Rapid detection and identification of metallo-beta-lactamase-encoding genes by multiplex real-time PCR assay and melt curve analysis

J

OURNAL OF

C

LINICAL

M

ICROBIOLOGY

, Feb. 2007, p. 544–547

Vol. 45, No. 2

0095-1137/07/$08.00

⫹

0 doi:10.1128/JCM.01728-06

Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Rapid Detection and Identification of Metallo-

␤

-Lactamase-Encoding

Genes by Multiplex Real-Time PCR Assay and Melt Curve Analysis

䌤

Rodrigo E. Mendes,

1,2

* Katia A. Kiyota,

2

Jussimara Monteiro,

1,2

Mariana Castanheira,

1

Soraya S. Andrade,

1,2

Ana C. Gales,

1

Antonio C. C. Pignatari,

1

and Sergio Tufik

2,3

Laborato

´rio Especial de Microbiologia Clı´nica and Laborato

´rio ALERTA, Division of Infectious Disease,

Federal University of Sa

˜o Paulo,

1

_{AFIP—Medicina Laboratorial, Sa}

_{˜o Paulo,}

2

_{and Department of}

Psychobiology, Federal University of Sa

˜o Paulo,

3

_Sa

_{˜o Paulo, Brazil}

Received 21 August 2006/Returned for modification 26 September 2006/Accepted 30 October 2006

Metallo-

␤

-lactamase enzymes (M

␤

L) are encoded by transferable genes, which appear to spread rapidly

among gram-negative bacteria. The objective of this study was to develop a multiplex real-time PCR assay

followed by a melt curve step for rapid detection and identification of genes encoding M

␤

L-type enzymes based

on the amplicon melting peak. The reference sequences of all genes encoding IMP and VIM types, SPM-1,

GIM-1, and SIM-1 were downloaded from GenBank, and primers were designed to obtain amplicons showing

different sizes and melting peak temperatures (

T

m

). The real-time PCR assay was able to detect all M

␤

L-harboring clinical isolates, and the

T

m

-assigned genotypes were 100% coincident with previous sequencing

results. This assay could be suitable for identification of M

␤

L-producing gram-negative bacteria by molecular

diagnostic laboratories.

Since the first report of acquired metallo-

␤

-lactamase

(M

␤

L) in Japan in 1994 (15), genes encoding IMP- and

VIM-type enzymes have spread rapidly among

Pseudomonas

spp. (1,

5, 10, 13, 14, 16, 18, 22–24),

Acinetobacter

spp. (3, 4, 17, 21, 29),

and strains of

Enterobacteriaceae

(6, 8, 11, 12, 20, 28).

More-over, new M

␤

L types have been described, such as SPM (25),

GIM (2), and, more recently, SIM (9).

The prevalence of M

␤

L-producing gram-negative bacilli has

increased in some hospitals, particularly among clinical isolates of

Pseudomonas aeruginosa

and

Acinetobacter

spp. (21, 23, 27). Since

M

␤

L production may confer phenotypic resistance to virtually all

clinically available

␤

-lactams, the continued spread of M

␤

L is a

major clinical concern (26). The aim of this study was to develop

a multiplex real-time PCR assay followed by a melt curve step for

rapid detection and identification of genes encoding the M

␤

L-type enzymes so far described. The M

␤

L type identification was

based on the characteristic amplicon melting peak.

MATERIALS AND METHODS

M␤L-harboring clinical isolates and M␤L-negative control strains. The strains used in this study are listed in Tables 1 and 2. The M␤L genotypes of the clinical isolates of gram-negative nonfermentative and fermentative bacteria harboring M␤L were previously characterized by PCR and sequencing. When applicable, these clinical isolates were also previously molecularly typed to en-sure that genetically unrelated strains were used. Additionally, several American Type Culture Collection (ATCC; Manassas, VA) reference strains and labora-tory strains were used as M␤L-negative controls (Table 2).

