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Occurrence and antibioticsusceptibility profiles of Burkholderiacepaciacomplex in coastal marineenvironmentAna Maravić a , Mirjana Skočibušić a , Matilda Šprung b , Ivica

Šamanić a , Jasna Puizina a & Maja Pavela-Vrančić ba Department of Biology, Faculty of Science, University of Split,Nikole Tesle 12, Split, 21000, Croatiab Department of Chemistry, Faculty of Science, University of Split,Nikole Tesle 12, Split, 21000, CroatiaVersion of record first published: 19 Mar 2012.

To cite this article: Ana Maravić , Mirjana Skočibušić , Matilda Šprung , Ivica Šamanić ,Jasna Puizina & Maja Pavela-Vrančić (2012): Occurrence and antibiotic susceptibility profilesof Burkholderia cepaciacomplex in coastal marine environment, International Journal ofEnvironmental Health Research, 22:6, 531-542

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Occurrence and antibiotic susceptibility profiles of Burkholderiacepacia complex in coastal marine environment

Ana Maravica*, Mirjana Sko�cibusica, Matilda Sprungb, Ivica Samanica,Jasna Puizinaa and Maja Pavela-Vran�cicb

aDepartment of Biology, Faculty of Science, University of Split, Nikole Tesle 12, Split 21000,Croatia; bDepartment of Chemistry, Faculty of Science, University of Split, Nikole Tesle 12,Split 21000, Croatia

(Received 13 December 2011; final version received 1 February 2012)

During an environmental study of bacterial resistance to antibiotics in coastalwaters of the Kastela Bay, Adriatic Sea, Croatia, 47 Burkholderia cepacia complex(Bcc) isolates were recovered from seawater and mussel (Mytilus galloprovincialis)samples. All isolates showed multiple antibiotic resistance. Among the isolates, twoBurkholderia cenocepacia isolates produced chromosomally encoded TEM-116extended-spectrum b-lactamase (ESBL). Analysis of outer membrane proteinsrevealed that decreased expression of a 36-kDa protein could be associated with ahigh level of b-lactam resistance in both isolates. Phenotypic study of efflux systemalso indicated an over-expression of Resistance-Nodulation-Cell Division (RND)efflux-mediated mechanism in one of the isolates. This study demonstrated thepresence of Bcc in seawater and M. galloprovincialis, which gives evidence thatcoastal marine environment, including mussels, could be considered as a reservoirfor Bcc species. Detection of ESBL-encoding genes indicates the potential role ofthese bacteria in the maintenance and dispersion of antibiotic resistance genes.

Keywords: Burkholderia cepacia complex; TEM-116; ESBL; Mytilusgalloprovincialis; coastal marine environment

1. Introduction

The Burkholderia cepacia complex (Bcc) is a heterogeneous group of Gram-negativebacteria currently consisting of 17 closely related species or genomovars which arewidely distributed in the natural environments, such as soil, plant rhizosphere andwater (Vial et al. 2011). They have been studied for various purposes includingbiocontrol, plant growth promotion and bioremediation (Parke and Gurian-Sherman 2001) but have also been identified as important opportunistic pathogensin patients with cystic fibrosis (Bernhardt et al. 2003) and others with a compromisedimmune system (Mann et al. 2010).

The treatment of Bcc infections is difficult, because these bacteria demonstratea high level of intrinsic resistance against most clinically available antibiotics,such as quinolones, aminoglycosides, polymyxins and b-lactam agents, includingmonobactams and carbapenems (Waters and Ratjen 2006). This property is nowrecognized to result from reduced permeability of the bacterial outer membrane(Moore and Hancock 1986; Parr et al. 1987; Aronoff 1988), expression of antibiotic

*Corresponding author. Email: ana.maravic@pmfst.hr

International Journal of Environmental Health Research

Vol. 22, No. 6, December 2012, 531–542

ISSN 0960-3123 print/ISSN 1369-1619 online

� 2012 Taylor & Francis

http://dx.doi.org/10.1080/09603123.2012.667797

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modifying enzymes (Trepanier et al. 1997; Poirel et al. 2009) and active drug efflux(Burns et al. 1996; Wigfield et al. 2002).

