Determination of synergy between sulbactam,meropenem and colistin in carbapenem-resistant Klebsiella pneumoniae and Acinetobacter baumannii isolates and correlation with the molecular mechanism of resistance

Treatment of infections with carbapenem-resistant Gram negative organism is a major challenge especially among intensive care patients. Combinations of sulbactam, meropenem and colistin was studied for its synergistic activity against 100 invasive isolates of carbapenem-resistant Klebsiella pneumoniae and Acinetobacter baumannii-calcoaceticus complex by checkerboard assay and time kill assay (TKA). In addition, presence of carbapenemase production was determined by multiplex PCR. Time kill assay detected more synergy than checkerboard assay. Good bactericidal activity of 70–100% was noted with the combinations tested. Among K. pneumoniae, isolates producing NDM carbapenemase alone showed significantly more synergy than isolates producing OXA-48-like carbapenemases. In treatment of infection with carbapenem-resistant organisms, the site of infection and the type of carbapenemase produced may help to determine the most effective combination of antimicrobials.     

In vitro synergy of colistin combinations against extensively drug-resistant Acinetobacter baumannii producing OXA-23 carbapenemase

Fifty extensively drug-resistant Acinetobacter baumannii (XDRAB) were isolated from patients. The chequerboard microdilution method was used to determine the in vitro activities of five colistin (COL)-based combinations including COL+fosfomycin (FOS), COL+rifampicin (RIF), COL+imipenem (IMP), COL+sulbactam (SUP) and COL+levofloxacin (LVX). The synergistic activity was evaluated by the fractional inhibitory concentration index (FICI). According to our results, the combination of COL was synergistic with FOS, RIF, IMP, SUP and LVX with the ratios of 50, 72, 88, 92 and 64%, respectively. When combined with COL, the other five agents showed increased antimicrobial activities. In addition, two of the combinations, COL+RIF and COL+IMP, were more active than the combinations of COL+FOS, COL+SUP and COL+LVX. More importantly, these combination regimens could exert synergistic effects at the sub-minimum inhibitory concentration (MIC) levels against XDRAB strains.     

In vitro bactericidal activity of antibiotic combinations against clinical isolates of Mycobacterium chelonae

Mycobacterium chelonae is one of the most antibiotic-resistant species of the pathogenic, rapidly-growing mycobacteria. The objective of this study was to evaluate the bactericidal activity of classical and new potential drugs, alone and in combinations, against clinical isolates of Mycobacterium chelonae. Clarithromycin was seen to be the most bactericidal drug. In drug combinations, this compound is most useful with gentamicin, fluoroquinolones, rifampicin, linezolid, other aminoglycosides, doxycyline and carbapenems. The combination of tobramycin with ciprofloxacin or moxifloxacin also exhibits similar activity. In conclusion, this study on bactericidal activity of drug combinations against Mycobacterium chelonae confirms the utility of clarithromycin associated with other drugs, and, of the new drugs, the value of moxifloxacin and linezolid.     

In vitro effect of subminimal inhibitory concentrations of antibiotics on the biofilm formation ability of Acinetobacter baumannii clinical isolates

The ability of A cinetobacter baumannii strains to form biofilm is one of the most important virulence factor which enables bacterial survival in a harsh environment and decreases antibiotic concentration as well. Subminimal inhibitory concentrations (subMICs) of antibiotics may change bacterial ultrastructure or have an influence on some different molecular mechanisms resulting in morphological or physiological changes in bacteria itself. The aim of this study was to determine effects of 1/2, 1/4, 1/8 and 1/16 minimal inhibitory concentrationsof imipenem, ampicillin-sulbactam, azithromycin, rifampicin and colistin on biofilm formation ability of 22 biofilm non-producing and 46 biofilm producing A. baumannii strains (30 weak producing strains and 16 moderate producing strains). Results of this study indicate that 1/2–1/16 MICs of imipenem, azithromycin, and rifampicin can reduce bacterial biofilm formation ability in moderate producing strains (p < 0.05), whereas 1/16 MIC of imipenem and 1/4–1/8 MICs of rifampicin reduce the biofilm formation in weak producing strains (p < 0.05). Statisticaly significant effect was detected among biofilm non-producing strains after their exposure to 1/16 MIC of azithromycin (p = 0.039). SubMICs of ampicillin-sulbactam and colistin did not have any significant effect on biofilm formation among tested A. baumannii strains.     

