In vitro interaction of fluconazole and trimethoprim-sulfamethoxazole against Candida auris using ETEST and checkerboard methods.

Candida auris was discovered in 2009 and has rapidly emerged as a serious public health threat with cases reported in over 20 countries worldwide. As of May 8, 2020, the Centers for Disease Control and Prevention reported a total of 1122 US cases. C. auris is often multidrug resistant, leaving few options for treatment. Sulfonamides are known to inhibit a bacterial enzyme involved in folate synthesis and may also inhibit yeast organisms by a similar mechanism. The combination of trimethoprim and sulfamethoxazole is more commonly used than either drug alone. The objective of this study was to evaluate the combination of fluconazole and trimethoprim-sulfamethoxazole against C. auris Minimum inhibitory concentrations (MICs) of fluconazole and trimethoprim-sulfamethoxazole were determined by ETEST and broth microdilution for 11 Cauris strains. Fluconazole MICs (µg/mL) were 4->256 by ETEST and 2->256 by broth microdilution (73% resistant); trimethoprim-sulfamethoxazole MICs were >32 by ETEST and 32->128 by broth microdilution (no interpretive guidelines for C. auris). Using our MIC: MIC ETEST method and a checkerboard method, we investigated the interaction of fluconazole and trimethoprim-sulfamethoxazole against all isolates. These interactions were analyzed by calculating the summation fractional inhibitory concentration with synergyof ≤0.5, additivity of >0.5-1.0, indifference of >1-4, and antagonism of >4. The combination of fluconazole and trimethoprim-sulfamethoxazole revealed synergy with three (27%) and additivity with one (9%) isolate. Indifference was found for the remaining seven (64%) isolates. With the checkerboard method, synergy was seen in 1/11 (9%) isolates with fluconazole (½ MIC) plus trimethoprim-sulfamethoxazole (1/64 MIC); additivity, in 7/11 (64%) isolates with fluconazole (1/8 MIC-1×MIC) plus trimethoprim-sulfamethoxazole (1/128 MIC-½ MIC); and indifference in 3/11 (27%) isolates. Regardless, in vitro interactions may or may not correlate with clinical outcomes. Synergy testing with additional drug combinations and isolates should be performed.

Evaluation of the in vitro interaction of fosfomycin and meropenem against metallo-ß-lactamase-producing Pseudomonas aeruginosa using Etest and time-kill assay.

Pseudomonas aeruginosa is a nosocomial pathogen containing various resistance mechanisms. Among them, metallo-ß-lactamase (MBL)-producing Pseudomonas are difficult to treat. Fosfomycin is an older antibiotic that has recently seen increased usage due to its activity against a broad spectrum of multidrug-resistant organisms. Our aim was to evaluate the combination of fosfomycin and meropenem against 20 MBL-producing P. aeruginosa (100% meropenem-resistant and 20% fosfomycin-resistant) using both an Etest minimal inhibitory concentration (MIC): MIC method and time-kill assay. MICs for fosfomycin and meropenem were determined by Etest and by broth microdilution method for the latter. The combination demonstrated synergy by Etest in 3/20 (15%) isolates and 5/20 (25%) isolates by time-kill assay. Results from the Etest method and time-kill assay were in agreement for 14/20 (70%) of isolates. No antagonism was found. Comparing both methods, Etest MIC: MIC method may be useful to rapidly evaluate other antimicrobial combinations.

Synergistic effect between nisin and polymyxin B against pandrug-resistant and extensively drug-resistant Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic pathogen predominantly associated with nosocomial infections. The World Health Organization's data on antibiotic-resistant 'priority pathogens' reports carbapenem-resistant A. baumannii as a pathogen which is in critical need of research and development of new antimicrobials. Emerging resistance against polymyxins, last-resort drugs for carbapenem-resistant A. baumannii, increases the need for new therapeutic approaches such as synergistic combinations. Nisin, an antibacterial peptide produced by the Gram-positive bacteria L. lactis, is a US Food and Drug Administration approved food preservative with bactericidal action predominantly against other Gram-positive bacteria. A 2008 study reported that topical nisin was effective against staphylococcal mastitis in humans. Additionally, nisin has shown activity against Gram-negative bacteria in combination with antimicrobials such as polymyxin B. A recent in vitro study reported that nisin and polymyxin B exhibited synergistic activity against one isolate each of A. baumannii, Acinetobacter lwoffii and Acinetobacter calcoaceticus using time-kill assay and checkerboard technique. We evaluated the synergistic potential of nisin and polymyxin B against 15 unique clinical A. baumannii isolates using time-kill assay. Three of eight (38%) extensively drug-resistant and six of seven (86%) pandrug-resistant A. baumannii isolates showed synergy with one or more combinations of nisin and polymyxin B. The synergy seen with the use of lower concentrations of polymyxin B may help in reducing the dose-dependent side effects. Additional studies involving pharmacokinetics and pharmacodynamics of nisin are required to explore clinical possibilities.

