Antimicrobial resistance of Pseudomonas aeruginosa in a cystic fibrosis population after introduction of a novel cephalosporin/ß-lactamase inhibitor combination.

Ceftolozane-tazobactam is a new ß-lactam/ß-lactamase inhibitor combination approved by the U.S. Food and Drug Administration in 2019 for the treatment of hospital-acquired and ventilator-associated pneumonia. The combination is a particularly potent inhibitor of penicillin-binding proteins with higher affinity than other ß-lactam agents. Persons with cystic fibrosis (pwCF) often harbour resistant Gram-negative bacteria in the airways and need antibiotics to prevent declining lung function. To test whether the introduction of ceftolozane-tazobactam in the period 2015-2020 led to a bacterial population level increase in cephalosporin resistance in a Danish CF population. In vitro, activity of ceftolozane-tazobactam was evaluated by susceptibility testing of clinical Pseudomonas aeruginosa isolated from pwCF from January 1, 2015, to June 1, 2020. Six thousand three hundred thirty two isolates collected from 210 adult pwCF were included. Thirty pwCF were treated with ceftolozane-tazobactam at least once. Ceftolozane-tazobactam exposure did not increase cephalosporin resistance on an individual or population level. However, resistance to ceftolozane-tazobactam was recorded despite no prior exposure in four pwCF. Compared to ceftazidime, ceftolozane-tazobactam had a better in vitro activity on P. aeruginosa. The percentage of non-mucoid P. aeruginosa isolates susceptible to ceftolozane-tazobactam were higher or equal to 5 other ß-lactams. Ceftolozane-tazobactam expands the armamentaria against P. aeruginosa with acceptable levels for a selection of drug resistance.

Synthesis of a Novel Boronic Acid Transition State Inhibitor, MB076: A Heterocyclic Triazole Effectively Inhibits Acinetobacter-Derived Cephalosporinase Variants with an Expanded-Substrate Spectrum.

Class C Acinetobacter-derived cephalosporinases (ADCs) represent an important target for inhibition in the multidrug-resistant pathogen Acinetobacter baumannii. Many ADC variants have emerged, and characterization of their structural and functional differences is essential. Equally as important is the development of compounds that inhibit all prevalent ADCs despite these differences. The boronic acid transition state inhibitor, MB076, a novel heterocyclic triazole with improved plasma stability, was synthesized and inhibits seven different ADC ß-lactamase variants with Ki values <1 µM. MB076 acted synergistically in combination with multiple cephalosporins to restore susceptibility. ADC variants containing an alanine duplication in the Ω-loop, specifically ADC-33, exhibited increased activity for larger cephalosporins, such as ceftazidime, cefiderocol, and ceftolozane. X-ray crystal structures of ADC variants in this study provide a structural context for substrate profile differences and show that the inhibitor adopts a similar conformation in all ADC variants, despite small changes near their active sites.

Role of Enzymatic Activity in the Biological Cost Associated with the Production of AmpC ß-Lactamases in Pseudomonas aeruginosa.

In the current scenario of growing antibiotic resistance, understanding the interplay between resistance mechanisms and biological costs is crucial for designing therapeutic strategies. In this regard, intrinsic AmpC ß-lactamase hyperproduction is probably the most important resistance mechanism of Pseudomonas aeruginosa, proven to entail important biological burdens that attenuate virulence mostly under peptidoglycan recycling alterations. P. aeruginosa can acquire resistance to new ß-lactam-ß-lactamase inhibitor combinations (ceftazidime-avibactam and ceftolozane-tazobactam) through mutations affecting ampC and its regulatory genes, but the impact of these mutations on the associated biological cost and the role that ß-lactamase activity plays per se in contributing to the above-mentioned virulence attenuation are unknown. The same questions remain unsolved for plasmid-encoded AmpC-type ß-lactamases such as FOX enzymes, some of which also provide resistance to new ß-lactam-ß-lactamase inhibitor combinations. Here, we assessed from different perspectives the effects of changes in the active center and, thus, in the hydrolytic spectrum resistance to inhibitors of AmpC-type ß-lactamases on the fitness and virulence of P. aeruginosa, using site-directed mutagenesis; the previously described AmpC variants T96I, G183D, and ΔG229-E247; and, finally, blaFOX-4 versus blaFOX-8. Our results indicate the essential role of AmpC activity per se in causing the reported full virulence attenuation (in terms of growth, motility, cytotoxicity, and Galleria mellonella larvae killing), although the biological cost of the above-mentioned AmpC-type variants was similar to that of the wild-type enzymes. This suggests that there is not an important biological burden that may limit the selection/spread of these variants, which could progressively compromise the future effectiveness of the above-mentioned drug combinations. IMPORTANCE The growing antibiotic resistance of the top nosocomial pathogen Pseudomonas aeruginosa pushes research to explore new therapeutic strategies, for which the resistance-versus-virulence balance is a promising source of targets. While resistance often entails significant biological costs, little is known about the bases of the virulence attenuations associated with a resistance mechanism as extraordinarily relevant as ß-lactamase production. We demonstrate that besides potential energy and cell wall alterations, the enzymatic activity of the P. aeruginosa cephalosporinase AmpC is essential for causing the full attenuation associated with its hyperproduction by affecting different features related to pathogenesis, a fact exploitable from the antivirulence perspective. Less encouraging, we also show that the production of different chromosomal/plasmid-encoded AmpC derivatives conferring resistance to some of the newest antibiotic combinations causes no significantly increased biological burdens, which suggests a free way for the selection/spread of these types of variants, potentially compromising the future effectiveness of these antipseudomonal therapies.

