Environmental selection of antibiotic resistance genes : Minireview

Environmental Microbiology (2001) 3(1), 1±9

Minireview

Environmental selection of antibiotic resistance genes

Ana Alonso, Patricia SaÂnchez and JoseÂ L. MartõÂnez*

Departamento de BiotecnologõÂa Microbiana, Centro

Nacional de BiotecnologõÂa, CSIC, Campus Universidad

AutoÂnoma de Madrid, Cantoblanco, 28049-Madrid, Spain.

Introduction

Acquisition and further spread of antibiotic resistance

determinants among virulent bacterial populations is the

most relevant problem for the treatment of infectious

diseases. Although mutations in antibiotic target genes

(Martinez and Baquero, 2000) were supposed to be

the primary cause of antibiotic resistance in the early

antibiotic era, it soon became evident that acquisition

of antibiotic resistance determinants by horizontal gene

transfer has a major role on the development and

spread of antibiotic resistance among pathogenic bacteria

(Davies, 1994; 1997). What is the origin of these genes?

The analysis of bacterial isolates from the preantibiotic era

demonstrated that the incompatibility groups and the

amount of plasmids carried by pathogenic bacteria were

essentially the same that can be found today. However,

the preantibiotic plasmids did not carry antibiotic resis-

tance genes, so it has been assumed that the acquisition

and further dissemination among pathogenic bacterial

populations of antibiotic resistance is the consequence of

strong antibiotic selective pressure as a result of antibiotic

therapy (Datta and Hughes, 1983; Hughes and Datta,

1983). If these genes were not present in the pathogenic

bacteria, they must have originated in the environmental

bacteria and the most obvious microorganisms in which

they might have a functional role are antibiotic producers.

In fact, it is widely accepted that antibiotic resistance

determinants originated in the antibiotic-producing organ-

isms (Benveniste and Davies, 1973; Webb and Davies,

1993), in which they play an obvious protective role.

Although the origin of some antibiotic resistance genes

from the antibiotic producers is clear, this is not the case

for some other determinants. For instance, it is difficult to

accept that chromosomal beta-lactamases (Bush et al.,

1995), multidrug resistance (MDR) determinants (Paulsen

et al., 1996; Nikaido, 1998) or some aminoglycoside-

inactivating enzymes (Ainsa et al., 1997; Lambert et al.,

1999; Macinga and Rather, 1999) that are present in all

isolates of a given non-antibiotic producer bacterial species

have been selected by antibiotic selective pressure. Another

concern resides in the environmental selection of bacteria

intrinsically resistant to antibiotics. Some of these bacterial

species are relevant opportunistic pathogens and, apart

from their environmental origin, they are refractory to

treatment by several antibiotics (Quinn, 1998). As those

antibiotics are not always present in the environmental

habitat of these bacterial species, we might assume that the

driving force selecting intrinsic resistance prior to infection

must differ from antibiotic selective pressure.

Alternatively, even for those genes with a clear primary

role in antibiotic resistance, selection without antibiotic

selective pressure might occur if they are present in a

replicon that also carries some other `selectable elements'

(see below). Selection of antibiotic resistance determin-

ants might then occur in the environment by means of

chemical or heavy metal pollution, or because the

presence of the determinant accompanying the antibiotic

resistance gene gives an ecological advantage to the

bacteria for colonizing their environmental habitat (Fig. 1).

Thus, antibiotic resistance genes have an environmental

origin, sometimes as an antibiotic protective mechanism and

sometimes with a different function. The environment

also has a role in their selection that is not always the

consequence of antibiotic selective pressure. We will

discuss these concepts throughout this review.

The physiological role of antibiotic resistance genes

As previously stated, several antibiotic resistance deter-

minants probably originated in antibiotic producers as

bona fide antibiotic resistance genes. This is the case with

the tetracycline resistance determinants otrA and otrB

(Pang et al., 1994) that are present in mycobacteria and

also in the tetracycline-producing bacterium Strepto-

myces rimosus. However, for most antibiotic resistance

genes described to date in pathogenic bacteria, an

identical counterpart has not been found in antibiotic

producers. It can be argued that less than 1% of

environmental species have been isolated to date, so

that the antibiotic producers carrying these genes will be

Q 2001 Blackwell Science Ltd

Received 12 September, 2000; revised 3 November, 2000; accepted9 November, 2000. *For correspondence. E-mail [email protected];Tel. (134) 91 5854551; Fax (134) 91 5854506.

found sooner or later. Nevertheless, increasing evidence

supports the notion that some antibiotic resistance genes

might have a physiological role different to antibiotic

resistance, even in the case of antibiotic producers

(Piepesberg et al., 1988). In the case of non-antibiotic

producers, if all isolates of a bacterial species carry a

number of identical antibiotic resistance genes, it might be

supposed that these determinants should have a role

different from antibiotic resistance because non-producer

species are not under constant antibiotic selective

pressure in the environment. As previously stated, the

most conspicuous examples of those genes are chromo-

somal beta-lactamases, some chromosomally encoded

aminoglycoside-modifying enzymes and MDR efflux

pumps.

