“Switching Partners”: Piperacillin Avibactam Is a Highly Potent Combination against Multidrug Resistant Burkholderia cepacia Complex and Burkholderia gladioli Cystic Fibrosis Isolates

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“Switching Partners”: Piperacillin-Avibactam Is a Highly Potent

Combination against Multidrug-Resistant

Burkholderia cepacia

Complex and

Burkholderia gladioli

Cystic Fibrosis Isolates

Elise T. Zeiser,

a

_{Scott A. Becka,}

a

_{Brigid M. Wilson,}

a

_{Melissa D. Barnes,}

a,b

_{John J. LiPuma,}

d

_{Krisztina M. Papp-Wallace}

a,b,c

a_{Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA} b_{Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA}

c_{Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, USA} d_{Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan, USA}

ABSTRACT

In persons with cystic ﬁbrosis (CF), airway infection with

Burkholderia

ce-pacia

complex (Bcc) species or

Burkholderia gladioli

presents a signiﬁcant challenge

due to inherent resistance to multiple antibiotics. Two chromosomally encoded

in-ducible

␤

-lactamases, a Pen-like class A and AmpC are produced in Bcc and

B. gladi-oli

. Previously, ceftazidime-avibactam demonstrated signiﬁcant potency against Bcc and

B. gladioli

isolated from the sputum of individuals with CF; however, 10% of the isolates

tested resistant to ceftazidime-avibactam. Here, we describe an alternative antibiotic

combination to overcome ceftazidime-avibactam resistance. Antimicrobial susceptibility

testing was performed on Bcc and

B. gladioli

clinical and control isolates. Biochemical

analysis was conducted on puriﬁed PenA1 and AmpC1

␤

-lactamases from

Burkhold-eria multivorans

ATCC 17616. Analytic isoelectric focusing and immunoblotting were

conducted on cellular extracts of

B. multivorans

induced by various

␤

-lactams or

␤

-lactam-

␤

-lactamase inhibitor combinations. Combinations of piperacillin-avibactam, as

well as piperacillin-tazobactam plus ceftazidime-avibactam (the clinically available

coun-terpart), were tested against a panel of ceftazidime-avibactam nonsusceptible Bcc and

B. gladioli

. The piperacillin-avibactam and piperacillin-tazobactam-ceftazidime-avibactam

combinations restored susceptibility to 99% of the isolates tested. Avibactam is a

potent inhibitor of PenA1 (apparent inhibitory constant [

K

iapp

]

⫽

0.5 ␮

M), while

pip-eracillin was found to inhibit AmpC1 (

K

i app

⫽

2.6 ␮

M). Moreover, piperacillin,

tazo-bactam, ceftazidime, and avitazo-bactam, as well as combinations thereof, did not induce

expression of

bla

penA1

and

bla

ampC1

in the

B. multivorans

ATCC 17616 background.

When ceftazidime-avibactam is combined with piperacillin-tazobactam, the susceptibility

of Bcc and

B. gladioli

to ceftazidime and piperacillin is restored

in vitro

. Both the lack of

blapenA1

induction and potent inactivation of PenA1 by avibactam likely provide the

ma-jor contributions toward susceptibility. With

in vivo

validation,

piperacillin-tazobactam-ceftazidime-avibactam may represent salvage therapy for individuals with CF and highly

drug-resistant Bcc and

B. gladioli

infections.

KEYWORDS

Burkholderia cepacia

complex, avibactam, beta-lactam, beta-lactamase

T

he

Burkholderia

genus currently includes

⬎

100 species, some of which are

opportunistic human pathogens. People particularly susceptible to infection with

Burkholderia

spp. include immunocompromised individuals, as well as persons with

cystic ﬁbrosis (CF) or chronic granulomatous disease (1–5). Members of the

Burkholderia

cepacia

complex (Bcc) and

Burkholderia gladioli

account for the great majority of

Burkholderia

infections in people. Select

␤

-lactam antibiotics (e.g., meropenem and

CitationZeiser ET, Becka SA, Wilson BM, Barnes MD, LiPuma JJ, Papp-Wallace KM. 2019. “Switching partners”: piperacillin-avibactam is a highly potent combination against multidrug-resistantBurkholderia cepaciacomplex and

Burkholderia gladiolicystic ﬁbrosis isolates. J Clin Microbiol 57:e00181-19.https://doi.org/ 10.1128/JCM.00181-19.

