Microb Ecol DOI 10.1007/s00248-013-0361-6

ENVIRONMENTAL MICROBIOLOGY

Investigating the Link Between Imipenem Resistance and Biofilm Formation by Pseudomonas aeruginosa Hadeel K. Musafer & Sherry L. Kuchma & Amanda A. Naimie & Joseph D. Schwartzman & Harith J. Fahad AL-Mathkhury & George A. O’Toole

Received: 7 June 2013 / Accepted: 23 December 2013 # Springer Science+Business Media New York 2014

Abstract Pseudomonas aeruginosa, a ubiquitous environmental organism, is a difficult-to-treat opportunistic pathogen due to its broad-spectrum antibiotic resistance and its ability to form biofilms. In this study, we investigate the link between resistance to a clinically important antibiotic, imipenem, and biofilm formation. First, we observed that the laboratory strain P. aeruginosa PAO1 carrying a mutation in the oprD gene, which confers resistance to imipenem, showed a modest reduction in biofilm formation. We also observed an inverse relationship between imipenem resistance and biofilm formation for imipenem-resistant strains selected in vitro, as well as for clinical isolates. We identified two clinical isolates of P. aeruginosa from the sputum of cystic fibrosis patients that formed robust biofilms, but were sensitive to imipenem (MIC≤2 μg/ml). To test the hypothesis that there is a general link between imipenem resistance and biofilm formation, we performed transposon mutagenesis of these two clinical strains to identify mutants defective in biofilm formation, and then tested these mutants for imipenem resistance. Analysis of the transposon mutants revealed a role for previously described biofilm factors in these clinical isolates of P. aeruginosa, including mutations in the H. K. Musafer : H. J. F. AL-Mathkhury Department of Biology, College of Science, University of Baghdad, Baghdad, Iraq J. D. Schwartzman Department of Pathology, Dartmouth-Hitchcock Medical Center, Lebanon, NH 03756, USA H. K. Musafer : S. L. Kuchma : A. A. Naimie : G. A. O’Toole Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA G. A. O’Toole (*) Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Rm 202 Remsen Building, Hanover, NH 03755, USA e-mail: [email protected]

pilY1, pilX, pilW, algC, and pslI genes, but none of the biofilmdeficient mutants became imipenem resistant (MIC≥8 μg/ml), arguing against a general link between biofilm formation and resistance to imipenem. Thus, assessing biofilm formation capabilities of environmental isolates is unlikely to serve as a good predictor of imipenem resistance. We also discuss our findings in light of the limited literature addressing planktonic antibiotic resistance factors that impact biofilm formation.

Introduction Pseudomonas aeruginosa is an important opportunistic human pathogen that can cause life-threatening infections, especially in patients with cystic fibrosis (CF) and individuals with a compromised immune system. This environmental bacterium is able to survive both in free-swimming planktonic form and in surfaceassociated communities known as biofilms. P. aeruginosa biofilms can form on both biotic and abiotic surfaces in a wide range of environments, thus likely contributing to this microbe’s ability to cause disease in clinical settings [1, 2]. Although there are several antimicrobial agents that continue to be effective against P. aeruginosa (i.e., carbapenems, cefepime, ceftazidime, tobramycin, and amikacin), in the last few years this bacterium’s increasing resistance to antibiotics has been reported [3, 4]. For example, carbapenems, particularly imipenem, are a suitable alternative for treating multidrugresistant P. aeruginosa, yet the emergence and spread of carbapenem-resistant strains have compromised the effectiveness of therapeutic and control efforts using this antibiotic [5]. The OprD porin of P. aeruginosa facilitates the uptake across the outer membrane of basic amino acids, small peptides that contain these amino acids, and their structural analogue, the antibiotic imipenem. Indeed, prolonged treatment of patients with P. aeruginosa infections with this antibiotic leads to imipenem-resistant mutants that lack OprD due to an