Chromosomal AmpC beta-lactamases contribute to

resistance to beta-lactam antibiotics in Enterobacteria-

ceae. However, the fact that they share a common

ancestor and are present in all members of each bacterial

species indicates that they were acquired by Enterobac-

teriaceae before the evolutionary differentiation of this

genus into species, hundreds of thousands of years

before the discovery of antibiotics. Beta-lactamases

have evolved from transpeptidases involved in cell wall

synthesis (Adachi et al., 1992; Knox et al., 1996).

Although a role in such process has not been demon-

strated, chromosomal beta-lactamases could be involved

in peptidoglycan metabolism or be remnant molecules

without a clear role in bacterial metabolism. However,

chromosomal beta-lactamases are probably house-keep-

ing genes and their activity against antibiotics is a side-

effect of their actual (unknown) physiological activity.

Some aminoglycoside-modifying enzymes might have

evolved from sugar kinases and acyltransferases (Udou

et al., 1989; Macinga and Rather, 1999). In the case of

chromosomal aminoglycoside-modifying enzymes present

in the isolates of given species, such as Providencia stuartii

(Macinga and Rather, 1999), Stenotrophomonas maltophilia

(Lambert et al., 1999), Serratia (Shaw et al., 1992) or

Mycobacteria (Ainsa et al., 1997), a metabolic role has been

suggested. A structural role has been described for the

chromosomal acetyltransferase (AAC(2 0)-Ia) from P. stuartii.

Apart from acetylating aminoglycosides, the enzyme has at

least one physiological function, which is the acetylation of

peptidoglycan (Payie et al., 1995). As P. stuartii isolates are

not always in contact with aminoglycosides, the probable

physiological role for this enzyme is cell wall metabolism.

A similar situation must happen with MDR pumps.

These determinants are ubiquitously found in all bacterial

species (Nikaido, 1998) and also in eukaryotic organisms.

The genome of a single bacterial isolate might contain

more than 20 putative MDR pumps (Stover et al., 2000).

Although they contribute to the intrinsic resistance to

antibiotics (Nikaido, 1994), they should also have different

functional roles, such as protection against toxic com-

pounds (Alekshun and Levy, 1999) or the involvement

in cell/environment signalling pathways. For example,

Escherichia coli presents MDR determinants involved in

the extrusion of bile salts (Thanassi et al., 1997) and

bile salts are part of the natural environment of E. coli,

whereas antibiotics have only appeared in this environ-

ment during the last 50 years. It is clear, therefore, that

these MDR determinants have probably been selected by

the presence of bile salts, even if they now contribute to

intrinsic antibiotic resistance in E. coli. The most remark-

able example of selection of intrinsically resistant micro-

organisms by the environment is that of Gram-negative

opportunistic pathogens with an environmental origin

(e.g., Pseudomonas aeruginosa, Burkholderia cepacia

and Stenotrophomonas maltophilia). Several of these

Fig. 1. Selection of clinically relevantantibiotic-resistant bacteria. Pathogenicbacteria can be resistant to antibiotics eitherbecause they contain the determinants forresistance in their genome or because theyacquire antibiotic resistance genes from anexogenous source. The most straightforwardselection is antibiotic treatment. Antibiotics willselect intrinsically resistant bacterial species,bacteria that have acquired antibioticresistance genes by horizontal gene transferon antibiotic-resistant mutants. However, non-antibiotic compounds can also select antibioticresistance bacteria. Thus, biocides anddetergents can select resistant strains as theconsequence of the expression of MDRdeterminants, or antibiotic resistance genescan present in replicons that contain otherselectable markers, such as heavy metalresistance or production of siderophores.

