Environmental and clinical isolates of Pseudomonas aeruginosa show pathogenic and biodegradative properties irrespective of their origin

Environmental and clinical isolates of Pseudomonasaeruginosa show pathogenic and biodegradativeproperties irrespective of their origin

Ana Alonso, Fernando Rojo* and JoseÂ L. MartõÂnez

Departamento de BiotecnologõÂa Microbiana, Centro

Nacional de BiotecnologõÂa, CSIC, Campus UAM,

Cantoblanco, 28049 Madrid, Spain.

Summary

Virulence properties of pathogenic bacteria, as well

as resistance to antibiotics, are thought to arise

through a specialization process favoured by the

strong selection pressure imposed in clinical treat-

ments. Nevertheless, in the case of opportunistic

pathogens, it is unclear whether strains can be classi-

®ed into virulent and non-virulent isolates. Clones of

the opportunistic pathogen Pseudomonas aerugin-

osa do not seem to be associated to a particular

biovar or pathovar, which suggests that virulence

characteristics in opportunistic pathogens may

already be present in environmental (non-clinical)

isolates. We have explored this possibility, studying

environmental isolates (mainly from oil-contaminated

soils) and clinical isolates (from bacteraemia and

cystic ®brosis patients) of P. aeruginosa. All environ-

mental strains were found to actively ef¯ux quino-

lones, which are synthetic antibiotics not expected

to be present in the environment. These strains con-

tained multidrug resistance determinants, were

capable of invading epithelial cells and presented

genes from the quorum-sensing and type III secretion

systems. Some of them expressed either haemolytic

or proteolytic activities or both, characteristics con-

sidered to be typical of virulent strains. All the strains

tested, of clinical or environmental origin, could use

alkanes (oil hydrocarbons) as a carbon source. Our

results suggest that clinical and non-clinical P. aeru-

ginosa strains might be functionally equivalent in sev-

eral traits relevant for their virulence or environmental

properties. Selection of clinically relevant traits, such

as antibiotic resistance or cellular invasiveness, in

opportunistic pathogens present in soil ecosystems

is discussed.

Introduction

Hospital-acquired bacterial infections caused by opportu-

nistic pathogens are a problem of great concern. The

consequences are dramatic, because most of these

pathogens are naturally resistant, or present a low sus-

ceptibility level, to a wide range of antibiotics (Neu, 1993;

Swartz, 1994; Dennesen et al., 1998). Several opportunis-

tic pathogens are found in many different ecosystems, and

the mechanisms favouring the selection of antibiotic-resis-

tant variants remain obscure. Current knowledge supports

the idea that virulent bacteria have acquired their clinically

relevant phenotype through a specialization process. Viru-

lence determinants are frequently grouped in DNA regions

with a G �C content different from that of the bacterial

genome, suggesting that they have been acquired through

horizontal transfer. These regions have received the name

of `pathogenicity islands' and are well characterized in

bacterial species such as Escherichia coli, Yersinia pestis,

Vibrio cholerae or Salmonella typhimurium (Blum et al.,

1994; Mills et al., 1995; Buchrieser et al., 1998; Karaolis

et al., 1998), among others. Nevertheless, an increasing

body of evidence suggests that, in the case of opportunis-

tic pathogens with a broad-range ecological distribution,

the difference between virulent and non-virulent strains

may not be so clear. A recent review pointed out that

`many people assume that antibiotic-resistant strains are

arising primarily in hospitals or clinics, but is this really

true?' (Salyers and Amabile-Cuevas, 1997).

We have used Pseudomonas aeruginosa as a model

microorganism to address this question. P. aeruginosa

can be found ubiquitously in nature from sources as

diverse as water, soil and plants. Indeed, Pseudomonas

has long been known to have a broad metabolic versatility

(Clarke, 1982), and P. aeruginosa is no exception to this.

Nevertheless, P. aeruginosa is more widely known as an

opportunistic pathogen for humans and animals than as

a soil bacteria. In this regard, P. aeruginosa can produce

severe infections in immunocompromised hosts (Quinn,

1998) and is the major factor for morbidity and mortality

in cystic ®brosis patients (Govan and Nelson, 1992).