EditorKaren C. Carroll, Johns Hopkins University School of Medicine

Address correspondence to Krisztina M. Papp-Wallace, krisztina.papp@va.gov.

Received4 February 2019

Returned for modiﬁcation23 February 2019

Accepted31 May 2019

Accepted manuscript posted online5 June 2019

Published

crossm

26 July 2019

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ceftazidime) have been used as treatment options for infections due to Bcc and

B. gladioli

; unfortunately, their potency against these species is declining (6–9).

␤

-Lactamases are enzymes that hydrolyze

␤

-lactam antibiotics, inactivating them

and preventing them from reaching their target, the penicillin-binding protein in

bacterial cell membranes.

Burkholderia

spp. produce at least two chromosomal

␤

-lactamases, a Pen-like family class A

␤

-lactamase and an AmpC class C

␤

-lactamase

(10–14). In addition, some strains of Bcc also express an OXA class D

␤

-lactamase. In Bcc

and

B. gladioli

, the expression of the genes encoding the Pen-like and AmpC

␤

-lactamases,

bla

pen

and

bla

ampC

, respectively

,

is regulated by a LysR-type

transcrip-tional regulator, PenR through a pathway similar to the AmpC/AmpR regulon found

in

Enterobacteriaceae

(13, 15). However, comprehensive characterization of these

␤

-lactamases, as well as the PenR regulatory pathway, is otherwise lacking, with only

PenA1, AmpC1, and PenR

A

from

Burkholderia multivorans

ATCC 17616 being well

studied to date (13, 16–18). Adding to the complexity, the

bla

penA

and

bla

ampC

genes

are highly heterogeneric among

B. multivorans

strains with PenA1 to PenA38 and

AmpC1 to AmpC28 annotated thus far (whole-genome sequencing was conducted on

50 B. multivorans

strains; of these 50 isolates, we identiﬁed 38 different PenAs and 28

different AmpCs by primary amino acid sequence comparisons) (16, 17). The spectrum

of activity of PenA2-38 and AmpC2-28 variants is under investigation.

Ceftazidime-avibactam is a novel cephalosporin-diazabicyclooctane

␤

-lactamase

inhibitor combination that was initially approved by the U.S. Food and Drug

Adminis-tration in 2015 for the treatment of complicated urinary tract infections caused by

Enterobacteriaceae

. Previously, Papp-Wallace et al. found that avibactam was highly

effective at restoring the susceptibility of 90% of Bcc and

B. gladioli

to ceftazidime

among strains obtained from persons with CF in the United States (8). Subsequently,

two case reports revealed the efﬁcacy of ceftazidime-avibactam in humans with Bcc

infections, including two individuals with CF after double lung transplantation (19, 20).

However, in addition to the 10% of strains that were found to be nonsusceptible to the

ceftazidime-avibactam combination (8), recent studies from Belgium and France found

that 19% and 12% of Bcc and

B. gladioli

isolates, respectively, obtained from individuals

with CF were resistant to ceftazidime-avibactam (9, 21). Another report from France

indicates that 6 of 13 (46%) of Bcc CF isolates tested were nonsusceptible to

ceftazidime-avibactam (22). Thus, a need exists to develop alternative strategies to

restore the activity of the ceftazidime-avibactam combination against Bcc and

B. gladioli

. Here, by “switching” the

␤

-lactam partner, we identiﬁed a novel combination,

piperacillin-avibactam, that overcomes most ceftazidime-avibactam-nonsusceptible

Bcc and

B. gladioli

. Only a single

B. multivorans

isolate AU28442 remained resistant to

all agents tested.

