to be strain dependent. In vitro results indicate that vancomycin and ciprofloxacin may represent valid antimicrobials in the treatment of B. cereus infections, although with different bacteriostatic rates. Fluoroquinolones exhibit a particularly high-penetration capacity into the cells of bacteria and tissues as well as a binding capacity with endocellular components and
PAEs of vancomycin and ciprofloxacin against B. cereus
prolonged permanence in comparison to other classes of antibiotics. To confirm our results, it would be necessary to include more B. cereus isolates in the study and to perform molecular typing of antibiotic resistance. In cases of severe infections, the doses of ciprofloxacin should be higher than those obtained with 500 mg by oral route or administered by I.V. determining serum levels.
Acknowledgements We would like to thank the Ministry of Science, Education and Sports, Agency for Mobility and EU Programmes of Croatia for financial support.
Disclaimer Statements Contributors All authors (Branka Bedenič, Vanda Plečko and Karmen Godič Torkar) contributed to the manuscript. Funding None. Conflict of Interest statement The authors declare that they have no conflict of interest. Ethical approval Ethical approval was not required.
References 1 Notermans S, Dufrenne J, Teunis P, Beumer R, Giffel M, Weem P. A risk assessment study of Bacillus cereus present in pasteurized milk. Food Microbiol. 1997;14(2):143–51. 2 Kiel JL, Parker JE, Holwitt EA, McCreary RP, Andrews CJ, De Los Santos A, et al. Geographical distribution of genotypic and phenotypic markers among Bacillus anthracis isolates and related species by historical movement and horizontal transfer. Folia Microbiol. 2008;53(6):472–8. 3 Adler A, Gottesman G, Dolfin T, Arnon S, Regev R, Bauer S, et al. Bacillus species sepsis in the neonatal intensive care unit. J Infect. 2005;51(5):390–5. 4 Dubouix A, Bonnet E, Alvarez M, Bensafi H, Archambaud M, Chaminade B, et al. Bacillus cereus infections in traumatologyorthopaedics department: retrospective investigation and improvement of healthcare practices. J Infect. 2005;50(1):22–30. 5 Ginsburg AS, Salazar LG, True LD, Disis ML. Fatal Bacillus cereus sepsis following resolving neutropenic enterocolitis during the treatment of acute leukemia. Am J Hematol. 2003; 72(3):204–8. 6 Katsuya H, Takata T, Ishikawa T, Sasaki H, Ishitsuka K, Takamatsu Y, et al. A patient with acute myeloid leukemia who developed fatal pneumonia caused by carbapenem-resistant Bacillus cereus. J Infect Chemother. 2009;15(1):39–41. 7 Savini V, Favaro M, Fontana C, Catavitello C, Balbinot A, Talia M, et al. Bacillus cereus heteroresistant to carbapenems in a cancer patient. J Hosp Infect. 2009;71(3):288–90. 8 Savini V, Berhardt LV. Bacillus cereus pneumonia. Advances in medicine and biology. 67. Italy: Nova Science Publisher Inc city; 2013; p.16. 9 Bottone EJ. Bacillus cereus, a volatile human pathogen. Clin Microbiol Rev. 2010;23(2):382–98. 10 Strauss R, Mueller A, Wehler M, Neureiter D, Fischer E, Gramatzki M, et al. Pseudomembranous tracheobronchitis due to Bacillus cereus. Clin Infect Dis. 2001;33(5):e39–e41. 11 Hanberger H, Nilsson LE, Kihlstrom E, Maller R. Postantibiotic effect of b-lactam antibiotics on Escherichia coli by bioluminescence assay of bacterial ATP. Antimicrob Agents Chemother. 1990;34(1):102–6. 12 Munckhof WJ, Giles C, Turnidge JD. Post-antibiotic growth suppression of linezolid against Gram-positive bacteria. J Antimicrob Chemother. 2001;47(6):879–83. 13 International Standard Organization: Microbiology of food and animal feeding stuffs – horizontal method for the enumeration of presumptive Bacillus cereus – colony-count technique at 30 ˚C. Brussels, Belgium: 7 [ISO document] International Standard Organization; 2004.
