tazobactam against clinical isolates of Pseudomonas aeruginosa in the planktonic and biofilm states

tazobactam against clinical isolates of Pseudomonas aeruginosa in the planktonic and biofilm states

    In vitro activity of ceftolozane/tazobactam against clinical isolates of Pseudomonas aeruginosa in the planktonic and biofilm states ...

496KB Sizes 10 Downloads 65 Views

    In vitro activity of ceftolozane/tazobactam against clinical isolates of Pseudomonas aeruginosa in the planktonic and biofilm states Antonio L. Velez Perez, Suzannah M. Schmidt-Malan, Peggy C. Kohner, Melissa J. Karau, Kerryl E. Greenwood Quaintance, Robin Patel PII: DOI: Reference:

S0732-8893(16)30015-3 doi: 10.1016/j.diagmicrobio.2016.02.014 DMB 14023

To appear in:

Diagnostic Microbiology and Infectious Disease

Received date: Revised date: Accepted date:

25 November 2015 8 February 2016 13 February 2016

Please cite this article as: Velez Perez Antonio L., Schmidt-Malan Suzannah M., Kohner Peggy C., Karau Melissa J., Greenwood Quaintance Kerryl E., Patel Robin, In vitro activity of ceftolozane/tazobactam against clinical isolates of Pseudomonas aeruginosa in the planktonic and biofilm states, Diagnostic Microbiology and Infectious Disease (2016), doi: 10.1016/j.diagmicrobio.2016.02.014

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT In vitro activity of ceftolozane/tazobactam against clinical isolates of Pseudomonas aeruginosa in the planktonic and biofilm states

RI P

T

Antonio L. Velez Perez, 1, 2 Suzannah M. Schmidt-Malan, 1 Peggy C. Kohner,1 Melissa J. Karau, 1 Kerryl E. Greenwood Quaintance, 1 Robin Patel, M.D.1# Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology,

SC

1

Mayo Clinic, Rochester, MN

University of Puerto Rico School of Medicine, San Juan, PR

CE

Robin Patel, M.D.

PT

#Correspondence to:

ED

MA

NU

2

AC

Division of Clinical Microbiology, Mayo Clinic 200 First St S.W., Rochester, MN 55905, USA 507-538-0579

507-284-4272 (fax) Email: [email protected]

1

ACCEPTED MANUSCRIPT ABSTRACT Pseudomonas aeruginosa causes a variety of life-threatening infections, some of which

RI P

T

are associated with planktonic and others with biofilm states. Herein, we tested the combination of the novel cephalosporin, ceftolozane, with the -lactamase inhibitor, tazobactam, against

SC

planktonic and biofilm forms of 54 clinical isolates of P. aeruginosa, using cefepime as a comparator. Minimum inhibitory concentration (MIC) values were determined following

NU

Clinical and Laboratory Standards Institute (CLSI) guidelines. Minimum biofilm inhibitory

MA

concentration (MBIC) values were determined using biofilm-laden pegged lids incubated in antimicrobial challenge plates containing varying concentrations of ceftolozane/tazobactam.

ED

Pegged lids were then incubated in growth recovery plates containing cation-adjusted MuellerHinton broth to determine the minimum biofilm bactericidal concentration (MBBC).

PT

Ceftolozane/tazobactam was highly active against planktonic P. aeruginosa, with all 54 isolates

CE

studied testing susceptible (MIC ≤4/4 µg/mL). On the other hand, 51/54 biofilm P. aeruginosa had MBICs ≥16/4 µg/mL, and all 54 isolates had MBBCs >32 µg/mL. Of the 54 isolates, 45

AC

(83.3%) tested susceptible to cefepime, with the MIC50/MIC90 being 4/16 µg/mL, respectively, and the MBIC90 and MBBC90 both being >256 µg/mL. Although ceftolozane/tazobactam is a promising antimicrobial agent for the treatment of P. aeruginosa infections, it is not highly active against P. aeruginosa biofilms.