Several studies suggest the ongoing acquisition of pathogenic strains from theenvironment (LiPuma et al. 2002; Coenye and LiPuma 2003), but the studiesaddressing this issue have been hampered by difficulties in isolating these bacteriafrom the environmental samples mostly due to limited selectivity and sensitivity ofculture media (Miller et al. 2002; Vermis et al. 2003). Moreover, the distribution ofBcc species in different habitats has not been equally investigated, as most studieshave focused on clinical and plant rhizosphere isolates (Vial et al. 2011). Thus, littleinformation exists concerning the distribution of different genomovars in the aquaticenvironments. Few studies documented the presence of Bcc bacteria in freshwaterecosystems (Vermis et al. 2003; Olapade et al. 2005), whereas oceans or seas are notconsidered to be their natural habitats (Vial et al. 2011).

Here, we aimed to investigate the antibiotic susceptibility profiles and to identifythe potential mechanisms accounting for multidrug resistance in Bcc isolates fromseawater and mussels (Mytilus galloprovincialis) collected in the Kastela Bay(Adriatic Sea, Croatia). This isolation occurred during a survey aimed to study thepresence of multiple resistant Gram-negative bacterial pathogens in the marineenvironment. Cultural and biochemical analyses, following antibiotic susceptibilitytesting, were conducted for each isolate. The production of extended-spectrumb-lactamases (ESBLs) was phenotypically tested, and the presence of the ESBL-encoding genes was confirmed by polymerase chain reaction (PCR) amplificationand DNA sequencing. Additionally, the study of the active efflux system using effluxpump inhibitor, Phe-Arg- b-naphthylamide dihydrochloride (PAbN), and outermembrane protein analysis were conducted for the ESBL-producing isolates.

2. Materials and methods

2.1. Study area

The Kastela Bay is a semi-enclosed bay (61 km2, average depth 23 m) located in thecentral part of the eastern Adriatic coast, in Croatia. The coastal belt around the bayis urbanized and industrialized, covering the municipalities of Split, Solin, Kastelaand Trogir with a population of 380,000 inhabitants (Knezic and Margeta 2002).This highly eutrophic basin is used as a recipient of untreated urban and partiallytreated industrial wastewater as well as agricultural runoff of the area. Despite thesignificant pollution level (Jaksic et al. 2005), coastal waters are extensively used forrecreational activities, fishing and shellfish harvesting. During the 2-year research(from September 2008 to July 2010), we analysed seawater (n¼ 97), mussels (n¼ 78)and fish (n¼ 31) samples for the presence of multidrug-resistant Gram-negativebacteria in coastal waters of the Kastela Bay.

2.2. Sample collection and preparation

Seawater samples were collected in sterile water sampling bottles (APHA 2005) fromthree sampling sites in the Kastela Bay (Kastel Gomilica 438330N, 168240E; KastelSucurac 438330N, 168250E and Kastel Stari 438330N, 168210E). Samples of theMediterranean mussel (M. galloprovincialis) were collected near the town of Trogir(438290N, 168100E), where natural beds of mussels are known for harvesting.Fresh fish caught in the Kastela Bay waters were purchased from the local fishermen

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as follows: conger (Conger conger; n¼ 3), sole (Solea solea; n¼ 7 samples), salema(Sarpa salpa; n¼ 12) and red mullet (Mullus barbatus, n¼ 9). Mussels and fishsamples were placed into sterile bags and together with seawater samples transportedto a laboratory under refrigerated conditions (48C). All samples were examinedwithin 3 h after collection. Mussel and fish samples were prepared according toprotocols described elsewhere (Ottaviani et al. 2005; Herrera et al. 2006).