In vitro synergy with fluconazole plus doxycycline or tigecycline against clinical Candida glabrata isolates.

Candida species, traditionally viewed as opportunistic agents, are increasingly seen as a cause of infection in hospitalized patients. Treatment options are limited to a few classes of drugs. Increased resistance, especially by Candida glabrata, is problematic. We investigated the interaction between fluconazole and doxycycline or tigecycline, using clinically unique blood culture C. glabrata isolates. Eighteen isolates were screened using an Etest® MIC:MIC synergy method. With the doxycycline plus fluconazole combination, 28% of isolates showed synergy; tigecycline plus fluconazole showed 94% synergy. No antagonism was seen. The mechanisms of these interactions are unclear. Further research is warranted to assess clinical utility.

In vitro Synergistic Activity of Caspofungin Plus Polymyxin B Against Fluconazole-Resistant Candida glabrata.

BACKGROUND: Candida species account for most invasive fungal infections, and the emergence of fluconazole and caspofungin resistance is problematic. Overcoming resistance with synergism between 2 drugs may be useful. In a 2013 in vitro study, caspofungin plus colistin (polymyxin E) was found to act synergistically against fluconazole-resistant and susceptible Candida albicans isolates. The purpose of our study was to extend this finding by evaluating caspofungin plus polymyxin B for in vitro synergy against fluconazole-resistant Candida glabrata isolates. MATERIALS AND METHODS: A total of 7 fluconazole-resistant C. glabrata bloodstream infection isolates were obtained from 2010-2011. Of these, 2 isolates were also resistant to caspofungin. Minimum inhibitory concentrations (MICs) for caspofungin and polymyxin B were determined by Etest and broth microdilution. Clinical and Laboratory Standards Institute breakpoints were used for fluconazole and caspofungin MIC interpretations. No interpretive guidelines exist for testing polymyxin B against C. glabrata. Synergy testing with caspofungin (1 × MIC) and polymyxin B (½MIC) was performed using a modified bacterial Etest synergy method and time-kill assay. RESULTS: With the Etest synergy method, 4 out of 7 isolates showed in vitro synergy and 1 out of 7 showed additivity. The remaining isolates (both caspofungin resistant) showed indifference. Using the time-kill assay, 1 out of 7 isolates showed synergy, 1 showed additivity and the remaining 5 (including both caspofungin-resistant isolates) showed indifference. CONCLUSIONS: Caspofungin susceptibility may be required for synergism between caspofungin and polymyxin B. Further synergy testing with caspofungin plus polymyxin B and additional fluconazole-resistant C. glabrata isolates should be performed. In vitro synergy/additivity may or may not correlate with in vivo benefit.

Detection of a New cfr-Like Gene, cfr(B), in Enterococcus faecium Isolates Recovered from Human Specimens in the United States as Part of the SENTRY Antimicrobial Surveillance Program.

Two linezolid-resistant Enterococcus faecium isolates (MICs, 8 µg/ml) from unique patients of a medical center in New Orleans were included in this study. Isolates were initially investigated for the presence of mutations in the V domain of 23S rRNA genes and L3, L4, and L22 ribosomal proteins, as well as cfr. Isolates were subjected to pulsed-field gel electrophoresis (just one band difference), and one representative strain was submitted to whole-genome sequencing. Gene location was also determined by hybridization, and cfr genes were cloned and expressed in a Staphylococcus aureus background. The two isolates had one out of six 23S rRNA alleles mutated (G2576T), had wild-type L3, L4, and L22 sequences, and were positive for a cfr-like gene. The sequence of the protein encoded by the cfr-like gene was most similar (99.7%) to that found in Peptoclostridium difficile, which shared only 74.9% amino acid identity with the proteins encoded by genes previously identified in staphylococci and non-faecium enterococci and was, therefore, denominated Cfr(B). When expressed in S. aureus, the protein conferred a resistance profile similar to that of Cfr. Two copies of cfr(B) were chromosomally located and embedded in a Tn6218 similar to the cfr-carrying transposon described in P. difficile. This study reports the first detection of cfr genes in E. faecium clinical isolates in the United States and characterization of a new cfr variant, cfr(B). cfr(B) has been observed in mobile genetic elements in E. faecium and P. difficile, suggesting potential for dissemination. However, further analysis is necessary to access the resistance levels conferred by cfr(B) when expressed in enterococci.