2 A. Alonso, P. SaÂnchez and J. L. MartõÂnez

Q 2001 Blackwell Science Ltd, Environmental Microbiology, 3, 1±9

opportunistic pathogens have their habitat in the soil, in

close contact with plants. Soil is an environment that

contain several potentially toxic aromatic compounds,

derived from degradation processes (VicunÄa, 2000) and

plant exudates (Canto-Canche and Loyola-Vargas, 1999).

It has been demonstrated that MDR determinants can

extrude an ample range of substances that include

solvents, detergents and aromatic compounds (Isken

and de Bont, 1996; Li et al., 1998; Segura et al., 1999).

Therefore, at least some of these MDR efflux pumps may

have been selected to avoid the effect of toxic compounds

present in their natural environment. A role in quorum

sensing has been suggested for others (Evan et al., 1998;

Pearson et al., 1999). It has been recently described that

P. aeruginosa synthesizes a natural 4-quinolone involved

in quorum sensing and that the MDR system MexABOprM

is capable of extruding this molecule (Pesci et al., 1999). It

has also been shown that environmental P. aeruginosa

strains isolated before quinolones (a family of synthetic

antibiotics) were discovered are capable of extruding

these drugs (Alonso et al., 1999). It is possible that the

capability for extruding quinolones is a side-effect of the

physiological role of this pump, which is the extrusion of

this quorum-sensing signal molecule.

We have seen that several antibiotic resistance

determinants have a primary physiological role other

than antibiotic resistance. In fact, they have been selected

for metabolic, biosynthetic or signalling purposes. How-

ever, once antibiotic selective pressure is applied,

mutants that overproduce these determinants can be

selected in this way, reinforcing their adaptive role as

antibiotic resistance determinants. Also, the genes can

enter an independent replicon and further disseminate

among pathogenic bacteria (Davies, 1994). Once these

genes enter heterologous hosts outside their physiologi-

cal context, they are not selected further for their primary

physiological role and their function becomes antibiotic

resistance alone (Fig. 2). This is a good example of the

evolutionary mechanism that Brosius and Gould (1992)

named `exaptation'.

For example, chromosomal AmpC beta-lactamases are

now present in plasmids (Coudron et al., 2000) that

disseminate among bacterial populations contributing to

the acquisition of an antibiotic resistance phenotype

by previously susceptible bacteria. What is the origin of

some other plasmid-encoded antibiotic resistance deter-

minants? Some of them are probably native antibiotic

resistance determinants and others may be metabolic

genes. For example, we cannot know whether prototypic

plasmidic beta-lactamases, such as the TEM family (Bush

et al., 1995), have originated in the beta-lactam producers

as antibiotic resistance genes or are enzymes that were

first involved in cell wall metabolism in an unknown

environmental bacterial species.

Selection of antibiotic resistance without antibiotic

selective pressure

We have seen that intrinsic antibiotic resistance might

have been selected in the course of bacterial evolution,

without antibiotic selective pressure, for covering func-

tions other than antibiotic resistance. Can this also

happen for acquired antibiotic resistance?. The expres-

sion of MDR determinants is frequently downregulated

under standard laboratory conditions. However, de-

repressed mutants can easily be obtained in vitro and

are frequently found clinical isolates (Nikaido, 1998) that

contribute to acquired antibiotic resistance. As MDR

determinants are capable of conferring simultaneous

resistance to toxic compounds belonging to several

different families, detergents and antiseptics included, an

obvious question is whether selection with non-antibiotic

compounds might select for antibiotic resistance.

For example, an efflux pump has been described in

Listeria monocytogenes that can extrude both antibiotics

and heavy metals (Mata et al., 2000). As L. monocyto-

genes has an environmental habitat, its growth in heavy

metal-contaminated soils might also select for antibiotic

resistance. It has also been demonstrated that biocides,

organic solvents and detergents are capable of selecting

mutants with increased expression of MDR determinants.

Triclosan and pine oil might select low-level antibiotic-

resistant E. coli strains as the consequence of over-

production of chromosomally encoded efflux systems

(Moken et al., 1997). It is true that the selective

concentrations of both biocides are low compared with

those used in clinical practice. However, low concentra-

tions of these biocides can be achieved both in clinical

settings and in the home, and might then select low-level

antibiotic-resistant bacteria.

Antibiotic selective pressure is not required for the

selection of antibiotic resistance genes carried by repli-

cons that contain not only antibiotic resistance determi-

nants but also any other selectable marker (see Table 1).