2.3. Isolation and identification of B. cepacia complex

Each of the seawater samples was mixed thoroughly, and 5-, 10-, 25- and 50-mlsamples were filtered through a 0.22-mm pore membrane (Pall Corporation LifeSciences, Ann Arbor, USA) which was then placed on marine agar (MA; FlukaBiochemika, Steinheim, Switzerland) and incubated at 378C for 24 h. Twenty-fivegrams of each mussel and fish samples were blended with 225 ml of trypticasesoy broth (TSB; Oxoid, Basingstoke, UK) and incubated at 288C for 8 h. A 100-mlaliquot was then inoculated on MA plates and incubated at 378C for 24 h. Thepresumptive identification of Bcc isolates was based on Gram staining andcytochrome oxidase activity, and Gram-negative, oxidase-positive colonies wereselected for further biochemical analyses. The API-20 NE system (BioMerieux,Marcy-l’Etoile, France) and additional biochemical tests such as lysine and ornithinedecarboxylase activity, oxidation of sucrose, lactose and adonitol, hemolysis, pigmentproduction and growth at 428C were used to identify the Bcc species (Coenye et al.2001). The ESBL-positive Bcc isolates were identified to a genomovar status usingduplex PCR using specific primers targeting recA and 16S rRNA sequences aspreviously described (Ramette et al. 2005). All Bcc isolates were stored at 7808C inglycerol-containing Luria–Bertani (LB) liquid media and were freshly subcultured ontrypticase soy agar (Oxoid) and incubated for 24–48 h at 378C for further analysis.

2.4. Antibiotic susceptibility testing

Susceptibilities to 13 therapy-relevant antibiotics were determined by disc-diffusion andbroth microdilution methods according to the Clinical and Laboratory StandardsInstitute (CLSI 2008) protocols. The following antibiotics were tested: amikacin,aztreonam, ceftazidime, ciprofloxacin, gentamicin, imipenem, meropenem, piperacillin,piperacillin/tazobactam, polymyxin B, tetracycline, tobramycin and trimethoprim/sulfamethoxazole. Minimal inhibitory concentration (MIC) breakpoints for ceftazi-dime, meropenem and trimetoprim/sulphamethoxazole were applied according to theCLSI guidelines for Bcc bacteria, and for other antibiotics, breakpoints forPseudomonas aeruginosa (CLSI 2008). P. aeruginosa ATCC 27853 was used as areference strain, because there is no reference strain for the Bcc bacteria relating to invitro antibiotic susceptibility testing. The antibiotic discs were purchased from BectonDickinson (Sparks, MD, USA). For microdilution method, antibiotics were purchasedin the form of powder from Sigma-Aldrich (St. Louis, MI, USA) except meropenem(Zeneca Pharmaceutical Group, Wilmington, DE, USA).

2.5. Screening tests for the detection of ESBL and metallo- b-lactamase production

Ceftazidime-resistant isolates were screened for the production of ESBLs using theESBL Etest strips (AB Biodisk, Solna, Sweden) and the double-disk synergy test as

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described previously (Aggarwal et al. 2008). Carbapenem-resistant isolates weretested by the MBL Etest (AB Biodisk) for possible production of metallo-b-lactamases (MBLs).

2.6. PCR amplification and DNA sequencing

Ceftazidime-resistant isolates were studied by PCR for the presence of genesencoding blaTEM, blaSHV, blaPER, blaVEB, blaGES and blaCTX-M using specific primersand conditions previously reported (Girlich et al. 2011). Total DNA was extractedby boiling a colony suspension at 1008C for 10 min, and supernatant was used as atemplate DNA. All reactions were performed in a 20-ml volume containing 1 ml oftemplate DNA, 1 U Taq DNA polymerase (Invitrogen, Barcelona, Spain), 16Taqbuffer (Invitrogen), 0.5 mM of each primer, 200 mM deoxyribonucleotide triphos-phates (dNTPs) and 3.0 mM MgCl2. Amplification products were separatedelectrophoretically in 1% (w/v) agarose gels, purified with a QIAEX II GelExtraction Kit (Qiagen, Hilden, Germany) and subjected to sequencing on 3730XLDNA sequencer (Applied Biosystems, CA, USA). The nucleotide and deducedprotein sequences were analysed using the GenBank database (http://www.ncbi.nlm.nih.gov). The environmental isolates that were found negative forESBL-encoding genes in the previous researches were used as negative controls in allPCR assays in this work.