DNA preparation. The microorganisms were grown on blood agar plates overnight at 37°C to ensure colony purity. Three or four bacterial colonies were taken from the blood agar plates and suspended in 200␮l of DNase/RNase-free distilled water (Invitrogen, CA). Two microliters of this suspension was used as templates for further amplification.

Primer design.The currently available reference sequences of the M␤L-encoding IMP- and VIM-type (http://www.lahey.org/studies/), SPM-1 (AJ492820), GIM-1 (AJ620678), and SIM-1 (AY887066) genes were downloaded from GenBank (Na-tional Center for Biotechnology Information, Na(Na-tional Institutes of Health, Be-thesda, MD). Based on the comprehensive analyses and alignments of each M␤L type, primers were designed to yield amplicons showing different sizes and melting peak temperatures (Tm) separated by at least 1°C. Predicted

amplicon sizes andTmwere determined by the Lasergene software package

(DNASTAR, Madison, WI).

Additionally, a primer pair targeting the consensus region of the bacterial 16S rRNA gene was included in the reaction mixture as a PCR internal-control target. Primer pairs were evaluated in a single format (using a primer concen-tration of 0.5␮M) to ensure that they correctly amplified their respective loci and that the amplicons showed the expectedTm. Subsequently, the multiplex format

was optimized by assaying different primer pair concentrations. The primer sequences, positions, and concentrations, and the sizes of the corresponding amplicons, are given in Table 3.

Multiplex real-time PCR.Amplification was performed in a 48-␮l mixture containing 25␮l of Platinum SYBR Green qPCR SuperMix (PlatinumTaqDNA polymerase, SYBR Green I dye, Tris-HCl, KCl, 6 mM MgCl2, 400␮M dGTP, 400␮M dATP, 400␮M dCTP, 800␮M dUTP, uracil DNA glycosylase, and stabilizers) (Invitrogen, CA), six pairs of primers at their respective concentra-tions (Table 3), and 2␮l of the template by using the DNA Engine Opticon 2 system (Bio-Rad Laboratories, CA). The PCR conditions were as follows: initial denaturation at 94°C for 5 min; 35 cycles of 94°C for 20 s, 53°C for 45 s, and 72°C for 30 s; and a melt curve step (from 68°C, gradually increasing 0.5°C/s to 95°C, with acquisition data every 1 s). Melt curves were then converted into melting peaks by plotting the negative derivative of fluorescence versus temperature (⫺dF2/dTversusTand⫺dF3/dTversusT).

Multiplex real-time PCR validation.In order to assess the accuracy of the assay, 44 bacterial strains were blindly tested after real-time PCR optimization (Tables 1 and 2).

Multiplex real-time PCR sensitivity.The sensitivity of the reaction was esti-mated by dilution experiments. Briefly, one representative of each M␤ L-harbor-ing clinical isolate was suspended in DNase/RNase-free distilled water to a density corresponding to a McFarland turbidity standard of 1.0 (3⫻108_CFU/ ml). These suspensions were used to prepare serial 10-fold dilutions using DNase/RNase-free distilled water.

RESULTS AND DISCUSSION

When different strains were submitted to the real-time PCR

assay, differences in the

T

m

of the amplicons were observed for

* Corresponding author. Mailing address: Division of Infectious

Diseases, Federal University of Sa˜o Paulo, Rua Leandro Dupret, 188,

Sa˜o Paulo, SP, Brazil CEP 04025-010. Phone: (55-11) 5081-2819 or

-2965. Fax: (55-11) 5571-5180. E-mail: [email protected].

䌤

_{Published ahead of print on 8 November 2006.}

strains harboring

bla

IMP

-type allelic variants (from 76.0°C to

77.5°C) as well as for those harboring

bla

VIM

-type allelic variants

(from 87.5°C to 88.5°C) (Table 1). These differences in

T

m

will be

observed mainly for amplicons generated from

bla

IMP

-type genes,

since the GC contents of the amplicons generated will be more

divergent than those for

bla

VIM

-type genes (Table 1).

