Double Antibiotic Combinations Research Articles

Intracellular bacterial eradication using a novel peptide in vitro.

To determine the effect of a novel antimicrobial peptide (AMP; OP145) and cell-penetrating peptide (Octa-arginine/R8) conjugate on the killing of intracellular Enterococcus faecalis, compared to OP145 and an antibiotic combination recommended for regenerative endodontic procedures. The biocompatible concentrations of OP145 and OP145-R8 were determined by assessing their cytotoxicity against human macrophages and red blood cells. Spatiotemporal internalization of the peptides into macrophages was investigated qualitatively and quantitatively by confocal laser scanning microscopy and flow cytometry respectively. Killing of extracellular and intracellular E. faecalis OG1RF by the peptides was determined by counting the colony-forming units (CFU). Intracellular antibacterial activity of the peptides was compared to a double antibiotic combination. Confocal microscopy was used to confirm the intracellular bacterial eradication. Significant differences between the different test groups were analysed using one-way analysis of variance. p < .05 was considered to be statistically significant. Peptides at a concentration of 7.5 μmol/L were chosen for subsequent experiments based on the results of the alamarBlue™ cell viability assay and haemolytic assay. OP145-R8 selectively internalized into lysosomal compartments and the cytosol of macrophages. Conjugation with R8 improved the internalization of OP145 into macrophages in a temporal manner (70.53% at 1 h to 77.13% at 2 h), while no temporal increase was observed for OP145 alone (60.53% at 1 h with no increase at 2 h). OP145-R8 demonstrated significantly greater extracellular and intracellular antibacterial activity compared to OP145 at all investigated time-points and concentrations (p < .05). OP145-R8 at 7.5 μmol/L eradicated intracellular E. faecalis after 2 h (3.5 log reduction compared to the control; p < .05), while the antibiotics could not reduce more than 0.5 log CFU compared to the control (p > .05). Confocal microscopy showed complete absence of E. faecalis within the OP145-R8 treated macrophages. The results of this study demonstrated that the conjugation of an AMP OP145 to a cell-penetrating peptide R8 eradicated extracellular and intracellular E. faecalis OG1RF without toxic effects on the host cells.

Susceptibility to antibiotic combinations of &lt;i&gt;Klebsiella pneumoniae&lt;/i&gt; and &lt;i&gt;Acinetobacter baumannii&lt;/i&gt; strains isolated from COVID-19 patients

Objective. To assess the susceptibility of K.pneumoniae and A.baumanii strains isolated from hospitalized COVID-19 patients to antibiotics and their combinations.Materials and methods. The minimum inhibitory concentrations (MICs) of meropenem and colistin were determined for 47 A.baumannii and 51K.pneumoniaestrains isolated from the hospitalized COVID-19 patients by the broth microdilution method. The susceptibility to 11 antibiotic combinations was assessed using the method of multiple combination bactericidal testing.Results. Colistin resistance was detected in 31.9 % of A.baumannii strains (MIC50 — 0.5 mg/l, MIC90 — 16 mg/l) and in 80.4 % of K.pneumoniaestrains (MIC50 — 16 mg/l, MIC90 — 256 mg/l). It has been shown that double antibiotic combinations with the inclusion of colistin exhibit bactericidal or bacteriostatic activity against 76.6–87.2 % of A.baumannii strains. Combinations with the addition of meropenem, colistin and macrolides exhibited bactericidal activity against 78.4–80.4 % of K.pneumoniae strains. Combinations of two carbapenems were not active, the combination of meropenem-colistin had a bactericidal effect only in 13.7 % of K.pneumoniae strains.Conclusion. Widespread colistin resistance was found in carbapenem-resistant K.pneumoniae and A.baumannii strains isolated from the hospitalized COVID-19 patients. The combinations of antibiotics that have a synergistic antibacterial effect in their pharmacokinetic/pharmacodynamic concentrations have been determined.