In Vitro Interactions of Aminoglycosides with Imipenem or Ciprofloxacin against Aminoglycoside Resistant Acinetobacter baumannii

Summary

The in vitro interactions between gentamicin, tobramycin, netilmicin and amikacin with imipenem and ciprofloxacin were evaluated by the killing curve technique against 20 clinical isolates of Acinetobacter baumannii highly resistant to aminoglycosides which were susceptible or moderately susceptible to imipenem and resistant or moderately susceptible to ciprofloxacin. Imipenem enhanced killing by gentamicin, tobramycin, netilmicin and amikacin in tests with 9, 12, 10 and 15 strains (45-75%) while ciprofloxacin with 3, 7, 5 and 6 strains (15-35%) respectively. Interaction results were influenced by the height of aminoglycoside minimum bactericidal concentrations (MBCs) but were independent of imipenem or ciprofloxacin MBCs and the presence of aminoglycoside modifying enzymes. It is concluded that enhanced killing after aminoglycoside interaction with imipenem or ciprofloxacin versus A. baumannii cannot be predicted but it should be carefully tested in vitro. The in vivo significance of the reported findings mandates clinical studies in humans.     

Detection of the plasmid-mediated quinolone resistance determinants in clinical isolates of Acinetobacter baumannii in China

We examined the prevalence of plasmid–mediated quinolone resistance (PMQR) determinants among Acinetobacter baumannii in Anhui, China. And β–lactamase genes and mutations in quinolone resistance–determining regions (QRDRs) were also investigated among the PMQR-positive isolates. Among the 39 A. baumannii isolates, 3 (7.7%) isolates harbored qnrB and 1 (2.6%) harbored qnrS. Mutations in the QRDRs of gyrA were identified in 2 isolates amongst the 4 PMQR-positive isolates. Quinolone resistance could be transferred by conjugation from all 4 PMQR-positive donors. In conclusion, more attention should be taken to prevent the transmission of PMQR genes among A. baumannii.     

DNA fingerprinting and antimicrobial susceptibility pattern of clinical and environmental Acinetobacter baumannii isolates: a multicentre study

Background and aim: The aims of this study were to establish antibiotic profile and the molecular epidemiology of Acinetobacter baumannii isolates, with considering the effectiveness of control infection measures across three hospitals in the Kurdistan, west part of Iran.

Methods: Fifty-four A. baumannii isolates were collected from patients and environmental specimens. Antibiotic susceptibility patterns (Antibio-type) were evaluated for 17 different antibiotics and MIC for imipenem was done. Isolates were assessed for the presence of metallo-beta-lactamases (MBLs), class 1 and 2 integrons, and integrated gene cassettes and bla_{OXA-likefamilies} genes. Repetitive-sequence-based PCR (REP-PCR) was done for analysing clonality and relativeness of isolates (REP-type).

Results: Antibiotic susceptibility patterns distinguished 11 distinct Antibio-types and REP-PCR showed three clusters with 20 subclusters, mostly belonged to two clonal subgroups, A1 and B1. bla_OXA-51 and bla_OXA-23 were detected in 100% (54/54) and 52% (28/54), respectively, while bla_OXA-24-like and bla_OXA-58 were not present in isolates. MBLs were not detected, but, however, high rate of imipenem resistance was observed (52%). MIC₉₀ of imipenem was 16 mg/ml. Class 1 integrons were detected in 11% (6/54) of isolates followed by 24% (13/54) of class 2. Both classes of integron genes were detected in 15% (8/54) of isolates. Integrated gene cassettes were in low level (11% of class 1 harboring isolates). Two arrays of gene cassettes were revealed, dfrA5-like and dfrA17-aadA5.

Conclusion: Infection control surveillance should be considered as a serious manner, even the superficial eradication of hospital acquired pathogens. MBL genes were not induced carbapenem resistance in studied hospital settings, but bla_{OXA-51 & 23} contributed in imipenem resistant. Integrons had a little share in resistance of A. baumannii isolates.     

Assessment of the combination of doripenem plus a fluoroquinolone against non-susceptible Acinetobacter baumannii isolates from nosocomial pneumonia patients

Abstract

Carbapenem- and fluoroquinolone-non-susceptible Acinetobacter baumannii were obtained from four nosocomial pneumonia patients who were clinically cured following combination therapy with doripenem/levofloxacin or ciprofloxacin. In vitro synergy of doripenem/levofloxacin or ciprofloxacin was evaluated using time-kill analysis. In vivo synergy was tested using a mouse lethal infection model. In time-kill studies, doripenem and levofloxacin were both bactericidal when tested at C_max; at ½C_max, the combination showed synergy up to 8 hours. Ciprofloxacin, alone or combined with doripenem, was not bactericidal. For mouse septicemia, doripenem (100 mg/kg) was ≧90% effective in preventing death in all four isolates. Levofloxacin (200 mg/kg) was 73% effective, and ciprofloxacin (35 mg/kg) was ineffective in preventing death. At lower drug concentrations, increased efficacy was observed for doripenem/levofloxacin, but not for doripenem/ciprofloxacin. Overall, the results suggest that a doripenem/levofloxacin combination may have clinical utility in treating some non-susceptible A. baumannii infections.     