H. K. Musafer et al.

oprD gene mutation [6], including disruption of the oprD structural gene by insertion of large IS elements [7–10], missense mutations or insertions [11], deletions creating frame shifts [12], or premature stop codons [12, 13]. Inactivating mutations in the oprD gene have been documented to confer resistance to imipenem, and to a lesser extent to meropenem and doripenem [14, 15]. Alternatively, the pathway to OprDmediated resistance can involve mechanisms that decrease the transcriptional expression of the oprD gene. For example, reduced OprD levels can be caused by a mutation in the gene encoding mexT, which encodes a LysR-family transcriptional regulator. Loss of MexT function results in reduced oprD expression and upregulation of the operon encoding the MexEF-OprN multidrug efflux pump [16, 17], thus contributing to imipenem resistance. By analyzing laboratory strains and clinical isolates of P. aeruginosa, we noticed a link between increased OprDmediated imipenem resistance and reduced biofilm formation. Based on this observation, we hypothesized that loss of biofilm formation might be generally linked to increased imipenem resistance, and to test this idea, we identified and characterized biofilm-deficient variants of two different clinical isolates. Our studies, while identifying well-conserved biofilm-promoting factors, do not support the hypothesis that loss of biofilm formation in general spurs increased resistance to imipenem. We discuss our findings in light of the limited literature addressing planktonic antibiotic resistance factors that impact biofilm formation.

Materials and Methods Strains and Media The strains, plasmids, and primers used in this study are listed in Table 1. All strains were routinely cultured on lysogeny broth (LB) medium, which was solidified with 1.5 % agar when necessary, and supplemented with antibiotics as indicated. Gentamicin (Gm) was used from 25 to 50 μg/ml for P. aeruginosa and at 10 μg/ml for Escherichia coli. Carbenicillin (Cb) was used at 50 μg/ml for P. aeruginosa and 10 μg/ml for E. coli. Nalidixic acid (NA) was used at 20 μg/ml for E. coli. For all phenotypic assays, M63 minimal salts medium was supplemented with MgSO4 (1 mM), glucose (0.2 %), and casamino acids (CAA; 0.5 %). Arabinose was added to cultures at a final concentration of 0.05 % for strains carrying expression plasmids harboring the pBAD promoter. Saccharomyces cerevisiae strain InvSc1 (Invitrogen), used for plasmid construction via in vivo homologous recombination, was grown with yeast extractpeptone-dextrose (1 % Bacto yeast extract, 2 % Bacto peptone, and 2 % dextrose) [18]. Selections with InvSc1 were performed using synthetic defined agar-uracil (catalog no. 4813-065; Qbiogene).

Determination of Imipenem Minimal Inhibitory Concentration The Etest method was used for minimal inhibitory concentration (MIC) determination according to the manufactures instructions. In brief, bacterial suspensions were prepared from fresh colonies, the concentration adjusted to 0.5 McFarland turbidity, each isolate was uniformly spread on the surface of a Mueller Hinton agar (MHA) plate, and an imipenem Etest strip (from 0.002 to 32 μg/mL; bioMérieux, France) placed on the surface of agar plate. After overnight incubation at 37 °C, MIC was determined and categorized as sensitive (≤2 μg/ml), intermediate (4 μg/ml) or resistant (≥8 μg/ml) according to Clinical and Laboratory Standards Institute (CLSI) guidelines [19]. In select cases, we also determined the MIC by a microdilution assay in microtiter plates to confirm the Etest findings [20, 21]. Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as quality control strains for Etest and microdilution assay, respectively. Mariner Transposon Mutagenesis of Clinical Strains The strain E. coli S17-1λpir carrying the pBT20 plasmid served as the donor and the P. aeruginosa clinical isolates (SMC576 and SMC 214) as recipient strains for conjugations. From overnight LB-grown cultures (with the appropriate antibiotic), 1 ml of donor and 1 ml of recipient was centrifuged to pellet the cells. Cell pellets were washed twice with LB and resuspended in 100 μl of fresh LB. The cell suspensions of each strain were then mixed, 60 μl of the mixture was plated to two LB plates and the conjugation was allowed to proceed for 1 h at 30 °C. Each conjugation mix was harvested, resuspended in 100 μl of LB, and then plated on LB agar plates containing Gm to select for the transposon-carrying P. aeruginosa strains, as well as NA to select against growth of the E. coli donor. Libraries of mutants were generated by inoculating individual colonies into 96-well plates containing 100 μl LB medium supplemented with Gm, and the plates incubated overnight at 37 °C to allow outgrowth of the candidate mutants. Mutant strains were screened for biofilm formation defects by inoculating fresh 96-well microtiter plates containing M63 minimal medium supplemented with MgSO4, glucose, and casamino acids, as reported [22]. The inoculum (2–3 μl) was transferred from the library plate containing LB-grown candidate mutants to the screening plates using a 48-pin multiprong device. The clinical isolates (SMC576 or SMC214) were used as controls in each microtiter plate, respectively. After inoculation, the screening plates were incubated at 37 °C for 16 h, and biofilm formation assessed by the crystal violet assay, as reported [22]. We screened ∼1,500 Mariner transposon mutants for each clinical isolate background. The mutations were mapped by determining the DNA sequences flanking the transposon insertions using an arbitrary-primed PCR, as