If virulence of P. aeruginosa is directly related to the

acquisition of virulence genes, different specialized popu-

lations should exist that have evolved either to degrade

environmental substrates or to colonize their hosts in the

Environmental Microbiology (1999) 1(5), 421±430

Q 1999 Blackwell Science Ltd

Received 11 March, 1999; revised 17 May, 1999; accepted 20 May,1999. *For correspondence. E-mail [email protected]; Tel. (�34) 91585 4539; Fax (�34) 91 585 4506.

course of an infection process. In fact, clinical isolates of

this bacterial species have been demonstrated to express

well-known virulence factors, such as haemolysins (Ostr-

off et al., 1989), proteases (Kharazmi, 1991), cytotoxic

activity (Fleiszig et al., 1997a; Kang et al., 1997), invasion

of epithelial cells (Fleiszig et al., 1994, 1997a), or to pre-

sent a low susceptibility to clinically useful antibiotics

(Nikaido, 1994). In spite of these characteristic virulence

properties, P. aeruginosa scarcely infects individuals

other than those with a basal pathology. In this respect,

P. aeruginosa is an archetype of nosocomial pathogens.

The relationship between clinical (pathogenic) and non-

clinical (environmental) isolates of P. aeruginosa has been

analysed recently. Molecular analyses have demonstrated

that P. aeruginosa clones indistinguishable by macrores-

triction ®ngerprinting patterns and other taxonomic criteria

can be present in the lung of cystic ®brosis patients as well

as in aquatic environments not related to medical habitats

(Romling et al., 1994). In addition, the analysis of a number

of environmental P. aeruginosa gasoline-using isolates

showed that they were taxonomically indistinguishable

from clinical strains (Foght et al., 1996). From these taxo-

nomic studies, it was concluded that `potential reservoirs

outside of clinics might play a role in the acquisition of

infections' (Romling et al., 1994). Nevertheless, in these

reports, no analyses were made about the virulence of

non-clinical P. aeruginosa isolates, an important subject

that needs to be addressed.

In this work, we explore the virulence properties of

some environmental strains of P. aeruginosa, most of

them isolated from oil-contaminated soils and able to use

crude oil hydrocarbons as the sole source of carbon and

energy. For comparison, all assays were performed in

parallel with the well-known virulent strain P. aeruginosa

PAO1 (Ostroff et al., 1989). On the other hand, we

explored the biodegradative properties of a collection of

clinical isolates of this bacterial species. The results

obtained indicate that environmental and clinical P. aeru-

ginosa strains are functionally equivalent with respect to

several clinically and biodegradative relevant properties.

Results

Antibiotic susceptibility of environmental

P. aeruginosa strains

To analyse the virulence characteristics of environmental

P. aeruginosa isolates, a total of seven strains was col-

lected from diverse sources: ®ve from oil-contaminated

soils, one from oil-contaminated sea water and another

from a water bottle (see Table 1). Their susceptibility to

several different antibiotics was determined and compared

with that of the well-characterized clinical isolate P. aeru-

ginosa PAO1. Many of the environmental strains were

more resistant to several antibiotics than the clinical strain

PAO1 (Table 2). For some strains, the differences were

10-fold in the case of chloramphenicol, eightfold for strep-

tomycin and between two- and threefold for erythromycin,

ceftazidime, cipro¯oxacin, o¯oxacin and nor¯oxacin. It

should be noted that neither these values nor those from

Q 1999 Blackwell Science Ltd, Environmental Microbiology, 1, 421±430

Table 1. P. aeruginosa strains used in this work.

Strain Origin Isolation Reference or source

RR1 Oil-contaminated soil 1996 F. Rojo, unpublishedCECT116 Ex animal room water bottle Before 1967 Spanish Type Culture CollectionCECT119 Oil-contaminated sea water Before 1979 Spanish Type Culture CollectionATCC14886 Soil Before 1958 American Type Culture CollectionATCC15524 Soil (alkane degrader) Before 1967 American Type Culture CollectionATCC15528 Soil (alkane degrader) Before 1967 American Type Culture CollectionATCC21472 Soil from an oilfield Before 1973 American Type Culture CollectionPAO1 Infected wound Before 1952 Ostroff et al. (1989)RYC25616 Cystic fibrosis 1997 This workRYC27028 Cystic fibrosis 1997 This workRYC27219 Cystic fibrosis 1997 This workRYC28290 Cystic fibrosis 1997 This workRYC28516 Cystic fibrosis 1997 This workRYC43442 Bacteraemia 1997 This workRYC97083283 Bacteraemia 1997 This workRYC96044911 Bacteraemia 1996 This workRYC9779653 Bacteraemia 1997 This workRYC46762 Bacteraemia 1997 This workRYC10355 Bacteraemia 1997 This workRYC97054493 Bacteraemia 1997 This workRYC16489 Bacteraemia 1997 This workRYC16469 Bacteraemia 1997 This workRYC111475 Bacteraemia 1997 This workRYC61343 Bacteraemia 1997 This workRYC98076143 Bacteraemia 1998 This work

422 A. Alonso, F. Rojo and J. L. MartõÂnez

the clinical isolate PAO1 are particularly relevant from a

clinical standpoint. However, the observed differences

indicate that environmental strains may present determi-

nants for low-level antibiotic resistance without the need

for antibiotic selective pressure (see below).