In vitro interactions of aminoglycosides with imipenem or ciprofloxacin against aminoglycoside resistant Acinetobacter baumannii.

The in vitro interactions between gentamicin, tobramycin, netilmicin and amikacin with imipenem and ciprofloxacin were evaluated by the killing curve technique against 20 clinical isolates of Acinetobacter baumannii highly resistant to aminoglycosides which were susceptible or moderately susceptible to imipenem and resistant or moderately susceptible to ciprofloxacin. Imipenem enhanced killing by gentamicin, tobramycin, netilmicin and amikacin in tests with 9, 12, 10 and 15 strains (45-75%) while ciprofloxacin with 3, 7, 5 and 6 strains (15-35%) respectively. Interaction results were influenced by the height of aminoglycoside minimum bactericidal concentrations (MBCs) but were independent of imipenem or ciprofloxacin MBCs and the presence of aminoglycoside modifying enzymes. It is concluded that enhanced killing after aminoglycoside interaction with imipenem or ciprofloxacin versus A. baumannii cannot be predicted but it should be carefully tested in vitro. The in vivo significance of the reported findings mandates clinical studies in humans.     

The resistance and transmission mechanism of Acinetobacter baumannii isolates in a tertiary care hospital,China

The aim of this study was to analyse the resistance and epidemiological data of 117 Acinetobacter baumannii isolates from Southwest Hospital, Chongqing, China. Except for polymyxin B, tigecycline, minocycline, cefoperazone/sulbactam, amikacin and levofloxacin, the resistance rates of other antimicrobial agents were above 90%. All the clinical isolates had the bla_OXA-51 gene and 114 isolates had the bla_OXA-23 gene. Forty-nine isolates were found to contain the bla_IMP-4 gene. PFGE data showed that 117 isolates were divided into 25 groups. Sixty-three (53.85%) were found to carry the class 1 integron, and the sequence analysis of the class 1 integron internal variable regions – five types, one of which had the bla_IMP-4 gene. For the bla_IMP-4 positive strain without class 1 integron, we found the flanking sequence had the TnpA gene. The result suggested that the resistance gene was widely distributed in our hospital; moreover, the modes of presence and transmission are different and complicated. The results of our study can improve the infection empirical treatment method and infection control programme.     

In vitro activity of nitroxoline against clinical isolates of Candida species

The in vitro antifungal activity of the quinoline nitroxoline has been compared with those of amphotericin B, flucytosine, and two azoles, miconazole and ketoconazole, against clinical isolates of Candida spp. A total of 186 isolates of 10 species of Candida and two culture collection strains were tested by an agar-dilution technique. Nitroxoline was highly active against Candida spp. MICs for nitroxoline ranged between 0.25-2 micrograms ml-1 for 186 representative strains. With MIC90 as the measure of antifungal activity, nitroxoline appeared to be superior to the imidazoles studied. Data for individual species of Candida revealed that the activities of nitroxoline and amphotericin B were generally just as effective against C. albicans, whereas flucytosine was the most active agent against Candida spp.     

In vitro activity of beta-lactam antimicrobial agents in combination with aztreonam tested against metallo-beta-lactamase-producing Pseudomonas aeruginosa and Acinetobacter baumannii

We evaluate the antimicrobial interactions between aztreonam and selected beta-lactams when tested against metallo-beta-lactamase (MbetaL)-producing clinical strains. Ten Pseudomonsa aeruginosa strains, including nine MbetaL-producers (IMP-1, -2, -13, -16, VIM-1, -2, -7, SPM-1 and GIM-1) and five Acinetobacter baumannii strains, including three MbetaL-producers (IMP-1 and -2) were tested using time kill/bactericidal activity methods. Aztreonam at 4, 8 and 16 mg/L was combined with four other beta-lactam antimicrobials (cefepime, ceftazidime, meropenem and piperacillin/tazobactam or ampicillin/sulbactam), each tested at the recognized susceptible breakpoint concentration. Enhanced activity (synergism or additive effect) was observed with four P. aeruginosa strains (IMP-16, VIM-2, SPM-1 and GIM-1 containing strains) and four A. baumannii strains, while antagonism was observed with two P. aeruginosa (IMP-16 and SPM-1-producing strains) and one A. baumannii (non-MbetaL) strain. All other strains showed indifferent interaction (variation of +/- 1 log10 CFU/ml) with any combination evaluated.     

