Cephalosporinases associated with outer membrane vesicles released by Bacteroides spp. protect gut pathogens and commensals against beta-lactam antibiotics

Objectives: To identify β-lactamase genes in gut commensal Bacteroides species and to assess the impact of these enzymes, when carried by outer membrane vesicles (OMVs), in protecting enteric pathogens and commensals. Methods: A deletion mutant of the putative class A β-lactamase gene (locus tag BT_4507) found in the genome of the human commensal Bacteroides thetaiotaomicron was constructed and a phenotypic analysis performed. A phylogenetic tree was built from an alignment of nine Bacteroides cephalosporinase protein sequences, using the maximum likelihood method. The rate of cefotaxime degradation after incubation with OMVs produced by different Bacteroides species was quantified using a disc susceptibility test. The resistance of Salmonella Typhimurium and Bifidobacterium breve to cefotaxime in liquid culture in the presence of B. thetaiotaomicron OMVs was evaluated by measuring bacterial growth. Results: The B. thetaiotaomicron BT_4507 gene encodes a β-lactamase related to the CepA cephalosporinase of Bacteroides fragilis. OMVs produced by B. thetaiotaomicron and several other Bacteroides species, except Bacteroides ovatus, carried surface-associated β-lactamases that could degrade cefotaxime. β-Lactamase-harbouring OMVs from B. thetaiotaomicron protected Salmonella Typhimurium and B. breve from an otherwise lethal dose of cefotaxime. Conclusions: The production of membrane vesicles carrying surface-associated β-lactamases by Bacteroides species, which constitute a major part of the human colonic microbiota, may protect commensal bacteria and enteric pathogens, such as Salmonella Typhimurium, against β-lactam antibiotics

Potentiation of imipenem by relebactam for Pseudomonas aeruginosa from bacteraemia and respiratory infections

BACKGROUND: Imipenem resistance in Pseudomonas aeruginosa most often entails loss of the 'carbapenem-specific' porin OprD; more rarely it reflects acquired carbapenemases. Loss of OprD only confers resistance to imipenem if AmpC β-lactamase is expressed, and we investigated whether this mechanism was overcome by relebactam, a developmental diazabicyclooctane β-lactamase inhibitor. METHODS: Consecutive P. aeruginosa isolates causing bacteraemia or hospital-onset lower respiratory tract infections were collected between 2014 and 2016 under the aegis of the BSAC Resistance Surveillance Programme. Imipenem MICs were determined centrally by BSAC agar dilution, with relebactam at a fixed concentration (4 mg/L). RESULTS: For most imipenem-susceptible P. aeruginosa (726/759, 95.7%), the MICs of imipenem alone were 0.5-2 mg/L and were decreased 3- to 4-fold by addition of relebactam, as based on geometric means or modes. For most imipenem-resistant P. aeruginosa (82/92, 89%), imipenem MICs were 8-16 mg/L, and were reduced to 1-2 mg/L by relebactam. These patterns applied regardless of whether the isolates were susceptible to penicillins and cephalosporins or had phenotypes suggesting derepressed AmpC or up-regulated efflux. Imipenem MICs for five P. aeruginosa with MBLs remained high (≥16 mg/L) regardless of relebactam. CONCLUSIONS: Potentiation of imipenem by relebactam was almost universal, in accordance with the view that endogenous pseudomonal AmpC ordinarily protects against this carbapenem to a small degree. Imipenem MICs were reduced to the current breakpoint, or lower, except for MBL producers. Potentiation was not compromised by derepression of AmpC or up-regulation of efflux

Activity of OP0595-β-lactam combination against Gram-negative bacteria with extended-spectrum, AmpC and carbapenem-hydrolysing β-lactama