Trends in beta-lactam resistance among Enterobacteriaceae.

beta-Lactam resistance among Enterobacteriaceae is related primarily to the emergence of novel beta-lactamases. The class A extended-spectrum beta-lactamases hydrolyze extended-spectrum beta-lactams and are inhibited by clavulanic acid. These beta-lactamases are divided in two groups: TEM and SHV derivatives and non-TEM and non-SHV extended-spectrum beta-lactamases (CTX-M1, CTX-M2, MEN-1, PER-1, PER-2, TOHO-1, and VEB-1). The plasmid-mediated cephalosporinases (MIR-1, FOX-1, MOX-1, BIL-1, CMY-1, CMY-2, and LAT-1) hydrolyze extended-spectrum cephalosporins and cephamycins and are not inhibited by clavulanic acid. They have been reported in Europe and in the United States. The 15 inhibitor-resistant penicillinases are TEM derivatives (except for SHV-10) and plasmid mediated, and they are mainly from Escherichia coli isolates. The carbapenemases noted among Enterobacteriaceae are either the chromosomally located penicillinases (Sme-1, NmcA, IMI-1) found in rare Enterobacter cloacae or Serratia marcescens isolates or the plasmid-mediated metalloenzyme IMP-1 that is widespread in Japan. The incidence of resistance among Enterobacteriaceae related to the other more common beta-lactam-resistance mechanisms has continued to rise worldwide.

Cephalosporinase interactions and antimicrobial activity of BMY-28142, ceftazidime and cefotaxime.

Cephalosporinases of Enterobacter cloacae and Citrobacter freundii were responsible for resistance to newer cephalosporins such as cefotaxime and ceftazidime but not BMY-28142. Interaction of these cephalosporins including hydrolysis, binding, inhibition, and inactivation with cephalosporinases from E. cloacae GN7471 and C. freundii GN7391 were studied. BMY-28142 was much more stable against the both enzymes than cephalothin, but more hydrolyzable than cefotaxime and ceftazidime at higher concentration such as 100 microM. Because of low affinity for the enzymes, i.e. large Km and Ki, the calculated hydrolysis rate of BMY-28142 at 0.1 microM was smaller than those of cefotaxime and ceftazidime, that explained the difference in activity against cephalosporinase-producing strains between BMY-28142 and cefotaxime or ceftazidime. The effects of cephalosporinase production on susceptibility of Escherichia coli omp mutants were examined using a plasmid having cephalosporinase gene of C. freundii GN346. Decrease in susceptibility of E. coli by cephalosporinase production was larger in the strain lacking outer membrane proteins (Omp) F and C, and smaller in the strain producing OmpF constitutively.

A comparative study of the activity of cefamandole and other cephalosporins and analysis of the beta-lactamase stability and synergy of cefamandole with aminoglycosides.