In vitro antifungal activity of sertaconazole against 309 dermatophyte clinical isolates

Three hundred and nine strains belonging to 11 species of dermatophyte moulds were tested against sertaconazole following mainly the National Committee for Clinical Laboratory Standards (M38-P) for filamentous fungi. However, several important factors such as the temperature (28 degrees C vs 35 degrees C) and time of incubation (4-10 d vs 21-74 h), have been modified. Sertaconazole was active against all the clinically important dermatophyte moulds involved in human infections tested. Overall geometric mean MIC of sertaconazole was 0.21 microg/ml with a MIC range of 0.01-8 microg/ml. MIC50 and MIC90 were respectively of 0.25 and 1 microg/ml. Sertaconazole was very active against Epidermophyton floccosum, Trichophyton rubrum, Trichophyton tonsurans and Microsporum canis (geometric means 0.08, 0.13, 0.13 and 0.19 microg/ml respectively). Microsporum audouinii had the lowest susceptibility in the study (geometric mean 0.59 microg/ml). Considering MIC50 and MIC90 these differences were significantly in favor of the activity of sertaconazole against E. floccosum (0.06 and 0.5 microg/ml respectively).     

In Vitro Evaluation of the Antimicrobial Activity of Meropenem in Combination with Polymyxin B and Gatifloxacin Against Pseudomonas aeruginosa and Acinetobacter baumannii

Abstract

The antimicrobial activity of meropenem combined with either polymyxin B or gatifloxacin was evaluated by the checkerboard method against Pseudomonas aeruginosa (10 strains) and Acinetobacter baumannii (10 strains). In addition, the triple combination of polymyxin B, gatifloxacin, and meropenem was also studied as well as the polymyxin B and gatifloxacin combination. A partial synergism interaction between meropenem and polymyxin B was observed for 80% of the A. baumannii strains. In contrast, this combination showed an indifferent effect for 80% of the P. aeruginosa strains tested. The combination of meropenem and gatifloxacin showed synergism only for two strains of A. baumannii, and partial synergism and additive effect for seven strains and indifference for four strains of both species. For the strains of P. aeruginosa, the double combination of polymyxin B and gatifloxacin and the triple combination of meropenem, polymyxin B and gatifloxacin were indifferent for the majority of the strains tested, that is, 90 and 80% respectively.     

In vitro evaluation of griseofulvin against clinical isolates of dermatophytes from Isfahan

Fifty dermatophyte isolates, recently obtained from clinical materials, belonging to Trichophyton mentagrophytes, T. verrucosum, Microsporum canis and Epidermophyton floccosum were examined for their susceptibility to griseofulvin. The minimum inhibitory concentration (MIC) values were obtained using the modified microdilution method. All 100% tested isolates had MIC geometric mean at a concentration between 0.43 and 0.95 microg ml(-1) The MIC(90)s and MIC(50)s were 8 microg ml(-1) and <0.25-1 microg ml(-1) respectively. From all isolates, 12% including three T. verrucosum, one M. canis and two T. mentagrophytes isolates had MIC values out of the standardized range, therefore, they were considered as relatively griseofulvin-resistant. At least some of the isolates tested might be difficult to eradicate in clinical terms with griseofulvin treatment in Isfahan.     

In vitro activity of fluconazole, voriconazole and caspofungin against clinical yeast isolates

Predicting the clinical outcome of a systemic mycosis is often a difficult task, especially when microbiological resistance is one of the factors contributing to therapeutic failure. Some of these factors are host-related--e.g. immune state, site and severity of infection, poor compliance to therapy--while others are associated with the drug's characteristics--e.g. dosage, type of compound (fungistatic/fungicidal), pharmacokinetic properties and drug-drug interactions. In the last few years, clinicians have been confronted with the problem of selecting the most appropriate antifungal therapy for systemic infections and have highlighted the need for a reliable method to assay the in vitro susceptibility of yeasts and molds to different antifungal agents, which would allow them to institute a tailored therapy. Using the CLSI micromethod--the reference method for clinically relevant yeast testing--we assayed 70 clinical yeast isolates ( Candida spp., collected from patients with systemic mycosis) for susceptibility against fluconazole, voriconazole and caspofungin. Data obtained from our in vitro susceptibility assays revealed good activity of azoles against the majority of Candida spp. In particular, 88.6% of the assayed isolates were susceptible to fluconazole, with minimum inhibitory concentrations (MICs) ranging from =0.125 microg/mL to 8 microg/mL; 97.1% of the isolates were susceptible to voriconazole, with MICs ranging from 0.008 microg/mL to 1 microg/mL; regarding caspofungin 72.9% of the isolates had MICs ranging from 0.25 microg/mL to 1 microg/mL.     

