Arbitrary primed PCR ¢ngerprinting and serotyping of clinical

Pseudomonas aeruginosa strains

Javier Hernaèndez

a;

*, Mar|èa A. Ferruès

a, Manuel Hernaèndez

a, Robert J. Owen

b

aDepartamento de Biotecnolog|èa, Universidad Politeècnica, Camino de Vera 14, 46022 Valencia, Spain

bNational Collection of Type Cultures, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK

Received 16 August 1996; revised 30 October 1996; accepted 31 October 1996

Abstract

Arbitrary primed PCR (AP-PCR) analysis was compared with serotyping as a means of high-resolution typing of

Pseudomonas aeruginosa. Seventy-four isolates from 3 different hospitals and 18 reference strains were studied. Serotyping

provided good index of discrimination, although eleven isolates could not be serotyped. Genomic DNA was amplified with a

single 10 nucleotide primer (sequence 5P-AGG GGT CTT G-3P). The strains were genetically diverse and 61 different AP-PCR

profiles of 2^7 bands between 0.3 and 2.4 kb were obtained. AP-PCR profiles were not consistently associated with serotypes,

but they clearly subtyped strains of the same serotype. Numerical analysis of AP-PCR patterns defined 7 groups at the 55%

similarity level, and identified predominant strains in each hospital. The results show that AP-PCR analysis provides a simple

and practical approach to typing P. aeruginosa that is more discriminatory than traditional serotyping scheme. We suggest that

maximum discrimination can be achieved by a combination of both methods.

Keywords: Pseudomonas aeruginosa ; DNA; Polymerase chain reaction; AP-PCR; Serotyping; Fingerprinting

1. Introduction

Pseudomonas aeruginosa is a Gram-negative bac-

terium widely distributed in nature and causing op-

portunistic infections in humans. The organism is an

important nosocomial pathogen of hospitalized pa-

tients, frequently associated with infections, such as

meningitis, septicemia, ocular and burn wound infec-

tions, otitis, urinary tract infections and cystic ¢bro-

sis (CF). Colonized patients can be an important

reservoir, and the organism is maintained in the hos-

pital environment by person-to-person transmission

and by its saprophytic survival and growth in a vari-

ety of hospital environments [1].

P. aeruginosa strains are identi¢ed on the basis of

phenotypic characteristics, but the nosocomial epide-

miology was hampered until recently by the impreci-

sion of most typing methods available. The most

commonly used epidemiological technique is serotyp-

ing, based on an agglutination reaction between bac-

terial lipopolysaccharide (LPS) and type-speci¢c rab-

bit antisera [2]. In 1983, an International Antigenic

Typing System (IATS) was developed [3], establish-

ing the existence of at least 17 major heat-stable so-

matic antigens, and 3 new antigens have been re-

cently added to the existing groups [4]. This

serotyping scheme characterizes most strains of

0928-8244 / 97 / $17.00 Copyright ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V.

PII S 0 9 2 8 - 8 2 4 4 ( 9 6 ) 0 0 1 0 3 - 4

FEMSIM 713 9-4-97

* Corresponding author. Tel. : +34 (6) 387 7423;

fax: +34 (6) 387 7429; e-mail: jhernand@upvnet.upv.es

FEMS Immunology and Medical Microbiology 17 (1997) 37^47

P. aeruginosa possessing smooth LPS, although

shows poor discrimination against rough LPS iso-

lates which are common in patients with cystic ¢bro-

sis [5,6]. Other conventional available systems based

on phenotypic markers, such as biotyping, pyocin-

typing, phage-typing, or antimicrobial susceptibility

may not give satisfactory answers to basic epidemio-

logical questions as they rely on the analysis of char-

acteristics that are not stably expressed [7,8].

In contrast, high-discriminative DNA-based meth-

ods, have been recently used to ¢ngerprint P. aeru-

ginosa isolates, particularly, analysis of DNA restric-

tion fragment length polymorphism of rRNA genes

(ribotyping) [9^14], restriction endonuclease analysis

(REA) [15], and macrorestriction of digested DNA

by pulsed-¢eld gel electrophoresis (PFGE) [16^18].

However, the use of those methods in the clinical

microbiology laboratory is restricted to specialized

centres because of the technical complexity and their

labour-intensive nature [19].