One of the most conspicuous examples of this situation is

the linkage between antibiotic resistance and heavy metal

resistance genes, which is frequently encountered in

environmental bacterial isolates (Davison, 1999). The co-

existence of both types of determinants in the same

genetic element allows antibiotic resistance to be selected

upon heavy metal selective pressure in contaminated

environments. This linkage might explain the selection of

determinants that would otherwise not be selected. For

example, an erythromycin resistance determinant with a

Gram-positive origin has been found in the genome of S.

maltophilia (Alonso et al., 2000). S. maltophilia, like all

Gram-negative bacteria, is intrinsically resistant to macro-

lides, so that erythromycin resistance genes are not

selectable determinants for S. maltophilia. The reason for

Role of the environment on antibiotic resistance 3


the presence of the erythromycin resistance determinant

in the genome of S. maltophilia must, therefore, rely on

accompanying selectable markers. In fact, this erythro-

mycin resistance determinant is flanked by a cadmium

resistance efflux pump, so that selection might have

occurred as a result of heavy metal antibiotic selective

pressure in the environment, prior to infection. Another

remarkable example of clinically relevant heavy metal/

antibiotic resistance co-selection is the linkage between

silver and antibiotic resistance in the same replicon. Silver

has historically been used as a biocide in the treatment of

burns (Klasen, 2000). Burn wounds are easily colonized/

infected by opportunistic pathogens (Pruitt et al., 1998).

Treatment with silver ions might then co-select, in the

case of a genetic linkage between silver and antibiotic

resistance, those bacteria carrying these type of deter-

minants, which are then more resistant to antibiotics

(McHugh et al., 1975). It has been suggested that

mercury, which is present in the amalgams used in

odontology, might select for microorganisms with

enhanced antibiotic resistance in the oral cavity and the

intestine (Summers et al., 1993; Edlund et al., 1996).

However, some recently published work casts doubts on

this hypothesis (Osterblad et al., 1995; Leistevuo et al.,

2000). More work is needed to clearly establish the actual

role of dental fillings in antibiotic resistance.

Selection pressure might be based not only on the

toxicity of the potential selector but on stringent growing

conditions. In this case, the metabolic capabilities

required for growing under such conditions will be good

selective markers. The linkage between aerobactin and

antibiotic resistance genes in different plasmids has been

described (Gonzalo et al., 1988). Aerobactin is a side-

rophore that allows bacteria to grow in environments

where iron is scarcely available (de Lorenzo and Martinez,

1988), so that its presence in antibiotic resistance

plasmids might contribute to the dissemination of anti-

biotic resistance (Delgado-Iribarren et al., 1987). This

Fig. 2. Environmental origin of antibiotic resistance genes. Antibiotic resistance genes are present in environmental antibiotic producer andnon-producer microorganisms. Some of them serve in the original organism to resist the action of an antibiotic, being the most conspicuousexample of the antibiotic resistance genes from antibiotic producers. They have thus been selected by antibiotic pressure and are bona fideantibiotic resistance genes. Other genes can be involved in other cellular processes such as antibiotic biosynthesis, signalling, metabolism ordetoxification, and were selected in the original organisms to cover these functions in the absence, therefore, of antibiotic selective pressure.However, once they are introduced into a heterologous host and selected by means of antibiotic selective pressure, they behave as antibioticresistance genes. Selection of a gene to cover a different function from that it was previously selected for is known as an exaptationmechanism of natural selection.

Table 1. Examples of selectable determinants carried by antibiotic resistance plasmids.

Determinant Conditions for the selection Reference

Aerobactin Low iron Delgado-Iribarren et al. (1987)Heavy metal resistance Heavy metal contamination Davison (1999)Adhesin Adhesion to surfaces in liquid environments Franklin et al. (1981)Cytolysin Infective conditions (environmental interactions with amoebas?) Barja et al. (1990)Microcin Competition with other bacteria Martinez and Perez-Diaz (1990)Colicin Competition with other bacteria Franklin et al. (1981)Resistance to disinfectants Decontamination of clinical settings Russell (2000)



situation may also happen with antibiotic resistance

plasmids containing other determinants of ecological

value, such as adhesins or citolysins (Franklin et al.,

1981).