MATERIALS AND METHODS

Bacterial strains and plasmids.TheBurkholderiasp. (i.e., Bcc andB. gladioli) clinical isolates used in this study were obtained from theBurkholderia cepacia Research Laboratory and Repository strain collection, as previously described (8, 23). The construction of strains that were used for susceptibly testing, as well as protein expression, was previously described (16, 18).

Antibiotic susceptibility testing.Mueller-Hinton agar dilution MICs, according to the Clinical and Laboratory Standards Institute (CLSI), were used to phenotypically characterize strains as previously described (8, 24, 25). The sources of piperacillin, avibactam, ceftazidime, nitroceﬁn, and piperacillin-tazobactam were as follows: Sigma, Advanced ChemBlocks, RPI International, Oxoid, and Fresenius Kabi USA, LLC, respectively.

Puriﬁcation of PenA1 and AmpC1.Escherichia coliDE3 Origami 2 cells carrying pGEX-6p2blapenA1

orblaampC1were used for protein expression and puriﬁcation. Brieﬂy, cells were grown in superoptimal

broth and then IPTG (isopropyl-␤-D-thiogalactopyranoside) was added to induce expression of thebla genes. Cells were pelleted and frozen at – 80°C. Subsequently, the cells were lysed, and the␤-lactamases were puriﬁed and veriﬁed by electrospray ionization-mass spectroscopy (ESI-MS), as previously described (16, 18, 26).

Steady-state kinetic assays.Steady-state kinetic parameters for piperacillin were determined using an Agilent 8453 diode array spectrophotometer with 10 mM phosphate-buffered saline (pH 7.4; PBS) at room temperature as previously described (16). As PenA1 hydrolyzed piperacillin,Kmandkcatvalues were

obtained by ﬁtting the initial velocities versus piperacillin concentration using the Henri-Michaelis-Menten equation in Enzﬁtter. Since AmpC1 was unable to hydrolyze piperacillin, a direct-competition

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assay under steady-state conditions was used to determine the apparent inhibitor constant (Kiapp) of

piperacillin for AmpC1.

ESI-MS.ESI-MS was conducted on a Waters Synapt G2-Si as previously described (8). Puriﬁed AmpC1 was incubated with a 1:150 enzyme/inhibitor ratio with piperacillin. Reactions were terminated at 15 min and 24 h by the addition of 0.1% formic acid and acetonitrile.

Analytical isoelectric focusing.B. multivoransATCC 17616 was grown in 50 ml of lysogeny broth (LB) to an optical density at 600 nm (OD600) of 0.6. To test for the induction of the expression of

␤-lactamases, the 50-ml culture was split into 5-ml cultures, and ﬁnal concentrations of 1␮g/ml of imipenem, 1␮g/ml of ceftazidime, 0.25␮g/ml of avibactam, 0.5␮g/ml of piperacillin, and/or 0.0625␮g/ml of tazobactam in various combinations were added to the cultures. The cultures were grown for 1 h in the presence of the different antibiotics and processed for analytical isoelectric focusing (aIEF) as previously described (16). Brieﬂy, 1.0 OD600of cells was pelleted by centrifugation at 12,000 rpm

for 10 min and frozen for 18 h at –20°C. Pellets were lysed for 30 min in 70␮l of lysis buffer (50 mM Tris-chloride [pH 7.4] with 1 mM magnesium sulfate, 40 mg/liter lysozyme [Sigma], and 1.0 U/ml benzo-nase nuclease [Novagen]). To complete the extraction, extracts were treated for 5 min with 2.0 mM EDTA. To remove cellular debris, extracts were centrifuged at 12,000 rpm for 10 min. Then, 15␮l (i.e.,⬃0.2 OD600of lysed cell supernatant) of crude extract was loaded into each lane of the aIEF gel. The aIEF gel

was run for 1.5 h at 4°C at a constant power of 8 W on a Multiphor II isoelectric focusing apparatus. The gel was overlaid with 2 mM nitroceﬁn to observe regions with␤-lactamase activity.