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14 Clinical and Laboratory Standards Institute: Performance standards for antimicrobial susceptibility testing; 23rd informational supplement. Wayne, PA: [CLSI document M100-S23] Clinical and Laboratory Standards Institute; 2013; p. 70–86. 15 Clinical and Laboratory Standards Institute: Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard. 9th edn. Wayne, PA: Clinical and Laboratory Standards Institute; 2012; p. 10–9. 48–57; 146–61 [CLSI document MO7-A9, 32]. 16 Bedenic´ B, Vranesˇ J, Sviben M, Beader N, Kalenic´ S. Postantibiotic and post-beta-lactamase inhibitor effect of carbapenems combined with EDTA against Pseudomonas aeruginosa strains producing VIM-metallo beta-lactamases. Chemother. 2004; 54(3):188–93. 17 Murbach V, Dhoyen N, Kinger H, Jehl F. Evidence for a true post-b-lactamase-inhibitor effect of clavulanic acid against Klebsiella pneumoniae and Haemophilus influenzae. Clin Microbiol Infect. 2008;7(12):661–5. 18 Giamarellos-Bourboulis EJ, Kentepozidis N, Antonopoulou A, Plachouras D, Tsaganos T, Giamarellou H. Postantibiotic effect of antimicrobial combinations on multidrug-resistant Pseudomonas aeruginosa. Diagn Microbiol Infect Dis. 2001;51(2):113–7. 19 Fritze D, Claus D. Media for Bacillus spp. and related genera relevant to food. In: Corry JEL, Curtis GDW, Baird RM, editors. Handbook of culture media for food microbiology. 2nd edn. New York: Elsevier Science; 2003; p. 61–78. 20 Mazoua S, Chauveheid E. Aerobic spore-forming bacteria for assessing quality of drinking water produced from surface water. Water Res. 2005;39(20):5186–98. 21 Louie A, VanScoy BD, Heine HS III, Weiguo L, Abshire T, Holman K, et al. Differential effects of linezolid and ciprofloxacin on toxin production by Bacillus anthracis in an in vitro pharmacodynamic system. Antimicrob Agents Chemother. 2012;56(1):513–7. 22 Lavigne JP, Bonnet R, Charachon SM, Jourdan J, Caillon J, Sotto A. Post-antibiotic and post-b-lactamase inhibitor effects of ceftazidime plus sulbactam on extended-spectrum-b-lactamase producing Gram-negative bacteria. J Antimicrob Chemother. 2004;53(4):616–9. 23 Mohammed MJ, Marston CK, Popovic T, Weyant RS, Tenover FC. Antimicrobial susceptibility testing of Bacillus anthracis: comparison of results obtained by using the National Committee for Clinical Laboratory Standards broth microdilution reference and Etest agar gradient diffusion methods. J Clin Microbiol. 2002;40(6):1902–7. 24 Tascini C, Flammini S, Leonildi A, Ciullo I, Tagliaferri E, Menichetti F. Comparison of teicoplanin and vancomycin in vitro activity on clinical isolates of Staphylococcus aureus. J Chemother. 2012;24(4):187–90. 25 Horii T, Notake S, Tamai K, Yanagisawa H. Bacillus cereus from blood cultures: virulence genes, antimicrobial susceptibility and risk factors for blood stream infection. FEMS Immunol Med Microbiol. 2011;63(2):202–9. 26 Ozkocaman V, Ozcelik T, Ali R, Ozkalemkas F, Ozkan A, Ozakin C, et al. Bacillus spp. among hospitalized patients with haematological malignancies: clinical features, epidemics and outcomes. J Hosp Infect. 2006;64(2):169–76. 27 Turnbull PCB, Sirianni NM, LeBron CL, Samaan MN, Sutton FN, Reyes AE, et al. MICs of selected antibiotics for Bacillus
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anthracis, Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides from a range of clinical and environmental sources as determined by Etest. J Clin Microbiol. 2004;42(8):3626–34. Athamna A, Athamna M, Medlej B, Bast DJ, Rubinstein E. In vitro post-antibiotic effect of fluoroquinolones, macrolides, b-lactams, tetracyclines, vancomycin, clindamycin, linezolid, chloramphenicol, quinupristin/dalfopristin and rifampicin on Bacillus anthracis. J Antimicrob Chemother. 2004;53(4): 609–15. Van Zanten ARH, Polderman KH, van Geijlswijk IM, van der Meer GYG, Schouten MA, Girbes ARJ. Ciprofloxacin pharmacokinetics in critically ill patients: a prospective cohort study. J Crit Care. 2008;23(3):422–30. D’Espine M, Bellido F, Peche`re JC, Auckenthaler R, Rohner P, Lew D, et al. Serum levels of ciprofloxacin after single oral doses in patients with septicemia. Eur J Clin Microbiol Infect Dis. 1989;8(12):1019–23. Yao JDC, Moellering RCJr. Antibacterial agents. In: Murray PR, Baron JH, Landry ML, Pfaller MA, editors. itors. Manual of clinical microbiology. 9th edn. Herndon: ASM Press; 2007; p. 1077–13. Berlanga M, Montero MT, Herna´ndez-Borrell J, Vin˜as M. Influence of the cell wall on ciprofloxacin susceptibility in selected wild-type Gram-negative and Gram-positive bacteria. Intern J Antimicrob Agents. 2004;23(6):627–30. Grohs P, Kitzis M-D, Gutma L. In vitro bactericidal activities of linezolid in combination with vancomycin, gentamicin, ciprofloxacin, fusidic acid, and rifampin against Staphylococcus aureus. Agents Chemother. 2003;47(1):418–20. Daniel RA, Errington J. Control of cell morphogenesis in bacteria: two distinct ways to make a rod-shaped cell. Cell. 2003;113(6):767–76. Tiyanont K, Doan T, Lazarus MB, Xiao Fang X, Rudner DZ, Walker S. Imaging peptidoglycan biosynthesis in Bacillus subtilis with fluorescent antibiotics. Proc Natl Acad Sci USA. 2006;103(29):11033–38. Aldred KJ, McPherson SA, Wang P, Kerns RJ, Graves DE, Turnbough CL Jr, et al. Drug interactions with Bacillus anthracis topoisomerase IV: biochemical basis for quinolone action and resistance. Biochemistry. 