2.7. Molecular analysis

For determination of the blaTEM-116 gene location, we adopted a direct approach byperforming PCR utilizing the purified chromosomal and plasmid DNA separately astemplates. Plasmid DNA of the isolates harbouring blaTEM-116 was extracted usingthe Plasmid Mini Kit (Qiagen) according to the manufacturer’s instruction.Chromosomal DNA was extracted using a standard protocol (Sambrook et al.1989). After denaturation at 958C for 10 min, DNA templates were used to amplifyblaTEM-116 gene as previously described (Girlich et al. 2011).

2.8. Transfer of resistance determinants

ESBL-producing isolates were investigated for the transferability of their resistancedeterminants. Conjugation experiments were performed as described previously(Picao et al. 2008) with sodium azide-resistant Escherichia coli J53 as the recipient.Equal volumes (4 ml) of cultures of each ESBL-producing isolate were individuallymixed with E. coli J53 (each at 109 cfu/ml) and grown in TSB at 378C for 24 h.Transconjugants were selected on Mueller–Hinton agar (Oxoid) supplemented withsodium azide (100 mg/ml; Sigma-Aldrich) to inhibit the growth of the donor strainsand with ceftazidime (10 mg/ml) to inhibit the growth of the recipient strain.

2.9. Study of efflux system

We employed the method of Lomovskaya et al. (2001) for the phenotypic study ofan active Resistance-Nodulation-Cell Division (RND) efflux system in ESBL-producing isolates. The following antibiotics that are substrates of RND effluxsystems were tested: meropenem, ceftazidime, piperacillin, aztreonam, tobramycin,

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chloramphenicol and tetracycline. MICs were determined using standard brothmicrodilution method (CLSI 2008) in the absence or presence of PAbN (Sigma-Aldrich) at 50 mg/ml. The reduction of MIC of the antibiotic in the presence of PabNwas an indication of resistance to this antibiotic mediated by an efflux system(Lomovskaya et al. 2001).

2.10. Preparation of outer membrane proteins

Outer membrane protein analyses of the ESBL-producing Burkholderia cenocepaciaisolates and a b-lactam-susceptible B. cenocepacia isolate BC122 were performedaccording to a previously described protocol (Quale et al. 2003) with modifications. Theisolates were grown overnight at 378C in 5 ml of LB medium and then diluted 100-foldinto fresh medium. Bacterial cells were then incubated for 5 h at 378C under shaking(350 rpm) to yield late-logarithmic phase of growth and harvested by centrifugation at50006 g for 20 min at 48C. The pellet was suspended in 10 ml of 30 mM Tris–HCl(pH 8.0) and sonicated. Unbroken cells were removed by centrifugation at 10,0006 gfor 10 min at 48C. The membranes were pelleted by centrifugation at 75,0006 g for1 h and resuspended in the same buffer. The membrane suspensions were thenincubated in 2% (wt/vol) sodium N-lauroyl sarkosinate (Sigma-Aldrich) at roomtemperature for 1 h. The insoluble outer membrane proteins were pelleted bycentrifugation at 75,0006 g for 30 min at 108C and resuspended in distilled water ata protein concentration of about 10 mg/ml. The outer membrane proteins were storedat 7208C. Protein concentrations were determined by the Braford protein assay(Bradford 1976). Bovine serum albumin was used as a standard.

2.11. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Protein fractions were solubilized in a sample buffer, heated at 958C for 5 min andanalysed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis(PAGE) on 10% acrylamide–0.26% bis (acrylamide)–SDS slab gel at a constantcurrent of 10 mA. The gels were stained with Coomassie Blue solution (Sigma-Aldrich).

2.12. Genetic typing

Genetic relatedness of the ESBL-producing isolates was determined by randomlyamplified polymorphic DNA (RAPD) PCR typing as described previously (Sevillanoet al. 2006).

2.13. Statistical analysis

Differences between the antibiotic susceptibility patterns of isolates recovered fromseawater and mussel samples were analysed using the w2 test, and a P value 50.05was taken to indicate significance.