Immunoblotting for PenA1.B. multivoransATCC 17616 and selectB. multivoransclinical isolates were grown in LB to log phase of an OD600between 0.6 and 0.7. Two different immunoblotting assays

were conducted: immunoblotting was conducted on the crude extracts prepared for aIEF as described above, and another immunoblot assay was used to assess the effects of piperacillin on induction ofbla expression. To measure the effects of piperacillin, the isolates were either continued in LB or treated with 32␮g/ml (sub-MICs) of piperacillin for 1 h to induce expression ofblapenA. Subsequently, both sets of

cells and samples were prepared for immunoblotting as previously described (27). For detection of PenA, the polyclonal anti-PenA-peptide antibody was used as previously described (17).

Nitrocefinase activity assay.B. multivoransATCC 17616 and selectB. multivoransclinical isolates were prepared as described in the immunoblotting section above. Cells were pelleted, and crude extracts were generated as described for aIEF. Crude extracts were added to 100␮M nitrocefin diluted in PBS and nitrocefin hydrolysis was monitored over time in a 96-well plate.

RESULTS AND DISCUSSION

Piperacillin-avibactam demonstrates potent activity against Bcc and

B. gladioli

.

To address the Bcc and

B. gladioli

that were not susceptible to ceftazidime-avibactam,

the efﬁcacy of piperacillin combined with avibactam was assessed as piperacillin

demonstrates potent activity against other nonfermenting Gram-negative organisms

(e.g.,

Pseudomonas

spp.) (28, 29). By switching avibactam’s

␤

-lactam partner from

ceftazidime to piperacillin, susceptibility was restored to 150 of 151 Bcc and

B. gladioli

isolates tested (Fig. 1 and see Table S1 in the supplemental material). Speciﬁcally, 13 of

14 Burkholderia

isolates that tested nonsusceptible to ceftazidime-avibactam were

susceptible when avibactam was paired piperacillin (Table 1). Since the combination of

piperacillin-avibactam is not clinically available, the combination of

ceftazidime-0 20 40 60 80 100 120 140 160

PIP-TAZO C-A-P-T PIP-AVI CAZ-AVI

Number of isolates Non-susceptible Susceptible

FIG 1Susceptibility testing results for 151 Bcc andB. gladioliisolates for ceftazidime-avibactam (CAZ-AVI), piperacillin-avibactam (PIP-(CAZ-AVI), ceftazidime-avibactam plus piperacillin-tazobactam (C-A-P-T), and piperacillin-tazobactam (PIP-TAZO).

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avibactam plus piperacillin-tazobactam was also tested, and 99% of the isolates were

susceptible (Fig. 1). Conversely, only 58% of isolates were susceptible to

piperacillin-tazobactam (Fig. 1). Thus, avibactam is critical for the activity of this combination.

Mechanistic basis for the activity of piperacillin-avibactam: analyzing

␤

-lactamases

from

Burkholderia

.

To evaluate the mechanistic considerations for the potency of

the piperacillin-avibactam combination, further biochemical and

␤

-lactamase

ex-pression/activity assays were conducted using the two most prevalent chromosomal

␤

-lactamases produced by

Burkholderia

spp.: the Pen-like and AmpC

␤

-lactamases.

Since the

␤

-lactamases (PenA1 and AmpC1) and PenR from

B. multivorans

ATCC 17616

were the ﬁrst described from Bcc, our laboratory uses this strain as a wild-type isolate

for comparison. PenA1 and AmpC1 are also the most characterized

␤

-lactamases of

those present in other Bcc spp. as well as

B. gladioli

. A limitation of selecting PenA1 and

AmpC1 is that these enzymes are highly heterogeneous within

B. multivorans

(16, 17).