2012;51(1):370–81. Citron DM, Appleman MD. In vitro activities of daptomycin, ciprofloxacin, and other antimicrobial agents against the cells and spores of clinical isolates of Bacillus species. J Clin Microbiol. 2006;44(10):3814–8. Tourasse NJ, Helgason E, Økstad OA, Hegna IK, Kolstø AB. The Bacillus cereus group: novel aspects of population structure and genome dynamics. J Appl Microbiol. 2006;101(3):579–93. Deziel MR, Heine H, Louie A, Kao M, Byrne WR, Basset J, et al. Effective antimicrobial regimens for use in humans for therapy of Bacillus anthracis infections and postexposure prophylaxis. Antimicrob Agents Chemother. 2005;49(12): 5099–06. Swaney SM, Aoki H, Ganoza MC, Shinabarger DL. The oxazolidinone linezolid inhibits initiation of protein synthesis in bacteria. Antimicrob Agents Chemother. 1998;42(12):3251–5. Smith RP, Baltch AL, Michelsen PB, Ritz WJ, Alteri R. Use of the microbial growth curve in postantibiotic effect studies of Legionella pneumophila. Antimicrob Agents Chemother. 2003; 47(3):1081–7.
ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20
Antimicrobial susceptibility and the in vitro postantibiotic effects of vancomycin and ciprofloxacin against Bacillus cereus isolates Karmen Godič Torkar, Branka Bedenić & Vanda Plečko To cite this article: Karmen Godič Torkar, Branka Bedenić & Vanda Plečko (2016) Antimicrobial susceptibility and the in vitro postantibiotic effects of vancomycin and ciprofloxacin against Bacillus cereus isolates, Journal of Chemotherapy, 28:3, 151-158, DOI: 10.1179/1973947815Y.0000000069 To link to this article: http://dx.doi.org/10.1179/1973947815Y.0000000069
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Date: 07 August 2016, At: 20:39
Antimicrobial Original Research Paper
Antimicrobial susceptibility and the in vitro postantibiotic effects of vancomycin and ciprofloxacin against Bacillus cereus isolates Karmen Godicˇ Torkar1, Branka Bedenic´2, Vanda Plecˇko2 University in Ljubljana, Faculty of Health Sciences, Slovenia, 2Clinical Department of Clinical and Molecular Microbiology, School of Medicine, University Hospital Center, Zagreb, Croatia
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Background: Vancomycin and ciprofloxacin were often used in the therapy of infections associated with Bacillus cereus. Methods: Four B. cereus food and clinical isolates were chosen for determination of time–kill curves and postantibiotic effects (PAE) of ciprofloxacin and vancomycin. Results: According to the minimum inhibition concentration (MIC), breakpoints defined by CLSI for Staphylococcus spp. were all four strains intermediate for vancomycin (MIC 5 4 mg/ml) and sensitive to ciprofloxacin (MIC 5 0.2 mg/ml) except the strain Bc63 resistant to the last antimicrobial (MIC 5 1.6 mg/ml). The lowest CFU values of tested strains were reached after 3–5 hours of exposure to 4| MIC of vancomycin, and after 6–7 hours exposure to 10| MIC of ciprofloxacin. The maximum reduction of the CFU in the presence of vancomycin and ciprofloxacin was about 2.46 log10 and 2.48 log10, respectively. The average duration of the PAE of vancomycin and ciprofloxacin was 0.94 and 1.60 hours, respectively. The statistically significant differences between PAEs induced with 3| MIC, 4| MIC and 8| MIC of vancomycin were observed (P v 0.05). Both antibiotics did not affect the sporulation of tested bacterial strains. Conclusions: The differences in PAE duration were strain and antimicrobial dependent. Keywords: Bacillus cereus, Antibiotic susceptibility, Postantibiotic effect, Time-killing effect, Sporulation
Introduction Bacillus cereus is a Gram-positive rod-shaped bacteria, which is frequently found as a saprophyte in the environment.1,2 Its endospores survive pasteurization and are resistant to various disinfectants.3–5 It grows easily during food storage and may be responsible for food poisoning and related emetic or diarrhoeal syndromes.6 It may also cause ocular and wound infections, bacteraemia, septicaemia, meningitis, endocarditis, endophthalmitis, osteomyelitis, necrotizing fasciitis, salpingitis, myeloaplasia and pneumonia, mainly among immunocompromised subjects.5–8 In general, most B. cereus isolates are resistant to penicillins and cephalosporins as a consequence of beta–lactamase production. It seems that susceptibility to clindamycin, erythromycin, chloramphenicol, ciprofloxacin, vancomycin, aminoglycosides and tetracycline is common. Specifically, vancomycin, ciprofloxacin, clindamycin, imipenem and aminoglycosides have been recommended as treatment options for severe B. cereus diseases.6,9,10
Correspondence to: Karmen Godicˇ Torkar, University in Ljubljana, Faculty of Health Sciences, Ljubljana, Slovenia. Email: [email protected]
ß 201 6 Edizioni Scientifiche per l’Informazione su Farmaci e Terapia DOI 10.1179/1973947815Y.0000000069
Persistent suppression of bacterial growth after short antimicrobial exposure is called a postantibiotic effect (PAE). It is the lag phase or recovery period of bacterial growth after a brief exposure to antibiotics.11 Studies on the pharmacodynamics of different antibiotics against Gram-positive bacteria are focussed mostly on staphylococci, streptococci and enterococci,12 but there are no available bibliographical references on PAE on B. cereus. The aim of this study was to investigate the time– kill kinetic and PAE of vancomycin and ciprofloxacin on B. cereus isolates.
