Pseudomonas aeruginosa Ceftolozane-Tazobactam Resistance Development Requires Multiple Mutations Leading to Overexpression and Structural Modification of AmpC

We compared the dynamics and mechanisms of resistance development to ceftazidime, meropenem, ciprofloxacin, and ceftolozane-tazobactam in wild-type (PAO1) and mutator (PAOMS, ΔmutS) P. aeruginosa. The strains were incubated for 24 h with 0.5 to 64× MICs of each antibiotic in triplicate experiments. The tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64× MIC for 7 consecutive days. The susceptibility profiles and resistance mechanisms were assessed in two isolated colonies from each step, antibiotic, and strain. Ceftolozane-tazobactam-resistant mutants were further characterized by whole-genome analysis through RNA sequencing (RNA-seq). The development of high-level resistance was fastest for ceftazidime, followed by meropenem and ciprofloxacin. None of the mutants selected with these antibiotics showed cross-resistance to ceftolozane-tazobactam. On the other hand, ceftolozane-tazobactam resistance development was much slower, and high-level resistance was observed for the mutator strain only. PAO1 derivatives that were moderately resistant (MICs, 4 to 8 μg/ml) to ceftolozane-tazobactam showed only 2 to 4 mutations, which determined global pleiotropic effects associated with a severe fitness cost. High-level-resistant (MICs, 32 to 128 μg/ml) PAOMS derivatives showed 45 to 53 mutations. Major changes in the global gene expression profiles were detected in all mutants, but only PAOMS mutants showed ampC overexpression, which was caused by dacB or ampR mutations. Moreover, all PAOMS mutants contained 1 to 4 mutations in the conserved residues of AmpC (F147L, Q157R, G183D, E247K, or V356I). Complementation studies revealed that these mutations greatly increased ceftolozane-tazobactam and ceftazidime MICs but reduced those of piperacillin-tazobactam and imipenem, compared to those in wild-type ampC. Therefore, the development of high-level resistance to ceftolozane-tazobactam appears to occur efficiently only in a P. aeruginosa mutator background, in which multiple mutations lead to overexpression and structural modifications of AmpC.     

Development of resistance in wild-type and hypermutable Pseudomonas aeruginosa strains exposed to clinical pharmacokinetic profiles of meropenem and ceftazidime simulated in vitro

In this study we investigated the interplay of antibiotic pharmacokinetic profiles and the development of mutation-mediated resistance in wild-type and hypermutable Pseudomonas aeruginosa strains. We used in vitro models simulating profiles of the commonly used therapeutic drugs meropenem and ceftazidime, two agents with high levels of antipseudomonal activity said to have different potentials for stimulating resistance development. During ceftazidime treatment of the wild-type strain (PAO1), fully resistant mutants overproducing AmpC were selected rapidly and they completely replaced wild-type cells in the population. During treatment with meropenem, mutants of PAO1 were not selected as rapidly and showed only intermediate resistance due to the loss of OprD. These mutants also replaced the parent strain in the population. During the treatment of the mutator P. aeruginosa strain with meropenem, the slowly selected mutants did not accumulate several resistance mechanisms but only lost OprD and did not completely replace the parent strain in the population. Our results indicate that the commonly used dosing regimens for meropenem and ceftazidime cannot avoid the selection of mutants of wild-type and hypermutable P. aeruginosa strains. For the treatment outcome, including the prevention of resistance development, it would be beneficial for the antibiotic concentration to remain above the mutant prevention concentration for a longer period of time than it does in present regimens.     

Impact of susceptibility profiles of Gram-negative bacteria before and after the introduction of ertapenem at a medical center in northern Taiwan from 2004 to 2010