In addition, between

Burkholderia

spp., the Pen-like

␤

-lactamases vary in amino acid

identity from 8% up to 37%. Thus, data obtained with PenA1 and AmpC1 may or may

not extrapolate to other

Burkholderia

spp. Further studies on Pen-like and AmpC

␤

-lactamases in

Burkholderia

spp. is critical.

(i) Biochemical analyses.

To assess the activity of piperacillin and avibactam

against PenA1 and AmpC1, the enzymes were subjected to steady-state kinetic analysis.

Piperacillin was found to be a substrate for PenA1, but inhibited AmpC1 (Table 2).

Hydrolysis of piperacillin by AmpC1 was not measurable. Previously, avibactam was

shown to be a potent inactivator of PenA1 (8). Conversely, avibactam was unable to

inhibit AmpC1 (16). The kinetic observations were supported by mass spectrometric

analysis of AmpC1 with piperacillin. At 15 min, the majority of AmpC1 was in a

covalent complex with piperacillin (40,266

⫾

5 Da) (Fig. 2A). By 24 h, only apo-AmpC1

(38,748

⫾

5 Da) was observed, which suggests the piperacillin may be slowly

hydro-lyzed by AmpC1 (Fig. 2B).

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(ii)

␤

-Lactamase expression.

Since the expression of

blapenA1

and

blaampC1

is

regulated by the transcription factor PenR

A

in response to exposure to select

␤

-lactams,

aIEF was used to assess the effect of various

␤

-lactams and

␤

-lactamase inhibitors on

PenA1 and AmpC1 nitroceﬁnase activity in the wild-type

B. multivorans

ATCC 17616

TABLE 1MIC results for ceftazidime-avibactam-nonsusceptible Bcc andB. gladiolia

Strain

MIC (␮g/ml)

Ceftazidime-avibactam

Piperacillin-avibactam

Ceftazidime-avibactam plus piperacillin-tazobactam

B. multivoransATCC 17616 2 1 1

B. ambifariaAU20319 16 2 2

B. ambifariaAU11161 16 2 2

B. cenocepaciaAU0756 32 4 4

B. cenocepaciaAU19684 16 4 2

B. contaminansAU20979 32 8 8

B. dolosaAU12872 16 8 8

B. dolosaAU29985 64 16 16

B. gladioliAU0032 16 2 2

B. multivoransAU28442 ⬎128 ⬎128 ⬎128

B. stabilisAU10235 32 8 8

B. vietnamiensisAU3578 16 4 4

a_{CLSI breakpoints for piperacillin-tazobactam, piperacillin-avibactam, and}

ceftazidime-avibactam-piperacillin-tazobactam are based on those for piperacillin-ceftazidime-avibactam-piperacillin-tazobactam forPseudomonas aeruginosa(susceptible [S], ⱕ16␮g/ml; intermediate [I], 32 to 64␮g/ml; resistant [R],ⱖ128␮g/ml) since piperacillin breakpoints for Bcc are not available. CLSI breakpoints for ceftazidime-avibactam againstBurkholderiaspp. are based on those for ceftazidime for Bcc (Sⱕ8␮g/ml; I⫽16␮g/ml; Rⱖ32␮g/ml) as breakpoints for ceftazidime-avibactam for Bcc are not available. Avibactam held constant at 4␮g/ml. Piperacillin-tazobactam was used at a 8:1 ratio.

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background.

B. multivorans

ATCC 17616 was grown in LB only or in LB with the addition

imipenem, a known inducer of

bla

penA1

and

bla

ampC1

(30). The proteins were extracted,

and an aIEF gel revealed that imipenem results in robust hydrolysis of nitroceﬁn by

PenA1 and AmpC1 (Fig. 3A). All the single and multiple combinations of piperacillin,

tazobactam, ceftazidime, and avibactam do not result in nitroceﬁnase activity by PenA1

or AmpC1 (Fig. 3B). Thus, these

␤

-lactams and

␤

-lactamase inhibitors do not induce the

expression of

bla

penA1

and

bla

ampC1

, which supports their potency in susceptibility

testing. The nitroceﬁn hydrolysis band observed for AmpC1 in all lanes is always

observed, suggesting that the regulation of

bla

ampC1

expression is leaky.