Environmental P. aeruginosa strains are able to

extrude quinolones

The increased resistance of environmental strains to cipro-

¯oxacin, nor¯oxacin and o¯oxacin was intriguing, as these

antibiotics belong to the family of quinolones, which are syn-

thetic drugs not present in the environment. For this reason,

soil habitats should not be expected to impose a selective

pressure favouring the development of quinolone-resistant

strains. Moreover, most of the environmental strains ana-

lysed in our work were isolated long before quinolones

began to be used in therapy (Table 1). Multidrug resistance

(MDR) in P. aeruginosa is associated with the expression of

ef¯ux pump systems that actively transport antibiotics (qui-

nolones included; Gotoh et al., 1994; 1998; Poole et al.,

1996; Kohler et al., 1997a) and related molecules outside

the cell. As a ®rst approach to investigate whether low sus-

ceptibility of environmental isolates to quinolones could be

ascribed to the expression of MDR system(s), we analysed

whether these P. aeruginosa strains had ef¯ux system(s) for

quinolones that could be inactivated by CCCP, an uncoupler

of membrane potential. Accumulation of higher amounts of

antibiotics inside the cell in the presence of CCCP than in

its absence indicates that bacteria can actively extrude the

tested antibiotic (Li et al., 1994). As shown in Fig. 1, all P.

aeruginosa isolates were able to extrude the tested quino-

lones through an energy-dependent mechanism that could

be inactivated by CCCP. Depending on the strain and anti-

biotic tested, the ef¯ux system reduced the antibiotic con-

centration inside the cell from two- to sixfold. These results

strongly suggest the presence of multidrug ef¯ux systems

in the strains analysed.

Environmental P. aeruginosa strains contain genes

for multidrug ef¯ux systems

In Gram-negative bacteria, multidrug resistance ef¯ux

pump systems are formed by three proteins: an inner

membrane transporter, an outer membrane protein and

a third protein, which acts as a bridge between the ®rst

two. P. aeruginosa PAO1 has three well-characterized

ef¯ux pump MDR systems encoded by the chromosomal

gene clusters mexA±mexB±oprM (Poole et al., 1993),

mexC±mexD±oprJ (Poole et al., 1996), and mexE±

mexF±oprN (Kohler et al., 1997a). However, sequence

analysis of the P. aeruginosa chromosome indicates that

this bacterial species might present additional MDR trans-

porters (http://www.bit.uq.edu.au/pseudomonas; http://

www.pseudomonas.com).

In the case of the systems functionally analysed so far, it

has been shown that their expression increases the level

of resistance to quinolones (Gotoh et al., 1994; 1998;

Poole et al., 1996; Kohler et al., 1997a) and to several

other unrelated antibiotics. The presence of similar MDR

systems in the environmental P. aeruginosa strains was

analysed by polymerase chain reaction (PCR). The pre-

viously described primers oprM1 and oprM2 (Bianco et

al., 1997), which amplify an internal 848 bp fragment of

the oprM gene, were used to detect the presence of the

mexA±mexB±oprM MDR system. The primers used to

detect the mexC±mexD±oprJ and mexE±mexF±oprN

genes were designed to amplify DNA regions comprising

part of the last two genes of the cluster to ensure the pre-

sence of both the outer membrane and the linker proteins.

In the case of the mexC±mexD±oprJ operon, the primers

(oprJ1 and oprJ2) annealed 100 bp upstream and 469 bp

downstream, respectively, of the 58 end of oprJ. The

primers used to detect the mexE±mexF±oprN genes

(oprN1 and oprN2) annealed 96 bp upstream and 642 bp

downstream, respectively, of the 58 end of oprN. As shown

in Fig. 2, these sets of primers allowed the ampli®cation of


Table 2. Susceptibility of P. aeruginosa strains to different antibiotics.

Antibiotic PAO1 RR1 CECT 116 CECT 119 ATCC 14886 ATCC 15524 ATCC15528 ATCC 21472

Tetracycline 6 8 8 8 6 6 4 12Chloramphenicol 24 48 > 256 > 256 > 256 96 64 196Erythromycin 96 > 256 > 256 > 256 96 > 256 128 > 256Ceftazidime 1 3 3 1 3 1.5 1.5 6Imipenem 2 3 1 1 1 0.75 0.5 1.5Trimethoprim/ 2 1.5 3 1 3 0.75 1 1sulphamethoxazoleStreptomycin 16 16 128 48 32 19 12 24Amikacin 2 4 4 4 6 3 3 3Ciprofloxacin 0.064 0.19 0.19 0.125 0.25 0.125 0.125 0.094Norfloxacin 0.25 0.5 0.75 0.5 1 0.5 0.5 0.38Ofloxacin 0.75 1.5 2 1 2 1 1.5 0.38Nalidixic acid 192 96 > 256 96 > 256 > 256 > 256 128

Minimal inhibitory concentrations were determined on Mueller±Hinton agar plates as indicated in Experimental procedures, and are expressed inmg mlÿ1.

