Improved multiplex PCR method for the rapid detection of β-lactamase genes in Escherichia coli of animal origin

Diagnostic Microbiology and Infectious Disease 56 (2006) 103 – 106 www.elsevier.com/locate/diagmicrobio

Improved multiplex PCR method for the rapid detection of h-lactamase genes in Escherichia coli of animal originB Constanc¸a Pombaa,4, Nuno Mendonc¸ab, Marta Costaa, Deolinda Lourob, Bruno Baptistaa, Mo´nica Ferreirab, Jose´ Duarte Correiaa, Manuela Canic¸ab b

a CIISA, Faculty of Veterinary Medicine, Universidade Te´cnica de Lisboa, Av. da Universidade Te´cnica, 1300-477 Lisboa, Portugal Antibiotic Resistance Unit, Centre of Bacteriology, National Institute of Health Dr Ricardo Jorge, Av. Padre Cruz, 1649-016 Lisboa, Portugal Received 22 February 2006; accepted 10 March 2006

Abstract We developed 2 variants, A and B, of a multiplex polymerase chain reaction method for detecting the h-lactam resistance genes bla TEM, bla SHV, and bla OXA in 122 uropathogenic Escherichia coli animal strains. Method B yielded 98% specificity and 100% sensitivity, and method A yielded 100% and 89%, respectively. Variant B was more accurate (99%) than A (94%). D 2006 Elsevier Inc. All rights reserved. Keywords: PCR; h-Lactamase genes; Escherichia coli

Detection and characterization of h-lactamase genes from animal isolates has been based on DNA hybridization, simplex polymerase chain reaction (PCR), and nucleotide sequencing methods. This has limited the number of clinical Escherichia coli strains characterized for h-lactam resistance in veterinary laboratories (Bradford et al., 1999; Brin˜as et al., 2003, 2005; Carattoli et al., 2005; Fe´ria et al., 2002; Maynard et al., 2004; Teshager et al., 2000). To detect the emergence of resistance to 3rd generation cephalosporins, we have to screen many isolates. The aim of this study is to develop and evaluate multiplex PCR assays as tools for characterization of E. coli h-lactam resistance in veterinary isolates. Sixty-two amoxicillin-resistant and 60 amoxicillin-susceptible E. coli strains were isolated from dogs (n = 96), swine (n = 10), and cats (n = 16) with urinary tract infection. Strains for PCR-positive controls, isoelectrofocusing (IEF) controls, and negative controls for ampC gene amplification were those previously used (Fe´ria et al., B

This work was financially supported by the POCTI/CVT/36253/99 (FEDER 38%) grant from Fundac¸a˜o para a Cieˆncia e Tecnologia, Lisbon, Portugal. 4 Corresponding author. Faculdade de Medicina Veterinaria, Av. da Universidade Te´cuica, 1300-477 Lisboa, Portugal. Tel.: +351-21-3652837; fax: +351-21-3652897. E-mail address: [email protected] (C. Pomba). 0732-8893/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2006.03.005

2002). The MIC agar dilution method, the susceptibility criteria, and the IEF were performed as previously described (Fe´ria et al., 2002). Two multiplex PCR variants, A and B, were developed from previously described simplex PCR assays for the detection of h-lactam resistance genes bla TEM, bla SHV, bla OXA-1, and ampC, yielding amplification fragments of 716, 471, 199, and 634 base pairs (bp), respectively (Fe´ria et al., 2002). Both protocols used final concentrations of each of the B/K, oxa1f/oxa1r, or ampCf/ampCr primer pairs at 0.2 Amol/L, the shvf/shvr primer pair at 0.6 Amol/L, and MgCl2 at 3.0 mmol/L. Multiplex PCR amplification for methods A and B comprised a 1st cycle of 7 min of denaturation at 94 8C, 5 min annealing at 61 8C, and 60 s extension at 72 8C, followed by 30 cycles of 60 s at 94 8C, 2 min at 61 8C, and 60 s at 72 8C, and a final extension step of 5 min at 72 8C. All E. coli strains were tested by both methods (Table 1), and there were some discrepancies for amoxicillin-resistant E. coli. Method B amplified bla TEM genes in 3 strains classified as negative by the method A; 1 harbored a bla OXA gene (Table 2). Method B correctly identified all 49 isolates with this gene, but also gave a falsepositive result by amplifying a bla TEM fragment from a strain harboring only the bla SHV gene (Table 2). Specificity for bla TEM gene detection was thus 100% for method A and 98% for method B (Table 3).