Materials and Methods Bacterial isolates Four representative B. cereus strains out of 63 isolates from food and clinical samples were selected for time–kill studies and the determination of PAE: Bc10, Bc16, Bc63 and Bc100. The isolates were obtained and identified from 2006 to 2008. The strains, Bc10 and Bc16, were isolated from clinical specimens (wound and faeces) collected from patients hospitalized in University Medical Centre Ljubljana. The strains, Bc63 and Bc100, were isolated from raw milk and milk product from Slovenian dairy.
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2.22 ^ 0.71 2.13 ^ 0.40 1.58 ^ 0.38
1.21 ^ 0.13 0.74 ^ 0.28
0.43 0.56 0.58 0.28 0.61 0.49 0.49 ^ ^ ^ ^ ^ ^ ^ 4 (4) 4 (4) Bc63 Bc100 Mean values
MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; PAE, postantibiotic effect; SD, standard deviation.
0.48 ^ 0.30 1.84 ^ 0.20 1.56 ^ 0.48 1.6 (1.6) 0.2 (0.2)
1.25 £ 10 £
2.15 1.23 0.61 1.72 2.89 2.22 1.96 2.47 ^ 0.64 1.44 ^ 0.79 10 £ 10 £ 0.2 (0.2) 0.2 (0.2)
0.63 2.97 0.13 0.42 0.42 0.60 0.45 ^ ^ ^ ^ ^ ^ ^ 1.49 3.74 3.85 1.44 0.24 1.08 1.67 4 (4) 4 (4) Bc10 Bc16
4£ 3£ 4£ 8£ 4£ 4£ 4£
Ciprofloxacin Vancomycin PAE (hours) ^ SD MIC (MBC) (mg/ml)
Conc., £ MIC at PAE
According to the results of susceptibility testing, the B. cereus strains Bc10, Bc16, Bc63 and Bc100 were exposed to 16 mg/ml (app. 4| MIC) for vancomycin and 2 mg/ml (10| MIC) for ciprofloxacin, except for the strain Bc63 with MIC 1.6 mg/ml of ciprofloxacin, for which this concentration represented 1.25| MIC (Table 1). The antibiotics were added to the logarithmic-phase broth culture, obtained by the dilution of the overnight culture, containing approximately 107 CFU/ml (1 : 100 dilution).16 Viable counting was performed on samples collected every hour for 8 hours and then at 24 hours. The samples were decimal diluted in phosphate-buffered saline (PBS), and 100 ml of dilutions and the original cultures were seeded on MH agar for viable counting. The growing colonies were counted after 24 hours of incubation at 37uuC. All experiments were performed in triplicate for each of four chosen B. cereus isolates, as described in previous studies.16,17
PAE (hours) ^ SD
Time –kill assay
Conc., £ MIC at PAE
The minimum inhibition concentration (MIC) and the minimum bactericidal concentration (MBC) of vancomycin and ciprofloxacin of four B. cereus strains (Bc10, Bc16, Bc63 and Bc100) were determined with a microdilution method, using Mueller–Hinton broth (MH, Merck, Germany) in accordance with the national standards.15 The concentrations of vancomycin and ciprofloxacin after dilution and the addition of the bacterial suspension ranged from 0.06 to 128 mg/ml and from 0.05 to 100 mg/ml, respectively.