2

ACCEPTED MANUSCRIPT INTRODUCTION Pseudomonas aeruginosa is a biofilm-producing Gram-negative bacillus frequently

RI P

T

observed in life-threatening infections including but not limited to pulmonary infections in cystic fibrosis patients (Kuti, et al., 2015), intra-abdominal infections (Skalweit, 2015), endocarditis

SC

(Polovina, et al., 2014), prosthetic joint infections (Rodríguez‐Pardo, et al., 2014), and ventilatorassociated pneumonia (Zhanel, et al., 2014). Of growing concern, a rise in P. aeruginosa strains

NU

resistant to commonly used antimicrobial agents, including carbapenems,

MA

piperacillin/tazobactam, ceftazidime and cefepime, has been reported (Zhanel, et al., 2014). This highlights the need for novel antimicrobial agents with specificity against P. aeruginosa. One

ED

such compound is the recently United States Food and Drug Administration-approved cephalosporin and -lactamase inhibitor combination, ceftolozane/tazobactam. Ceftolozane has a

PT

modified 3-position side chain, providing enhanced anti-pseudomonal activity (Zhanel, et al.,

CE

2014). Unlike other cephalosporins, a bulky pyrazole ring is present on the ceftolozane structure, providing protection against hydrolysis and adding stability against AmpC -lactamases.

AC

Although the addition of the -lactamase inhibitor tazobactam is not known to enhance the antipseudomonal activity of ceftolozane (Shlaes, 2013; Soliman, et al., 2015), it broadens the activity of ceftolozane to include extended-spectrum β-lactamases (ESBLs) (Zhanel, et al., 2014). Biofilms, which can be described as surface-attached layers of microorganisms with selfproduced extracellular polymeric substances, are a major cause of morbidity and mortality in clinical practice. Biofilms may grow on a variety of surfaces, such as prosthetic devices, subvenous catheters, and cardiac pacemakers, as well as on human tissues; their treatment can be difficult and costly, since they are often inherently resistant to high levels of antimicrobial agents (Frank, et al., 2007). Mechanisms of biofilm resistance to antimicrobials include the presence of 3

ACCEPTED MANUSCRIPT “persister-cells” and, in some cases, impenetrability of their matrix. Today, surgical removal of biofilm-laden devices is often the only effective treatment for curing such infections, which

T

subjects patients to the risks of an invasive procedure. Herein, we assessed the activity of

RI P

ceftolozane/tazobactam against biofilm and planktonic forms of P. aeruginosa and compared the activity of ceftolozane/tazobactam with that of the commonly used anti-pseudomonal agent,

AC

CE

PT

ED

MA

NU

SC

cefepime.

4

ACCEPTED MANUSCRIPT MATERIALS AND METHODS Microorganisms and growth conditions. 54 Pseudomonas aeruginosa clinical isolates

RI P

T

collected at Mayo Clinic, Rochester, MN between 1990 and 2014, were studied. The sources of the isolates included prosthetic joint infection (n=17), bloodstream (n=8), respiratory (n=5),

SC

penile prosthesis (n=1), aortic valve tissue (n=1), urine (n=1), and unknown (n=21). P. aeruginosa ATCC 27853 was used as a quality control strain. Organisms were stored at -70°C in

NU

Microbank vials (Pro-Lab Diagnostics, Round Rock, TX) and cultured on blood agar plates

MA

before each assay.

Antimicrobial agents. Ceftolozane (Merck, Kenilworth, NJ), tazobactam (Merck), and cefepime

ED

(USP, Rockville, MD) powders were placed into solution before each planktonic and biofilm susceptibility experiment, as described in the Clinical and Laboratory Institutes (CLSI)

PT

guidelines (Clinical and Laboratory Standards Institute, 2015b).