Background: OP0595 is a diazabicyclooctane that (i) acts as a PBP2-ctive antibacterial, (ii) inhibits Class A and C β-lactamases and (iii), like mecillinam, gives β-lactamase-independent potentiation of β-lactams targeting other PBPs. We tested its behaviour against β-lactam-resistant Enterobacteriaceae and non-fermenters. Methods: Organisms were UK clinical isolates; MICs were determined by CLSI agar dilution for OP0595 alone or combined at 1–4 mg/L with aztreonam, biapenem, cefepime or piperacillin. Results: MICs of OP0595 for Escherichia coli, Enterobacter, Citrobacter and Klebsiella spp. were mostly 1–4 mg/L but values >4 mg/L were seen for minorities of isolates irrespective of other resistances, and for 50%–60% of those with ertapenem resistance involving porin loss plus ESBL or AmpC activity. OP0595 MICs for Serratia, Proteeae and non-fermenters mostly were >4 mg/L. When its MIC was ≤4 mg/L, OP0595's antibacterial activity dominated combination activity. For ‘OP0595-resistant’ (MIC >4 mg/L) isolates with Class A or C β-lactamases OP0595 achieved strong potentiation of substrate β-lactams, contingent on β-lactamase inhibition. β-Lactamase-independent potentiation was evident with aztreonam, cefepime and piperacillin—less so for biapenem—for many OP0595-resistant Enterobacteriaceae with Class B carbapenemases, which are not inhibited by OP0595. OP0595 acted solely as a β-lactamase inhibitor for non-fermenters. Conclusions: OP0595 inhibited Enterobacteriaceae, not non-fermenters; its combinations had broad activity versus Enterobacteriaceae, largely contingent on OP0595's antibacterial activity but also on inhibition of Class A and C β-lactamases and on the β-lactam-enhancer effect, which allowed activity against many OP0595-resistant metallo-β-lactamase-producing Enterobacteriaceae. For non-fermenters OP0595 acted only as a β-lactamase inhibitor

Successful Treatment of Acute Prostatitis Caused by Multidrug-Resistant Escherichia coli With Tigecycline Monotherapy

We present a successful treatment, with tigecycline monotherapy, of acute prostatitis caused by multidrug-resistant Escherichia coli harboring an NDM-1 carbapemenase along with a CMY-2 cephalosporinase and a TEM ESBL

In vitro activity of cefepime/zidebactam (WCK 5222) against Gram-negative bacteria

Background: Diazabicyclooctanes (DBOs) inhibit class A, class C and some class D β-lactamases. A few also bind PBP2, conferring direct antibacterial activity and a β-lactamase-independent ‘enhancer' effect, potentiating β-lactams targeting PBP3. We tested a novel DBO, zidebactam, combined with cefepime. Methods: CLSI agar dilution MICs were determined with cefepime/zidebactam in a chequerboard format. Bactericidal activity was also measured. Results: Zidebactam MICs were ≤2 mg/L (mostly 0.12–0.5 mg/L) for most Escherichia coli, Klebsiella, Citrobacter and Enterobacter spp., but were >32 mg/L for Proteeae, most Serratia and a few E. coli, Klebsiella and Enterobacter/Citrobacter. The antibacterial activity of zidebactam dominated chequerboard studies for Enterobacteriaceae, but potentiation of cefepime was apparent for zidebactam-resistant isolates with class A and C enzymes, illustrating β-lactamase inhibition. Overall, cefepime/zidebactam inhibited almost all Enterobacteriaceae with AmpC, ESBL, K1, KPC and OXA-48-like β-lactamases at 1 + 1 mg/L and also 29 of 35 isolates with metallo-carbapenemases, including several resistant to zidebactam alone. Zidebactam MICs for 36 of 50 Pseudomonas aeruginosa were 4–16 mg/L, and the majority of AmpC, metallo-β-lactamase-producing and cystic fibrosis isolates were susceptible to cefepime/zidebactam at 8 + 8 mg/L. Zidebactam MICs for Acinetobacter baumannii and Stenotrophomonas maltophilia were >32 mg/L; potentiation of cefepime was frequent for S. maltophilia, but minimal for A. baumannii. Kill curve results largely supported MICs. Conclusion: Zidebactam represents a second triple-action DBO following RG6080, with lower MICs for Enterobacteriaceae and P. aeruginosa. Clinical evaluation of cefepime/zidebactam must critically evaluate the reliance that can be placed on this direct antibacterial activity and on the enhancer effect as well as β-lactamase inhibition