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C. Pomba et al. / Diagnostic Microbiology and Infectious Disease 56 (2006) 103 – 106

Table 1 Multiplex PCR methods A and B differences: reaction components and thermocyclers Component or PCR steps

Water sterile Millipore (volume up to) Buffer

Deoxynucleoside triphosphate (each nucleoside) Dimethylsufoxide

Q solution Taq DNA polymerase DNA template Thermocyclers

Final concentration (composition or brand) or PCR conditions Method A

Method B

50 AL total reaction

25 AL total reaction

1 (10 mmol/L Tris–HCl, pH 8.8, 50 mmol/L KCl, 0.08% Nonidet P40)

1 (unknown concentrations of Tris–HCl, KCl, (NH4)2SO4, and 1.5 mmol/L MgCl2, pH 8.7) 500 Amol/L (Amersham Biosciences) –

200 Amol/L (Amersham Biosciences, Lisbon, Portugal) 5% (vol/vol) (Hibri-max; Sigma, Lisbon, Portugal) – 1.25 U (MBI Fermentas) 2 AL (unknown concentration) Eppendorf Mastercycler (Eppendorf, Hamburg, Germany)

1 (Qiagen) 1.25 U (Qiagen) 100 ng Stratagene Robocycler (Stratagene, Madrid, Spain)

Method B was more sensitive than method A for bla SHV genes, detecting all genes present in clinical E. coli penicillinase-producing strains with a pI compatible with SHV enzymes and confirmed by nucleotide sequencing (Table 2). Method A was less sensitive (67%) and had a lower negative predictive value (95%) than method B (Table 3). Polymerase chain reaction DNA fragments of 1092 bp from 7 bla TEM genes and of 860 bp from 6 bla SHV were purified and sequenced as previously described (PombaFe´ria and Canic¸a, 2003). Nucleotide sequencing identified 7 genes as bla TEM-1 (pI 5.4), 5 as bla SHV-1 (pI 7.6), and 1 as bla SHV-12 (pI 6 8.0) (accession no. AJ920369, EMBL Nucleotide database). These findings confirmed the specificity of the multiplex PCR methods (Table 2). As expected, amplifications were negative with bla OXA-2and bla OXA-3-positive reference strains: the primers were specific for the bla OXA-1 gene, which encodes the OXA enzyme most frequently found in Enterobacteriaceae of animal and human origin (Fe´ria et al., 2002; Livermore, 1995). However, these primers may also identify bla OXA-4, bla OXA-30, and bla OXA-47 (http://www.ncbi.nlm.nih.gov). Method B was better than method A for detecting bla OXA genes (Table 3). Qiagen (IZASA, Lisbon, Portugal) reagents performed better than MBI Fermentas (Lisbon, Portugal) reagents, identifying 2 strains as bla OXA and bla TEM positive (Table 2). Sensitivity and negative predictive values for the

detection of bla OXA genes by method A were 67% and 98%, respectively (Table 3). Chromosomal ampC gene amplification was positive in all E. coli control strains and negative with 4 other bacterial species (Salmonella typhimurium, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa) not harboring this gene. The presence of the ubiquitary ampC chromosomal gene in all E. coli strains allows it to serve as a positive internal PCR control (Table 2). Both methods scored only 1 ampC gene coding for a cephalosporinase (pI 9.0) producer strain (Table 2), such that the positive predictive value was low (0.8%, Table 3). These PCR assays are therefore unable to differentiate between cefoxitin-resistant, AmpC-producing E. coli strains and cefoxitinresistant, non-AmpC producing strains (extended-spectrum h-lactamases, ESBL) (Coudron et al., 2003). Chromosomal and plasmid-encoded AmpC-mediated resistance mechanisms remain difficult to evaluate in clinical laboratories (Hanson, 2003). The 2 multiplex PCR assays were sensitive for detecting the bla TEM, bla SHV, bla OXA-1, and ampC genes in positive control strains (TEM-1, TEM-2, TEM-3, IRT-1, IRT-2, IRT14, OXA-1, SHV-1, SHV-3, SHV-4, SHV-5, AmpC, and TEM-8 producer strains). The primers did not cross-react with other genes. Because the findings for the control strains were in as expected, discrepancies between methods A and B are unlikely to have been due to the use of different models of thermal cycler from different manufactures. The reason for the poorer performance of method A was due to the use of dimethylsufoxide and variable amounts of template DNA, as demonstrated by Henegariu et al. (1997), and was confirmed by us (data not shown). The smaller amount of DNA used in method B, together with the higher MgCl2 (4.5 mmol/L) and of each deoxynucleoside triphosphate (500 Amol/L) concentrations, improved amplification through greater DNA polymerization. The DNA polymerase-buffer systems are also important, as demonstrated for quantitative real-time PCR (Wolffs et al., 2004). In our study, we do not know how the composition of solution Q, the concentration of KCl in the buffer for method B, or the effect of Nonidet P40 could affect the performance of the multiplex assays tested and contribute to the lower sensitivity of method A (89%) than B (100%). Overall, for E. coli h-lactamase genotype detection, multiplex B was more accurate (99%) than variant A (94%) (Table 3). Genotyping results were in agreement with the resistance phenotypes and the corresponding enzymatic characteristics. We demonstrated that multiplex PCR variant B allowed rapid, sensitive, and efficient characterization of h-lactam resistance mechanisms of E. coli strains isolated from animals and may be also used in detecting h-lactam resistance genes in human E. coli strains. This novel method has the advantage, over the previously described assay by Colom et al. (2003), of using ampC gene as a positive internal PCR control. This type of approach has already been proved valuable for evaluating