This retrospective observational study evaluated the impact of antimicrobial consumption on antimicrobial susceptibility among aerobic Gram-negative bacteria after introducing ertapenem to the formulary of a teaching hospital (1130 beds) in northern Taiwan. Data on consumption of various antimicrobial agents, expressed as defined daily dose/1000 patient-days (DDD/1000 PD), were collected retrospectively from hospital pharmacy records 2 years before and 5 years after the introduction of ertapenem (October 2005). During the study period, the consumption of ampicillin and aminoglycosides decreased significantly. In contrast, the consumption of cefoxitin, ceftazidime, cefpirome, piperacillin–tazobactam, carbapenems (ertapenem, imipenem, and meropenem), and fluoroquinolones (ciprofloxacin, levofloxacin, and moxifloxacin) increased significantly over time. There was a significant increase in the rate of susceptibility of Escherichia coli to ampicillin, cefotaxime, ceftazidime, piperacillin–tazobactam, cefpirome, amikacin, and levofloxacin; an increase in the rate of susceptibility of Klebsiella pneumoniae to ceftazidime, cefepime, cefpirome, piperacillin–tazobactam, meropenem, levofloxacin, and amikacin; a significant decrease in the rate of susceptibility of Pseudomonas aeruginosa to meropenem; and a significant decrease in the rate of susceptibility of Acinetobacter baumannii to ceftazidime, carbapenems, ciprofloxacin, and levofloxacin. The rate of antibiotic susceptibility to ertapenem of extended spectrum β-lactamase producers, including E. coli and K. pneumoniae, remained stable. Usage of ertapenem was found to be negatively and significantly associated with the susceptibility rates of P. aeruginosa to meropenem and gentamicin. Significantly negative correlations were noted between the use of ertapenem and the rates of susceptibility of A. baumannii to ceftazidime, piperacillin–tazobactam, carbapenems (imipenem and meropenem), ciprofloxacin, and levofloxacin.     

Antimicrobial usage and resistance trend relationships from the MYSTIC Programme in North America (1999-2001)

BACKGROUND: The MYSTIC Programme is a global, longitudinal antimicrobial surveillance network of hospitals that frequently utilize carbapenems. One aspect of the programme is the ability to capture antimicrobial consumption data from participating institutions. The current report evaluates these relationships for Enterobacteriaceae and Pseudomonas aeruginosa over the initial 3 year period of the programme in the USA. METHODS: Between 10 and 15 medical centres participated during 1999-2001, each submitting up to 200 isolates/year (7003 strains overall). Evaluations of the relationship between drug usage and antimicrobial resistance in P. aeruginosa and Enterobacteriaceae for the carbapenems (imipenem and meropenem), cefepime, ceftazidime, ciprofloxacin, gentamicin and piperacillin-tazobactam were determined. Data were analysed based on: (1) aggregate usage results; (2) medical centre-specific usage compared with resistance rates; and (3) medical centre-specific usage results compared with yearly changes in resistance rates (DeltaR). The parameter of drug usage was the defined daily dose (DDD)/100 patient days calculated from total grams administered, using WHO definitions. RESULTS: Resistance (1999-2001) among Enterobacteriaceae did not change significantly for beta-lactams, but tended to increase slightly for gentamicin (+1.1%) and ciprofloxacin (+3.1%). P. aeruginosa resistance rates (1999-2001) for gentamicin (+9.0%) and ciprofloxacin (+10.2%) increased, in contrast to a significantly decreased resistance rate for meropenem (-7.7%). Formulary-use changes were noted: increased meropenem and ciprofloxacin use and decreased consumption for imipenem, aminoglycosides, ceftazidime and cefepime. Aggregate ciprofloxacin DDD/100 days rates were directly related (+3.3 DDD) to Enterobacteriaceae and P. aeruginosa resistance changes, whereas among P. aeruginosa, usage and resistance were inversely correlated for gentamicin (-3.8 DDD; +9.0% resistant). Medical centre-specific antimicrobial usage calculations did not demonstrate a correlation to rates of resistance (r = -0.38 to 0.61) or yearly changes in resistance rates (r = -0.56 to 0.43). CONCLUSIONS: The availability of aggregate USA medical centre antimicrobial usage data enabled us to identify several important trends in the incidence of resistance among P. aeruginosa and Enterobacteriaceae: (1) increased use of ciprofloxacin associated with a higher resistance among Enterobacteriaceae; and (2) a correlation between ciprofloxacin categories of resistance and levels of resistance to other antimicrobial classes in P. aeruginosa. Medical centre-specific antimicrobial usage and resistance did not demonstrate direct statistical relationships, and require a continued search for other monitoring methods that can better identify antimicrobial/environmental factors that lead to resistance.     