A final example resides in the selection of the qac

genes present in staphylococci plasmids. qacB specifies

resistance to quaternary amines, acridine diamidines and

ethidium bromide, whereas qacA additionally encodes

resistance to chlorhexidine and is frequently carried on

penicillinase plasmids (Rouch et al., 1990). The qacB

gene has been found in plasmids isolated 50 years ago

(Paulsen et al., 1998), whereas most isolates from 1980

contain qacA (Leelaporn et al., 1994). In the 1980s,

chlorhexidine was introduced in hospitals, so it has been

suggested that a replacement of one gene by the other

occurred in staphylococci populations, as the conse-

quence of the introduction of a biocide (Russell, 2000). As

those plasmids also carry beta-lactamase genes, the

introduction of chlorhexidine might have contributed to

the selection of beta-lactam-resistant staphylococci as

the consequence of beta-lactamase production in hospital

settings. Interestingly, qac genes are also present in

integrons of Gram-negative bacteria carrying multiple

antibiotic resistance cassettes (Paulsen et al., 1993; Bass

et al., 1999). This may contribute to the successful

selection of such determinants in the presence of

biocides, without antibiotic selective pressure.

Human intervention in the environment and antibiotic

resistance

Chemical pollution

Industrial activities, minery and intensive farming are

causing dramatic changes in natural ecosystems. Among

the novel selective pressures that face environmental

bacterial populations from the industrial revolution, dis-

charges of heavy metals, xenobiotic compounds, anti-

biotics and organic solvents can have a remarkable role

on the environmental selection of antibiotic resistance

genes. Also, intensive farming requires the utilization of

high amounts of probiotics and antibiotics (as far as 50%

of total antibiotic consumption in developed countries) and

contributes to the selection of antibiotic resistance genes

in bacteria that colonize animals (Piddock, 1996; Witte,

1998). A good example of such a problem is the utilization

of the glycopeptide antibiotic avoparcin as a growth

promoter. In those countries in which the antibiotic has

been extensively used, vancomycin-resistant enterococci

are frequently encountered, not only in animals, but also

in the human population (Van den Boggard and Stobber-

ingh, 2000). Alternatively, the ban of avoparcin in animal

feeding has curbed the development of resistance in

European Union countries (Bager et al., 2000), which

shows the relevant role that the utilization of antibiotics for

animal feeding may have in the selection of antibiotic-

resistant bacteria in the clinical setting.

We have to mention here that synthetic antibiotics

are xenobiotic compounds that can also be considered

as important pollutants. For example, quinolones are

extremely stable in the environment (Halling-Sorensen

et al., 1998), so their presence might produce dramatic

effects on bacterial populations in natural habitats, the

most prominent being the selection of antibiotic-resistant

bacteria. Of note, quinolones are the most used synthetic

antibiotics in aquaculture (Grave et al., 1996) and

selection of quinolone resistance in indigenous river

water bacterial populations as the consequence of

contamination by run-off waters containing quinolones

has been suggested (GonÄ i-Urriza et al., 2000). Never-

theless, in spite of the constant release of these

xenobiotic non-degradable compounds in the environ-

ment, the effect of quinolones on the environmental

bacterial populations has not been properly analysed.

The effects of industrial pollution on environmental

bacterial communities have not been extensively studied.

Most published work relies on the analysis of heavy

metal-contaminated environments. Release of toxic metal

species is the most relevant pollution problem since the

industrial revolution (Ayres, 1992), mainly because heavy

metals cannot be degraded and, therefore, they remain in

the environment. In this situation, heavy metal-contami-

nated environments maintain the selective pressure on

indigenous bacterial populations for long periods of time.

Natural ecosystems containing high concentrations of

heavy metals are also frequent. Not surprisingly, heavy

metal resistance genes are easily found in environmental

bacteria (Silver and Phung, 1996). It has been documen-

ted that heavy metal-contaminated environments also

contain a higher percentage of antibiotic-resistant strains

than non-contaminated ones, and bacteria isolated from

contaminated soils contain more plasmids than those

isolated from non-contaminated places (Rasmussen and

Sorensen, 1998). Finally, under mercury stress, the gene-

mobilizing capacity of soil bacterial populations increases.

As heavy metal and antibiotic resistance are frequently

linked in the same plasmid (see before), increased

mobilization under metal selective conditions might also

increase the mobilization of antibiotic resistance genes

among environmental bacterial populations.

If contaminated environments might contribute to the

selection of antibiotic-resistant bacteria, cleaning of these

habitats may contribute to the restoration of an antibiotic-

susceptible population. If this was the case, bioremedia-

tion of contaminated environments might be of help in

reducing environmentally selected antibiotic resistance.