3. Results

Of the various Gram-negative bacteria isolated during an environmental studycarried out from 2008 to 2010 in coastal waters of the Kastela Bay, Croatia, a total

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of 4.5% (47/1035) of isolates were identified as Bcc species (Table 1). The isolateswere recovered from seawater (n¼ 11) and samples of M. galloprovincialis (n¼ 36),whereas no Bcc isolates were recovered from fish samples. Overall, 9.3% (9/97) of theseawater samples and 21.8% (17/78) of the mussel samples yielded growth of the Bccspecies. The results of in vitro susceptibility testing are shown in Figure 1. All theisolates were multidrug-resistant, demonstrating resistance to at least three differentclasses of antibiotics. No significant differences (P4 0.05) were found between theisolates recovered from seawater and mussel samples regarding the number ofantibiotics to which they were resistant. Among the antibiotics tested, highestresistance was recorded against tetracycline (97.8%, 46/47), following polymyxin B(93.6%, 44/47), tobramycin (87.2%, 41/47), gentamicin (76.6%, 36/47) and amikacin(72.3%, 34/47). The resistance to ciprofloxacin and aztreonam was revealed in asignificant and equal percentage (57.4%, 27/47). Lowest rates of resistance wereobserved for ceftazidime (6.4%, 3/47), meropenem (8.5%, 4/47), trimethoprim/sulphamethoxazole (12.7%, 6/47), piperacillin/tazobactam (17.0%, 8/47) andpiperacillin (27.6%, 13/47). Although 20 (42.5%) of the isolates were resistant toimipenem, the production of MBLs was not detected.

Two out of three (6.4%, 3/47) ceftazidime-resistant isolates were identified asESBL producers by the results of phenotypic tests. PCR and sequence analyses

Table 1. Distribution of Bcc isolates in various samples collected for this study.

SourceNo. ofsamples

No. of sampleswith Bcc isolates

No. of Bccisolates

Water 97 9 11ShellfishMediterranean mussel (M. galloprovincialis) 78 17 36

FishConger (C. conger) 3 0 0Sole (S. solea) 7 0 0Salema (S. salpa) 12 0 0Red mullet (M. barbatus) 9 0 0

Total 206 26 47

Figure 1. Percentages of multiple resistance to antibiotics detected in Bcc isolates fromseawater and mussel samples collected from the Kastela Bay, Croatia.

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revealed the presence of identical nucleotide sequences corresponding to blaTEM-typegene and shared 99.7% similarity to blaTEM-1 at the nucleotide level. In comparisonto deduced amino acid sequence of blaTEM-1, two point mutations were revealed.These mutations consisted of a replacement of valine by isoleucine at position 84(Val84Ile) and that of alanine by valine at position 184 (Ala184Val). The nucleotidesequences were 100% identical to the previously reported blaTEM-116 gene fromB. cenocepacia (GenBank accession number GU169419). Both isolates (BC154 andBC206) were recovered from mussels, one in June and the other in July 2010, andwere identified as B. cepacia genomovar III (B. cenocepacia) by a recA and16S rRNA gene-targeted duplex PCR assay. MIC determinations of TEM-116-producing B. cenocepacia strains and an ESBL-negative and b-lactam-susceptiblestrain BC122 are given in Table 2. When compared to a strain BC122, bothTEM-116-producing isolates were resistant to a broad spectrum of b-lactamantibiotics, including piperacillin, ceftazidime and aztreonam, while remainedsusceptible to piperacillin in combination with tazobactam. The strain BC206 alsoexhibited resistance to imipenem. Among the tested agents, the TEM-116-producingisolates were most susceptible to amikacin, gentamicin and ciprofloxacin.

Despite several attempts, PCR amplification utilizing plasmid DNA as thetemplate yielded no respective amplicon of blaTEM gene, and we were unable toobtain transconjugants in conjugation experiments, suggesting the chromosomallocation of the blaTEM-116 gene in two B. cenocepacia isolates. This was furtherconfirmed by the sequence analysis of the PCR product amplified from thechromosomal DNA.

Attempts at inhibiting possible drug efflux systems in TEM-116-producingB. cenocepacia strains were performed by the determination of MICs of selected

Table 2. MIC determinations of selected antibiotics for B. cenocepacia strains.