Activity of RX-04 Pyrrolocytosine Protein Synthesis Inhibitors against Multidrug-Resistant Gram-Negative Bacteria

Pyrrolocytosines RX-04A-D are designed to bind to the bacterial 50S ribosomal subunit differently from currently-used antibiotics. The four analogs had broad anti-Gram-negative activity: RX-04A inhibited 94.7% of clinical Enterobacteriaceae, Acinetobacter baumannii and Pseudomonas aeruginosa at 0.5-4 μg/ml, with no MICs >8 μg/ml. MICs for multi-resistant carbapenemase producers were up to two-fold higher than for control strains, with values ≥8 μg/ml for one Serratia isolate with porin and efflux lesions. mcr-1 did not affect MICs

Potential of high-dose cefepime/tazobactam against multi-resistant Gram-negative pathogens

Background: Early β-lactamase inhibitors were combined with established penicillins, but different combinations may be more appropriate to counter current β-lactamase threats, with development facilitated by the US Generating Antibiotic Incentives Now (GAIN) Act. Cefepime/tazobactam is especially attractive, combining an AmpC-stable cephalosporin with a clinically established inhibitor, active against ESBLs and suitable for high-dose administration. Methods: Organisms (n = 563) were clinical isolates submitted to the UK national reference laboratory. MICs were determined by CLSI agar dilution with tazobactam at 4 mg/L and, for a subset, at 8 mg/L. Results: Cefepime/tazobactam 8 + 4 mg/L achieved coverage of 96%–100% of Enterobacteriaceae with penicillinases, AmpC, ESBL, K1 or OXA-48 β-lactamases. Even at 1 + 4 mg/L, the combination inhibited >94% of isolates with penicillinases, AmpC enzymes or ESBLs. Most Enterobacteriaceae with KPC and NDM carbapenemase were resistant at current cefepime breakpoints but 80% of those with VIM types were susceptible at 8 + 4 mg/L. Tazobactam did little to potentiate cefepime against non-fermenter groups, though gains were seen against AmpC-producing Acinetobacter spp. and Stenotrophomonas maltophilia. Increasing the tazobactam concentration to 8 mg/L gave further small increases in activity against Enterobacteriaceae groups. Conclusions: High-dose cefepime/tazobactam, justifying an 8 + 4 or 8 + 8 mg/L breakpoint, can achieve a carbapenem-like spectrum, with some additional coverage of OXA-48 (and maybe VIM) Enterobacteriaceae. Clinical evaluation is warranted

Susceptibility testing challenges with ceftaroline, MRSA, and a 1-mg/L breakpoint

Objectives A 1 mg/L susceptibility breakpoint for ceftaroline and staphylococci is universally agreed; EUCAST counts MIC >1 mg/L as resistant whereas CLSI and FDA count 2 mg/L as intermediate and >2 mg/L as resistant. We investigated whether routine diagnostic tests reliably distinguish MICs of 1 versus 2 mg/L. Methods Thirty-five UK laboratories collected Staphylococcus aureus isolates and performed tests with 5 μg (as EUCAST) or 30 μg (as CLSI) discs and either confluent growth on Mueller–Hinton agar (as EUCAST and CLSI) or semi-confluent growth on Iso-Sensitest agar (as BSAC). They also ran Etests for MRSA. Reference MICs were determined centrally by CLSI and BSAC agar dilution. Results We obtained paired local disc and central MIC results for 1607 S. aureus (33% MRSA). EUCAST's zone breakpoint recognized 56% of isolates found resistant in MIC tests, but the positive predictive value (PPV) for resistance was 11.0%; corresponding proportions by CLSI testing were 28.0% and 13.4%. The BSAC disc method detected 25% of resistant isolates, with a PPV of 18.2%. Essential agreement, ±1 dilution, of local Etests and central agar MICs was >95%, but only 20% of the isolates found non-susceptible by agar dilution were found non-susceptible by Etest and vice versa. Review for isolates with the modal MIC (0.25 mg/L) indicated that the same laboratories reported large or small zones irrespective of disc and method, implying systematic bias. Conclusions MRSA with ceftaroline MICs of 1 and 2 mg/L were poorly discriminated by routine methods. Solutions lie in greater standardization, automation or dosages justifying a higher breakpoint