MIC (MBC) (mg/ml)
Susceptibility testing
Bacterial strains
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Vancomycin and ciprofloxacin (American Pharmocopoeia, Rockville, MD, USA) were used for the study of antibiotic susceptibility, the PAE and the time–kill effect. Each agent was freshly prepared according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) in the appropriate solvent. The stock solutions of vancomycin and ciprofloxacin were prepared by diluting the antibiotic powder with distilled water.15
Ciprofloxacin
Antimicrobial agents
Vancomycin
These four strains were selected based on their resistance to chosen antimicrobials. Colony morphology, cell morphological and physiological characteristics, as well as haemolytic activity were determined using conventional procedures.13 The isolates were identified with the API 50 CHB and API 10S test systems using the API WEB identification programme (bioMerieux, Marcy-I’Etoile, France). The control strain used to validate the antimicrobial susceptibility testing was Staphylococcus aureus ATCC 29213.14
Reduction of log CFU/ml after 2 hours exposure to antibiotics
PAEs of vancomycin and ciprofloxacin against B. cereus
Table 1 Postantibiotic effects (PAEs) of vancomycin induced by concentrations of 16 μg/ml and of ciprofloxacin in concentration 2 μg/ml, on four B. cereus strains. Additionally, the PAE of vancomycin on concentrations of 12 and 32 μg/ml on the strain Bc16 was also tested. The reduction in viable bacterial cells after 2hours exposure to antibiotics was represented as well
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The bactericidal activity and bacteriostatic activity were determined. Bactericidal activity was defined as a 3 log10 decrease in CFU/ml (99.9% kill). Bacteriostatic activity was defined as v99.9% kill of bacterial cells after exposure to an antimicrobial.18
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The effect of antimicrobials to sporulation Vancomycin and ciprofloxacin in concentrations 16 and 2 mg/ml, respectively, were added to the logarithmic-phase broth cultures of four tested strains, prepared as for time–kill assay. The samples were divided into two, and one of them was thermized at 80uuC for 10 min.19 The heat shock procedure killed the vegetative bacteria without affecting the viability of the spore population. Then the samples that were and were not subjected to heat shock were quantitatively cultured on antibiotic-free MH agar. The number of colonies of thermized and unthermized samples was counted after incubation at 30uuC for 72 hours to determine the effect of each regimen on the spore and total B. cereus populations.19,20 The control cultures without antimicrobials were tested using the same procedure. The number of spores and the percentage of sporulation were calculated after recovery of viable spores and bacteria on MH agar from three independent experiments. The colony-forming units per millilitre of unthermized samples represented the number of spores and vegetative cells, while the colony-forming units per millilitre in thermized samples represented the number of spores.21
Determination of PAE
The strains were exposed to 16 mg/ml (app. 4| MIC) for vancomycin and 2 mg/ml (10| MIC for ciprofloxacin for 2 hours. The exception was Bc63, where the concentration of ciprofloxacin used in the experiment represented 1.25| MIC. Additionally, the exposure to 3| MIC (12 mg/ml) and 8| MIC (32 mg/ml) of vancomycin was implemented with the strain Bc16. The lower concentrations with about 4| MIC and 8| MIC of ciprofloxacin in our preliminary studies had no bacteriostatic effect. The antibiotic was added to the logarithmic-phase broth culture, obtained by dilution of the overnight culture, containing approximately 107 CFU/ml. After 2 hours, the antibiotic was removed by centrifugation at 4000 | g/10 minutes and the washing of the pellet twice in 5 ml of saline; the recovery of the tested strains was followed for 8 hours and then at 24 hours. Viable counting was performed in the same manner as for time–kill assay.16,17 The PAE was calculated using the following formula: PAE 5 T { C, where T was the time necessary for the number of viable organisms in the test culture to increase by 1 log10 (10-fold) above the number, observed immediately after removal of
PAEs of vancomycin and ciprofloxacin against B. cereus
antibiotics. C was the time necessary for the number of viable organisms in the untreated culture to increase by 1 log10 above the number observed immediately after the completion of the same procedure used in the test procedure for drug removal (centrifugation).16,22
Statistical analysis The data were log-transformed prior to analysis. The IBS SPSS Statistics 22 Programme was used for statistical analyses. Descriptive statistics for calculating the mean, standard deviation, variance minimum and maximum values were chosen. Comparisons between the groups were done with an analysis of variance (ANOVA) with post hoc tests. Comparisons between the PAE of different antimicrobials and their concentrations were calculated with a Wilcoxon signed rank test. Any P value below 0.05 was considered significant.
Results Susceptibility testing The MIC and MBC of each antimicrobial agent of four tested isolates are shown in Table 1. The interpretive standards for staphylococci14 or S. aureus (vancomycin)23,24 were used as no interpretive standards have been established for B. cereus. Among the 63 strains, four of them were chosen (Bc10, Bc16, Bc63 and Bc100) based on their resistance to vancomycin (MIC54 mg/ml) and the highest MIC values of ciprofloxacin. The only resistant strain to ciprofloxacin was Bc63 (MIC 5 1.6 mg/ml).