Allelic variants for the remaining M

␤

L types (

bla

SPM-1

,

bla

GIM-1

, and

bla

SIM-1

) have not been found yet. For this

rea-son, only one clinical isolate harboring

bla

GIM-1

and one

har-boring

bla

SIM-1

were used during the validation process. The

theoretical and practical

T

m

obtained were very similar, and no

T

m

differences were observed when several genetically

unre-lated

bla

SPM-1

-harboring

P. aeruginosa

isolates were submitted

to the assay (Table 1).

When the negative-control ATCC reference strains and

lab-oratory strains were submitted to the assay, the melt curve

analysis showed only one melting peak varying from 85.5°C to

86.5°C (Table 2). These melting peaks were consistent with the

T

m

of the amplicon generated by the primers targeting the

conserved sequences of the 16S rRNA gene. This

internal-control primer pair was used at a lower concentration than the

primers targeting the M

␤

L genes; thus, the latter would have

preference during the amplification reaction. This strategy was

employed to avoid double amplification, which could

compro-mise the melt curve analysis.

The real-time PCR sensitivity experiment showed that the

assay was capable of detecting the16S rRNA target gene at

a dilution corresponding to 6

⫻

10

3

CFU per reaction;

bla

SPM

,

bla

VIM

,

bla

SIM

, and

bla

IMP

at 6

⫻

10

2

CFU per

reaction; and

bla

GIM

at 6

⫻

10

1

CFU per reaction (data not

shown). Additionally, the lowest detection limits of the

tar-get genes were represented by the cycle threshold values of

34.62, 34.11, 33.66, 32.69, 32.58, and 28.86 for the16S rRNA,

bla

SPM

,

bla

GIM

,

bla

IMP

,

bla

SIM

, and

bla

VIM

genes,

respec-tively. This suggests that the assay as developed is

suffi-TABLE 1. M

␤

L-harboring clinical isolates used during the validation process

Clinical isolate M␤L-encoding_gene Reference or_{accession no.} Strain no. Ribogroup Tmof amplicon Amplicon GC_{content (%)} Theoretical Practical

