Emergence of Resistance to Novel β-Lactam–β-Lactamase Inhibitor Combinations Due to Horizontally Acquired AmpC (FOX-4) in Pseudomonas aeruginosa Sequence Type 308

Abstract

T he growing prevalence of nosocomial infections produced by multidrug-resistant (MDR) and extensively multidrug-resistant (XDR) Pseudomonas aeruginosa strains is associated with significantly increased morbidity and mortality (1). This increasing threat results from the extraordinary capacity of P. aeruginosa for developing resistance to nearly all available antibiotics by the selection of mutations in chromosomal genes, the growing prevalence of transferable resistance determinants (such as carbapenemases or extended-spectrum ␤-lactamases [ESBLs]), and the global dissemination of MDR/XDR strains, the so-called high-risk clones (2, 3). The recent introduction of novel ␤-lactam-␤-lactamase inhibitor combinations, such as ceftolozane-tazobactam or ceftazidime-avibactam, has substantially improved our arsenal for combating infections by MDR/XDR P. aeruginosa (1, 4, 5). However, ceftolozanetazobactam or ceftazidime-avibactam are not effective against horizontally acquired class B carbapenemases (metallo-␤-lactamases), which are a growing threat in hospitals worldwide (2). Moreover, while ceftolozane-tazobactam or ceftazidime-avibactam are stable against hydrolysis by the intrinsic chromosomal cephalosporinase AmpC from P. aeruginosa, resistance may emerge during treatment due to the selection of mutations in the ⍀-loop of the ␤-lactamase (6, 7). Here, we describe a case of ceftolozanetazobactam and ceftazidime-avibactam resistance caused by the production of a horizontally acquired AmpC ␤-lactamase, adding further complexity and concern to the current scenario of P. aeruginosa resistance mechanisms. In 2017, an XDR P. aeruginosa strain (HUIGC-PA1) was isolated from a urine sample obtained from a urinary catheter of a female spine trauma patient transferred for rehabilitation to Hospital Universitario Insular de Gran Canaria (Gran Canaria, Spain) 1 week previously. During the prior 2 months, the patient had received several broadspectrum antibiotics, including cefotaxime and cefepime, for hospital-acquired pneumonia (Escherichia coli) and wound infections (Staphylococcus epidermidis). The isolation of XDR P. aeruginosa from the urine was considered colonization, and the patient was discharged 2 months later without antibiotic treatment. The susceptibility profile of HUIGC-PA1 is shown in Table 1. The strain was resistant or showed borderline susceptibility to all ␤-lactams tested (including ceftolozanetazobactam and ceftazidime-avibactam), tobramycin, and ciprofloxacin, remaining only susceptible to amikacin and colistin. Phenotypic and molecular assays for horizontally acquired carbapenemases and ESBLs (8) yielded negative results. Therefore, the strain was subjected to whole-genome sequencing (WGS) using previously described approaches (9) to determine the potential mechanisms involved. The presence of hori-Citation Fraile-Ribot PA, del Rosario-Quintana C, López-Causapé C, Gomis-Font MA, Ojeda-Vargas M, Oliver A. 2020. Emergence of resistance to novel β-lactam-β-lactamase inhibitor combinations due to horizontally ...

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

“…The P. aeruginosa chromosomal AmpC β-lactamase variants studied in this work were previously described to confer resistance to TOL-TAZ and CAZ-AVI (T96I [PDC-222], G183D [PDC-322], and ΔG229-E247 [PDC-223] [ 32 , 33 , 38 ]). In the case of the plasmid-mediated AmpC-type bla FOX-4 and bla FOX-8 enzymes, whereas the former was shown to enable resistance to TOL-TAZ and CAZ-AVI, the latter is a bla FOX-3 derivative with an impaired β-lactam hydrolysis capacity ( 34 , 35 ). As shown in Table 2 , the antibiograms of engineered strains producing the above-mentioned variants in pUCP24 relative to the parent strains demonstrate the associated phenotypes.…”

“…An asterisk over the horizontal bars indicates a P value of <0.05 by Tukey’s post hoc test for multiple comparisons between the calculated LD 50 s. Strains are grouped with horizontal lines when there is no statistical difference between them, whereas the symbols for obvious statistical significance are omitted to declutter the figure. (C) Relative increase in the mRNA levels of ampC or bla FOX (black columns) genes cloned into the pUCP24 vector and transformed into PAΔAG, considering the expression level of ampC from PAO1 or bla FOX-4 from the clinical strain HUIGC-PA1 ( 34 ), respectively, as 1. Horizontal columns represent mean values from experimental replicates, whereas the error bars correspond to the SD (log scale).…”

“…Next, 5 ng of purified RNA was used for one-step reverse transcription and real-time PCR using the QuantiTect SYBR green RT-PCR kit (Qiagen) in a CFX Connect device (Bio-Rad). The rpsL housekeeping gene was used to normalize mRNA levels using previously described primers ( 21 ), and the results were referred to PAO1 (to quantify ampC variant expression) or a previously described P. aeruginosa clinical strain harboring bla FOX-4 (HUIGC-PA1 [ 34 ]) in order to quantify bla FOX-4 -like gene expression. All RT-PCRs were performed in duplicate, and mean expression levels from three independent RNA extractions were considered.…”

“…Moreover, we wanted to ascertain whether several previously described AmpC variants involving P. aeruginosa resistance to CAZ-AVI and TOL-TAZ (T96I [PDC-222], G183D [PDC-322], and ΔG229-E247 [PDC-223]) ( 32 , 33 ) could carry differential biological burdens compared to the wild-type enzyme, similarly to what happens for OXA-2-like enzymes ( 25 ). Finally, we also applied this reasoning to the comparison of two closely related plasmid-encoded AmpC-type β-lactamases, namely, FOX-4 and FOX-8, which display different hydrolytic behaviors, with the former causing resistance to CAZ-AVI and TOL-TAZ and the latter showing a reduced capacity for CAZ hydrolysis compared to its progenitor FOX-3 ( 34 , 35 ).…”

β-lactam Resistance in Pseudomonas aeruginosa: Current Status, Future Prospects

“…These enzymes are thought to have originated from chromosomally-encoded enzymes that have been transferred to mobile elements [257]. Class C β-lactamases present in P. aeruginosa as a result of horizontal gene transfer include FOX-4 in a high-risk strain of P. aeruginosa (ST308) [258] and CMY-2 that was found in a variety of clinical isolates [259].…”