Comparison of 3 Etest(®) methods and time-kill assay for determination of antimicrobial synergy against carbapenemase-producing Klebsiella species.

Increasing global antibiotic resistance has resulted in more use of antibiotic combinations. There is a lack of a gold standard for in vitro testing of these combinations for synergy or antagonism. Time-kill assay (TKA) may be used but is labor intensive and not practical for clinical use. Etest® synergy methods are more rapid and easier to perform, but there is no agreement regarding which method is best. We tested 31 clinical genetically unique Klebsiella pneumoniae carbapenemase-producing Klebsiella isolates with the combination of meropenem and polymyxin B by TKA and 3 Etest methods, each in triplicate: Method 1, MIC:MIC; Method 2, direct overlay; and Method 3, cross. Overall, testing with Etest synergy methods showed the following agreement with TKA: Method 1: 25/31 (80.6%), Method 2: 7/31 (22.6%), and Method 3: 8/31 (25.8%). The MIC:MIC method had the highest agreement (80.6%, κ = 0.59, P < 0.001) and should be evaluated more extensively.

In Vitro Synergy of Telavancin and Rifampin Against Enterococcus faecium Resistant to Both Linezolid and Vancomycin.

BACKGROUND: An emerging pathogen is Enterococcus faecium resistant to both linezolid and vancomycin (LRVRE). Antimicrobial combinations may be required for therapy and need to be evaluated. The combination of daptomycin and rifampin has demonstrated good in vitro activity against gram-positive bacteria, including E faecium. Telavancin, a newer lipoglycopeptide, has shown in vitro activity against E faecium. We evaluated the combination of telavancin and rifampin and compared the results to the combination of daptomycin and rifampin used previously on the same isolates. METHODS: Twenty-four genetically unique (by pulsed-field gel electrophoresis), clinical LRVRE isolates were collected in the United States from 2001-2004. Etest minimal inhibitory concentrations (MICs) (µg/mL) were 0.064-8 for telavancin, 1-4 for daptomycin, and 0.012 to >32 for rifampin. In vitro synergy testing was performed in triplicate by an Etest MIC:MIC ratio method, and summation fractional inhibitory concentration (ΣFIC) was calculated: synergy ≤0.5; indifference >0.5-4; and antagonism >4. RESULTS: The Etest method showed synergy (ΣFICs of 0.1-0.5) with telavancin + rifampin in 20/24 (83%) isolates and indifference (ΣFICs of 0.6-0.8) in 4/24 (17%) isolates. Similarly, the daptomycin + rifampin combination showed synergy (ΣFICs of 0.1-0.5) in 21/24 (88%) isolates and indifference (ΣFICs of 0.6-1.0) in 3/24 (12%) isolates by the Etest method. No antagonism was found. CONCLUSIONS: In vitro synergy with both combinations (rifampin + telavancin or daptomycin) was 83% and 88%, respectively, by Etest against these LRVRE isolates. Although both daptomycin and telavancin in combination with rifampin showed a high incidence of synergistic activity, further in vitro synergy testing with this combination should be performed against additional E faecium isolates. In vitro synergy may or may not translate into in vivo effectiveness.

Detection of synergy using the combination of polymyxin B with either meropenem or rifampin against carbapenemase-producing Klebsiella pneumoniae.

Polymyxin B (PB) plus meropenem (MER) or rifampin (RIF) was tested by Etest® method and time-kill assay (TKA) against 14 genetically unique clinical Klebsiella pneumoniae carbapenemase-producing K. pneumoniae. PB + MER: Etest, 43% synergy; TKA, 64% synergy. Concordance between methods was 79%. For PB + RIF: Etest, 21% synergy; TKA, 100% synergy. Concordance between methods was 21%.

In vitro synergistic/additive activity of levofloxacin with meropenem against Stenotrophomonas maltophilia.

Synergy testing of levofloxacin and meropenem by Etest and time-kill assay (TKA) was performed against 30 genetically unique clinical Stenotrophomonas maltophilia isolates. Synergy was demonstrated in 18/30 (60%) isolates by Etest and in 13/30 (43%) by TKA; the remaining isolates were indifferent. Methods showed agreement for 25/30 (83%) of isolates.