Serratia marcescens

bla

_IMP-1

15

TN9106

NA

a

_77.9

_77.5

_38.30

Pseudomonas putida

bla

_IMP-1

AM283489

48-12346A

NA

77.9

77.5

38.30

Enterobacter cloacae

bla

_IMP-1

2a

A199

NA

77.9

77.5

38.30

Acinetobacter baumannii

bla

_IMP-1

AJ640197

48-696D

NA

77.9

77.5

38.30

P. aeruginosa

bla

_IMP-1

19

A5386

NA

77.9

77.5

38.30

Klebsiella pneumoniae

bla

_IMP-1

11

A13309

NA

77.9

77.5

38.30

P. aeruginosa

bla

_IMP-5

19a

115-10639A

NA

77.5

76.5

37.23

P. aeruginosa

bla

_IMP-13

22

86-14571

NA

77.1

76.0

36.17

P. aeruginosa

bla

_IMP-16

14

101-4704

164-7

77.3

76.5

36.70

P. aeruginosa

bla

_IMP-16

This study

P3987

164-8

77.3

76.5

36.70

P. aeruginosa

bla

_IMP-18

This study

A3486

NA

76.4

76.0

34.57

P. aeruginosa

bla

_VIM-1

23

75-3636C

NA

87.1

88.5

57.33

E. cloacae

bla

_VIM-1

AM183120

75-10433A

NA

87.1

88.5

57.33

P. aeruginosa

bla

_VIM-2

23

81-11963A

NA

86.9

88.0

56.81

P. aeruginosa

bla

_VIM-2

13

49-4583C

NA

86.9

88.0

56.81

P. aeruginosa

bla

_VIM-2

This study

A1254

NA

86.9

88.0

56.81

P. aeruginosa

bla

_VIM-7

24

7-406

NA

86.2

87.5

55.00

P. aeruginosa

bla

_SPM-1

25

48-1997A

72-3

83.8

83.5

47.62

P. aeruginosa

bla

_SPM-1

7

A3488

88-2

83.8

83.5

47.62

P. aeruginosa

bla

_SPM-1

7

A2535

77-2

83.8

83.5

47.62

P. aeruginosa

bla

_SPM-1

7

A3301

105-3

83.8

83.5

47.62

P. aeruginosa

bla

_SPM-1

7

A3307

106-4

83.8

83.5

47.62

P. aeruginosa

bla

_SPM-1

7

A3302

105-4

83.8

83.5

47.62

P. aeruginosa

bla

_SPM-1

7

A3304

97-7

83.8

83.5

47.62

P. aeruginosa

bla

_SPM-1

7

A3300

105-1

83.8

83.5

47.62

P. aeruginosa

bla

_SPM-1

7

A2839

88-1

83.8

83.5

47.62

P. aeruginosa

bla

_SPM-1

7

A3309

105-5

83.8

83.5

47.62

P. aeruginosa

bla

_SPM-1

7

A2526

78-4

83.8

83.5

47.62

P. aeruginosa

bla

_GIM-1

2

73-5671

NA

72.2

72.0

34.72

A. baumannii

bla

_SIM-1

9

03-9-T104

NA

80.4

80.5

39.89

a_{NA, not applicable.}

TABLE 2. ATCC reference and laboratory strains of gram-negative

bacteria used during the validation process as M

␤

L-negative

control strains

a

Organism Strain no. _ampliconPractical_T

m

P. aeruginosa

PA01

86.0

Escherichia coli

DH5

␣

87.0

E. coli

DH10B

86.5

E. coli

K-12

86.5

E. coli

ATCC 25922

85.5

Acinetobacter calcoaceticus

ATCC 33305

85.5

Enterobacter aerogenes

ATCC 13048

86.5

P. aeruginosa

ATCC 27853

86.0

Klebsiella pneumoniae

ATCC 700603

86.5

Neisseria meningitidis

ATCC 13090

86.0

Neisseria perflava

ATCC 14799

86.0

Neisseria lactamica

ATCC 49142

86.0

Neisseria sicca

ATCC 29193

86.0

Salmonella

serovar Typhimurium

ATCC 14028

86.0

a_{The 16S rRNA gene was the target gene in each case.}

ciently robust, even when the bacterial cells suspended in

water are used as the template.

Although the assay was developed to detect all M

␤

L-encod-ing genes, we could not submit strains harborL-encod-ing all the

bla

IMP

and

bla

VIM

allelic variants, since we do not have access to all of

them. We also acknowledge the possibility of future assay

limitations once more M

␤

L types or newly emerging M

␤

L

allelic variants are detected, requiring a possible assay

recon-figuration.

The assay was able to detect and identify all M

␤

L-harboring

strains evaluated. It is a single-tube reaction, technically

sim-ple, performed in only 2 h after colony selection. The

T

m

-assigned M

␤

L genotypes are easily interpreted (Fig. 1a) and

may be suitable for the detection of M

␤

L-producing

gram-negative bacteria by molecular diagnostic laboratories.

Fur-thermore, the assay may also be performed through a

conven-tional amplification reaction, followed by visualization of the

amplicons by using a UV light box after electrophoresis on

a 1.5% agarose gel containing 0.5

␮

g/ml ethidium bromide

(Fig. 1b).

The rapid detection of M

␤

L-producing isolates could be

helpful for epidemiological purposes and for monitoring the

emergence of M

␤

L-producing isolates in clinical settings. The

detection of such isolates could help rapidly establish

stan-dards for hospital infection control measures to minimize the

spreading of these resistant determinants.