Mutations in β-Lactamase AmpC Increase Resistance of Pseudomonas aeruginosa Isolates to Antipseudomonal Cephalosporins

Mutation-dependent overproduction of intrinsic β-lactamase AmpC is considered the main cause of resistance of clinical strains of Pseudomonas aeruginosa to antipseudomonal penicillins and cephalosporins. Analysis of 31 AmpC-overproducing clinical isolates exhibiting a greater resistance to ceftazidime than to piperacillin-tazobactam revealed the presence of 17 mutations in the β-lactamase, combined with various polymorphic amino acid substitutions. When overexpressed in AmpC-deficient P. aeruginosa 4098, the genes coding for 20/23 of these AmpC variants were found to confer a higher (2-fold to >64-fold) resistance to ceftazidime and ceftolozane-tazobactam than did the gene from reference strain PAO1. The mutations had variable effects on the MICs of ticarcillin, piperacillin-tazobactam, aztreonam, and cefepime. Depending on their location in the AmpC structure and their impact on β-lactam MICs, they could be assigned to 4 distinct groups. Most of the mutations affecting the omega loop, the R2 domain, and the C-terminal end of the protein were shared with extended-spectrum AmpCs (ESACs) from other Gram-negative species. Interestingly, two new mutations (F121L and P154L) were predicted to enlarge the substrate binding pocket by disrupting the stacking between residues F121 and P154. We also found that the reported ESACs emerged locally in a variety of clones, some of which are epidemic and did not require hypermutability. Taken together, our results show that P. aeruginosa is able to adapt to efficacious β-lactams, including the newer cephalosporin ceftolozane, through a variety of mutations affecting its intrinsic β-lactamase, AmpC. Data suggest that the rates of ESAC-producing mutants are ≥1.5% in the clinical setting.     

阴沟肠杆菌的产酶性及抗菌药物的体外联合抗菌活性研究

目的 探讨咸阳地区阴沟肠杆菌的产酶现状及其抗菌药物的应用.方法 收集2012年6月-2013年5月临床分离无重复的100株阴沟肠杆菌,采用KB琼脂扩散法检测超广谱β内酰胺酶（ESBLs）和通过改良三维试验检测AmpC,碳青霉烯酶的检测采用亚胺培南＋ EDTA法,MIC检测采用微量倍比稀释法,按CLSI法规进行.结果 在100株阴沟肠杆菌中,产ESBLs酶的为49％,产AmpC酶的为29％,碳青霉烯酶的为0％,同时产ESBLs和AmpC酶为29％.药敏结果是哌拉西林/他唑巴坦、头孢哌酮/舒巴坦、美洛培南、头孢吡肟、环丙沙星、阿米卡星和头孢他啶的抑菌率分别为64.7％,58.8％,100％,70.6％,70.6％,64.7％,41.1％.联合药物头孢吡肟、哌拉西林/他唑巴坦、美洛培南、头孢哌酮/舒巴坦与阿米卡星对阴沟肠杆菌的协同及相加作用分别为（17.64-41.18）％,（11.76-47.06）％,（5.88-52.94）％和（0-58.83）％.联合药物头孢吡肟、哌拉西林/他唑巴坦、美洛培南、头孢哌酮/舒巴坦与环丙沙星对阴沟肠杆菌协同及相加作用分别为（11.76-23.54）％,（5.88-11.77）％,（11.77-47.06）％和（5.88-17.65）％.结论 产酶的阴沟肠杆菌在该地区处于一个相对较高的水平,因此临床医师应根据药敏结果选用抗菌药物.     

Surveillance for antimicrobial susceptibility among clinical isolates of Pseudomonas aeruginosa and Acinetobacter baumannii from hospitalized patients in the United States, 1998 to 2001