An alternative and simpler typing technique based

on PCR with a single short arbitrary primer (AP-

PCR) has been used successfully for the typing of

various medically important microorganisms [20] in-

cluding P. aeruginosa [17,21,22]. In AP-PCR analy-

sis, the genomic DNA is ampli¢ed using single oli-

gonucleotides of arbitrarily chosen sequences and

non-stringent annealing temperatures, resulting in a

series of PCR ampli¢cation products with di¡erent

sizes that provide a characteristic electrophoretic ¢n-

gerprint. Although some factors need to be standard-

ized to achieve high reproducibility, it is one of the

easiest typing methods, because the template DNA

need not be of high molecular weight, double-

stranded, or highly puri¢ed, and only ng quantities

of DNA are required [23,24].

A comparison of phenotypic and genotypic meth-

ods for typing clinical strains of P. aeruginosa has

been done by The International Pseudomonas aeru-

ginosa Typing Study Group [25], but none PCR-

based typing technique was investigated in that

study.

We report here the use of AP-PCR ¢ngerprinting

and serotyping to study reference strains of the dif-

ferent P. aeruginosa serotypes, and clinical isolates

from hospitalized patients in Spain. The aim was

to determine the genetic diversity among such bac-

teria in order to detect DNA polymorphisms which

can indicate if there were any common strain types

associated with P. aeruginosa infections in 3 hospi-

tals in the Valencia area, and to evaluate the discri-

minatory power of both typing methods for future

epidemiological studies.

2. Materials and methods

2.1. Bacterial strains and culture conditions

A total of 92 strains of P. aeruginosa were used in

this study. Eighteen reference strains, including the

type strain P. aeruginosa NCTC 10332T, representing

di¡erent serotypes were obtained from the NCTC

(National Collection of Type Cultures, London,

UK). Table 1 provides information about their strain

numbers, serotypes, and AP-PCR pro¢les. The re-

maining 74 strains were isolated in 1991 and 1992

from patients in 3 hospitals (referred to as A, B,

and C) in Valencia (Spain). Table 2 shows the num-

ber of isolates from each hospital and their typing

results. There was no clinical evidence to indicate the

isolates were implicated in any de¢ned hospital in-

fection outbreaks. The following type strains of al-

lied species were also included for comparative pur-

FEMSIM 713 9-4-97

Table 1

P. aeruginosa reference strains studied and typing results

Strain Serotype AP-PCR pro¢lea

NCTC 11440 O1 P1

NCTC 11441 O2 P2

NCTC 11442 O3 P3

NCTC 11443 O4 P4

NCTC 11444 O5 P5

NCTC 11446 O6 P6

NCTC 11445 O7 P7

NCTC 11447 O8 P1

NCTC 11448 O9 P8

NCTC 11449 O10 P9

NCTC 11450 O11 P10

NCTC 11451 O12 P11

NCTC 11452 O13 P12

NCTC 11453 O14 P13

NCTC 11663 O15 P7

NCTC 11455 O16 P2

NCTC 11456 O17 P14

NCTC 10332T

O6 P15

aAP-PCR types were de¢ned according to the results of the numer-

ical analysis.

J. Hernaèndez et al. / FEMS Immunology and Medical Microbiology 17 (1997) 37^4738

poses: P. £uorescens NCTC 10038

T, P. putida

NCTC 10936

T, and Burkholderia (Pseudomonas) ce-

pacia NCTC 10661

T.

All bacteria were cultivated at 37³C for 24 h on

Pseudomonas agar base (Oxoid CM559), with Pseu-

domonas C-N supplement (Oxoid SR102E) con-

taining cetrimide (200 mg/l) and sodium nalidixate

(15 mg/l). Strains were preserved at 370³C on glass

beads on nutrient broth No. 2 (Oxoid CM67) con-

taining 10% (v/v) glycerol.

2.2. Conventional bacteriological tests

The identity of all clinical isolates were con¢rmed

previously in a range of standard conventional bac-

teriological tests: gram stain, growth at 42³C, oxi-

dase production, DNA hydrolysis, pigment (pyocya-

nin and pyoverdin) production, haemolytic activity,

and the glucose oxidation-fermentation (O-F) test.

The isolates were identi¢ed by the API 20NE system

(bioMeérieux).