No. of strains

h-Lactamase detection by

PCR genotype (multiplex A and B) bla TEM gene

MIC

49 8 1 1 2 1 60

pI a

Penicillinase Penicillinasea Penicillinasea Penicillinasea Penicillinasea Cephalosporinaseg Negative

5.4 7.6 7.4 5.4, 68.0 5.4 – 5.6, 67.5 9.0

Identity of h-lactamase gene(s)

bla OXA gene

bla SHV gene

ampC gene

A

B

A

B

A

B

A

B

+

+

+

+

+

+

+

+

b

47 0

2 0

49 1e

0 0

1 1

0 1b

1 2

0 0

1

0

1

0

1

1b

2

0

5

3b

8

0

1

0

1

0

49 8 1 1 2 1 60

0 0 0 0 0 0 0

49 8 1 1 2 1 60

0 0 0 0 0 0 0

bla TEMc + ampC d bla SHVf + ampC d bla OXA + ampC d bla TEMc+ bla SHVf + ampC d bla TEMc + bla OXA + ampC d ampC h ampC d

a Resistance to amoxicillin (MIC from 512 to N 4096 Ag/mL), possibly diminished susceptibility to clavulanic acid (MIC from 4 to 2048 Ag/mL), cefoxitin (MIC from 2 to N 128 Ag/mL), and 3rd generation cephalosporins (cefotaxime MIC from 0.06 to 32 Ag/mL, ceftazidime MIC from 0.12 to 128 Ag/mL, ceftriaxone MIC from 0.03 to 64 Ag/mL). b False-negative results. c Nucleotide sequencing of 7 bla TEM genes confirmed multiplex results. d Constitutive chromosomal ampC gene (with absence of cephalosporinase resistance phenotype and bands in IEF). e False-positive result. f Nucleotide sequencing of 6 bla SHV genes confirmed multiplex results. g One strain with low resistance to amoxicillin (MIC 256 Ag/mL) and clavulanic acid (MIC 256 Ag/mL), and diminished susceptibility to cefoxitin (MIC 16 Ag/mL), aztreoname (MIC of 8 Ag/mL), and 3rd generation cephalosporins (cefotaxime 2 Ag/mL, ceftazidime 2 Ag/mL, ceftriaxone 0.5 Ag/mL) compatible with a chromosomal encoded hyperproduced AmpC h-lactamase (Fe´ria et al., 2002). h Gene coding for an AmpC h-lactamase caused a cephalosporin resistance phenotype and a pI of 9.0.

C. Pomba et al. / Diagnostic Microbiology and Infectious Disease 56 (2006) 103 – 106

Table 2 Detection by PCR genotyping with multiplex PCR (variants A and B) of h-lactamases encoded by amoxicillin-resistant and susceptible E. coli strains (n = 122)

105

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C. Pomba et al. / Diagnostic Microbiology and Infectious Disease 56 (2006) 103 – 106

Table 3 Comparison of the sensitivities, specificities, and positive and negative predictive values of multiplex PCR (methods A and B) for E. coli h-lactamase genotype detection using phenotypic detection (MIC and pI) as reference Performance of protocols A and B on different h-lactamase gene types bla TEM gene Sensitivity (%)b Specificity (%)c Positive predictive value (%)b Negative predictive value (%)c

bla OXA gene

bla SHV gene

All genesa

ampC gene

A

B

A

B

A

B

A

B

A

B

94 100 100

100 98 98

67 100 100

100 100 100

67 100 100

100 100 100

100 0d 0.8

100 0d 0.8

89 100 100

100 98 98

95

100

98

100

95

100

NDd

NDd

90

100

ND = not determined. a Values for bla TEM, bla OXA, and bla SHV genes. b A h-lactamase–producing E. coli strain with a penicillinase or cephalosporinase phenotype and a known pI is considered a true positive. c An E. coli strain with a h-lactam–susceptible phenotype is considered a true negative. d Specificity was 0 for the ampC gene, and the negative predictive value could not be determined because of the ubiquitous nature of the chromosomal ampC gene in E. coli.

aminoglycoside resistance in enterococci (Vakulenko et al., 2003). Multiplex PCR with phenotypic resistance data is suitable for the rapid detection of the emergence of ESBL enzymes in animals, allowing a subsequent oriented nucleotide sequencing approach, and finally h-lactamase identification. The combination of high MIC for 3rd generation cephalosporins and negative multiplex results is suggestive of the presence of AmpC h-lactamases or other ESBLs, such as CTX-M enzymes. This approach can be used to improve surveillance in veterinary laboratories, involving screening of many E. coli strains of different origins (companion or food animals), and may have an impact on future therapeutic and prophylactic choices in veterinary medicine.

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