CE

Biofilm index growth assay. To confirm that the isolates used were able to form biofilms, we

AC

measured a biofilm index as previously described (Frank, et al., 2007). Briefly, isolates were grown in trypticase soy broth (TSB), adjusted to 0.5 McFarland turbidity (approximately 108 cfu/mL), and then inoculated into flat bottom 96-well microtiter plates with 200 µL of TSB containing 106 cfu or TSB alone (blank). After incubation for 24 hours at 37°C in room air, optical density at 600 (OD600) was read on a Multiskan Plus (Thermo Scientific, Waltham, MA) spectrophotometer plate reader. Plates were rinsed with deionized (DI) water and allowed to air dry overnight. To measure the biofilm density, plates were stained with 0.1% safranin for one minute, rinsed with tap water, and allowed to air dry. Then, 30% glacial acetic acid was added to the stained wells; plates were read on the spectrophotometer at OD492. Four replicates were

5

ACCEPTED MANUSCRIPT performed for each isolate. Results were normalized against blank wells and graphed as biofilm indices (OD492/OD600).

RI P

T

Planktonic antimicrobial susceptibility assay. To determine planktonic P. aeruginosa antimicrobial susceptibility, CLSI guidelines for both broth microdilution and agar dilution

SC

susceptibility testing were followed (Clinical and Laboratory Standards Institute, 2015a). Briefly, for broth microdilution, isolates were grown in TSB to a 0.5 McFarland turbidity, diluted in

NU

CAMHB for a final concentration of 104 cfu/well, and then incubated for approximately 20 hours

MA

in round-bottom antimicrobial challenge plates containing 128/4 to 0.125/4 µg/mL of ceftolozane/tazobactam or 128 to 0.125 µg/mL of cefepime. The minimum inhibitory

ED

concentration (MIC) was defined as the lowest concentration without turbidity in the well. For agar dilution, isolates were grown to a 0.5 McFarland turbidity and inoculated onto agar plates

PT

containing 128/4 to 0.06/4 µg/mL of ceftolozane/tazobactam, and incubated for approximately

CE

16 hours. The MIC was defined as the lowest concentration that inhibited growth. The CLSI susceptibility breakpoint for cefepime (≤8 µg/mL) and the FDA susceptibility breakpoint for

AC

ceftolozane/tazobactam (≤4/4 µg/mL) were applied. Biofilm antimicrobial susceptibility assay. We modified our previously-described in vitro biofilm assay (Frank, et al., 2007) to determine the minimum biofilm inhibitory concentration (MBIC) and minimum biofilm bactericidal concentration (MBBC). Briefly, isolates were grown in TSB to a 0.5 McFarland turbidity. Biofilms were allowed to grow on pegged lids by submerging the lids in 96-well microtiter plates containing 150 µL aliquots of bacteria in TSB; the plates were incubated at 37°C for 5 hours. After incubation, the pegged lids were rinsed with phosphate-buffered saline (PBS) for 30 seconds, and then placed in microtiter plates containing antimicrobial dilutions ranging from 256/4 µg/mL to 0.25/4 µg/mL for ceftolozane/tazobactam, 6

ACCEPTED MANUSCRIPT and 256 to 0.25 µg/mL for cefepime. CLSI broth microdilution guidelines were followed (Clinical and Laboratory Standards Institute, 2015a), except that the final volume of cation-

T

adjusted Mueller Hinton broth (CAMHB) containing ceftolozane/tazobactam or cefepime was

RI P

200 µL per well. The biofilm-laden pegged lids were incubated in antimicrobial challenge plates at 37°C in room air for approximately 20 hours. The MBIC was defined as the lowest

SC

concentration without turbidity in the well. Pegged lids were then rinsed with PBS for 30

NU

seconds, and incubated in CAMHB-containing growth recovery plates at 37°C in room air for approximately 20 hours. The MBBC was defined as the lowest concentration without turbidity in

AC

CE

PT

ED

MA

the well.