2.3. Serotyping

All strains were serotyped according to the scheme

proposed by the International Antigenic Typing Sys-

tem [3]. Qualitative slide agglutination of each strain

was done with autoclaved cells (120³C for 30 min)

and commercial antisera (Sano¢ Diagnostics Pasteur,

Madrid, Spain). All agglutinations were repeated 3

times to assess reproducibility.

Strains showing agglutination with standard saline

were coded as autoagglutinable. Strains reacting with

s 1 antiserum were referred as polyagglutinable.

Non-typeable strains were those which did not react

with any antiserum.

FEMSIM 713 9-4-97

Fig. 1. Arbitrarily primed polymerase chain reaction (AP-PCR) patterns of Pseudomonas aeruginosa strains representing all serotypes of

the scheme proposed by IATS. Genomic DNA was ampli¢ed using a 10-mer primer with sequence 5P-AGGGGTCTTG-3P and electro-

phoresed on a 1.2% agarose gel. Lanes 1^17 contain reference strains of serotypes O1-O17 (see Table 1 for NCTC strain numbers). Sizes

(in kb) of characteristic fragments are indicated on the right.

Table 2

Clinical strains studied and typing results

Source

aStrains (n) O serotypes

bAP-PCR pro¢les

c

Hospital A 13 1, 6^9, 11, PA 16^26

Hospital B 37 1^8, 10^12, NA, PA 1, 7^9, 19, 26^49

Hospital C 24 1, 2, 5^7, 10^13, 16, AA, PA, NA 1, 7, 15, 27, 28, 33, 49^61

aA, Hospital La Fe; B, Hospital General Universitario; C, Hospital Cl|ènico Universitario (Valencia, Spain).

bAA, autoagglutinable; NA, non-agglutinable; PA, polyagglutinable.

cAP-PCR types were de¢ned according to the results of the numerical analysis.

J. Hernaèndez et al. / FEMS Immunology and Medical Microbiology 17 (1997) 37^47 39

2.4. AP-PCR analysis

Bacterial cells were cultivated at 37³C for 24 h on

plate count agar (Oxoid) and suspended in 200 Wl of

ultrapure water. Each suspension was boiled for 10

min and centrifuged for 5 min. The concentration of

DNA in the supernatant was adjusted to 1.5 Wg/ml

(A260 =0.03). DNA ampli¢cation was performed

using 20 di¡erent primers (OPA 1^20, kit A; Operon

Technologies, Alameda, CA, USA) according to the

FEMSIM 713 9-4-97

Fig. 2. AP-PCR patterns of representative Pseudomonas aeruginosa strains from infected patients. Lanes 1^5 contain strains from hospital

A; lanes 6^11 contain strains from hospital B; lanes 12^17 contain strains from hospital C. Sizes (in kb) of characteristic fragments are

indicated on the right.

Table 3

Relation between serotypes and the origin of strains

Strains (n) from:

Serotypea

NCTC Hospital A Hospital B Hospital C Total

O1 1 3 5 4 13

O2 1 1 2 4

O3 1 3 4

O4 1 1 2

O5 1 1 2 4

O6 2 1 2 3 8

O7 1 1 3 1 6

O8 1 2 1 4

O9 1 1 2

O10 1 3 2 6

O11 1 4 8 3 16

O12 1 2 1 4

O13 1 1 2

O14 1 1

O15 1 1

O16 1 2 3

O17 1 1

AA 1 1

NA 2 1 3

PA 1 5 1 7

aAA, autoagglutinable; NA, non-agglutinable; PA, polyagglutinable.

J. Hernaèndez et al. / FEMS Immunology and Medical Microbiology 17 (1997) 37^4740

manufacturer's instructions.

A reaction volume of 100 Wl contained 2.5 U of

Taq polymerase (DynaZyme thermostable DNA

polymerase; Finnzymes, Finland), 10 mM Tris-

HCl, pH 8.8, at 25³C, 50 mM KCl, 1.5 mM MgCl2,

0.1% Triton-X100, 200 WM of each deoxynucleotide

triphosphate, 0.8 WM of the primer, 10 Wl of the cell

suspension (15 Wg of DNA) and sterile distilled

water. A negative control in which DNA was re-

placed with sterile distilled water was also included.