Are antibiotic prescription practices in Eastern Uganda concordant with the national standard treatment guidelines? A cross-sectional retrospective study

This study aimed to evaluate the antibiotic prescription patterns of health workers in Eastern Uganda and more specifically whether they are in accordance with the Ugandan standard treatment guidelines and other indicators of appropriate antimicrobial prescription. Patient data were obtained from the health management information system of the outpatient department registers of Soroti and Mbale Regional Referral Hospitals from 2016-2018. The prevalence of non-adherence to treatment guidelines when prescribing antibiotics was 82.6% (95% CI 81.4-83.7%). Guidelines were more likely to be adhered to when prescribing antibiotics for individuals aged 13-19 years compared with their counterparts aged 0-12 years [adjusted odds ratio (aOR)=0.55, 95% CI 0.40-0.74]. When prescribing antibiotics for males, health workers were twice as likely not to adhere to guidelines compared with when prescribing for females (aOR=2.09, 95% CI 1.61-2.72). When prescribing cephalosporins and nitroimidazoles, health workers were likely not to adhere to guidelines compared with when prescribing penicillin (cephalosporins, aOR=1.92, 95% CI 1.28-2.86; nitroimidazoles, aOR=1.70, 95% CI 1.09-2.65). Health workers were most likely not to follow guidelines when prescribing antibiotics in combination (two antibiotics, aOR=1.27, 95% CI 1.03-1.56). Non-adherence to treatment guidelines for an indicated diagnosis and inappropriate antibiotic prescription are significantly prevalent in Eastern Uganda. Health workers were more likely not to follow guidelines when prescribing for males, children up to 12 years of age and when prescribing cephalosporins, nitroimidazoles or double antibiotic combinations.

In Vitro Activity of Fosfomycin in Double and Triple Combinations with Imipenem, Ciprofloxacin and Tobramycin Against Multidrug-Resistant Escherichia coli.

The rates of urinary tract infection with multidrug-resistant (MDR) Escherichia coli have dramatically increased and the treatment of these infections with single and double antibiotic combinations became limited or ineffective. The present study aimed at finding effective antibiotic combinations against MDR uropathogenicE. coli. Antibiotic susceptibility testing of uropathogenic E. coli isolates (n = 29) showed that all the examined isolates were found to be MDR. The interaction of double and triple combinations of fosfomycin (FOS) with imipenem (IPM), ciprofloxacin (CIP) and tobramycin (TOB) against selected isolates (n = 8) by checkerboard method showed that all the examined combinations exhibited synergistic effects (FIC index < 1) against tested isolate. However, 1/8, 5/8 and 6/8 of the isolates remained resistant to the constituent antibiotics in FOS/IPM, FOS/CIP and FOS/TOB combinations, respectively. Notably, the triple combinations (FOS/IPM/CIP, FOS/IPM/TOB and FOS/CIP/TOB) increased the synergism against all selected isolates at MIC levels lower than the susceptible breakpoints. Furthermore, time-kill analysis demonstrated that FOS/IPM combination exhibited synergistic and bactericidal effects with UTI-9. However, the combination had no effect on UTI-13. The highest synergistic and bactericidal effects against both representative isolates were achieved by FOS/IPM/CIP, FOS/IPM/TOB and FOS/CIP/TOB combinations after 2h of post-treatment and lasted up to 24h. Therefore, we report here that the combinations of FOS with IPM, CIP and TOB could be beneficial against MDR uropathogenic E. coli at least in vitro. The effectiveness of these antibiotics increased in combination with FOS compared to individual antibiotics acting alone.

In vitro activities of colistin, tigecycline and tobramycin, alone or in combination, against carbapenem-resistant Enterobacteriaceae strains.