7

ACCEPTED MANUSCRIPT RESULTS Biofilm growth. All 54 isolates of P. aeruginosa formed biofilms, albeit some more robustly

RI P

T

than others (Figure 1); all indices were above 0.05.

Susceptibility assays. Results are shown in Table 1. Ceftolozane/tazobactam was highly active

SC

in inhibiting P. aeruginosa growth in its planktonic form, but was less active against the same

NU

isolates in the biofilm state. In the planktonic state, all 54 (100%) P. aeruginosa isolates were susceptible to ceftolozane/tazobactam, with MIC50 and MIC90 values of 1/4 and 2/4 µg/mL,

MA

respectively, for broth microdilution and 1/4 and 1/4 µg/mL, respectively, for agar dilution. For cefepime, 45 (83.3%) tested susceptible, with the MIC50 and MIC90 being 4 and 16 µg/mL,

ED

respectively. When tested against organisms in the biofilm state, ceftolozane/tazobactam was not highly active in inhibiting P. aeruginosa in the biofilm assay (MBIC90, >256/4 µg/mL) or against

PT

established biofilms (MBBC90, >256/4 µg/mL). Cefepime showed similar results with a MBIC90

CE

>256 µg/mL and a MBBC90 >256 µg/mL. In the biofilm state, 51/54 isolates showed

AC

ceftolozane/tazobactam MBIC values ≥16/4 µg/mL, and all 54 isolates showed MBBC values >32 µg/mL. The quality control strain had MBIC and MBBC values of >256/4 µg/mL for ceftolozane/tazobactam and >256 µg/mL for cefepime.

8

ACCEPTED MANUSCRIPT DISCUSSION In this study, we showed that in the planktonic state, all 54 (100%) P. aeruginosa isolates

RI P

T

tested susceptible to ceftolozane/tazobactam. In contrast, only 45 (83.3%) testing susceptible to cefepime; another 7 (13%) tested intermediate to cefepime, while the remaining 2 (3.7%) tested

SC

resistant. Tazobactam does not have anti-pseudomonal activity, but is combined with ceftolozane

NU

to broaden its coverage to include ESBL-producing Enterobacteriaceae (Sader, et al., 2014). Both ceftolozane/tazobactam and cefepime showed poor activity against P. aeruginosa

MA

biofilms, with high MBICs and MBBCs. This is not surprising given that bacteria in biofilms are slow growing; further, there are hypoxic conditions in the deep layers of the biofilms, with

ED

overexpression of efflux pumps (Hill, et al., 2005). A study by Ceri et al., done in a similar manner as ours, tested the susceptibility of several antibiotics including ceftazidime (a third

PT

generation cephalosporin) against P. aeruginosa ATCC 27853 biofilms and concluded that the

CE

susceptibility for ceftazidime was 1,000-fold or more greater than the MIC (Ceri, et al., 1999);

AC

these results are similar to our findings that the MBIC is high for ceftolozane/tazobactam and cefepime. Further research is needed to develop better treatments for biofilm-mediated infections caused by P. aeruginosa. In conclusion, ceftolozane/tazobactam is a promising novel antimicrobial that is highly active against the planktonic form of P. aeruginosa clinical isolates (Farrell, et al., 2013; Sader, et al., 2014; Zhanel, et al., 2014), although, similar to cefepime, it does not exhibit marked activity against established biofilms.