In vitro evaluation of the antimicrobial activity of meropenem in combination with polymyxin B and gatifloxacin against Pseudomonas aeruginosa and Acinetobacter baumannii

The antimicrobial activity of meropenem combined with either polymyxin B or gatifloxacin was evaluated by the checkerboard method against Pseudomonas aeruginosa (10 strains) and Acinetobacter baumannii (10 strains). In addition, the triple combination of polymyxin B, gatifloxacin, and meropenem was also studied as well as the polymyxin B and gatifloxacin combination. A partial synergism interaction between meropenem and polymyxin B was observed for 80% of the A. baumannii strains. In contrast, this combination showed an indifferent effect for 80% of the P. aeruginosa strains tested. The combination of meropenem and gatifloxacin showed synergism only for two strains of A. baumannii, and partial synergism and additive effect for seven strains and indifference for four strains of both species. For the strains of P. aeruginosa, the double combination of polymyxin B and gatifloxacin and the triple combination of meropenem, polymyxin B and gatifloxacin were indifferent for the majority of the strains tested, that is, 90 and 80% respectively.     

In vitro activity of ertapenem against common clinical isolates in relation to human pharmacokinetics

The in vitro activity of ertapenem against bacterial pathogens isolated from patients with moderate to severe complicated intra-abdominal, complicated skin/skin structure, acute pelvic, or complicated urinary tract infection or community acquired pneumonia was compared to ceftriaxone and piperacillin-tazobactam and related to known plasma concentrations of the three agents. Ertapenem was more potent against methicillin-susceptible Staphylococcus aureus (MSSA) than ceftriaxone and piperacillin-tazobactam and was more potent and more active than both of these agents against Enterobacteriaceae and anaerobes. Piperacillin-tazobactam was the most active agent against enterococci and Pseudomonas aeruginosa. All isolates of Enterobacteriaceae (n=1088), Streptococcus pyogenes (n=37), Streptococcus agalactiae (n=48), MSSA (n=187), Haemophilus influenzae (n=59), and Moraxella catarrhalis (n=9) were susceptible to ertapenem; < 1% of 1284 anaerobes and only 1 of 113 Streptococcus pneumoniae (a penicillin-resistant isolate) were resistant to ertapenem. The MIC value at which 90% of all Enterobacteriaceae, streptococci, MSSA, H. influenzae, M. catarrhalis, and anaerobes were inhibited (MIC90) was < or = 1 microg/ml and below the mean plasma concentration of total ertapenem following a 1 g intravenous infusion for at least 24 hours, i.e., the entire recommended dosing interval, and below the free drug concentration for at least 8 h.     

In vitro synergy of pseudolaric acid B and fluconazole against clinical isolates of Candida albicans

Candida albicans is the most common fungal pathogen in humans. The emergence of resistance to azole antifungals has raised the issue of using such antifungals in combination to optimise therapeutic outcome. The objective of this study was to evaluate in vitro synergy of pseudolaric acid B (PAB) and fluconazole (FLC) against clinical isolates of C. albicans. The in vitro antifungal activity of PAB, a diterpene acid from Pseudolarix kaempferi Gordon, was evaluated alone and in combination with FLC against 22 FLC-resistant (FLC-R) and 12 FLC-susceptible (FLC-S) C. albicans using the chequerboard microdilution method and time-killing test assays. Synergism was observed in all 22 (100%) FLC-R strains tested as determined by both fractional inhibitory concentration index (FICI) with values ranging from 0.02 to 0.13 and bliss independence (BI) models. Synergism was observed in two of 12 (17%) FLC-S strains as determined by FICI model with values ranging from 0.25 to 0.5 and in three of 12 (18%) FLC-S strains as determined by BI model. For FLC-R strains, the drug concentrations of FLC and PAB, where synergistic interactions were found, ranged from 0.06 to 4 μg ml(-1) and 0.5 to 4 μg ml(-1) respectively. For FLC-S strains, the drug concentrations of FLC and PAB were 1-8 μg ml(-1) and 0.5-4 μg ml(-1) respectively. The BI model gave results consistent with FICI, but no antagonistic activity was observed in any of the strains tested. These interactions between PAB and FLC were confirmed using the time-killing test for the selected strains. Fluconazole and PAB exhibited a good synergism against azole-R isolates of C. albicans.