Time–kill assay The kill curve profiles following vancomycin and ciprofloxacin exposure are shown in Fig. 1A–D. The untreated control sample showed exponential growth reaching a maximum of 6.7–7.6 log10 CFU/ml 4–6 hours after the start of the experiment. We found statistically significant correlations in the reduction of viable counts between ciprofloxacin and vancomycin with all tested strains. However, the difference in duration of inhibitory activity between the two antibiotics was not statistically significant (P 5 0.064). The lowest CFU value of the cultures treated with antibiotics was reached after approximately 4 hours 4 of exposure to 4| MIC of vancomycin and 6.6 hours of exposure to 10| MIC of ciprofloxacin. These data were the mean values for the strains Bc10, Bc16 and Bc100. The strain Bc63, resistant to ciprofloxacin, exhibited a bacteriostatic effect after 4 hours (vancomycin) to 5 hours (ciprofloxacin), and the lowest CFU values were reached after 6 hours of exposure to each of both antimicrobials. This strain only demonstrated a reduction of viable counts for w3 log10 in the presence of the ciprofloxacin compared with the
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reduction of 3 log10 CFU/ml. The viable count after 6 hours incubation was w3 log10 CFU/ml lower for all strains in comparison with the untreated controls (Fig. 1A–D).
The effect of antimicrobials to sporulation
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The average number of viable cells in all four control unthermized samples (Bc10, Bc16, Bc63 and Bc100) increased to 7.7 log10 CFU/ml over 24 hours. The spore population in the thermized samples slowly decreased to the sixth hour of incubation and then rose by about 2 log10 CFU/ml within 24 hours, when their number reached 4 log10 CFU/ml and represented 0.02% of the total bacterial count. The total number of cells in cultures with added antimicrobials decreased to 3.5 log10 CFU/ml after 6 hours ofincubation and rose to 5.3 log10 CFU/ml in 24 hours. The spores represented only 0.6% (after 6 hours) and 0.01% (after 24 hours) of the total (vegetative and spore) bacterial count. The results did not differ significantly between the cultures exposed to vancomycin and ciprofloxacin (P 5 0.064). After 48 hours incubation, the number of spores and the total number of cells in control samples without antibiotics reached 5.2 log10 CFU/ ml and 7.2 log10 CFU/ml, respectively. The values of the spores and the total bacterial count in samples treated with antibiotics decreased to under 3 log10 CFU/ml (Fig. 2).
Figure 1 (A– D) Representative time–kill plots for B. cereus isolates Bc10 (A), Bc16 (B), Bc63 (C) and Bc100 (D), exposed to 4 3 MIC vancomycin (VAN) and 10 3 MIC ciprofloxacin (CIP). The exception was Bc63, where the concentration of ciprofloxacin used in the experiment represented 1.25 3 MIC. Bc10C, Bc16C, Bc63C and Bc100C were untreated control cultures for each strain.
starting inoculum, whereas this antimicrobial yielded a reduction from 2 log10 CFU/ml but not higher than 3 log10 for the other three strains. Vancomycin yielded a 2 log10 reduction or greater for all four tested strains, but none of them expressed the
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Figure 2 The average values of spore count and vegetative cell count in the cultures of four B. cereus strains (Bc10, Bc16, Bc63 and Bc100) in the absence of the antimicrobials and at the presence of 4 3 MIC of vancomycin and 10 3 MIC (1.25 3 MIC at Bc63) of ciprofloxacin during 48 hours incubation. VAN, total bacterial count (vegetative cells and spores), exposed to vancomycin; VANSP, the number of spores, exposed to vancomycin; CIP, total bacterial count (vegetative cells and spores), exposed to ciprofloxacin; CIPSP, the number of spores, exposed to ciprofloxacin; CON, total bacterial count (vegetative cells and spores), without antimicrobial; CONSP, the number of spores, without antimicrobial.
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Determination of PAEs The average duration of the PAE of 16 mg/ml of vancomycin against three tested B. cereus strains (Bc10, Bc63 and Bc100) ranged between 0.24 and 1.94 hours with a mean value of 0.94 hours. The PAE of 16 mg/ml of ciprofloxacin with a median value of 1.60 hours and range between 0.48 and 2.47 hours was longer than that caused by vancomycin. The extremely long PAE duration of 3.47 hours of vancomycin against the fourth strain, Bc16, was observed. Vancomycin produced a slightly longer PAE (1.67 hours) than ciprofloxacin did (1.56 hours). The PAEs of ciprofloxacin and vancomycin were the shortest with the strain Bc63, while the values of PAE with Bc10 and Bc100 of both antibiotics were quite similar (Table 1). The average reduction values of the bacterial count of all four tested strains after 2 hours exposure to vancomycin and ciprofloxacin were 1.96 log10 and 1.58 log10, respectively. After 6 hours of incubation following antibiotic removal, the average increase in the viable count was 3.08 log10 for vancomycin and 2.65 log10 for ciprofloxacin. The regrowth of the Bc16 culture was significantly longer in comparison to the other strains. The strain, Bc16, differed from other three tested strains in some characteristics. Without added antibiotics, the culture Bc16 grew slightly slower than other strains did. The average number of viable cells after 6 hours incubation increased only by 0.98 log10 CFU/ml in comparison with average 1.40 log10 CFU/ml of the other three untreated cultures (Table 1, Fig. 3B). Bc16 differed significantly in the CFU count during 24 hours growth in comparison to the other three strains (P 5 0.001). Furthermore, it also differed from other strains in the duration of PAE induced by vancomycin (P 5 0.03) and ciprofloxacin (P 5 0.001). As seen in Fig. 3B, the PAE of vancomycin of the strain Bc16 was induced by all three concentrations (3|, 4| or 8| S. aureus MIC). The reduction of CFU after 2 hours exposure to 3| MIC was 1.24 log10. The regrowth of bacterial culture was similar at both 3| MIC and 4| MIC (1.06 and 1.04 log10). At the 8| MIC, there was a significant (P v 0.05) reduction of the inoculum after 2 hours exposure to vancomycin, but the rate of regrowth was high, and the duration of PAE was short. The time of PAE was significantly longer after exposure to 8| MIC in comparison to those induced by lower concentrations (3| MIC and 4| MIC). The duration of PAE of vancomycin was not concentration dependent, while the reduction of the bacterial count was.