Imipenem Resistance and Biofilm Formation Table 1 Strains, plasmids, and primers used in this study Strain name

Relevant genotype, description or sequence

Source

S. cerevisiae InvSc1

MATa/MATα leu2/leu2 trp1-289/trp1-289 ura3-52/ura3-52 his3-Δ1/his3-Δ1

Invitrogen

E. coli S17-1(λpir)

thi pro hsdR-hsdM+ ΔrecA RP4-2::TcMu-Km::Tn7

[55]

E. coli DH5α

lˉ f80dlacZDM15D(lacZYA-argF)U169 recA1 endA hsdR17(rKˉ mKˉ) supE44 thi-1 gyrA relA1

Life Technologies

P. aeruginosa PAO1

Wild type

[54]

oprD::IsphoA/hah

PAO1 with isphoA/hah insertion in opr::Tcʳ

[54]

SMC214

Imipenem intermediate P. aeruginosa clinical isolate; robust biofilm former

This study

SMC576

Imipenem-sensitive P. aeruginosa clinical isolate; robust biofilm former

This study

SMC631

Imipenem-sensitive clinical isolate; robust biofilm former

This study

SMC4972

P. aeruginosa clinical isolate

This study

SMC4973

P. aeruginosa clinical isolate

This study

SMC4974

P. aeruginosa clinical isolate

This study

SMC4979

P. aeruginosa clinical isolate

This study

SMC5806

631-F/oprD; imipenem-resistant derivative of SMC631; premature stop mutation in oprD gene

This study

SMC5810

P. aeruginosa clinical isolate

This study

SMC5811

P. aeruginosa clinical isolate

This study

SMC5812

SMC576 pilW::Mar19, Mariner insertion in pilW (769)a

This study

SMC5813

SMC576 pilY1::Mar8, Mariner insertion in pilY1 (3214)

This study This study

SMC5814

SMC576 pilY1::Mar13, Mariner insertion in pilY1 (1933)

SMC5815

SMC576 pilY1::Mar18, Mariner insertion in pilY1 (3242)

This study

SMC5816

SMC576 pilY1::Mar20, Mariner insertion in pilY1(2156)

This study

SMC5817

SMC576 pilY1::Mar25, Mariner insertion in pilY1(1228)

This study

SMC5818

SMC576 pslI::Mar15, Mariner insertion in pslI

This study

SMC5819

SMC576 algC::Mar10, Mariner insertion in algC

This study

SMC5824

SMC214 pilX::Mar, Mariner insertion in pilX (84)

This study

SMC5825

SMC214 pilW::Mar, Mariner insertion in pilW (627)

This study

SMC5832

P. aeruginosa ATCC 27853

This study

SMC5833

Escherichia coli ATCC 25922

This study

Plasmid

Plasmid description

Reference

pMQ70

Shuttle vector for cloning in yeast and for arabinose-inducible gene expression; Apʳ

[18]

pMQ72

Shuttle vector for cloning in yeast and for arabinose-inducible gene expression; Gmʳ