Pseudomonas aeruginosa and Acinetobacter baumannii are the most prevalent nonfermentative bacterial species isolated from clinical specimens of hospitalized patients. A surveillance study of 65 laboratories in the United States from 1998 to 2001 found >90% of isolates of P. aeruginosa from hospitalized patients to be susceptible to amikacin and piperacillin-tazobactam; 80 to 90% of isolates to be susceptible to cefepime, ceftazidime, imipenem, and meropenem; and 70 to 80% of isolates to be susceptible to ciprofloxacin, gentamicin, levofloxacin, and ticarcillin-clavulanate. From 1998 to 2001, decreases in antimicrobial susceptibility (percents) among non-intensive-care-unit (non-ICU) inpatients and ICU patients, respectively, were greatest for ciprofloxacin (6.1 and 6.5), levofloxacin (6.6 and 3.5), and ceftazidime (4.8 and 3.3). Combined 1998 to 2001 results for A. baumannii isolated from non-ICU inpatients and ICU patients, respectively, demonstrated that >90% of isolates tested were susceptible to imipenem (96.5 and 96.6%) and meropenem (91.6 and 91.7%); fewer isolates from both non-ICU inpatients and ICU patients were susceptible to amikacin and ticarcillin-clavulanate (70 to 80% susceptible); and <60% of isolates were susceptible to ceftazidime, ciprofloxacin, gentamicin, or levofloxacin. From 1998 to 2001, rates of multidrug resistance (resistance to at least three of the drugs ceftazidime, ciprofloxacin, gentamicin, and imipenem) showed small increases among P. aeruginosa strains isolated from non-ICU inpatients (5.5 to 7.0%) and ICU patients (7.4 to 9.1%). From 1998 to 2001, rates of multidrug resistance among A. baumannii strains isolated from non-ICU inpatients (27.6 to 32.5%) and ICU patients (11.6 to 24.2%) were higher and more variable than those observed for P. aeruginosa. Isolates concurrently susceptible, intermediate, or resistant to both imipenem and meropenem accounted for 89.8 and 91.2% of P. aeruginosa and A. baumannii isolates, respectively, studied from 1998 to 2001. In conclusion, for aminoglycosides and most beta-lactams susceptibility rates for P. aeruginosa and A. baumannii were constant or decreased only marginally (相似文献   

Antibiotic resistance surveillance over a 4-year period (2000–2003) in Turkey: results of the MYSTIC Program

The Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) Program is a global study that provides antimicrobial susceptibility data in centers prescribing meropenem. The activity of meropenem and 7 broad-spectrum antimicrobials have been examined against 5208 bacterial isolates from 9 Turkish centers between 2000 and 2003. Cumulative susceptibility rates against all species of Enterobacteriaceae combined were ranked as follows: meropenem (99.3%), imipenem (97.6%), cefepime (80.0%), piperacillin–tazobactam (73.6%), ceftazidime (70.3%), ciprofloxacin (70.1%), cefotaxime (66.9%), and tobramycin (67.2%). The production of extended-spectrum β-lactamases (ESBLs) was detected in 48.7% of Klebsiella pneumoniae and in 19.5% of Escherichia coli isolates. Of ESBL producing K. pneumoniae isolates, 75.7% were resistant to tobramycin, 40.3% to ciprofloxacin, and 48.3% to piperacillin–tazobactam. Only piperacillin/tazobactam and carbapenems were active against more than 50% of Pseudomonas aeruginosa at the National Committee for Clinical Laboratory Standards-susceptible breakpoint, and the carbapenems were the most active compounds against Acinetobacter spp. These data confirm the continued potency of meropenem against Enterobacteriaceae in units where it is actively being prescribed.     

MYSTIC Europe 2007: activity of meropenem and other broad-spectrum agents against nosocomial isolates

The Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) Program is a longitudinal antimicrobial surveillance study that has been in existence since 1997 in centers that are actively prescribing meropenem. This report examines the results from the study in Europe in 2007. A total of 5208 isolates were examined for activity (MIC) of meropenem and other broad-spectrum antibacterial comparators. Cumulative susceptibility rates using Clinical and Laboratory Standards Institute criteria against all methicillin-susceptible staphylococci were imipenem (97.7%) > meropenem (97.3%) > piperacillin/tazobactam (96.2%) > tobramycin (94.2%) > gentamicin (92.0%) > ciprofloxacin (84.0%) > ceftazidime (39.8%). Against all species of Enterobacteriaceae, the rates were meropenem (99.4%) > imipenem (98.3%) > tobramycin (92.0%) > gentamicin (89.5%) > ceftazidime (86.2%) > piperacillin/tazobactam (85.5%) > ciprofloxacin (84.2%). Meropenem was most effective against the nonfermenters, although multidrug-resistant Acinetobacter spp. and imipenem-resistant Pseudomonas aeruginosa strains were reported. The continued need for surveillance studies such as MYSTIC is exemplified, and results from these types of surveillance can, hopefully, help in the correct choice of empiric therapy.     