In vitro antibacterial activity of tigecycline against resistant Gram-negative bacilli and enterococci by time-kill assay.

This time-kill study was performed with 65 genetically unique clinical isolates of Gram-negative bacilli and enterococci to further define the antibacterial activity of tigecycline. To our knowledge, this is the largest published time-kill study evaluating tigecycline activity to date. Isolates evaluated were 10 meropenem-resistant Acinetobacter baumannii; 15 Escherichia coli, including 10 extended-spectrum beta-lactamase (ESBL) producers; 15 Klebsiella pneumoniae, including 10 ESBL producers; 20 vancomycin-resistant Enterococcus faecium (VRE), including 10 that were linezolid resistant; and 5 vancomycin-susceptible Enterococcus faecalis. Time-kill testing was performed using tigecycline concentrations of 1x, 2x, and 4x MIC with colony-forming units (CFU) per milliliter determined at 0, 4, 8, 12, 24, 36, and 48 h. Tigecycline MICs (microg/mL) were < or =1 for E. coli and K. pneumoniae, regardless of the isolates' ESBL production; A. baumannii, 0.06 to 4; 9/10 (90%) were < or =2; E. faecalis < or =0.12; and VRE < or =0.25, regardless of linezolid susceptibility. In the time-kill assay, tigecycline significantly inhibited bacterial growth when compared with the growth control. The reduction in growth was <3 log(10) CFU/mL for all isolates, indicative of a bacteriostatic effect.

The detection of synergy between meropenem and polymyxin B against meropenem-resistant Acinetobacter baumannii using Etest and time-kill assay.

Time-kill assay and Etest testing for synergy of meropenem (MER) (1x MIC) plus polymyxin B (1/4, 1/2, and 1x MIC) were performed against 8 genetically unique MER-resistant clinical Acinetobacter baumannii isolates. Time-kill assay demonstrated synergy for all isolates, whereas Etest showed synergy in 5 isolates and indifference in 3.

In vitro testing of daptomycin plus rifampin against methicillin-resistant Staphylococcus aureus resistant to rifampin.

OBJECTIVE: To test for synergy between daptomycin (DAP) and rifampin (RIF) against RIF-resistant methicillin-resistant Staphylococcus aureus (MRSA) isolates. METHODS: Synergy testing using time-kill assay (TKA) was performed on 6 clinically, and genetically unique RIF-resistant MRSA isolates. The isolates were identified out of 489 (1.2%) samples collected during April 2003 to August 2006, from patients at the Ochsner Medical Center in New Orleans, Louisiana, United States of America. RESULTS: Synergy testing of DAP plus RIF by TKA showed that 5 isolates were indifferent, but one isolate was antagonistic. CONCLUSION: Our in vitro study failed to demonstrate synergy between DAP plus RIF, against our RIF-resistant MRSA isolates. Clinical failure of this combination should prompt the clinician to consider antagonism, as one of the potential causes.

In vitro synergy of ciprofloxacin and gatifloxacin against ciprofloxacin-resistant Pseudomonas aeruginosa.

Multidrug-resistant Pseudomonas aeruginosa with combined decreased susceptibility to ceftazidime, ciprofloxacin, imipenem, and piperacillin is increasingly being found as a cause of nosocomial infections. It is important to look for combinations of drugs that might be synergistic. Ciprofloxacin resistance by P. aeruginosa is mediated in part by an efflux pump mechanism. Gatifloxacin, an 8-methoxyfluoroquinolone, inhibits a staphylococcal efflux pump. An earlier in vitro study using an Etest synergy method and time-kill assay suggested synergy of ciprofloxacin and gatifloxacin against P. aeruginosa. Synergy testing was performed by Etest and time-kill assay for 31 clinically unique, plasmid DNA distinct, U.S. P. aeruginosa isolates. Etest MICs for ciprofloxacin were 4 to >32 microg/ml, and for gatifloxacin they were >32 microg/ml. Ciprofloxacin plus gatifloxacin showed synergy by the Etest method for 6 (19%) of the 31 P. aeruginosa isolates using a summation fractional inhibitory concentration of < or = 0.5 for synergy. Synergy was demonstrated for 13/31 (42%) of isolates by time-kill assay. No antagonism was detected. The remaining isolates were indifferent to the combination. The Etest method and time-kill assay were 65% (20/31) concordant. The mechanism of the in vitro synergy may include P. aeruginosa ciprofloxacin efflux pump inhibition by gatifloxacin.