The aim of this study was to investigate the activities of various antibiotics, alone or in combination, against carbapenem-resistant Enterobacteriaceae (CRE). Minimum inhibitory concentrations (MICs) of meropenem, colistin, tigecycline and tobramycin were determined by microbroth dilution against 40 clinical strains. Carbapenemase-encoding genes were detected by PCR using specific primers. The in vitro synergistic activities of tigecycline, colistin and tobramycin in double antibiotic combinations were determined by the microbroth chequerboard technique, and results were interpreted using the fractional inhibitory concentration index (FICI). To confirm the results acquired by the chequerboard method, time-kill assays were performed on eight isolates representing four different susceptibility patterns. Based on MIC90 values, colistin was the most potent agent, followed by tigecycline and tobramycin. According to PCR studies, carbapenem resistance in tested Enterobacteriaceae isolates was most often mediated by OXA-48-type carbapenemases. With an FICI of ≤0.5 as the cut-off, synergistic interactions were most frequent with tigecycline+tobramycin (30%); other results for synergistic interactions were 23% for colistin+tobramycin and 9% for colistin+tigecycline. By time-kill assays, all tested antibiotic combinations demonstrated synergistic activity against at least three of the eight strains at 1× or 4× MIC. Overall, the combinations used in this study were effective regimens, demonstrating synergy or no interaction (indifference) against all tested strains. No antagonism was observed with either of the techniques. The findings of this study might play a useful role in selecting appropriate combinations when a single agent is inadequate against CRE strains.

Çocuk Yoğun Bakım ve Yenidoğan Yoğun Bakımlarda Antibiyotik Kullanımı; Çok Merkezli Nokta Prevalans Çalışması

Objective: Identifying antibiotic use in pediatric and neonatal intensive care units with a point prevalence study in Adana, Turkey’s sixth largest city. Material and Methods: In this point prevalence study, demographic information and antibiotic treatment data were taken on the same day from patients in pediatric and neonatal intensive care units of 6 hospitals located in Adana’s city center. Results: Four pediatric intensive care units (two university, one research and training hospital, and one public hospital) and six neonatal intensive care units (two university, one research and training hospital, one public hospital, and two private hospital) were included in the study; 220 patients were at the intensive care units at the time of the study, 44 (20%) of the patients were in the pediatric intensive care units, and 176 (80%) of them were at the neonatal intensive care units. Also, 146 (66.4%) of the patients were using antibiotics. The frequency of antibiotic use was 72.7% in the pediatric intensive care units and 64.8% in neonatal intensive care units. There was a pediatric infectious disease physician at the university and research and training hospital. Antibiotic usage was lower (p=0.002) in clinics where pediatric infectious disease physician consultations could be done. Double antibiotic combination was applied most frequently. Mostly, ampicillin was preferred at neonatal intensive care units. Clarithromycin was used as a second choice because of seasonal lower respiratory tract infections. Vancomycin was the most preferred antibiotic in pediatric intensive care units, and meropenem and linezolid were the second and third choices. At both intensive care units, use of empiric antibiotic treatment was more frequent. Empiric treatment was applied in 22 (68.7%) patients in the pediatric intensive care units and 95 (83.3%) in neonatal intensive care units. Antibiotics were given to 14.3% of the patients in line with the resulting cultures. Conclusion: Intensive care units are services where antibiotics are used most frequently both in Turkey and in the world. In our opinion, protocols need to be established in clinics, national and international guides should be followed; and pediatric infectious disease physician consultations should be increased in order to reduce the frequency of antibiotic use, inappropriate indications, and inappropriate doses. (J Pediatr Inf 2014; 8: 56-63)

Evaluation of Double- and Triple-Antibiotic Combinations for VIM- and NDM-Producing Klebsiella pneumoniae by In Vitro Time-Kill Experiments