Mutator genes giving rise to decreased antibiotic susceptibility in Pseudomonas aeruginosa

Screening of the PA14 genomic transposon mutant library for resistance to ceftazidime, tobramycin, and ciprofloxacin led to the discovery of several mutants that appeared in more than one screen. Testing of the frequency of mutation to ciprofloxacin resistance revealed previously known mutator genes, including mutS and mutL, as well as mutators that have not yet been described for P. aeruginosa, including PA3958 and RadA (PA4609).     

Activity of meropenem and comparators against Pseudomonas aeruginosa and Acinetobacter spp. isolated in the MYSTIC Program, 2002-2004

This study examines the susceptibilities of meropenem and other broad-spectrum antimicrobials tested against bacterial isolates collected from hospitalized patients during 2002-2004 from worldwide medical centers participating in the Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) Program. The in vitro activity of meropenem and 5 comparator antimicrobial agents was assessed against Pseudomonas aeruginosa and Acinetobacter spp. Generally, the susceptibility of Australasian and North American isolates was higher than that of the European and South American isolates. The rank order of activity of the antimicrobial agents tested against a worldwide collection of P. aeruginosa was piperacillin/tazobactam (77.7% susceptible) > meropenem (75.4%) > ceftazidime (70.0%) > imipenem (69.7%) > gentamicin (66.1%) > ciprofloxacin (62.0%). Against a worldwide collection of Acinetobacter spp. meropenem (76.1% susceptible) was the most active compound followed by imipenem (74.7%) > gentamicin (51.9%) > ciprofloxacin (40.5%) > piperacillin/tazobactam (39.8%) > ceftazidime (38.1%). The carbapenems appear to be a valuable option for the treatment of serious nosocomial infections caused by P. aeruginosa or Acinetobacter spp. over a broad geographical region.     

Differential Selection of Single-Step AmpC or Efflux Mutants of Pseudomonas aeruginosa by Using Cefepime,Ceftazidime, or Ceftobiprole

Single-step Pseudomonas aeruginosa mutants, selected with ceftobiprole, ceftazidime, or cefepime, were generated at frequencies of 10⁻⁶ to <10⁻⁹ at two and four times the MIC. The chromosomal AmpC β-lactamase activity was increased in all ceftazidime-selected mutants. Mutants selected with cefepime either increased AmpC activity or upregulated expression of the mexXY efflux genes. Mutants selected with ceftobiprole did not overexpress AmpC; 90% of these produced elevated levels of mexXY RNA, indicating that increased efflux, not AmpC derepression, is the predominant response to ceftobiprole during first-step mutations in P. aeruginosa.Pseudomonas aeruginosa is a pathogen known for both intrinsic resistance and acquired resistance to antibiotics (15). A common cause of resistance to extended-spectrum cephalosporins in P. aeruginosa is overexpression of the chromosomal AmpC β-lactamase (5). In addition, downregulation of porins and increased expression of efflux pumps have roles in resistance to β-lactams and other types of antibiotics (2, 8, 27). Recent studies with clinical isolates indicated that some extended-spectrum cephalosporins, such as ceftobiprole and cefepime, but not ceftazidime, are substrates of the MexXY-OprM efflux pump (1). For these P. aeruginosa isolates, reduced susceptibility to cefepime and/or ceftobiprole was associated with overexpression of the MexXY efflux system (1, 9, 14). These substrate profiles from clinical isolates correlate with genetic experiments that showed that cefepime, and not ceftazidime, was extruded by MexXY (17).Ceftobiprole, an anti-methicillin-resistant Staphylococcus aureus (MRSA) cephalosporin with broad-spectrum activity, has successfully completed clinical trials for the treatment of complicated skin and skin structure infections and pneumonia (22-24). The activity of ceftobiprole against P. aeruginosa (MIC₅₀, 2 μg/ml) is similar to those of cefepime (MIC₅₀, 4 μg/ml) and ceftazidime (MIC₅₀, 2 μg/ml) (7). Surveillance studies have shown that MICs of cefepime and ceftobiprole are lower than those of ceftazidime for Enterobacteriaceae with AmpC hyperproduction (26); however, there are limited data for P. aeruginosa with overexpressed AmpC. The goal of this study was to analyze the resistance mechanisms present in P. aeruginosa mutants selected by exposure to ceftobiprole, ceftazidime, or cefepime and to evaluate the MIC profiles for mutants that overexpressed either AmpC β-lactamase or the MexXY efflux system.(Part of this study was previously presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, October 2008.)     