[18]

pBT20

Vector carrying mariner transposon; Apʳ; Gmʳ

[56]

poprD

oprD gene cloned in pMQ72; Gmʳ

This study

ppilY1

His-tagged pilY1 gene cloned in pMQ70; Apʳ

This study

Primersb

a

Primer sequence (5′–3′)

oprD comp 5′

ttctccatacccgtttttttggggaaggagatatacatATGAAAGTGATGAAGTGGAG

oprD comp 3′

taatctgtatcaggctgaaaatcttctctcatccgccTCACAGGATCGACAGCGGATAG

oprD seq a-f

ATGAAAGTGATGAAGTGGAGC

oprD seq a-r

AGGGAGGCGCTGAGGTT

oprD seq b-f

AACCTCAGCGCCTCCCT

pilY1 comp 5′

tctccatacccgtttttttgggctagcgaattcgaaggagatatacatATGAAATCGGTACTCCACCAG

pilY1 comp 3′

tcttctctcatccgccaaaacagccaagcttgcatgcctTCAgtggtgatggtggtggtgGTTCTTTCCGATGGGGC

pilY1 seq Rev 2

TGAACGGACAGGTACAGATCC

pilY1 seq 3

GGATCTGTACCTGTCCGTTC

pilY1 seq 4

GGCGAGTTTCTCAAGAAGACC

pilY1 seq 5

CTTCCAGGACATCCTCAACCG

pilY1 seq 6

AGCCCAGCGGTAACTACTCC

pilY1 seq 7

CAAGGTCAACCAGGACGATC

P730

GCAACTCTCTACTGTTTCTCC

The number in parentheses indicates the nucleotide after which the transposon has inserted in the open reading frame For primer sequences, lowercase letters indicate sequence identity to the cloning vector, uppercase letters indicate a Pseudomonas gene-specific sequence, boldface and lower case letters indicates a His tag sequence

b

H. K. Musafer et al.

described previously [23, 24], and the PCR products were sequenced at the Molecular Biology and Proteomics Core at Dartmouth College. The resulting DNA sequences were aligned to the P. aeruginosa PAO1 genomic sequence using the NCBI BLAST program. Construction of Mutant Strains and Plasmids Table 1 lists all plasmids constructed in this study and primers used in plasmid construction. Plasmids for complementation and overexpression were generated via homologous recombination in yeast [18]. The pMQ72 vector [18] was used as the backbone for the oprD complementation construct, and plasmid pMQ70 [18] served as the backbone for the pilY1 complementation construct. The poprD complementation constructs and ppilY1 complementation constructs were generated by PCR amplification of the respective genes using the highfidelity Phusion polymerase (Finnzyme, Espoo, Finland). Motility Assays Twitch motility plates consisted of M63 medium supplemented with glucose, MgSO4, and CAA solidified with 1.5 % agar. Twitch assays were performed as previously reported [25, 26]. Swarming motility plates were comprised of M8 medium supplemented with MgSO4, glucose, and CAA and solidified with 0.5 % agar. For each strain tested, 2 μl of LB grown overnight cultures was inoculated onto the surface of the swarm plates and incubated for 16 h at 37 °C. Biofilm Formation Assay Biofilm formation in 96-well microtiter plates was assayed and quantified as previously described [26, 27]. All biofilm assays were performed using M63 minimal medium supplemented with MgSO4, glucose, and CAA. Quantification of Polysaccharide Production We quantified Psl production via ELISA with some modifications from an existing protocol [28], using the anti-PSL antibody WapR-001: Class II anti-Psl mAb. MedImmune LLC previously reported this identification and characterization of this antibody [29], and generously provided this reagent for our studies. Flat-bottom 96-well MaxiSorp plates (Nunc) were coated, in triplicate, overnight at 4 °C with 100 μL/well of the indicated strains grown overnight in LB medium. The plate was washed thrice with PBS/0.1 % Tween-20 for 3 min each and tapped to mix, then blocked with 300 μL/well PBS+1 % BSA for 60 min at 4 °C. Primary anti-PSL antibody (WapR001: Class II anti-Psl mAb) diluted in PBS+0.1 % BSA to a concentration of 1 μg/mL was added for 1–2 h at room temp,

then washed three times as above. Next, a 1:5,000 dilution of HRP-conjugate secondary antibody diluted in PBS+0.1 % BSA was added to each well and incubated for 1 h, the plate washed three times with PBS, and 100 μL/well TMB SureBlue development reagents added for color development. Finally, 100 μL/well 0.2 N H2SO4 was added to terminate color development and the wells measured at 450 nm.