Pathogens of skin and skin-structure infections in the UK and their susceptibility to antibiotics, including ceftaroline

Objectives: Bacterial skin and skin-structure infections (SSSIs) are frequent settings for antibiotic use. We surveyed their UK aetiology and pathogen susceptibility, including susceptibility to ceftaroline. Methods: Consecutive SSSI isolates were collected at 35 UK hospitals, to a maximum of 60/site, together with 15 ‘supplementary’ MRSA/site. Isolates were re-identified and BSAC susceptibility testing was performed, with parallel CLSI agar testing for ceftaroline. Results: Isolates (n¼1908) were collected from 1756 hospitalized patients, predominantly with surgical and traumatic infections, abscesses and infected ulcers and largely from general medicine and general surgery patients. They included 1271 Staphylococcus aureus (201 MRSA), 162 b-haemolytic streptococci, 269 Enterobacteriaceae, 138 Pseudomonas aeruginosa and 37 enterococci. Most (944/1756) patients had monomicrobial MSSA infections. Rates of resistance to quinolones, gentamicin and cephalosporins were ,20% in Enterobacteriaceae and ,10% in P. aeruginosa. MRSA rates varied greatly among hospitals and were 2.5-fold higher in general medicine than in general surgery patients. At breakpoint, ceftaroline inhibited: (i) all MSSA and 97.6% of MRSA, with MICs of 2 mg/L for the few resistant MRSA; (ii) all b-haemolytic streptococci; and (iii) 83% of Enterobacteriaceae. High-level ceftaroline resistance in Enterobacteriaceae involved ESBLs or AmpCenzymes. Ceftaroline MICs by CLSI methodology generally equalled those by BSAC or were 2-fold higher, but this differential was 4–16-fold for P. aeruginosa. Conclusions: Irrespective of patient group, SSSIs were dominated by S. aureus. Most pathogens were susceptible, but 15.8% of S. aureus were MRSA, with locally higher prevalence

WCK 4234, a novel diazabicyclooctane potentiating carbapenems against Enterobacteriaceae, Pseudomonas and Acinetobacter with class A, C and D β-lactamases

Background: Several diazabicyclooctanes (DBOs) are under development as inhibitors of Class A and C -lactamases. Inhibition of OXA (Class D) carbapenemases is variable, with those of Acinetobacter spp. remaining notably resistant. We describe a novel DBO, WCK 4234 (Wockhardt), with distinctive activity against OXA carbapenemases.  Methods: MICs of imipenem and meropenem were determined by CLSI agar dilution with WCK 4234 added at 4 or 8 mg/L. Test organisms were clinical Enterobacteriaceae, Acinetobacter baumannii and Pseudomonas aeruginosa with carbapenemases or carbapenem resistance via porin loss plus AmpC or ESBL activity. AmpC mutants were also tested.  Results: WCK 4234, which lacked direct antibacterial activity, strongly potentiated imipenem and meropenem against Enterobacteriaceae with OXA-48/181, KPC enzymes, or with combinations of impermeability and AmpC or ESBL activity, with MICs reduced to <2 mg/L in almost all cases. Carbapenems likewise were potentiated against P. aeruginosa (n=2) with OXA-181 enzyme, with MICs reduced from 64-128 mg/L to 2-8 mg/L and against A. baumannii with OXA carbapenemases, particularly OXA-23 or hyperproduced OXA-51, with MICs reduced to <2 mg/L for 9/10 acinetobacters with OXA-23 enzyme. Carbapenems were not potentiated against Enterobacteriaceae or non-fermenters with metallo--lactamases.   Conclusion: WCK 4234 distinctively overcame resistance mediated by OXA-type carbapenemases, including in A. baumannii. It behaved similarly to other DBOs against strains with KPC carbapenemases or combinations of impermeability and ESBL or AmpC activity