MIC (mg/ml)

Antibiotic

MICbreakpoint(mg/ml)�R

BC122 BC154 BC206

7PAbN 7PAbN þPAbN* 7PAbN þPAbN*

Amikacin 32 4 16 ND 16 NDAztreonam 32 2 64 8 128 128Ceftazidime 32 2 128 1 128 128Ciprofloxacin 4 0.25 0.25 ND 2 NDChloramphenicol 32 16 64 2 128 128Gentamicin 8 2 2 ND 4 NDImipenem 16 1 0.125 ND 256 NDMeropenem 16 0.5 128 128 256 256Piperacillin 128 4 256 0.5 256 256Piperacillin/

tazobactam128/4 0.25 1 ND 0.25 ND

Polymyxin B 4 32 128 ND 128 NDTrimethoprim/

sulfamethoxazole8/152 2/38 64/1,216 ND 2/38 ND

Tobramycin 8 4 8 0.5 2 NDTetracycline 16 64 128 128 256 256

Notes: *MICs of seven antibiotics were also determined in the presence of PAbN (50 mg/ml) for the studyof efflux systems; ND, not determined.

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antibiotics in the absence or presence of PAbN (Table 2). PAbN showed a significantefflux inhibition effect for strain BC154 by reduction of MICs of ceftazidime(512-fold) and piperacillin (128-fold), following chloramphenicol (32-fold). Thepresence of PAbN also caused a reduction of MICs of tobramycin (16-fold) andaztreonam (8-fold), whereas PAbN did not affect the MICs of meropenem andtetracycline. Furthermore, other TEM-116-producing strain, BC206, did not showdiminished MICs of tested antibiotics after treatment with pump inhibitor.

The outer membrane protein patterns of the TEM-116-producing strains anda b-lactam-susceptible strain BC122 were analysed by SDS-PAGE and compared(Figure 2). The level of a 36-kDa outer membrane protein was markedly lower inboth TEM-116-producing strains than in strain BC122, while 27-kDa porin waspresent in the tested strains. Genetic typing was carried out by RAPD PCR analysisand showed no clonally relatedness between the TEM-116-producing strains.

4. Discussion

A total of 47 Bcc isolates were recovered from seawater and Mediterranean mussel(M. galloprovincialis) samples collected from the coastal waters of the Kastela Bay inCroatia. This finding is interesting because little is known about their distributionin marine environment. Bcc species are ubiquitous in nature and are typicallyconsidered a terrestrial rather than marine bacterial species (Vial et al. 2011). Theirgrowth was found to be poor in the conditions reflective of seawater, indicating thatthe ocean and coastal marine waters were not their natural habitats (Mahenthir-alingam et al. 2006). Moreover, the Burkholderia SAR-1 metagenome was recovered

Figure 2. SDS-PAGE of the outer membrane proteins of the B. cenocepacia strains. Lanes: 1,strain BC122; 2, strain BC154; 3, strain BC206. The migration positions of standard proteinsare shown on the left (in kilodaltons). Arrows indicate the 36-kDa (top) and 27-kDa (bottom)outer membrane proteins.

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from the Sargasso Sea metagenomic study (Venter et al. 2004), but later phylogeneticanalysis placed the metagenome within Burkholderia contaminans genomovar(Vanlaere et al. 2009), suggesting a sample contamination as the most probableexplanation for the presence of Bcc in the Sargasso Sea. Our study showed thatBcc bacteria are present in the marine environment, therefore pointing out coastalwaters and raw mussels as potential reservoirs for Bcc species, includingB. cenocepacia, a major opportunistic pathogen. The wide diffusion of Bcc speciesin clinical and variety of natural habitats raises speculations for environmentalsources of infection (Coenye and LiPuma 2003). Bevivino et al. (2002) reported thatdeterminants related to virulence and transmissibility are not confined solely toclinical isolates and are also spread among environmental isolates belonging todifferent Bcc species.