Acquisition of an antibiotic-resistant phenotype reduces

the fitness of bacteria (Andersson and Levin, 1999) so



that replacement of resistant populations by susceptible

ones can occur in the absence of selection. Nevertheless,

antibiotic-resistant bacteria accumulate mutations that

compensate for the effect of antibiotic resistance on

fitness (Andersson and Levin, 1999), making the acqui-

sition of antibiotic resistance a non-return evolution. It

is thus unclear whether the cleaning of contaminated

environments could restore the antibiotic-susceptible

populations. The analysis of antibiotic-resistant bacteria

in contaminated, non-contaminated and cleaned environ-

ments is thus an important topic that should be addressed

in the near future.

Introduction of organisms in the environment

In recent years, society is increasingly concerned with the

risks of dissemination of antibiotic resistance genes used

for the construction of genetically modified organisms.

For these reasons, safer systems that avoid the spread

of genes (Diaz et al., 1994) and that are based in non-

antibiotic markers (Herrero et al., 1990) or even in

markers that are eliminated after the organism has been

modified (Panke et al., 1998; Zubko et al., 2000) have

been implemented. Even for genetically modified organ-

isms that carry antibiotic resistance genes, we do not

believe that the potential release of the antibiotic

resistance genes currently used for the development of

genetically modified organisms constitutes a significant

risk for the dissemination of antibiotic resistance genes of

clinical importance for two reasons: (i) Current studies

indicate that the probability of dissemination of those

genes is very low; and (ii) the genes currently used in

genetic engineering, such as the beta-lactamase TEM1,

are already (unfortunately) widely disseminated among

pathogenic and commensal bacteria (Ferber, 1999).

Another concern could be the release of antibiotic

resistance genes from non-modified organisms used in

the field. It has been shown that the biopesticide

Paenibacillus popilliae has a vancomycin resistance gene

cluster homologous to the enterococcal vanA vancomycin

resistance gene cluster (Patel et al., 2000) and to

vancomycin resistance genes present in glycopeptide-

producing actinomycetes (Marshall et al., 1998). Biopestici-

dal powders containing spores of P. popilliae have been

used for more than 50 years in the United States for

suppression of Japanese beetle populations (Patel et al.,

2000). An identical counterpart of the P. popilliae vanA

gene in pathogenic bacteria has not been found, so it

seems that the use of P. popilliae biopesticidal prepara-

tions in agricultural practice have not had (at least at

present) an impact on bacterial resistance in the clinical

setting. However, this does not mean that it will not have

an impact in the near future and this illustrates the need

to analyse the effect of the introduction not only of

genetically modified microorganisms, but also of `natural'

bacterial populations in the field.

The risks for the utilization of intrinsically resistant

microorganisms, either genetically modified or not, for

bioremediation or biotransformation processes have also

been discussed (Holmes et al., 1998; LiPuma and

Mahenthiralingam, 1999). Most bacteria currently used

in bioremediation/biotransformation belong to the Pseu-

domonadacea family. Bacterial species belonging to this

family are intrinsically resistant to antibiotics and are

increasingly isolated from nosocomial infections (Quinn,

1998). Could the release of these intrinsically resistant

microorganisms increase the probability of infections

owing to antibiotic-resistant bacteria?. The archetype of

this situation is Burkholderia cepacia. This bacterial

species is being used both for bioremediation and as a

promoter of crop growth. B. cepacia is also a relevant

antibiotic-resistant opportunistic pathogen. Although the

probability of infection by B. cepacia introduced in the field

has not been analysed, we believe that it is low because:

(i) B. cepacia does not produce infection in the commu-

nity, but only in immunocompromised, hospitalized or

cystic fibrosis patients, so that the number of people at

risk of infection by B. cepacia used for agriculture or

bioremediation should be low, and (ii) B. cepacia strains

are already present in the field, so it is unclear whether

introducing some naturally occurring microorganisms

might increase the probability of infection. Alternatively,

even if the probability of infection is low, it must be

evaluated because infection by B. cepacia may have fatal

consequences for previously debilitated patients, there-

fore, the risk might be high. It may be that the utilization of

huge amounts of B. cepacia in areas with crowded human

populations might increase the probability of raising the

number of infections among unhealthy populations

(hospitals, AIDS patients, famine situations etc.). As

stated by other authors (Holmes et al., 1998; LiPuma

and Mahenthiralingam, 1999), this possibility must be

carefully evaluated.

Acknowledgements

The authors wish to thank Fernando Rojo for useful criticism and

comments on draft versions of this manuscript. A. Alonso is a

recipient of a fellowship from Gobierno Vasco. P. SaÂnchez is a

recipient of a fellowship from Ministerio de EducacioÂn y Cultura.

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