Discussion The systemic and wound infections caused by B. cereus are usually treated with antimicrobials,
Figure 3 (A –D) Postantibiotic effect (PAE) of 16 mg/ml of vancomycin (4 3 MIC) (VAN) and 2 mg/ml of ciprofloxacin (10 3 MIC) (CIP) on strains B. cereus Bc10 (A), Bc16 (B), Bc63 (C) and Bc100 (D). The exception was Bc63, where the concentration of ciprofloxacin used in the experiment represented 1.25 3 MIC. Bc10C, Bc16C, Bc63C and Bc100C were untreated control cultures for each strain. The PAE of 12 mg/ml (3 3 MIC, VAN12) and 32 mg/ml (8 3 MIC, VAN32) of vancomycin was additionally tested on the strain Bc16 (B).
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such as aminoglycosides, macrolides, lincosamides, tetracycline and particularly with carbapenems, glycopeptides (vancomycin) and quinolones (ciprofloxacin).25,26 Turnbull et al. (2004)27 also believed that susceptibility to ciprofloxacin was uniform; it has been shown to be highly effective in the treatment of B. cereus wound infections. We evaluated the time–kill curves and postantibiotic (PAE) effect of vancomycin and ciprofloxacin against four B. cereus isolates from food and clinical specimens, which displayed higher MICs for these two antibiotics out of 63 tested. The time–kill analysis was done with an antibiotic concentration corresponding to 4| the MIC of vancomycin and 10| the MIC of ciprofloxacin, according to Athamna et al. (2004).28 These concentrations are in the range of average peak levels in a serum after dosing 500 mg of these antimicrobials, which are used in the routine therapy.29–31 A bactericidal or killing effect, as defined by a 3 log10 reduction in the viable count, occurred with only one strain (Bc63) out of the four strains exposed to ciprofloxacin. In the experiment, we included the ciprofloxacin-resistant Bc63 strain (MIC 1.6 mg/ml, 1.25| MIC) to verify the potential concentrationdependent effect. A bacteriostatic effect, which is defined as 2 log10 reduction in viable count, was observed with other three strains (Bc10, Bc16 and Bc100) in 1–4 hours and 1–5 hours after exposure to vancomycin and ciprofloxacin, respectively. The rapidity of the effectiveness of the killing of both antimicrobials used in the experiment seems low in respect to Gram-negative32 and some other Gram-positive33 bacteria, and similar to that against B. anthracis.21 Some experiments suggest that vancomycin inhibits peptidoglycane biosynthesis by binding to its intermediates (i.e., a substrate-binding antibiotic) and that it has a bacteriostatic effect to B. subtilis.34,35 Berlanga et al. (2004)32 also reported that ciprofloxacin did not act as a bactericide against B. cereus and B. subtilis. Instead, it seems to have a bacteriostatic effect. The observation of low-level activity against Bacillus species may be the consequence of ciprofloxacin’s decreased interaction with the changed target enzymes DNA gyrase and topoisomerase IV rather than its diminished permeability.32,36 Their findings also suggests that ciprofloxacin might not serve as a good substrate for the Bacillus efflux pump, which depends on the proton-motive force.32 The tests carried out by Citron et al. (2006)37 show that doptamycin and ciprofloxacin are both rapidly bactericidal against the B. cereus group, while their killing effect is slower for representative strains of B. subtilis and B. pumilus for which the number of bacterial cells decreased only for 2 log10 during
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24 hours incubation. In our case, there were clearly expressed strain-dependent differences in the inhibitory effects of vancomycin and ciprofloxacin (Fig. 1A–D). The most pronounced killing effect of both antimicrobials was found on strain Bc63, in spite of it having the highest ciprofloxacin MIC. We cannot explain this phenomenon. The number of viable cells after 24 hours incubation was higher than that before the addition of the antibiotic to the culture with the strains, Bc16, Bc63 and Bc100. The strain Bc10 did not show the increase in the number of viable cells after the removal of the antibiotic (Fig. 1A–D). Published studies on the PAE of the antimicrobials against strains B. cereus in the available bibliography are rare. Some of them were performed on pathogen species B. anthracis, which belongs to the group B. cereus sensu lato as well as B. cereus.38 According to Deziel et al. (2005),39 the exposure to subinhibitory concentrations of antimicrobials, such as quinolones, might promote sporulation instead of the killing the bacterial cells. This might be the case in our study: the regrowth occurred in the presence of ciprofloxacin with all four tested strains. This antimicrobial is not a protein synthesis inhibitor,40 so it does not inhibit the production of the molecules and enzymes that are needed for vegetative bacteria to form spores. Louie et al.21 reported that ciprofloxacin in high concentrations rapidly killed vegetative B. anthracis so that