The solutions were overlaid with 100 Wl of para¤n

oil and cycled through the following temperature

pro¢le: an initial denaturation step at 95³C for

5 min, 45 cycles of 95³C for 1 min (denaturation),

37³C for 3 min (annealing) and 72³C for 7 min

(DNA chain extension), and a ¢nal elongation step

at 72³C for 10 min. Incubation was performed in a

thermocycler (PHC-3 Thermal Cycler, Techne Cor-

poration, Cambridge, UK). Standard procedures

were adopted to eliminate contamination [26]. Dupli-

cated AP-PCR analysis was performed for each

strain to assess reproducibility. The ampli¢ed DNA

products were electrophoresed on 1.2% (w/v) agarose

gels (ultrapure electrophoresis grade, Gibco-BRL) at

90 V for 3 h and stained in ethidium bromide. A 123

bp DNA ladder (Gibco-BRL) was used as a molec-

ular weight marker. Two lanes (outside left and

right) containing the markers were included on

each gel to correct for within and between gel dis-

tortions.

2.5. Fragment size estimation

Molecular size of fragments was estimated by

comparison with the molecular weight markers using

the computer program HOWBIG [27], based on the

reciprocal correlation of size to migration distance of

DNA. To perform the calculations, the program

chose the 3 marker bands migrating closest to the

test band, so reducing the error to 9 0.5%.

2.6. Computation of strain similarities

To compare AP-PCR patterns from di¡erent gels,

the bands were coded according to size to minimize

errors when determining similarities by computer-

assisted methods of analysis. The ampli¢cation pat-

terns were screened for bands within 16 di¡erent size

ranges from 0.3 to 2.4 kb and positive (presence) or

negative (absence) results were recorded. Computed

similarities among strains were estimated by means

of the Dice coe¤cient (negative matches excluded)

and clustering of strains was based on the un-

weighted average pair group method (UPGMA) to

facilitate the plotting of a dendrogram [28]. All com-

putations were performed using the NTSYS-PC pro-

gram [29].

2.7. Discrimination power

The discriminatory power of AP-PCR analysis

was calculated by application of Simpson numerical

index of diversity [30].

3. Results

3.1. Typeability and reproducibility

To assess primer e¤ciency and discrimination, an

initial screening with the 20 random primers was

FEMSIM 713 9-4-97

Table 4

Numbers of P. aeruginosa strains of di¡erent serotype in each

group based on numerical analysis of AP-PCR pro¢les

AP-PCR group

Serotype I II III IV V VI VII

O1 5 1 2 3 1 1

O2 3 1

O3 4

O4 1 1

O5 2 2

O6 5 3

O7 2 1 1 1 1

O8 2 1 1

O9 1 1

O10 6

O11 9 2 2 1 2

O12 2 1 1

O13 2

O14 1

O15 1

O16 1 2

O17 1

AA 1

NA 1 2

PA 5 2

Total 53 4 19 8 1 4 3

J. Hernaèndez et al. / FEMS Immunology and Medical Microbiology 17 (1997) 37^47 41

FEMSIM 713 9-4-97

J. Hernaèndez et al. / FEMS Immunology and Medical Microbiology 17 (1997) 37^4742

performed to determine di¡erences between 3 se-

lected P. aeruginosa strains. As primer OPA-5 (se-

quence 5P-AGG GGT CTT G-3P) gave a clear and

reproducible AP-PCR ¢ngerprint, it was selected for

use in comparative studies. All the 18 reference

strains and the 74 clinical isolates subjected to AP-

PCR analysis yielded ampli¢cation products (type-

ability of 100%). No product was detected in any

negative (distilled water) control. To check the re-

producibility of the method, DNA of each strain

was tested on at least 2 di¡erent subcultures. Iden-

tical AP-PCR banding patterns (relative position and

intensity of bands) were observed. Although similar

results were obtained for most repeated subcultures

of the same strain, some minor di¡erences were ob-

served in the relative amounts of certain fragments,

and variability in the presence of faint bands. There-

fore, only the major ampli¢cation fragments that

appeared in both analysis were used in the numerical

analysis. The reproducibility of the method was also

demonstrated by the fact that identical patterns were

obtained with epidemiologically related strains.