Virulence of environmental Pseudomonas aeruginosa 423

DNA fragments of the expected sizes in all strains. In the

case of strain RR1, the DNA fragments obtained in each

of the three ampli®cation reactions were puri®ed from the

agarose gel and sequenced using the same primers as

for the PCR reaction. In the three cases, the sequence

obtained con®rmed that the ampli®ed DNA indeed corre-

sponded to the expected MDR genes, suggesting that P.

aeruginosa RR1, as well as the other environmental

strains yielding DNA fragments of the same size, contain

the three mentioned MDR clusters.

Environmental P. aeruginosa strains are haemolytic

and proteolytic and have quorum-sensing genes

A characteristic of P. aeruginosa PAO1, believed to be

related to its behaviour as an opportunistic pathogen, is

its ability to lyse red blood cells (Ostroff et al., 1989) and

to excrete proteases (Kharazmi, 1991). Induction of

haemolysis and secretion of proteases were assayed by

growing cells in blood agar and milk agar plates, respec-

tively, using strain PAO1 and the non-haemolytic, non-pro-

teolytic E. coli strain HB101 as controls. Haemolytic or

proteolytic activities were detectable by the formation of

clear halos surrounding the bacterial colonies. As pre-

sented in Table 3, all the environmental P. aeruginosa

strains were proteolytic. Four of the environmental strains

were clearly haemolytic, while haemolysis required

extended incubation periods for three of them. Although

the control strain E. coli HB101 did not produce haemo-

lysis even after prolonged incubation times, we have con-

sidered as haemolytic only those P. aeruginosa strains

producing clear haemolysis within the ®rst 48 h of incuba-

tion. It should be noted that not all clinical P. aeruginosa

isolates are haemolytic; the percentage of environmental

strains being haemolytic was similar to that reported for

clinical P. aeruginosa isolates (VaÂzquez et al., 1992;

Puzova et al., 1994).

Several virulence factors, haemolysis and proteolysis

included, are at least partly under the control of the

quorum-sensing system in P. aeruginosa (van Delden

and Iglewski, 1998). The presence of genes rhlI, which

encodes the autoinducer synthase, and rhlR, which

encodes the transcriptional activator of the system, were

analysed in the P. aeruginosa environmental strains by

PCR. The primers used to detect rhlI (rhlId and rhlr)


Fig. 1. Extrusion of quinolones by P. aeruginosa environmentalstrains. The bars show the accumulation of quinolones inside thecells in the absence (open bar) or presence (®lled bar) of themembrane proton uncoupler CCCP. Higher accumulation in thepresence of CCCP indicates the expression of a quinolone ef¯uxpump system in the bacterial strain analysed. Antibioticconcentrations are expressed as pmol mgÿ1 cell protein. Valuesrepresent the mean of three independent assays; error bars areshown.


annealed 197 bp and 563 bp downstream, respectively, of

the 58 end of rhlI. The primers used to detect rhlR (rhlRd

and rhRr) annealed 23 bp and 455 bp downstream,

respectively, of the 58 end of rhlR. As shown in Fig. 3,

these sets of primers allowed the ampli®cation of DNA

fragments of the expected sizes in all strains.

Environmental P. aeruginosa isolates invade

epithelial cells and have Type III secretion genes

P. aeruginosa PAO1 is known to invade epithelial cells in

vitro (Fleiszig et al., 1994; 1997a). We tested whether the

non-clinical environmental strains could also invade

the epithelial cell line MDCK. As presented in Table 4, all

environmental strains were capable of invading epithelial

cells to different extents under conditions in which no inva-

sion was detected with the negative control E. coli TG1.

Type III secretion systems have an important role in

bacterial virulence (Hueck, 1998). In the case of P. aeru-

ginosa, this system belongs to the exoenzyme S regulon

(Frank, 1997). The ExoS regulon includes a family of

extracellular proteins, as well as their secretion system,

involved in the pathogenic properties of P. aeruginosa.

We thus analysed whether these genes were also present

in the environmental strains. The presence of pscJ, which

encodes a protein involved in the bacterial secretion appa-

ratus, was analysed by PCR. The primers used (pscJd

and pscJr) annealed, respectively, 304 bp and 568 bp

downstream of the 58 end of pscJ. As shown in Fig. 4,

this set of primers allowed the ampli®cation of DNA frag-

ments of the expected size in all strains.

Biodegradative properties of clinical P. aeruginosa

isolates

The environmental P. aeruginosa strains analysed were

isolated in most cases from the soil of water habitats

contaminated with crude oil, and were all able to grow at

the expense of oil hydrocarbons such as hexadecane.