9

ACCEPTED MANUSCRIPT REFERENCES

AC

CE

PT

ED

MA

NU

SC

RI P

T

Ceri, H, Olson, M, Stremick, C, Read, R, Morck, D, & Buret, A (1999) The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. Journal of clinical microbiology, 37, 1771-1776. Clinical and Laboratory Standards Institute (2015a) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard – tenth edition. CLSI document M07-A10, Clinical and Laboratory Standards Institute. Wayne, PA. Clinical and Laboratory Standards Institute (2015b) Performance standards for antimicrobial susceptibility testing; Twenty-Fifth Informational Supplement M100-S25. Clinical and Laboratory Standards Institute. Wayne, PA. Farrell, DJ, Flamm, RK, Sader, HS, & Jones, RN (2013) Antimicrobial activity of ceftolozane/tazobactam tested against Enterobacteriaceae and Pseudomonas aeruginosa with various resistance patterns isolated in US hospitals (2011-2012). Antimicrobial agents and chemotherapy, AAC. 01802-01813. Frank, KL, Reichert, EJ, Piper, KE, & Patel, R (2007) In vitro effects of antimicrobial agents on planktonic and biofilm forms of Staphylococcus lugdunensis clinical isolates. Antimicrobial agents and chemotherapy, 51, 888-895. Hill, D, Rose, B, Pajkos, A, Robinson, M, Bye, P, Bell, S, Elkins, M, Thompson, B, MacLeod, C, & Aaron, SD (2005) Antibiotic susceptibilities of Pseudomonas aeruginosa isolates derived from patients with cystic fibrosis under aerobic, anaerobic, and biofilm conditions. Journal of clinical microbiology, 43, 5085-5090. Kuti, JL, Pettit, RS, Neu, N, Cies, JJ, Lapin, C, Muhlebach, MS, Novak, KJ, Nguyen, ST, Saiman, L, & Nicolau, DP (2015) Microbiological activity of ceftolozane/tazobactam, ceftazidime, meropenem, and piperacillin/tazobactam against Pseudomonas aeruginosa isolated from children with cystic fibrosis. Diagnostic microbiology and infectious disease. Polovina, M, Potpara, T, Milosevic, I, Stepanovic, J, Jovanovic, M, & Pavlovic, M (2014) Mitral valve endocarditis caused by Pseudomonas aeruginosa: a case report. The Journal of Infection in Developing Countries, 8, 676-679. Rodríguez‐Pardo, D, Pigrau, C, Lora‐Tamayo, J, Soriano, A, Toro, M, Cobo, J, Palomino, J, Euba, G, Riera, M, & Sánchez‐Somolinos, M (2014) Gram‐negative prosthetic joint infection: outcome of a debridement, antibiotics and implant retention approach. A large multicentre study. Clinical Microbiology and Infection, 20, O911-O919. Sader, HS, Farrell, DJ, Castanheira, M, Flamm, RK, & Jones, RN (2014) Antimicrobial activity of ceftolozane/tazobactam tested against Pseudomonas aeruginosa and Enterobacteriaceae with various resistance patterns isolated in European hospitals (2011–12). Journal of Antimicrobial Chemotherapy, dku184. Shlaes, DM (2013) New β‐lactam–β‐lactamase inhibitor combinations in clinical development. Annals of the New York Academy of Sciences, 1277, 105-114. Skalweit, MJ (2015) Profile of ceftolozane/tazobactam and its potential in the treatment of complicated intra-abdominal infections. Drug design, Development and Therapy, 9, 2919. Soliman, R, Lynch, S, Meader, E, Pike, R, Turton, JF, Hill, RL, Woodford, N, & Livermore, DM (2015) Successful ceftolozane/tazobactam treatment of chronic pulmonary infection with pan‐resistant Pseudomonas aeruginosa. JMM Case Reports, 2, e000025. 10

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

MA

NU

SC

RI P

T

Zhanel, GG, Chung, P, Adam, H, Zelenitsky, S, Denisuik, A, Schweizer, F, Lagacé-Wiens, PR, Rubinstein, E, Gin, AS, & Walkty, A (2014) Ceftolozane/tazobactam: a novel cephalosporin/β-lactamase inhibitor combination with activity against multidrug-resistant gram-negative bacilli. Drugs, 74, 31-51.

11

ACCEPTED MANUSCRIPT Acknowledgements: This project was supported by CTSA Grant Number TL1 TR000137 from the National Center for Advancing Translational Science (NCATS) and by

T

Cubist/Merck. Its contents are solely the responsibility of the authors and do not necessarily

RI P

represent the official views of the NIH or Cubist/Merck. Presented in part at the 7th ASM

AC

CE

PT

ED

MA

NU

SC

Conference on Biofilms, 2015.