In vitro and in vivo activities of a new cephalosporin, FR264205, against Pseudomonas aeruginosa

FR264205 is a novel parenteral 3′-aminopyrazolium cephalosporin. This study evaluated the in vitro and in vivo activities of FR264205 against Pseudomonas aeruginosa. The MIC of FR264205 at which 90% of 193 clinical isolates of P. aeruginosa were inhibited was 1 μg/ml, 8- to 16-fold lower than those of ceftazidime (CAZ), imipenem (IPM), and ciprofloxacin (CIP). FR264205 also exhibited this level of activity against CAZ-, IPM-, and CIP-resistant P. aeruginosa. The reduction in the susceptibility of FR264205 by AmpC β-lactamase was lower than that of CAZ, indicating a relatively high stability of FR264205 against AmpC β-lactamase, the main resistance mechanism for cephalosporins. Neither expression of efflux pumps nor deficiency of OprD decreased the activity of FR264205. No spontaneous resistance mutants were selected in the presence of FR264205, and the reduction in susceptibility to FR264205 was lower than that to CAZ, IPM, and CIP after serial passage, suggesting that FR264205 has a low propensity for selecting resistance. In murine pulmonary, urinary tract, and burn wound models of infection caused by P. aeruginosa, the efficacy of FR264205 was superior or comparable to those of CAZ and IPM. These results indicate that FR264205 should have good potential as an antibacterial agent for P. aeruginosa.     

Antimicrobial spectrum of activity for meropenem and nine broad spectrum antimicrobials: report from the MYSTIC Program (2002) in North America

The Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) Program provides susceptibility data for participating medical centers where carbapenems are utilized. The activity of meropenem and nine broad-spectrum antimicrobial agents were assessed against 3,047 bacterial isolates collected during 2002 from 16 North American sites. The overall rank order of susceptibility of the 10 antimicrobial agents tested against Gram-negative isolates was: meropenem (98%) > imipenem (97%) > cefepime (95%) > tobramycin (93%) > piperacillin/tazobactam = gentamicin (92%) > ceftazidime (91%) > ciprofloxacin (87%) > aztreonam (86%) > ceftriaxone (74%). These results and those from previous years, demonstrate the continued excellent potency and spectrum of activity for meropenem. The utility of meropenem against Pseudomonas aeruginosa isolates has increased steadily with a rise in percent susceptibility each year from 78.2% in 1999 to a present rate of 93.1% susceptible. Conversely, we showed the susceptibility for ciprofloxacin against these same P. aeruginosa isolates has decreased from 82.9 to 72.3% susceptible over four years. Many medical centers have observed a decreased activity of some aminoglycosides, cephalosporins and fluoroquinolones due to increases in rates of extended-spectrum beta-lactamases, Amp C and other resistance mechanisms. Carbapenem resistance remains rarely documented and these beta-lactamase-stable agents appear to be an alternative treatment option for serious community-acquired or nosocomial infections in high risk patient populations.     

Comparative In Vitro Activities of Meropenem, Imipenem, Temocillin, Piperacillin, and Ceftazidime in Combination with Tobramycin, Rifampin, or Ciprofloxacin against Burkholderia cepacia Isolates from Patients with Cystic Fibrosis

We evaluated the activities of meropenem, imipenem, temocillin, piperacillin, and ceftazidime by determination of the MICs for 66 genotypically characterized Burkholderia cepacia isolates obtained from the sputum of cystic fibrosis patients. In vitro synergy assays, as performed by the time-kill methodology, of two- and three-drug combinations of the β-lactams with tobramycin, rifampin, and/or ciprofloxacin were also performed with 10 strains susceptible, intermediate, or resistant to fluoroquinolones. On the basis of the MICs, meropenem and temocillin were the most active β-lactam agents, with MICs at which 90% of isolates are inhibited of 8 and 32 μg/ml, respectively. The addition of ciprofloxacin significantly enhanced the killing activities of piperacillin, imipenem, and meropenem against the 10 strains tested (P < 0.05). The best killing activity was obtained with the combination of meropenem and ciprofloxacin, with bactericidal activity of 3.31 ± 0.36 log₁₀ CFU/ml (P < 0.05). Compared to the activity of the two-drug β-lactam–ciprofloxacin combination, the addition of rifampin or tobramycin did not significantly increase the killing activity (P > 0.05). The three-drug combinations (with or without ciprofloxacin) significantly enhanced the killing activities of piperacillin, imipenem, and meropenem relative to the activities of the β-lactams used alone (P < 0.05). The combination β-lactam–ciprofloxacin–tobramycin was the combination with the most consistently synergistic effect.     