Seventeen clinical isolates of P. aeruginosa obtained

from cystic ®brosis patients or from different bacterae-

mias, as well as strain PAO1, were tested for their ability

to grow using hexadecane as the sole source of carbon

and energy. With the exception of the isolate RYC61343,

all of them were able to grow at the expense of this

major component of crude oil. Visual inspection of the cul-

tures also showed that the alkane was emulsi®ed in the


Fig. 2. Analysis of multidrug ef¯ux systems in P. aeruginosaenvironmental strains. The presence of the MDR determinantsmexA±mexB±oprM, mexC±mexD±oprJ and mexE±mexF±oprNwas analysed by PCR with speci®c primers. The ampli®ed DNAfragments were resolved by electrophoresis in a 1% agarose gel. Inthe control lane, no bacterial DNA was added to the PCR.

Table 3. Haemolytic and proteolytic activity of environmental P.aeruginosa strains.

Strain Haemolysis Proteolysis

PAO1 � **

RR1 � **

CECT116 6 **

CECT119 6 *

ATCC14886 � **

ATCC15524 6 **

ATCC15528 � **

ATCC21472 � *

E. coli HB101 ± ±

�, Clear haemolysis after 48 h of incubation.6, Haemolysis visible only after 3±4 days of incubation.**Clear proteolytic halo of more than 1.5 cm after 48 h of incubation.*Clear proteolytic halo between 0.5 and 1 cm after 48 h of incubation.±, No halo.

Fig. 3. Analysis of the quorum-sensing system in P. aeruginosaenvironmental strains. The presence of genes rhlI, encoding theautoinducer synthase, and rhlR, which encodes the transcriptionalactivator, were analysed in the P. aeruginosa environmental strainsby PCR with speci®c primers. The ampli®ed DNA fragments wereresolved by electrophoresis in a 1% agarose gel. In the controllane, no bacterial DNA was added to the PCR.


course of the incubation, suggesting that the cells were

also able to produce tensioactive biosurfactants, which

allowed them to interact with the organic carbon source.

Discussion

The analyses described above were directed at investigat-

ing whether environmental P. aeruginosa strains could

behave as pathogens showing virulence properties similar

to those of the clinical isolate PAO1. Our results indicate

that environmental P. aeruginosa isolates present a `nat-

ural' reduced susceptibility to several antibiotics relative

to strain PAO1, in particular to the synthetic quinolones

cipro¯oxacin, nor¯oxacin and o¯oxacin. Quinolones are

synthetic drugs scarcely present in the environment, so

that a selective pressure with these antibiotics in natural

habitats is unlikely. Our results indicate that the increased

resistance of environmental P. aeruginosa strains to qui-

nolones is likely to arise from their ability to extrude

these compounds out of the bacterial cell by means of

ef¯ux pump(s) system(s). The resistance levels found

were low, having therefore little importance from a clinical

point of view. Nevertheless, the existence of these extru-

sion systems may provide the starting point for the

development of an ef®cient resistance mechanism

to quinolones under conditions of selective pressure.

The presence of actively working resistance mechanisms

in environmental isolates of P. aeruginosa, operative

against synthetic antibiotics, suggests that selection of

antibiotic resistance can occur by the selective pressure

of non-antibiotic compounds. In agreement with this

idea, in vitro analyses have demonstrated that the use of

disinfectant pine oil can select multiple antibiotic-resistant

mutants in E. coli (Moken et al., 1997). Recently, tolerance

to organic solvents in a strain of Pseudomonas was shown

to be associated with increased resistance towards some

natural or semi-synthetic antibiotics (Isken et al., 1997),

the effect being caused, at least in part, by the presence

of ef¯ux pumps similar to the P. aeruginosa MDR systems

(de Bont, 1998). In addition, a linkage between bacterial

resistance to antibiotics and the use of biocides has

been suggested (Russell et al., 1998). Altogether, these

results illustrate how opportunistic pathogens with `natural'

low susceptibility to antibiotics can arise even in the

absence of the strong antibiotic selective pressure that

exists in hospitals.

It has been shown that quinolones are good substrates

for MDR systems in P. aeruginosa (Gotoh et al., 1994;

1998; Poole et al., 1996; Kohler et al., 1997a). In fact,

most of the resistant P. aeruginosa mutants that emerge

upon quinolone selection are MDR-overproducing strains

(Kohler et al., 1997b). MDR systems consist of three pro-

teins, one located in the outer membrane, one in the inner

membrane and a third acting as a linker (Paulsen et al.,

1996; Nikaido, 1998). Our results show that all P. aerugin-

osa isolates analysed present in their genome the three

best-characterized MDR pumps described in PAO1:

mexA±mexB±oprM, mexC±mexD±oprJ and mexE±

mexF±oprN. The available sequence of the P. aeruginosa

PAO1 chromosome (http://www.bit.uq.edu.au/pseudo-

monas; http://www.pseudomonas.com) indicates that

this bacteria might possess additional MDR determinants.