12

ED

PT

CE

Figure 1. Biofilm growth indices of the study Pseudomonas aeruginosa isolates

AC

0.4

T

IP

0.3

CR

0.2

0.1

0

MA NU S

IDRL 3982 IDRL 4170 IDRL 4216 IDRL 4217 IDRL 4218 IDRL 4219 IDRL 4221 IDRL 4223 IDRL 4224 IDRL 4225 IDRL 4227 IDRL 4228 IDRL 4229 IDRL 4230 IDRL 4231 IDRL 4232 IDRL 4233 IDRL 4234 IDRL 4235 IDRL 4236 IDRL 4237 IDRL 4238 IDRL 4239 IDRL 4240 IDRL 4241 IDRL 4242 IDRL 4244 IDRL 5319 IDRL 5970 IDRL 6017 IDRL 6139 IDRL 6654 IDRL 6735 IDRL 6736 IDRL 6737 IDRL 7122 IDRL 7127 IDRL 7141 IDRL 7252 IDRL 7262 IDRL 7319 IDRL 7498 IDRL 7662 IDRL 7852 IDRL 8280 IDRL 8458 IDRL 8510 IDRL 8653 IDRL 9093 IDRL 9237 IDRL 9630 IDRL 9812 IDRL 9948 IDRL 9966 ATCC 27853 (QC)

OD492/OD600

ACCEPTED MANUSCRIPT

0.6

0.5

13

ACCEPTED MANUSCRIPT

Table 1: Cumulative distributions of ceftolozane/tazobactam and cefepime MICs, MBICs, and MBBCs against 54 Pseudomons aeruginosa isolates

MBIC

2

4

8

0 (0.0)

20 (37.0)

28 (88.9)

5 (98.1)

1 (100)

0 (100)

1/4

2/4

100

0.0

0.0

2 (3.7)

21 (42.6)

27 (92.6)

4 0 0 0 0 0 (100.0) (100.0) (100.0) (100.0) (100.0) (100.0) 1/4

1/4

100

0.0

0.0

0 (0.0)

1 (1.9)

0 (1.9)

12 (24.1)

16

83.3

13.0

3.7

Ceftolozane (with 4 µg/mL 0 tazobactam) (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

Cefepime

ED

1

18 (57.4)

PT

Cefepime

0.5

CE

Broth dilution Ceftolozane (with 4 µg/mL tazobactam) Agar dilution Ceftolozane (with 4 µg/mL tazobactam)

14 (83.3)

16

32

>32

0 (100)

0 (100)

0 (100)

% % % susceptible intermediate resistant

0.25

7 (96.3)

0 (96.3)

2 (100)

MIC50

4

MIC90

MBIC50 MBIC90

0 (0.0)

0 (0.0)

3 (5.6)

2 (9.3)

2 (13.0)

47 (100)

>256/4 >256/4

0 (0.0)

0 (0.0)

1 (1.9)

1 (3.7)

2 (7.4)

50 (100)

>256

AC

MIC

MA NU S

CR

IP

T

Number of P. aeruginosa isolates (cumulative %) inhibited at the specified concentrations of ceftolozane (tazobactam constant at 4 µg/mL) or cefepime (µg/mL)

>256

MBBC50 MBBC90 MBBC

Ceftolozane (with 4 µg/mL 0 tazobactam) (0.0) 0 Cefepime (0.0)

0 (0.0) 0 (0.0)

0 (0.0) 0 (0.0)

0 (0.0) 0 (0.0)

0 (0.0) 0 (0.0)

0 (0.0) 0 (0.0)

0 (0.0) 0 (0.0)

0 (0.0) 0 (0.0)

54 (100) 54 (100)

>256/4 >256/4 >256

>256

14