Furthermore, the frequency of multidrug resistance in our isolates was noticeablyhigh due to a remarkable adaptability of these bacteria to selective antibioticpressure, which is a result of the low permeability of the outer membrane (Mooreand Hancock 1986; Parr et al. 1987; Aronoff 1988), the constitutive expression ofvarious efflux pumps with wide substrate specificity (Burns et al. 1996; Wigfield et al.2002) and the naturally occurring class A b-lactamases (Trepanier et al. 1997; Poirelet al. 2009). In agreement with the results from other studies (Bevivino et al. 2002;Nzula et al. 2002), the antibiotic resistance profiles obtained emphasize the high ratesof resistance of the environmental Bcc isolates. The most effective antibiotics in vitrowere ceftazidime, meropenem and trimethoprim–sulfamethoxazole, confirming theprevious indications that these antibiotics are among the few antimicrobials effectiveagainst infections caused by Bcc bacteria (Mandell et al. 2005).

In this work, two B. cenocepacia strains isolated from the musselM. galloprovincialis produced TEM-116 ESBL, which confers resistance to a broadspectrum of b-lactam antibiotics, including piperacillin, ceftazidime and aztreonam.Although TEM-116 was chromosomally encoded, this b-lactamase is not a featureof the Bcc species, thus raising a question about the possible origin of blaTEM-116

gene and its dissemination among environmental B. cenocepacia isolates. TEM-typeESBLs are mostly found in the members of the Enterobacteriaceae family (Falagasand Karageorgopoulos 2009), indicating the possible horizontal gene transfer of theTEM genetic determinants, which provided B. cenocepacia an additional resistancemechanism with possible clinical and environmental consequences.

In many bacteria expressing multiple drug resistance, efflux systems areresponsible for active and relatively non-specific expelling of antibiotics and otherforeign substances. RND efflux systems are mainly involved in drug resistanceof Gram-negative bacteria (Poole 2004), but the information relating to thecontribution of efflux systems in Bcc bacteria is limited (Burns et al. 1996; Wigfieldet al. 2002; Nair et al. 2004; Guglierame et al. 2006). The results of the phenotypicstudy of the efflux systems in TEM-116-producing B. cenocepacia isolates wereintriguing. Strain BC154 was considered to have an active RND efflux-mediatedmechanism partially underlying its resistance phenotype, while this mechanism couldnot be phenotypically detected in strain BC206. The efflux pump inhibitor PAbNrestored susceptibilities to piperacillin, ceftazidime and aztreonam in strain BC154,indicating that an active RND efflux system, together with the production ofTEM-116 ESBL, is accounting for resistance to these antibiotics in this strain.Moreover, the resistance to tobramycin and chloramphenicol is considered to resultfrom the over-expression of efflux system in BC154 strain, as well.

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The outer membrane is another contributing factor in the b-lactam resistance inBcc bacteria, retarding the diffusion of b-lactams to their penicillin-binding proteintargets (Aronoff 1988). Two outer membrane proteins with molecular sizes of 36 and27 kDa have been identified as potential porins and are associated with decreasedpermeability (Parr et al. 1987). Analysis of outer membrane proteins by SDS-PAGEshowed that both TEM-116-producing strains had reduced amounts of a 36-kDaprotein, which is associated with a high level of b-lactam resistance, confirming theobservations from previous studies (Parr et al. 1987; Aronoff 1988).

In conclusion, the results of this study show that Bcc bacteria are present inmussels and coastal waters of the Kastela Bay in Croatia, which could thereforerepresent an environmental reservoirs and potential sources of infection. Highrates of multidrug resistance of environmental isolates as well as the detection ofESBL-encoding genes emphasize a need for more detailed knowledge about thedistribution of each of the Bcc species in different natural habitats and, in particular,for a more precise study to provide a full picture of antibiotic resistance in theseopportunistic bacteria.

Acknowledgments

This work was supported by Ministry of Science, Education and Sports, Republic of Croatia,project ‘‘Faecal indicator and potential pathogens in coastal and marine waters’’, Grant 177-0000000-3182 and project ‘‘Mechanisms of maintenance genome stability in higher plants’’,Grant 177-1191196-0829.

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