3.2. Distribution of serotypes

Table 3 lists the serotypes of all reference and

clinical strains and the distribution according their

origin. Eleven strains (12%) could not be assigned

to any international serotype and were coded as

autoagglutinable (1 strain), non-agglutinable (3

strains), or polyagglutinable (7 strains). The test re-

sults of the non-serotypeable strains were checked

for growth at 41³C, oxidation of gluconate, and ni-

trate reduction, to con¢rm the identity as typical P.

aeruginosa. The discrimination index of serotyping

analysis referred to clinical isolates was 0.886. Over-

all, the predominant serotypes were O11 and O1,

representing 24 and 19%, respectively, of serotype-

able strains. Three serotypes (O14, O15, and O17)

were not found among the strains isolated from in-

fected patients. The type strain of P. aeruginosa

(NCTC 10332) was serotype O6. No association

was observed between strain serotype and hospital.

3.3. AP-PCR patterns of clinical isolates and

reference strains

The 92 di¡erent strains of P. aeruginosa listed in

Tables 1 and 2 were subjected to AP-PCR analysis

using primer OPA-5. Examples of AP-PCR patterns

of reference strains from all 17 serotypes of the IATS

scheme, and of representative strains from infected

patients are shown diagrammatically in Figs. 1 and

2, respectively. The number of ampli¢ed DNA prod-

ucts detected by electrophoresis varied between 2

and 7, with most having relatively uniform intensi-

ties. The AP-PCR patterns of the reference strains of

3 allied species (P. £uorescens NCTC 10038T, P. pu-

tida NCTC 10936T, and B. cepacia NCTC 10661

T)

were di¡erent from each other and from all the

strains of P. aeruginosa (data not shown).

3.4. Analysis of AP-PCR patterns

Overall 16 di¡erent DNA fragments were detected

among the patterns from P. aeruginosa. The esti-

mated sizes ranged from 0.3 to 2.4 kb. The 1.48 kb

fragment was found in AP-PCR patterns of all iso-

lates. Two additional fragments of 0.96 and 1.71 kb

were found in 89 and 65% of strains, respectively.

The ampli¢cation products of 1.48 and 1.71 kb

were not detected in patterns of the type strains of

3 allied Pseudomonas species, but the 0.96 kb frag-

ment could be observed in the type strain of B. ce-

pacia.

Based on the AP-PCR patterns, a total of 61 dif-

ferent pro¢les were obtained. The pro¢le assigned to

each strain is listed in Tables 1 and 2. Most pro¢les

(45/61) were unique, but 16 pro¢les were common

for 2 or more strains that were not necessarily

from the same hospital. The 61 di¡erent AP-PCR

pro¢les were compared by numerical methods and

the resultant dendrogram (Fig. 3) based on the per-

centage similarity between isolates, shows a high de-

gree of genetic diversity. The numerical index of dis-

crimination was high (D=0.984). Seven major

groups were de¢ned at the 55% similarity level, and

FEMSIM 713 9-4-97

Fig. 3. Dendrogram of the numerical analysis based on the AP-PCR patterns of strains. The numbers on the horizontal axis indicate the

percentage similarities as determined by the Dice coe¤cient. The vertical axis shows the main groups de¢ned at the 55% similarity level.

P, AP-PCR pro¢le.

6

J. Hernaèndez et al. / FEMS Immunology and Medical Microbiology 17 (1997) 37^47 43

all strains of P. aeruginosa were linked at the 40%

homology level.

Group I, the overall predominant cluster, com-

prised 53 (58%) di¡erent strains representing 34

(56%) diverse AP-PCR pro¢les, linked at the 60%

similarity level. No strain isolated at Valencia hospi-

tal A fell in this group. The more frequent serotypes

represented were O11 (9 strains) and O10 (6 strains).

Only serotype O4 was not included.

Group II, de¢ned at the 70% similarity level, was

heterogeneous and contained only 4 (5%) clinical

strains isolated in the 3 hospitals. Serotypes O1, O7

and O11 were represented among the strains.

Group III was de¢ned at the 55% similarity level

and comprised 19 (21%) strains from all sources (ref-

erence and clinical isolates), with 13 (21%) di¡erent

AP-PCR pro¢les. The type strain of P. aeruginosa

(NCTC 10332) was included in this cluster. No single

serotype predominated among the isolates.

Groups IV (8 strains, 4 AP-PCR pro¢les), V

(1 strain), VI (4 strains, 3 AP-PCR pro¢les), and

VII (3 strains, 2 AP-PCR pro¢les), were relatively

homogeneous, comprising 1 reference strain and 15

di¡erent isolates from patients at hospitals A and B,

but not hospital C.