the total population consisted primarily of spores. In his study, the heat-shock studies demonstrated that there were spores in 10% of the population in 24-hour bacterial culture prior to the start of the antibiotic treatments, while in our tested cultures in log-phase the spores were almost absent (only 0.04%), whereas after 48 hours the number of spores was 2 log10 CFU/ml lower than the total bacterial count (1%). In all four B. cereus strains treated with antimicrobials, we detected the difference between the total (primarily vegetative bacteria) and the spore population after the first 6 hours, even though the total population was decreasing. At this point, the vegetative B. cereus represented 99.4% of the bacterial population (Fig. 2). There was no difference in the ratio between the spores and vegetative cells of the untreated culture and the two cultures with antibiotics, or between the cultures with vancomycin and ciprofloxacin (P 5 0.064). We examined the PAE effect induced by 10| MIC of ciprofloxacin and at 4| MIC of vancomycin. The duration of PAE of vancomycin was 1.67 hours, which is only slightly longer than that of ciprofloxacin (1.56 hours). Vancomycin induced longer PAE than ciprofloxacin did but only with the strain Bc16, which grew slower and weaker in
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the culture even without antimicrobials. Against other three tested strains, ciprofloxacin was more effective, and its PAEs took longer than those of vancomycin. The reason for such results was probably a higher concentration of ciprofloxacin used in the study (Table 1, Fig. 3A–D). Our preliminary experiments confirmed that the concentrations of 4| MIC and even 8| MIC of ciprofloxacin did not significantly reduce the number of viable bacteria of the tested B. cereus strains (data are not shown in this article). These results are comparable with the study by Athamna et al. (2004)28 in which PAEs of the 10| MIC ciprofloxacin and vancomycin against B. anthracis strains were 2–3 and 1–2 hours, respectively. The differences in PAEs of both antimicrobials were statistically significant (P 5 0.006). Moreover, the duration of PAE was strain dependent. The duration of PAE of ciprofloxacin and vancomycin was the shortest for the strain Bc63, due to its resistance to higher concentrations of ciprofloxacin. The PAEs of vancomycin in different concentrations of Bc16 were compared. Its activity in PAE might be characteristic of a concentration-dependent drug, because increasing drug concentration above 4| MIC had significant beneficial effects on bacterial counts and their re-growth (P v 0.05).
Conclusions This study confirms that some B. cereus isolates have developed resistance to vancomycin and ciprofloxacin, which are often used in the therapy of infections associated with this bacteria. The bactericidal effect was determined in only one strain exposed to ciprofloxacin, although the antimicrobial concentrations of even 10| MIC were used in the experiment. The bacteriostatic effect was observed after 1–5 hours, depending on the strain; the regrowth was significant for all tested strains. Vancomycin and ciprofloxacin did not have any significant effect on the sporulation on tested B. cereus strains. The differences in PAE were dependent upon the B. cereus strain, the antibiotic tested, and the antibiotic concentration. Organisms differ in their growth curves, and the rate of the resumption of more rapid growth and different drugs can cause different effects. This must be taken into consideration when comparisons of growth rates are made after antimicrobial treatment.41 Postantibiotic effect was found to be strain dependent. In vitro results indicate that vancomycin and ciprofloxacin may represent valid antimicrobials in the treatment of B. cereus infections, although with different bacteriostatic rates. Fluoroquinolones exhibit a particularly high-penetration capacity into the cells of bacteria and tissues as well as a binding capacity with endocellular components and
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prolonged permanence in comparison to other classes of antibiotics. To confirm our results, it would be necessary to include more B. cereus isolates in the study and to perform molecular typing of antibiotic resistance. In cases of severe infections, the doses of ciprofloxacin should be higher than those obtained with 500 mg by oral route or administered by I.V. determining serum levels.
Acknowledgements We would like to thank the Ministry of Science, Education and Sports, Agency for Mobility and EU Programmes of Croatia for financial support.
Disclaimer Statements Contributors All authors (Branka Bedenič, Vanda Plečko and Karmen Godič Torkar) contributed to the manuscript. Funding None. Conflict of Interest statement The authors declare that they have no conflict of interest. Ethical approval Ethical approval was not required.
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