The presence of oprM in environmental P. aeruginosa

strains has been reported previously (Bianco et al.,


Table 4. Invasion of epithelial MDCK cells byenvironmental isolates of P. aeruginosa. Strain Added bacteria Intracellular bacteria Extracellular bacteria

PAO1 1.8 ´ 106 16 500 6 1500 73 6 28RR1 2.1 ´ 106 8100 6 800 7 6 5CECT116 1.9 ´ 106 1100 6 300 3 6 5CECT119 1.2 ´ 106 5300 6 1100 10 6 8ATCC14886 2.9 ´ 106 5500 6 700 13 6 12ATCC15524 1.2 ´ 106 11 300 6 1800 < 3ATCC15528 1.9 ´ 106 4500 6 900 3 6 5ATCC21472 3.9 ´ 105 500 6 200 < 3E. coli TG1 2.0 ´ 106 < 3 < 3

Intracellular bacteria indicate the viable bacteria recovered from the inside of the epithelial cellsafter a 2 h infection period. Extracellular bacteria are those that remain in the cell culturemedium after 2 h of incubation with gentamicin. The values are expressed as colony-formingunits (cfu) mlÿ1.

Fig. 4. Analysis of type III secretion system in P. aeruginosaenvironmental strains. The presence of gene pscJ, belonging to thetype III secretion system of P. aeruginosa, was analysed by PCRwith speci®c oligonucleotides. The ampli®ed DNA fragments wereresolved by electrophoresis in a 1% agarose gel. In the controllane, no bacterial DNA was added to the PCR.


1997), and it has been suggested that this MDR system

could be a major determinant for the intrinsic ef¯ux of anti-

biotics by wild-type P. aeruginosa strains (Li et al., 1995).

Here, we show that both mexC±mexD±oprJ and mexE±

mexF±oprN are also present in environmental P. aerugin-

osa isolates. The presence of MDR determinants able to

extrude antibiotics in all our environmental isolates indi-

cates that quinolones are fortuitous substrates of those

systems. Altogether, these data support the idea that

MDR pumps can be selected in the environment without

antibiotic-selective pressure; these pumps could evolve

towards a new function as antibiotic resistance determi-

nants in hospital environments through an `exaptation'

(Brosius and Gould, 1992) process.

When looking for further evidence for the virulence

properties of P. aeruginosa environmental isolates, we

found that all of them produced proteases and could

invade epithelial cells in a cell culture model system.

Most of them were also haemolytic. These characteristics

are typical of virulent strains; however, they were also pre-

sent in the environmental strains analysed. PCR analysis

of genes relevant in virulence also demonstrated that all

the isolates carried the genes responsible for quorum sen-

sing, a major regulatory system in the virulence gene net-

work (van Delden and Iglewski, 1998), and genes from the

type III secretion system. Type III secretion systems are

involved in the pathogenic properties of several bacterial

pathogens (Hueck, 1998). In the case of P. aeruginosa,

this system is used for the translocation of exoenzymes

encoded by the ExoS regulon, contributing to the cytotoxic

properties of some bacterial isolates (Frank, 1997). The

ExoS regulon has been found in all P. aeruginosa clinical

isolates tested (Frank, 1997), irrespective of whether

they were invasive or cytotoxic. In fact, cytotoxic strains

of P. aeruginosa are inherently capable of invasion

(Fleiszig et al., 1997a; Evans et al., 1998), and muta-

tions in different genes of the ExoS regulon abolish cyto-

toxicity and increase invasion (Fleiszig et al., 1997b;

Evans et al., 1998; Hauser et al., 1998). Our results

show that environmental P. aeruginosa strains are also

capable of invading cells and possess type III secretion

genes.

The idea that pathogenic determinants have been

acquired by specialized pathogens in the form of

pathogenicity islands has strong experimental support.

Nonetheless, two important questions remain to be

answered: (i) what is the origin of these pathogenicity

islands; and (ii) does transformation of a non-pathogen

into a pathogen necessarily require the horizontal trans-

fer of virulence genes in all cases? In the case of non-

specialized opportunistic pathogens, it seems that viru-

lence determinants can also appear through selective

pressures unrelated to the use of antibiotics or to the

human host±pathogen interaction. In fact, the same

virulence factors account for the pathogenic properties

of plant and human isolates of P. aeruginosa (Rahme

et al., 1995), and the virulence properties of P. aerugin-

osa have been analysed successfully using the nema-

tode Caenorhabditis elegans as a suitable model

(Mahajan-Miklos et al., 1999). Whether the pathogenicity

islands present in specialized pathogens could originate

in environmental microorganisms needing those determi-

nants to survive in soil or plants is a question that has

not been addressed. However, our results suggest that

the presence of antibiotic resistance determinants and

virulence factors can be selected in P. aeruginosa by

the environment, without the need for the human host±

pathogen interaction habitat that occurs in hospitals.