A review of the in vitro activity of meropenem and comparative antimicrobial agents tested against 30,254 aerobic and anaerobic pathogens isolated world wide

The in vitro activity of meropenem (formerly SM-7738), a new carbapenem, was compared with that of imipenem and five other broad-spectrum antimicrobials (ceftazidime, cefotaxime, piperacillin, piperacillin/tazobactam, and ciprofloxacin) against 30,254 clinically significant pathogens isolated in nine countries worldwide. Overall, the carbapenems, meropenem and imipenem, were the most active drugs. Meropenem was four- to 64-fold more active than imipenem against Gram-negative bacteria, including the Enterobacteriaceae, Pseudomonas aeruginosa, Burkholderia cepacia, Haemophilus influenzae, and Neisseria meningitidis. Meropenem was also quite active against ceftazidime-resistant strains of Enterobacteriaceae, inhibiting 87.5 to 100% at ≤ 4 μg/ml. In contrast, imipenem was four- to eight-fold more active than meropenem against Gram-positive species, including methicillin-susceptible strains of Staphylococcus aureus and Staphylococcus epidermidis, Streptococcus pneumoniae, and Enterococcus faecalis. Among the anaerobes, strains resistant to meropenem or imipenem were encountered very rarely. These extensive data provide additional in vitro support for the clinical use of meropenem as a broad spectrum antimicrobial agent active against key pathogenic species of bacteria.     

Sublethal Ciprofloxacin Treatment Leads to Rapid Development of High-Level Ciprofloxacin Resistance during Long-Term Experimental Evolution of Pseudomonas aeruginosa

The dynamics of occurrence and the genetic basis of ciprofloxacin resistance were studied in a long-term evolution experiment (940 generations) in wild-type, reference strain (PAO1) and hypermutable (PAOΔmutS and PAOMY-Mgm) P. aeruginosa populations continuously exposed to sub-MICs (1/4) of ciprofloxacin. A rapid occurrence of ciprofloxacin-resistant mutants (MIC of ≥12 μg/ml, representing 100 times the MIC of the original population) were observed in all ciprofloxacin-exposed lineages of PAOΔmutS and PAOMY-Mgm populations after 100 and 170 generations, respectively, and in one of the PAO1 lineages after 240 generations. The genetic basis of resistance was mutations in gyrA (C248T and G259T) and gyrB (C1397A). Cross-resistance to beta-lactam antibiotics was observed in the bacterial populations that evolved during exposure to sublethal concentrations of ciprofloxacin. Our study shows that mutants with high-level ciprofloxacin resistance are selected in P. aeruginosa bacterial populations exposed to sub-MICs of ciprofloxacin. This can have implications for the long-term persistence of resistant bacteria and spread of antibiotic resistance by exposure of commensal bacterial flora to low antibiotic concentrations.     

Genomic Characterization of Ciprofloxacin Resistance in a Laboratory-Derived Mutant and a Clinical Isolate of Streptococcus pneumoniae