The inclusion in the study of reference strains re-

presenting all international serotypes revealed no re-

lationship between AP-PCR pro¢les and serotypes.

Some strains assigned to the same AP-PCR pro¢le

had di¡erent serogroups, and conversely, some

strains of the same serogroup had di¡erent AP-

PCR pro¢les.

3.5. Distribution of strain types

The distribution of the di¡erent serotypes and AP-

PCR types within and between each hospital are

shown in Table 3. The number of strains in each

group belonging to di¡erent serogroups is shown in

Table 4. The results show that AP-PCR ¢ngerprint-

ing provides signi¢cantly improved discrimination

within several serotypes; for example, see serotypes

O1, O7 and O11.

4. Discussion

Typing to evaluate the probability of cross-trans-

mission is commonly advised in epidemiological in-

vestigations, and the use of very discriminatory typ-

ing methods is recommended for di¡erentiating

epidemiologically unrelated isolates. It is widely ac-

cepted that traditional phenotypic methods of typing

isolates of P. aeruginosa [5,7,8] do not permit accu-

rate investigation of nosocomial infections because

phenotypic markers are relatively unstable. In con-

trast, molecular subtyping methods o¡er high type-

ability and reproducibility, can de¢ne clonal relation-

ships between isolates, and have contributed

signi¢cantly to a better understanding of the epide-

miology of P. aeruginosa infections [9^18,21,22,25,

31].

In our study, a high degree of technical reprodu-

cibility was achieved by both typing methods, but

AP-PCR ¢ngerprinting provided higher typeability

(100%) and discrimination power (0.984), and conse-

quently, was more useful for epidemiological inves-

tigations. AP-PCR-generated DNA fragments were

relatively easy to interpret because of the low num-

ber of bands. Also, visual comparisons of gels were

facilitated by the presence of common bands that

could be used as internal markers to allow normal-

ization of slight gel-to-gel variations. Our AP-PCR

¢ngerprinting results show considerable genomic di-

versity among the P. aeruginosa isolated from hospi-

tals in Valencia (Spain), and most strains have dis-

tinct genotypes.

The high degree of discrimination associated with

DNA-based typing methods is dependent upon an

evolutionary genetic divergence arising from point

mutations, insertions or deletions of chromosomal

DNA, and if such random genetic events occur fre-

quently in a strain, the applicability of these methods

would be decreased [32,33]. These features are espe-

cially relevant for typing P. aeruginosa isolates as

genetic rearrangements associated with the horizon-

tal transfer of genetic elements from habitats have

been demonstrated [34]. In fact, investigators have

recently proposed several criteria for interpreting

chromosomal DNA restriction patterns generated

by molecular methods [35].

Ribotyping has provided invaluable information

during the investigation of many nosocomial infec-

tions, but its apparently low sensitivity for the study

of P. aeruginosa strains might be related to an insuf-

¢cient search for the most appropriate restriction

FEMSIM 713 9-4-97

J. Hernaèndez et al. / FEMS Immunology and Medical Microbiology 17 (1997) 37^4744

endonuclease [10]. Generally, the use of single en-

zymes provide less discrimination to that reached

by other genotyping methods [9,10,14,25]. Addition-

ally, when the circumstances and results indicate the

need for further characterization, as in the investiga-

tion of strains from a suspected outbreaks, extra

discrimination must be obtained by combining data

from several endonucleases [10,12]. Thus, Blanc et al.

[11] critically assessed the typeability, reproducibility

and discriminatory power of ribotyping applied to

epidemiologically unrelated P. aeruginosa isolates in

Switzerland and concluded the system compared well

with serotyping. When they used single endonu-

cleases, a discriminatory index of W0.7 was re-

ported, which could be increased to 0.958 with com-

bined data of 4 enzymes.

In a study of 102 P. aeruginosa isolates infecting

23 CF patients in France [12], a comparison was

made of results from esterase electrophoresis and

combined ribopatterns for 3 enzymes. All strains

were typeable with this molecular approach, whereas

only 17% of the isolates could be serotyped. A total

of 16 ribotypes and 25 zymotypes were di¡eren-

tiated, but it was demonstrated that by combining

the 2 molecular methods, precise characterization

of P. aeruginosa could be achieved. An index of dis-

crimination was not calculated.