Combination therapy is recommended for infections with carbapenemase-producing Klebsiella pneumoniae. However, limited data exist on which antibiotic combinations are the most effective. The aim of this study was to find effective antibiotic combinations against metallo-beta-lactamase-producing K. pneumoniae (MBL-KP). Two VIM- and two NDM-producing K. pneumoniae strains, all susceptible to colistin, were exposed to antibiotics at clinically relevant static concentrations during 24-h time-kill experiments. Double- and triple-antibiotic combinations of aztreonam, ciprofloxacin, colistin, daptomycin, fosfomycin, meropenem, rifampin, telavancin, tigecycline, and vancomycin were used. Synergy was defined as a ≥2 log10 decrease in CFU/ml between the combination and its most active drug after 24 h, and bactericidal effect was defined as a ≥3 log10 decrease in CFU/ml after 24 h compared with the starting inoculum. Synergistic or bactericidal activity was demonstrated for aztreonam, fosfomycin, meropenem, and rifampin in double-antibiotic combinations with colistin and also for aztreonam, fosfomycin, and rifampin in triple-antibiotic combinations with meropenem and colistin. Overall, the combination of rifampin-meropenem-colistin was the most effective regimen, demonstrating synergistic and bactericidal effects against all four strains. Meropenem-colistin, meropenem-fosfomycin, and tigecycline-colistin combinations were not bactericidal against the strains used. The findings of this and other studies indicate that there is great potential of antibiotic combinations against carbapenemase-producing K. pneumoniae. However, our results deviate to some extent from those of previous studies, which might be because most studies to date have included KPC-producing rather than MBL-producing strains. More studies addressing MBL-KP are needed.

Biofilm Compared to Conventional Antimicrobial Susceptibility of Stenotrophomonas maltophilia Isolates from Cystic Fibrosis Patients

Stenotrophomonas maltophilia is a multidrug-resistant organism increasingly isolated from the lungs of cystic fibrosis (CF) patients. One hundred twenty-five S. maltophilia isolates from 85 CF patients underwent planktonic and biofilm susceptibility testing against 9 different antibiotics, alone and in double antibiotic combinations. When S. maltophilia isolates were grown as a biofilm, 4 of the 10 most effective antibiotic combinations included high-dose levofloxacin and 7 of the 10 combinations included colistin at doses achievable by aerosolization.

Combination antibiotic susceptibility of biofilm-grown Burkholderia cepacia and Pseudomonas aeruginosa isolated from patients with pulmonary exacerbations of cystic fibrosis

We identified double and triple antibiotic combinations effective against biofilm-grown Burkholderia cepacia and Pseudomonas aeruginosa sampled from cystic fibrosis (CF) patients undergoing acute pulmonary exacerbations. Sputum bacteria from 110 CF patients were grown as biofilms. Combination antibiotic susceptibility testing was used to test 94 double and triple antibiotic combinations. Biofilm-grown bacterial isolates were less susceptible to antibiotic combinations compared to the same bacterial isolates grown planktonically (P < 0.001). Fifty-nine percent of biofilm-grown B. cepacia isolates and 29% of P. aeruginosa isolates were resistant to all double antibiotic combinations tested. Triple antibiotic combinations were more effective than double antibiotic combinations against biofilms (P < 0.0001). For P. aeruginosa biofilms, the addition of azithromycin or rifampin to otherwise effective antibiotic combinations was frequently associated with antagonism. Bacterial biofilms of CF organisms are highly resistant to antibiotics. This study identified potentially effective antibiotic combinations to guide the empirical treatment of CF pulmonary exacerbations.