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A lingering question remained: if piperacillin does not induce

bla

penA1

in

B. multi-vorans

ATCC 17616, then why is avibactam needed for the efﬁcacy of this combination?

To address this question, a panel of piperacillin-resistant

B. multivorans

isolates were

grown with or without piperacillin, the protein expression of PenA was assessed via

immunoblotting, and the

␤

-lactamase activity was determined by measuring nitroceﬁn

hydrolysis. Of the 13 isolates tested, PenA was induced by piperacillin in 11 (Fig. 4).

Thus, even though piperacillin does not induce PenA1 in the wild-type

B. multivorans

ATCC 17616, piperacillin was able to induce PenA expression in clinical isolates of

B. multivorans

. Crude extracts from induced and noninduced isolates were tested with

nitroceﬁn, a chromogenic ﬁrst-generation cephalosporin. Indeed, strains that produce

more PenA via Western blot also possess more

␤

-lactamase activity (Fig. 4). One

exception was

B. multivorans

AU15814, which possesses a band upon induction with

piperacillin but has limited nitroceﬁnase activity. This lack of activity is likely due to the

N132S amino acid substitution present in PenA, that we previously reported (17). The

N132S substitution in PenA substantially diminishes the MIC (from 1,024 to 8

␮

g/ml)

toward cephalothin, which is also a ﬁrst-generation cephalosporin. The regulatory

processes that allow piperacillin to behave as an inducer in clinical isolates of

B. multivorans

are currently under further investigation. Alterations in PBPs, PenR

A

, or

TABLE 2Steady-state kinetic parameters for PenA1 and AmpC1 with piperacillin and avibactama

␤-Lactam ␤-Lactamaseb _K

morKi app(␮M) kcat(sⴚ1) kcat/Km(␮Mⴚ1sⴚ1)

Piperacillin PenA1 ⬍5 10 2.1

AmpC1 2.6⫾0.3 NA NA

Avibactam PenA1* 0.5⫾0.1 NA NA

AmpC1* ⬎600 NA NA

a_K

mandkcatwere determined under pseudo-ﬁrst-order conditions and a nonlinear least-square ﬁt of the data to the Henri-Michaelis-Menten equation determined the kinetic parameters. Each experiment was completed in triplicate, and the error values represent the standard errors of the mean. TheKi appwas determined using a direct-competition assay under steady-state conditions. Each experiment was completed in triplicate, and the error values represent the standard errors of the mean. NA, not applicable.

b_*_{, Some data were previously published (8, 16).}

FIG 2Mass spectrometry of AmpC1:piperacillin acyl-enzyme complex after incubation for 15 min (A) or 24 h (B) at 1:150 enzyme/piperacillin ratios.

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anywhere within the PenR

A

regulatory pathway (e.g., AmpD) could contribute to this

phenotype.

Conclusions.

In this study, by “switching” avibactam’s

␤

-lactam partner from

cef-tazidime to piperacillin, nonsusceptibility to cefcef-tazidime-avibactam in Bcc and

B. glad-ioli

was overcome. Because avibactam is not available clinically,

piperacillin-tazobactam would need to be given in conjugation with ceftazidime-avibactam. The