This suggests that clinical and non-clinical strains are

not different branches of P. aeruginosa that have

evolved to occupy different ecological niches. If

this hypothesis is true, the converse should also occur:

clinical P. aeruginosa isolates might present properties

important for their survival in soil and water ecosystems,

but unnecessary to colonize humans or animals. To test

this hypothesis, we have analysed whether several clin-

ical P. aeruginosa isolates obtained from cystic ®brosis

patients and from bacteraemias can grow using alkanes

(major components of crude oil) as the sole carbon and

energy source. Our results show that all but one of the

clinical isolates tested, including strain PAO1, can miner-

alize alkanes and most probably produce biosurfactants.

Alkane metabolism is a common feature in soil bacteria

and starts by oxidation of a terminal methyl group by an

alkane hydroxylase enzyme composed of three subunits:

a hydroxylase, a rubredoxin and a rubredoxin reductase

(van Beilen et al., 1994). Computer analysis of the avail-

able sequence of P. aeruginosa PAO1 (Pseudomonas

genome project; http://www.pseudomonas.com) shows

that this strain contains at least two unlinked genes cod-

ing for alkane hydroxylases, as well as genes coding for

a rubredoxin and a rubredoxin reductase that are also

unlinked to the former ones (our own unpublished obser-

vations). In addition, genes for the synthesis of rhamno-

lipid biosurfactants have been detected in P. aeruginosa

PAO1 (Campos-GarcõÂa et al., 1998). These observa-

tions further reinforce the idea that clinical P. aeruginosa

strains are not specialized pathogens but, rather, envir-

onmental strains able to infect immunocompromised

patients with the traits acquired to survive in natural eco-

systems.

A ®nal observation concerns the use of P. aeruginosa in

bioremediation. Some of the strains we have character-

ized were isolated in the course of bioremediation projects

and have been shown to present relevant virulence deter-

minants. Careful protocols must be implemented to check

the health risks associated with the use of these bacteria

in non-con®ned environments.



Experimental procedures

Bacterial strains and growth conditions

The origin of the P. aeruginosa isolates analysed in this workis shown in Table 1. Clinical isolates were obtained from Hos-pital RamoÂn y Cajal (Madrid). E. coli HB101 and TG1 havebeen described previously (Sambrook et al., 1989). Bacteriawere grown at 378C in Luria±Bertani (LB) medium (Atlas,1993) or in minimal salts M9 medium (Sambrook et al.,1989) supplemented with trace elements (Bauchop and Eld-sen, 1960) and hexadecane as a carbon source. Minimal inhi-bitory concentrations (MICs) of antibiotics were determined inMueller±Hinton (MH) medium (Atlas, 1993) by the E-testmethod (AB Biodisk).

Measurement of quinolone accumulation

Intracellular accumulation of nor¯oxacin, cipro¯oxacin ando¯oxacin was assayed by a ¯uorometric method describedpreviously (Chapman and Georgopapadekou, 1989). P. aeru-ginosa isolates were grown in LB until mid-exponential phase(OD600 of 0.4 ±0.5). Cells were harvested at 48C and washedwith 50 mM sodium phosphate buffer (pH 7.2). The cellsuspension was concentrated eightfold in 50 mM sodiumphosphate (pH 7.2), 1 mM MgSO4 and 0.2% glucose and incu-bated at 378C for 10 min. The suspension was aliquoted and,after the addition of each quinolone to a ®nal concentration of10 mg mlÿ1, samples were incubated for another 10 min.For each quinolone, the cell suspension was divided intotwo equal aliquots, and carbonyl cyanide m-chlorophenyl-hydrazone (CCCP, 100 mM ®nal concentration; Sigma) wasadded to one aliquot and incubated for another 10 min underthe same conditions. Triplicate samples (0.5 ml) were thenwithdrawn and added to 1 ml of ice-cold 50 mM sodium phos-phate (pH 7.2) buffer. Cells were harvested at 48C andwashed once in the same buffer. The pellet was resuspendedin 1 ml of 0.1 M glycine, pH 3.0, and incubated for 1 h at roomtemperature to break the cells. Samples were centrifuged at7000 ´ g for 5 min. Supernatants were recovered, and theamount of quinolone was measured in a ¯uorescence spec-trophotometer. The excitation and the emission slit widthswere both 4 nm. The amount of quinolone accumulated wasdetermined by comparing the ¯uorescence of the sampleswith samples containing known amounts of quinolones.Excitation/emission lights to determine nor¯oxacin, cipro-¯oxacin and o¯oxacin were 281/440 nm, 292/496 nm and275/448 nm respectively. Protein concentration was deter-mined by the BCA protein assay (Pierce) using bovineserum albumin (BSA) as standard. Speci®c quinolone accu-mulation is referred to as the amount of protein in eachsample.