143 The Effect of Various Antimicrobial Agents on Acinetobacter Baumannii: An In Vitro Evaluation

Background: Acinetobacter baumannii has recently emerged as an important hospital-acquired pathogen, especially in surgery, burn, and intensive care units. Due to its ability to develop resistance to antimicrobials, wound infection with A. baumannii is difficult to treat, and can lead to septicemia and even death. Use of appropriate topical antimicrobial agents in these circumstances could be one of the first steps in prevention of A. baumannii wound infection. Objectives: In this study, we will discuss the in vitro effects of seven common topical antimicrobial creams and dressings on A. baumannii. Methods: A. Baumannii ATCC# 6919 was subjected to sensitivity tests against mupirocin, silver sulfadiazine, mafenide acetate, a double antibiotic combination of polymyxin and bacitracin, a triple antibiotic combination of Neomycin, bacitracin and polymyxin, and two silver-containing dressings. Zones of inhibition were measured after 24 hours incubation period. Results and Conclusion: Of the evaluated antimicrobial agents, mafenide acetate was the most efficacious followed by mupirocin, triple and double antibiotic combinations in decreasing order. The silver-containing dressings yielded a lesser zone of inhibition as compared to the previously mentioned, and no zone of inhibition was observed using silver sulfadiazine. Further in vivo studies on the effect of antimicrobial agents against A. Baumannii are necessary to substantiate these findings and determine the potential clinical relevance of these therapies.

Effects of Commonly Used Topical Antimicrobial Agents onAcinetobacter baumannii: An In Vitro Study

Acinetobacter baumannii has recently emerged as an important pathogen among wounded soldiers in Iraq. Because of its ability to develop resistance to antimicrobial agents, wound infections with A. baumannii are difficult to treat and can lead to septicemia and even death. Use of appropriate topical antimicrobial agents in these circumstances could be one of the first steps in the prevention of A. baumannii wound infections. In this study, we present the in vitro effects of seven common topical antimicrobial creams and dressings on A. baumannii. A. baumannii was subjected to sensitivity tests with mupirocin, silver sulfadiazine, mafenide acetate, a double-antibiotic combination of polymyxin and bacitracin, a triple-antibiotic combination of neomycin, bacitracin, and polymyxin, and two silver-containing dressings. Zones of inhibition were measured after 24 hours of incubation. Of the evaluated antimicrobial agents, mafenide acetate was the most efficacious, followed by mupirocin and triple- and double-antibiotic combinations (in decreasing order). The silver-containing dressings yielded smaller zones of inhibition, compared to the previously mentioned agents, and no zone of inhibition was observed with silver sulfadiazine. Further in vivo studies on the effects of antimicrobial agents against A. baumannii are necessary to substantiate these findings and to determine the potential clinical relevance of these therapies.

Approach to resistant gram-negative bacterial pulmonary infections in patients with cystic fibrosis.

Patients with cystic fibrosis are living longer with chronic pulmonary bacterial infections. One consequence of antibiotic treatment of these chronic infections has been the increasing prevalence of antibiotic resistance seen in bacterial isolates recovered from patients with cystic fibrosis. Bacteria such as Pseudomonas aeruginosa and Burkholderia cepacia are able to acquire antibiotic resistance by either spontaneous mutation or gene transfer via plasmids or integrins. In addition, bacteria survive by forming antibiotic-resistant biofilms within the airways of patients with cystic fibrosis. Therapeutic approaches to dealing with antibiotic-resistant bacterial pulmonary infections include the use of in vitro synergy testing to determine optimal double antibiotic combinations or multiple-combination bactericidal testing to determine bactericidal double and triple antibiotic combinations to use against the bacteria in the clinical setting of acute exacerbations. Therapy for antibiotic-resistant bacterial infections in cystic fibrosis involves the use of new laboratory methods (synergy testing or multiple-combination bactericidal testing) to optimize antibiotic treatment strategies. Clinical trials are required to address whether treatment guided by susceptibility testing improves clinical outcomes. Future novel approaches will likely include drugs that can disrupt bacterial biofilm formation and the use of cationic peptide antimicrobial compounds.