The broad-spectrum fluoroquinolone ciprofloxacin is a bactericidal antibiotic targeting DNA topoisomerase IV and DNA gyrase encoded by the parC and gyrA genes. Resistance to ciprofloxacin in Streptococcus pneumoniae mainly occurs through the acquisition of mutations in the quinolone resistance-determining region (QRDR) of the ParC and GyrA targets. A role in low-level ciprofloxacin resistance has also been attributed to efflux systems. To look into ciprofloxacin resistance at a genome-wide scale and to discover additional mutations implicated in resistance, we performed whole-genome sequencing of an S. pneumoniae isolate selected for resistance to ciprofloxacin in vitro (128 μg/ml) and of a clinical isolate displaying low-level ciprofloxacin resistance (2 μg/ml). Gene disruption and DNA transformation experiments with PCR fragments harboring the mutations identified in the in vitro S. pneumoniae mutant revealed that resistance is mainly due to QRDR mutations in parC and gyrA and to the overexpression of the ABC transporters PatA and PatB. In contrast, no QRDR mutations were identified in the genome of the S. pneumoniae clinical isolate with low-level resistance to ciprofloxacin. Assays performed in the presence of the efflux pump inhibitor reserpine suggested that resistance is likely mediated by efflux. Interestingly, the genome sequence of this clinical isolate also revealed mutations in the coding region of patA and patB that we implicated in resistance. Finally, a mutation in the NAD(P)H-dependent glycerol-3-phosphate dehydrogenase identified in the S. pneumoniae clinical strain was shown to protect against ciprofloxacin-mediated reactive oxygen species.     

In vitro activities of antimicrobial agents,alone and in combinations,against Burkholderia cepacia isolated from blood

Burkholderia cepacia is a widespread, environmental gram-negative bacillus that is associated with nosocomial infections. This bacterium is considered to be an important pathogen in immunocompromised patients and is inherently resistant to multiple antimicrobial agents. To compare the activity of different antimicrobial agents and the potential of combinations against invasive strains of B. cepacia, we collected 36 isolates of B. cepacia from blood cultures and checked their susceptibilities to 13 antimicrobials by broth microdilution method. Most strains tested were susceptible to minocycline (94.4%), ceftazidime (86.1%), ciprofloxacin (83.3%), and trimethoprim-sulfamethoxazole (83.3%). All strains were resistant to aminoglycosides, and only some strains were susceptible to imipenem (16.7%), aztreonam (19.4%), moxalactam (25.0%), piperacillin (25.0%), and carbenicillin (47.2%). The effects of combinations of ceftazidime with amikacin, ceftazidime with ciprofloxacin, and ciprofloxacin with amikacin were assayed by checkerboard titration method. Synergistic effect was found in 28 out of 36 tested strains (77.8%), when ceftazidime was combined with amikacin, in 25 out of 36 strains (69.4%) when ceftazidime was combined with ciprofloxacin, and in only 8 out of 36 strains (22.2%) when ciprofloxacin was combined with amikacin.     

Dynamics of mutator and antibiotic-resistant populations in a pharmacokinetic/pharmacodynamic model of Pseudomonas aeruginosa biofilm treatment

Biofilm growth, antibiotic resistance, and mutator phenotypes are key components of chronic respiratory infections by Pseudomonas aeruginosa in cystic fibrosis patients. We examined the dynamics of mutator and antibiotic-resistant populations in P. aeruginosa flow-cell biofilms, using fluorescently tagged PAO1 and PAOMS (mutator [mutS] derivative) strains. Two-day-old biofilms were treated with ciprofloxacin (CIP) for 4 days (t4) at 2 μg/ml, which correlated with the mutant prevention concentration (MPC) and provided an AUC/MIC ratio of 384 that should predict therapeutic success. Biofilms were monitored by confocal laser scanning microscopy (CLSM), and the numbers of viable cells and resistant mutants (4- and 16-fold MICs) were determined. Despite optimized pharmacokinetic/pharmacodynamic (PK/PD) parameters, CIP treatment did not suppress resistance development in P. aeruginosa biofilms. One-step resistant mutants (MexCD-OprJ or MexEF-OprN overexpression) were selected for both strains, while two-step resistant mutants (additional GyrA or GyrB mutation) were readily selected only from the mutator strain. CLSM analysis of competition experiments revealed that PAOMS, even when inoculated at a 0.01 proportion, took over the whole biofilm after only 2 days of CIP treatment outnumbering PAO1 by 3 log at t4. Our results show that mutational mechanisms play a major role in biofilm antibiotic resistance and that theoretically optimized PK/PD parameters fail to suppress resistance development, suggesting that the increased antibiotic tolerance driven by the special biofilm physiology and architecture may raise the effective MPC, favoring gradual mutational resistance development, especially in mutator strains. Moreover, the amplification of mutator populations under antibiotic treatment by coselection with resistance mutations is for the first time demonstrated in situ for P. aeruginosa biofilms.