ACKNOWLEDGMENTS

We thank Timothy R. Walsh, Mark A. Toleman, Yoshichika Arakawa,

and Yunsop Chong for providing some of the M

␤

L-harboring clinical

isolates included in this study.

REFERENCES

1.Bahar, G., A. Mazzariol, R. Koncan, A. Mert, R. Fontana, G. M. Rossolini, and G. Cornaglia. 2004. Detection of VIM-5 metallo-␤-lactamase in a

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FIG. 1. (a) Characteristic melting peaks (colored lines) of the amplicons

generated by primers targeting the five M

␤

L types so far described when

M

␤

L-harboring clinical isolates were submitted to the real-time PCR assay.

Colors and genes targeted, from left to right, are as follows: blue,

bla

GIM-1

(

T

m

, 72.0°C); red,

bla

IMP

-type genes (

T

m

, 76.5°C); green,

bla

SIM-1

(

T

m

,

80.5°C); pink,

bla

SPM-1

(

T

m

, 83.5°C); orange,

bla

VIM

-type genes (

T

m

, 89.0°C).

(b) Amplicons generated by primers targeting the five M

␤

L types and the

internal-control gene (16S rRNA). Visualization was performed in a UV light

box after electrophoresis on a 1.5% agarose gel containing 0.5

␮

g/ml ethidium

bromide. Lane 1, SPM-1 amplicon (798 bp; strain 48-1997A); lane 2, SIM-1

amplicon (569 bp; strain 03-9-T104); lane 3, VIM-type amplicon (382-bp;

strain 7-406); lane 4, IMP-type amplicon (188 bp; strain 48-696D); lane 5,

GIM-1 amplicon (72 bp; strain 73-5671); lanes 6 to 9, the internal-control

amplicon (1,499 bp; strains

A. calcoaceticus

ATCC 33305,

P. aeruginosa

ATCC 27853,

Klebsiella pneumoniae

ATCC 700603, and

Enterobacter

aero-genes

ATCC 13048, respectively); lane 10, negative control; lanes M,

molec-ular size markers (50-bp DNA ladder; Invitrogen).

TABLE 3. Primers used in this study

Target Primer Oligonucleotide sequence (5⬘–3⬘) ₍Concn_␮M)a Amplicon_{size (bp) practical}Amplicon_T

mb Position c

bla

_IMP

type

IMPgen-F1

GAATAG(A/G)(A/G)TGGCTTAA(C/T)TCTC

1.0

188

76.0–77.5

308–328

IMPgen-R1

CCAAAC(C/T)ACTA(G/C)GTTATC

495–478

bla

_VIM

type

VIMgen-F2

GTTTGGTCGCATATCGCAAC

0.1

382

87.5–88.5

157–176

VIMgen-R2

AATGCGCAGCACCAGGATAG

538–519

bla

_GIM-1

GIM-F1

TCAATTAGCTCTTGGGCTGAC

0.1

72

72.0

574–594

GIM-R1

CGGAACGACCATTTGAATGG

645–626

bla

_SIM-1

SIM-F1

GTACAAGGGATTCGGCATCG

0.1

569

80.5

126–145

SIM-R1

TGGCCTGTTCCCATGTGAG

694–676

bla

_SPM-1

SPM-F1

CTAAATCGAGAGCCCTGCTTG

0.1

798

83.5

11–31

SPM-R1

CCTTTTCCGCGACCTTGATC

808–789

16S rRNA

16S-8F

AGAGTTTGATCCTGGCTCAG

0.04

1,499

86.0–87.0

8–27

16S-1493R

ACGGCTACCTTGTTACGACTT

1512–1492

a_{Final concentration in the multiplex real-time PCR.} b_PracticalT

mof the amplicon obtained from the evaluated strains. c_{Position numbers correspond to the nucleotides of the coding sequences.}

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