efﬁcacy of this combination is largely dependent on avibactam. Even though

pipera-cillin appears to have potent activity against

Burkholderia

clinical isolates, in select

isolates piperacillin induces expression of the class A carbapenemase,

blapenA

, requiring

FIG 3(A and B)B. multivoransATCC 17616 (Bm) was grown in LB to log phase, selected␤-lactam and␤-lactam/␤-lactamase inhibitor combinations were added at sub-MIC levels, and cells were grown for an additional hour. Crude extracts were prepared and run on an analytic isoelectric focusing gel. A nitroceﬁn overlay was used to develop the gel and visualize␤-lactamase activity. Puriﬁed PenA1 and AmpC1 were used as controls. Abbreviations: CAZ, ceftazidime; AVI, avibactam; PIP, piperacillin; TAZO, tazobactam. (C) Samples: pure PenA, 200 ng (lane 1); pure AmpC, 200 ng (lane 2); ATCC 17616, no induction (lane 3); ATCC 17616, with imipenem (lane 4); ATCC 17616, with piperacillin (lane 5); ATCC 17616, with piperacillin-tazobactam (lane 6); ATCC 17616, with ceftazidime (lane 7); ATCC 17616, with ceftazidime-avibactam (lane 8); ATCC 17616, with ceftazidime-piperacillin-tazobactam (lane 9); ATCC 17616, with piperacillin-tazobactam-avibactam (lane 10); ATCC 17616, with piperacillin-ceftazidime-piperacillin-tazobactam-avibactam (lane 11); ATCC 17616, with piperacillin-tazobactam-ceftazidime-piperacillin-tazobactam-avibactam (lane 12); ATCC 17616, with piperacillin-avibactam (lane 13). Cells were grown in LB to log phase, antibiotic(s) was added at sub-MIC levels, and cells were grown for an additional hour. Crude extracts were prepared, and immunoblotting was conducted with␣-PenA and␣-AmpC polyclonal antibodies.

FIG 4B. multivoransstrains ATCC 17616 (lane 1), AU21015 (lane 2), AU21596 (lane 3), AU23365 (lane 4), AU23995 (lane 5), AU24277 (lane 6), AU24362 (lane 7), AU11772 (lane 8), AU29198 (lane 9), AU11233 (lane 10), AU15814 (lane 11), AU14786 (lane 12), AU19659 (lane 13), and AU17534 (lane 14) were grown in LB to log phase. Piperacillin was added at sub-MIC levels, and cells were grown for an additional hour. Crude extracts were prepared, and immunoblotting was conducted with␣-PenA polyclonal antibody, or a nitrocefin hydrolysis assay was completed. The nonspecific higher-molecular-weight band was previously observed, and it likely represents unprocessed PenA protein prior to cleavage of the signal peptide; red arrows point to the processed PenA (27). Piperacillin MICs for selected isolates are presented on the right. CLSI breakpoints for piperacillin are based on those for piperacillin-tazobactam for Pseudomonas aeruginosa(susceptible [S],ⱕ16␮g/ml; intermediate [I], 32 to 64␮g/ml; resistant [R],ⱖ128␮g/ml) since piperacillin breakpoints for Bcc are not available. The thin black line in the nitrocefin hydrolysis assay indicates where two rows from a 96-well plate were spliced together; the splicing was necessary in order to represent all 12 samples next to each other.

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the addition of avibactam to achieve susceptibility. Further consideration of the

piperacillin-tazobactam-ceftazidime-avibactam combination is warranted. This study

supports the notion of making

␤

-lactams and

␤

-lactamase inhibitors commercially

available as separate agents and not only as combination drugs. This would allow

clinicians to choose more targeted therapies in “precision medicine” treatment

strate-gies. However, the prescribing of such combinations would also result in additional

challenges for clinicians since the mechanisms of resistance present in a particular

pathogen would likely need to be known.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at

https://doi.org/10.1128/JCM

.00181-19

.

SUPPLEMENTAL FILE 1

, PDF ﬁle, 0.3 MB.

ACKNOWLEDGMENTS

Research reported in this publication was supported in part by funds and/or facilities

provided by the Cleveland Department of Veterans Affairs, the Veterans Affairs Merit

Review Program BX002872, to K.M.P.-W. from the U.S. Department of Veterans Affairs

Biomedical Laboratory Research and Development Service. The contents do not

rep-resent the views of the U.S. Department of Veterans Affairs or the U.S. Government. This

study was also supported by funding (to J.J.L.) from the Cystic Fibrosis Foundation.

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