The antibacterial activity of cefamandole against 445 clinical isolates was investigated and compared with the activity of other known cephalosporins (cephalothin, cephaloridine, cephalexin, and cefazolin) and of two penicillins (ampicillin and carbenicillin). Cefamandole was the most active antibiotic against isolates of Citrobacter, Enterobacter, and Shigella, and its activity against Staphylococcus aureus, Bacteroides, and some members of the Enterobacteriaceae was comparable to that of the other antibiotics tested. The stability of cefamandole with respect to beta-lactamase was investigated and compared with that of cephalothin, cefazolin, and cephalexin. Cefamandole was stable with respect to the beta-lactamases of Enterobacter and some other members of the Enterobacteriaceae. No significant correlation was found between the antibacterial activity and the beta-lactamase stability of cefamandole, except with Enterobacter. The synergistic activity of cefamandole combined with gentamicin or amikacin was demonstrated by killing-curve techniques, isobolograms, and susceptibility data. Although 12%--46% of the isolates were synergistically inhibited by either combination, antagonism was not observed. No correlation between the hydrolysis of cefamandole by beta-lactamase and the synergistic activity of cefamandole combined with amikacin was demonstrated.

Rational choice of penicillins and cephalosporins based on parallel in-vitro and in-vivo tests.

Because of the unavailability of strictly comparable data, seven representative penicillins and the five cephalosporins currently used in Britain were evaluated in parallel, both in vitro and in vivo. Penicillin sensitive and resistant strains of Staphylococcus aureus and Proteus mirabilis were the main test organisms. Minimum bacteriocidal concentrations of cloxacillin, flucloxacillin, cephalothin, and cephazolin in serum were much higher than conventional minimum inhibitory concentrations in the absence of serum. Cephalexin and cephradine showed the smallest divergence in these values. Staph, aureus beta-lactamase caused least damage to methicillin and cephradine, whereas enzymes from Escherichia, Klebsiella, and Bacillus cereus had least effect against cephradine followed by cephalexin. In mouse protection experiments, highly protein-bound antibiotics had relatively low efficacy. Cephradine had the highest mean activity followed closely by cephaloridine and cephalexin. From the data, cephradine was the cephalosporin of choice, and firm decisions were also made about the choice of penicillins.

Cephamycins: a review, prospects and some original observations.

The cephamycins are a group with great potential. The first member of the group intended for therapeutic use offers the following advantages over existing cephalosporins: 1. Stability to various beta-lactamases; in an environment increasingly threatened by R-factors, this property may be of increasing value as time passes. 2. Possible lack of cross-allergenicity with other beta-lactam antibiotics. 3. Activity against anaerobic strains. Cefoxitin is only the first semi-synthetic derivative; presumably there are other compounds awaiting assessment which have even more favourable properties.

Characterization and prevalence of the different mechanisms of resistance to beta-lactam antibiotics in clinical isolates of Escherichia coli.

A survey of clinical isolates from a hospital laboratory showed that Escherichia coli could be grouped into three classes of beta-lactam-antibiotic resistance by results of routine susceptibility testing to ampicillin, cephalothin, and carbenicillin. E. coli highly resistant to ampicillin and carbenicillin but not to cephalothin (class I) were found to have one of two levels of R factor-mediated, periplasmic-beta-lactamase which resembled R(TEM) and was located behind a permeability barrier to penicillins but not to cephalosporins. This permeability barrier appeared to act synergistically with the beta-lactamase in producing high levels of resistance to penicillins. E. coli highly resistant to ampicillin and cephalothin but not carbenicillin (class II) were found to have a beta-lactamase with predominantly cephalosporinase activity which was neither transferable nor releasable by osmotic shock. E. coli moderately resistant to one or to all three of these antibiotics (class III) were found to have low levels of different beta-lactamases including a transferable beta-lactamase which resembled R(1818). Thus, different mechanisms producing resistance to beta-lactam antibiotics could be deduced from the patterns of resistance to ampicillin, cephalothin, and carbenicillin found on routine susceptibility testing. E. coli of class I were much more prevalent than the other classes and the proportion of E. coli that were class I increased with duration of patient hospitalization. The incidence of class I E. coli rose only slightly over the past 7 years and that of class II E. coli remained constant despite increased usage of both cephalothin and ampicillin. These observations emphasize that the properties of the apparently limited number of individual resistance mechanisms that exist in a bacterial flora, such as their genetic mobility and linkages and the spectrum of their antibiotic inactivating enzymes and permeability barriers, may govern the effect that usage of an antibiotic has upon the prevalence of resistance to it and to other antibiotics.