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Ceftolozane-Tazobactam Resistance in Multidrug-Resistant Pseudomonas aeruginosa Isolates Not Associated with AmpC Activity

Abstract Background Ceftolozane-tazobactam (CT) is a newly approved cephalosporin/β-lactamase inhibitor combination with excellent in vitro activity against multidrug-resistant (MDR) P. aeruginosa. Unfortunately, CT-resistance (CT-R) has already been reported. In this work, we evaluate mutational pathways associated with high level of CT-R and assess the role of AmpC in a clinical strain-pair of MDR P. aeruginosa. Methods A CT susceptible isolate of P. aeruginosa (2365) and its CT-R derivatives (2366 and 2367) were recovered from the infected device of a patient before and after treatment with CT. Minimum inhibitory concentrations (MICs) to CT were determined by Etest. Resistance mediated by AmpC hyperproduction was evaluated using ceftazidime (CAZ) and meropenem (MER) with and without cloxacillin (CLOX) at concentration of 1 mg/ml. In addition, the β-lactamase hydrolysis activity was determined for crude cell lysate of the isolates using a spectrophotometric assay for nitrocefin degradation. Furthermore, whole genome sequencing of the three strains was performed and compared (2365 vs. 2366 and 2367). Reads from each isolate were mapped against the genome of the reference strain PAO1. Variants identified by GATK, SamTools and CLC Genomics Workbench 8.5 were selected and annotated with SnpEff. Results Strain 2365 had a CT MIC of 0.75 mg/ml while 2366 and 2367 have MICs > 256 mg/ml. AmpC hyperproduction test was positive only for the susceptible isolate (2365). In concordance, the hydrolysis assay showed a lack of nitrocefin degradation by CT-R 2366 compared with its CT-susceptible isolate 2365. Notably, the three strains (S and R) exhibited a truncated AmpD. Comparison of the resistant derivatives vs. 2365 and 2367 showed a 7 amino acid deletion in the Ω-loop of the β-lactamase AmpC in both resistant derivatives and mutations in genes predicted to encode a hypothetical protein, an ABC transporter ATP-binding protein and a multidrug resistance operon repressor MexR. Conclusion Our results suggest that the deletion in the Ω-loop of AmpC in 2366 and 2367 does not contribute to CT-R in these P. aeruginosa strains. Further characterization of AmpC and other predicted proteins identified by WGS are needed to determine the mechanism of CT-R. Disclosures All authors: No reported disclosures.

Background. The aim of the present study was to assess the prevalence and characteristics of S. pneumoniae serotype 19A isolates from invasive and non-invasive pneumococcal disease (IPD) in children, before and after the introduction of pneumococcal conjugate vaccines (PCVs) in Greece.
Methods. All S. pneumoniae isolates collected between 1986 and 2015 were re-cultured for further testing. Serotyping was performed using the quellung reaction and antibiotic susceptibilities were determined by E-test. The altered pbp genes (pbp2b, pbp2x, and pbp1a) conferring resistance to penicillin and the macrolide-resistant genes [mef(A), mef(E), and erm(B)] were detected by polymerase chain reaction (PCR) using specific primers. The sequence types (ST) and clonal complexes were determined by multilocus sequence typing (MLST).
Conclusion. S. pneumoniae serotype 19A increased significantly after the introduction of PCV7 and a substantial proportion of these isolates exhibited MDR phenotype. The majority of the examined isolates belonged to limited number of CC.
Disclosures Background. Ceftolozane-tazobactam (CT) is a newly approved cephalosporin/β-lactamase inhibitor combination with excellent in vitro activity against multidrug-resistant (MDR) P. aeruginosa. Unfortunately, CT-resistance (CT-R) has already been reported. In this work, we evaluate mutational pathways associated with high level of CT-R and assess the role of AmpC in a clinical strain-pair of MDR P. aeruginosa.
Methods. A CT susceptible isolate of P. aeruginosa (2365) and its CT-R derivatives (2366 and 2367) were recovered from the infected device of a patient before and after treatment with CT. Minimum inhibitory concentrations (MICs) to CT were determined by Etest. Resistance mediated by AmpC hyperproduction was evaluated using ceftazidime (CAZ) and meropenem (MER) with and without cloxacillin (CLOX) at concentration of 1 mg/ml. In addition, the β-lactamase hydrolysis activity was determined for crude cell lysate of the isolates using a spectrophotometric assay for nitrocefin degradation. Furthermore, whole genome sequencing of the three strains was performed and compared (2365 vs. 2366 and 2367). Reads from each isolate were mapped against the genome of the reference strain PAO1. Variants identified by GATK, SamTools and CLC Genomics Workbench 8.5 were selected and annotated with SnpEff.
Results. Strain 2365 had a CT MIC of 0.75 mg/ml while 2366 and 2367 have MICs > 256 mg/ml. AmpC hyperproduction test was positive only for the susceptible isolate (2365). In concordance, the hydrolysis assay showed a lack of nitrocefin degradation by CT-R 2366 compared with its CT-susceptible isolate 2365. Notably, the three strains (S and R) exhibited a truncated AmpD. Comparison of the resistant derivatives vs. 2365 and 2367 showed a 7 amino acid deletion in the Ω-loop of the β-lactamase AmpC in both resistant derivatives and mutations in genes predicted to encode a hypothetical protein, an ABC transporter ATP-binding protein and a multidrug resistance operon repressor MexR.
Conclusion. Our results suggest that the deletion in the Ω-loop of AmpC in 2366 and 2367 does not contribute to CT-R in these P. aeruginosa strains. Further characterization of AmpC and other predicted proteins identified by WGS are needed to determine the mechanism of CT-R.
Disclosures. All authors: No reported disclosures. Background. OXA-2 is a class D β-lactamase which is primarily found in E. coli (Ec) and P. aeruginosa and confers resistance to penicillins as well as narrow-spectrum cephalosporins. However, recent reports suggest that OXA-2 also possesses carbapenem hydrolyzing activity. We report a case of K. pneumoniae that confers reduced susceptibility to ceftazidime and ertapenem due to production of OXA-2.

Reduced Ertapenem Susceptibility Due to OXA-2 Production in
Methods. K. pneumoniae (Kp) strain YDC787 was identified from the BAL culture of an inpatient in December 2016. The strain was initially reported as intermediate to ertapenem. MICs and carbapenemase production were confirmed by Etest and modified Hodge test (MHT) respectively. Ec TOP10 was transformed with purified plasmids followed by selection with ampicillin (TOP10 [pYDC787]), and the carbapenem resistance determinant was cloned into vector pBCSK using partial Sau3AI digestion (TOP10 [pOXA-2]).
Disclosures. All authors: No reported disclosures.