DNA manipulation

P. aeruginosa genomic DNAs were prepared using standardmethods (Bagdasarian and Bagdasarian, 1994). The threemultidrug ef¯ux operons analysed, the rhlI and rhlR genes(involved in quorum sensing) and gene pscJ (which formspart of the type III secretion system) were detected by PCR.Primers to detect the mexA±mexB±oprM genes were

oprM1 (58-CTGAACGTCGAGGCCTTCC-38) and oprM2 (58-CTGGATCTTCGCGTAGTCC-38) (Bianco et al., 1997). Todetect mexC±mexD±oprJ, the primers used were oprJ1 (58-GCGTGCTGTTCGTACCTA-38) and oprJ2 (58-TACTGTTG-CAGGGCTGCG-38). For genes mexE±mexF±oprN, primersoprN1 (58-GCTGACGCCGGTGTTCTA-38) and oprN2 (58-CGTCGCGAGTTGCTCGG-38) were used. Primers used todetect the rhlI gene were rhld (58-GCTGGGACGTGGTCTCCA-38) and rhlIr (58-GGAGGATCACGCCGTTGC-38). Pri-mers for rhlR were rhlRd (58-CTGTGGTGGGACGGTTTG-38) and rhlRr (58-CATGCAACGCAGCCACAG-38). Primersused to detect pscJ were pscJd (58-CATCGAAGAGCGCGCGCG-38) and pscJr (58-GCTGATGCGGTCGTAGCT-38).Reaction mixtures (50 ml) contained 0.2 mM each deoxynu-cleotide, 0.5 mM each primer, 1.5 mM MgCl2, 10% (v/v)DMSO, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 100 ng of geno-mic DNA and 2.5 U of Taq DNA polymerase. The mixtureswere heated for 90 s at 948C followed by 35 cycles of 30 s at948C, 60 s at 658C for oprM1/M2, at 648C for oprJ1/J2 andoprN1/N2, and 608C for the other primers, a 90 s extensionstep at 728C and, ®nally, a 10 min extension at 728C beforethe end of the reaction (Bianco et al., 1997). PCR productswere analysed on 1.0% (w/v) agarose gels and puri®edusing the Prep-A-Gene DNA puri®cation kit (Bio-Rad).Where indicated, puri®ed PCR products were sequencedusing primers oprM1, oprJ1 and oprN1.

Detection of proteolytic and haemolytic activities

Proteolytic activity was analysed on milk agar plates (Atlas,1993). Haemolytic activity was analysed on Columbia agarplates containing 5% defribrinated sheep blood (Oxoid).Bacteria were grown in LB broth at 378C to late exponen-tial phase, and 5 ml of each bacterial suspension waspoured on top of the plates. After 48 h of incubation at378C, haemolytic or proteolytic activities were detectedby the formation of clear halos surrounding the bacterialcolonies on the corresponding plates. For some of the iso-lates, the incubation was maintained for a longer time, asdescribed in Table 3.

Bacterial infection of epithelial cells

Tissue culture reagents were obtained from Gibco. Cells ofstrain I Madin Darby canine kidney (MDCK) were grown asdescribed previously (Finlay et al., 1989) in Eagle minimalessential medium (MEM) supplemented with 10% fetal bovineserum (FBS) obtained from Biowhittaker. MDCK cells wereseeded in 24-well tissue culture plates and grown overnightto 80% con¯uence. The next day, epithelial cells were washedwith PBS and incubated further with fresh MEM/10% FBS. Abacterial suspension [5 ml (around 3 ´ 106 cells)] from an over-night culture was added to each well. After 2 h, epithelial cellswere washed three times with PBS. MEM/10% FBS contain-ing gentamicin (100 mg mlÿ1) was then added to kill theremaining extracellular bacteria. Viable intracellular bacteriawere determined by lysing the infected cell monolayers 2 hlater with PBS containing Triton X-100 and seeding the bac-terial suspension in LB plates. Triplicates of the assayswere performed in all cases.



Acknowledgements

We are grateful to L. Yuste and E. Campanario for excellenttechnical assistance, and to Dr Rafael CantoÂn from HospitalRamoÂn y Cajal for the kind gift of clinical P. aeruginosaisolates. This research was aided by grants BIO97-0645-C02±01 from CICYT and 08.2/0022/1998 from CAM. A.Alonso is a recipient of a fellowship from Gobierno Vasco.

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