Macrorestriction analysis of digested DNA by

pulse-¢eld gel electrophoresis, as a molecular ap-

proach to studying P. aeruginosa epidemiology, was

found to be more discriminatory than ribotyping

[16], but it was equally e¡ective when compared to

AP-PCR ¢ngerprinting [17]. In a recent work, Ren-

ders et al. [18], demonstrated the enhanced resolution

of AP-PCR and PFGE when compared to serotyp-

ing and pyocin-typing. Both DNA typing techniques

generated concordant results, although di¡erences in

resolution (15%) are apparent.

Other investigators used AP-PCR analysis to show

genomic diversity among unrelated strains in hospi-

tal environments, and that it could be used to di¡er-

entiate such strains from other isolates in the same

hospital during an outbreak [21,22]. Pitt et al., using

ribotyping for the characterization of multiresistant

O12 P. aeruginosa strains, provided evidence of a

common strain in Europe [31].

In the present work, there were no common strain

AP-PCR types detected in the 3 hospitals in the Va-

lencia area, whereas the majority of types were only

associated with 1 hospital. For instance, just 2 AP-

PCR pro¢les (P19 and P26) were found in hospitals

A and B, whereas 6 di¡erent pro¢les (P1, P7, P27,

P28, P33, and P49) were associated with hospitals B

and C. Because of the relatively small sample size in

each hospital, it is di¤cult to draw any general con-

clusions about the signi¢cance of the strain type dis-

tributions among the hospitals. In the main, they

appear to be associated with sporadic randomly oc-

curring infections. However, it is uncertain from

these data if patient to patient spread of P. aerugi-

nosa with the same type occurred because such strain

types were found independently in 2 hospitals, and

no clinical outbreaks were reported. Nevertheless,

the distinctive feature of the AP-PCR-typing results

shown in Fig. 3 is that no strains from patients at

hospital A were in group I, even though it was the

predominant group with s 50% of strains. In addi-

tion, groups IV^VII were also relatively homoge-

neous, encompassing only strains isolated from pa-

tients at hospitals A and B.

Several parameters can alter AP-PCR pro¢les and

must be previously standardized in order to obtain

reproducible results. Selection of an appropriate

primer, the annealing temperature, the primer-tem-

plate concentration ratio, the magnesium concentra-

tion, and the type of thermocycler have been

reported to produce variations in the pro¢les

[36,37], and are of great importance for maximizing

the discriminatory power of AP-PCR analysis. For

the correct interpretation and classi¢cation of di¡er-

ent but often similar pro¢les, a set of major bands

must be previously de¢ned, and strains with similar

pro¢les have to be analyzed on the same gel. It is

possible to increase the discriminatory power of AP-

PCR analysis by using 2 primers together [38,39]

although we achieved good discrimination with just

1 primer.

This study shows that AP-PCR analysis is a mol-

ecular method that provides a useful and practical

means of typing P. aeruginosa strains. AP-PCR ana-

lysis is clearly more discriminatory and o¡ers higher

typeability than serotyping. It also has the advan-

tages of rapidity, technical simplicity, and relative

ease of ¢ngerprint interpretation compared with

other molecular methods, such as ribotyping and

PFGE. In addition, whole bacterial cells can be

FEMSIM 713 9-4-97

J. Hernaèndez et al. / FEMS Immunology and Medical Microbiology 17 (1997) 37^47 45

used without the requirement for DNA extraction,

and there is no need for multiple primer pairs, nested

PCR or DNA sequencing to di¡erentiate between P.

aeruginosa strains. Consequently, AP-PCR is an ex-

cellent ¢rst-line typing procedure, especially where

large numbers of strains need molecular typing.

Further investigations on outbreaks are needed to

validate AP-PCR in clinical investigations, even so

we would recommend its use in conjunction with

serotyping for improving discrimination in the epide-

miological surveillance of P. aeruginosa.

Acknowledgments

We are grateful to the following for providing

strains: Dr. M. Gobernado (Ciudad Sanitaria La

Fe); Dr. R. Llucian (Hospital General Universita-

rio); and Dr. J. Garc|èa de Lomas (Hospital Cl|ènico

Universitario, Valencia, Spain).

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