Antimicrobial Susceptibility and Synergistic Activity of Meropenem Against Gram-Negative Non-Fermentative Bacteria Isolated from Cystic Fibrosis Patients

The objective of the present study was to evaluate the activity of meropenem (a beta-lactam carbapenem with good bactericidal potency and a very wide spectrum of activity) and of ticarcillin, ceftazidime ciprofloxacin, tobramycin, cefepime, which are the most commonly used antimicrobial agents for treatment of pulmonary infections associated with CF. The effect of these antibiotics was tested on 27 multiresistant strains isolated from 24 CF patients during 2000 and 2001. Furthermore, the in vitro synergistic effect of meropenem in association with the other antibiotics was evaluated.Ciprofloxacin, ticarcillin, meropenem and ceftazidime had the most activity and inhibited 66%, 37%, 36% and 33% of strains respectively. The addition of a second antibiotic to meropenem resulted in a synergistic effect on 5 (18.5%) isolates; on average 2.8 synergistic combinations where determined per strain. Of these 27 isolates, antagonism was observed in 3 (11%) strains (1 antagonistic combination per strain).Our study suggests that selecting effective double antibiotic therapy cannot be made empirically for CF patients infected with Gram-negative multiresistant bacilli. Therefore in vitro methods for testing double antibiotic combinations are mandatory.

Multiple combination bactericidal antibiotic testing for patients with cystic fibrosis infected with multiresistant strains of Pseudomonas aeruginosa.

We developed a rapid in vitro antibiotic susceptibility test to screen double- and triple-antibiotic combinations for bactericidal activity against 75 multiresistant Pseudomonas aeruginosa isolates referred from 44 cystic fibrosis (CF) patients. When used alone, the most effective intravenous antibiotic, meropenem, was bactericidal against only 44% of the isolates. High-dose tobramycin (200 microg/ml; concentrations achievable by aerosol administration) was bactericidal against 72% of isolates. Adding a second antibiotic significantly improved bactericidal activity. The most effective double-antibiotic combinations contained high-dose tobramycin plus meropenem, piperacillin/tazobactam, or ciprofloxacin, and were bactericidal against 88 to 94% of the isolates. Excluding high-dose tobramycin, the most effective intravenous double-antibiotic combinations contained meropenem plus ciprofloxacin, tobramycin (4 microg/ml), or cefipime, and were bactericidal against 85%, 71%, and 70% of isolates, respectively. Adding a third antibiotic did not significantly improve inhibition in vitro. We conclude that double-antibiotic combinations containing meropenem or high-dose tobramycin show the best bactericidal activity in vitro against multiresistant strains of P. aeruginosa. Addition of a third antibiotic to these double-antibiotic combinations may be unnecessary.

Multiple combination bactericidal antibiotic testing for patients with cystic fibrosis infected with Burkholderia cepacia.

Most Burkholderia cepacia strains are resistant to many, or all, of the antibacterial agents commonly used in cystic fibrosis (CF), and selection of appropriate antibiotics for treatment of pulmonary exacerbations is therefore difficult. We developed a technique for rapid in vitro testing of multiple antibiotic combinations for B. cepacia isolates. For each of 119 multi-drug-resistant isolates of B. cepacia, our multiple combination bactericidal test (MCBT) studied the bactericidal activity of 10 to 15 antimicrobial agents using 225 +/- 97 single, double, and triple antibiotic combinations. Of the 119 isolates, 50% were resistant to all single antibiotics tested, 8% were resistant to all two-drug antibiotic combinations, but all were inhibited by at least one bactericidal triple-drug combination. When used alone, meropenem, ceftazidime and high-dose tobramycin (200 microg/ml) were bactericidal against only 47, 15, and 14% of in vitro isolates, respectively. Using a double antibiotic combination improved bactericidal activity; meropenem-minocycline, meropenem-amikacin, and meropenem-ceftazidime combinations were bactericidal against 76, 73, and 73% of isolates, respectively. However, 47% of isolates demonstrated antagonism (growth of an organism when a second antibiotic was added to a bactericidal single antibiotic). Triple antibiotic combinations that contained tobramycin, meropenem, and an additional antibiotic were most effective, and were bactericidal against 81 to 93% of isolates. We conclude that triple-antibiotic combinations are more likely than double and single antibiotic combinations to be bactericidal against B. cepacia in vitro. MCBT testing is a useful technique to help clinicians decide on appropriate nonantagonistic combination antibiotic therapy for patients with CF infected with B. cepacia.