Results OprD Participates in Biofilm Formation Given the critical role of OprD in imipenem resistance [30, 31], the well-established role of biofilms in antimicrobial tolerance [32], and the limited literature linking planktonic resistance mechanisms to biofilm formation, we assessed whether strains with a defect in the oprD gene might display an altered biofilm phenotype. To test this hypothesis, we assessed the ability of an oprD transposon mutant to form a biofilm compared to the parental P. aeruginosa PAO1 strain. The oprD mutant showed a significant, but modest reduction in biofilm formation using the 96-well microtiter assay compared to P. aeruginosa PAO1 (Fig. 1a). We observed no difference in the growth rate of the WT versus the oprD mutant (not shown), thus a growth defect could not explain the reduction in biofilm formation. The biofilm formation defect of the oprD mutant was complemented by introducing a wild-type copy of the oprD gene on a plasmid but the vector control (pMQ72) was not able to rescue this defect (Fig. 1a). To confirm that the oprD mutant did indeed confer imipenem resistance, we performed an Etest assay on the strains described in Fig. 1a. The oprD transposon mutant and the mutant carrying the vector control (pMQ72) showed significantly higher resistance to imipenem compared to the parental P. aeruginosa PAO1 and the oprD mutant complemented with a plasmid carrying the wild-type copy of this gene (Fig. 1b). To assess whether these phenotypes might be observed in a clinical strain as well as the PAO1 laboratory strain, we assessed the biofilm formation and imipenem resistance phenotypes of a clinical strain of P. aeruginosa isolated from the sputum of a cystic fibrosis patient. This non-mucoid strain, designated SMC631, could form a biofilm in the 96 well microtiter dish and was sensitive to imipenem (Table 2). We isolated several imipenem-resistant (ImR) variants of the SMC631 strain that grew as colonies in the zone of inhibition at 32 μg/mL while performing an MIC assay with an imipenem ETest strip. Upon retesting in a microdilution assay, these mutants were confirmed to be resistant to imipenem (MIC≥16 μg/L). Sequencing of the oprD gene in one of these

Imipenem Resistance and Biofilm Formation

Biofilm Formation (A550)

c

1.0 0.8

ns **

**

0.6 0.4 0.2 0.0

Imipenem MIC ( g/ml)

b 40 30

***

***

20 10

ns

0

d

0.20

ns

0.15 0.10

**

**

0.05 0.00

Imipenem MIC ( g/ml)

Biofilm Formation (A550)

a

40

***

***

***

30 20 10 0

Fig. 1 OprD participates in biofilm formation as well as imipenem resistance. a Quantification of biofilm formation for the indicated strains, including P. aeruginosa PAO1 (PAO1), the oprD::IsphoA/hah transposon mutant [54], and the oprD mutant complemented with the vector control (pMQ72) or a plasmid carrying a wild-type copy of oprD (poprD+). The biofilm was detected by crystal violet (CV) staining. To quantify the biofilm, CV was solubilized with 30 % glacial acetic acid and the absorbance was measured at 550 nm. Strains were grown in M63 medium with MgSO4, glucose, and CAA for 24 h at 37 °C prior to crystal violet staining. In this and all figures, each strain was tested in four wells per experiment. Error bars represent standard deviations of means from three separate experiments. Statistical analysis was performed using ANOVA with Tukey’s post-test comparison. ns not significantly different; **, P

Investigating the link between imipenem resistance and biofilm formation by Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a ubiquitous environmental organism, is a difficult-to-treat opportunistic pathogen due to its broad-spectrum antibiotic resis...
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