A practical molecular identification of nonfermenting Gram-negative bacteria from cystic fibrosis

Capizzani, Carolina Paulino da Costa; Caçador, Natália Candido; Marques, Elizabeth Andrade; Levy, Carlos Emílio; Tonani, Ludmilla; Torres, Lidia Alice Gomes Monteiro Marin; Darini, Ana Lúcia da Costa

doi:10.1016/j.bjm.2017.07.002

Carolina Paulino da Costa Capizzani

Universidade de São Paulo, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Ribeirão Preto, SP, Brazil

Natália Candido Caçador

Universidade de São Paulo, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Ribeirão Preto, SP, Brazil

Elizabeth Andrade Marques

Universidade Estadual do Rio de Janeiro, Faculdade de Ciências Médicas, Departamento de Microbiologia, Imunologia e Parasitologia, Rio de Janeiro, RJ, Brazil

Carlos Emílio Levy

Universidade Estadual de Campinas, Faculdade de Ciências Médicas, Departamento de Patologia Clínica, Campinas, SP, Brazil

Ludmilla Tonani

Universidade de São Paulo, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Ribeirão Preto, SP, Brazil

Lidia Alice Gomes Monteiro Marin Torres

Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Puericultura e Pediatria, Serviço de Referência de Fibrose Cística do Hospital das Clínicas, Ribeirão Preto, SP, Brazil

Ana Lúcia da Costa Darini ^**Corresponding author at: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Avenida do Café, s/no, Ribeirão Preto, SP, Brazil. E-mail: aldarini@usp.br (A.L. Darini).

Universidade de São Paulo, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Ribeirão Preto, SP, Brazil

*Corresponding author at: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Avenida do Café, s/n^o, Ribeirão Preto, SP, Brazil. E-mail: aldarini@usp.br (A.L. Darini).

Conflicts of interest

The authors declare that they have no conflict of interest.

Primer Target	Primers	Annealing temperature (ºC)	Product size (bp)^a a Size of amplified product by PCR.	References
B. cepacia (Genomovar I)	BCRG11 BCRG12	62	492	Mahenthiralingam et al.²²22 Mahenthiralingam E, Bischof J, Byrne SK, et al. DNA-based diagnostic approaches for identification of Burkholderia cepacia complex, Burkholderia vietnamiensis, Burkholderia multivorans, Burkholderia stabilis, and Burkholderia cepacia genomovars I and III. J Clin Microbiol. 2000;38(9):3165-3173.
B. multivorans (Genomovar II)	BCRBM1 BCRBM2	62	714	Mahenthiralingam et al.²²22 Mahenthiralingam E, Bischof J, Byrne SK, et al. DNA-based diagnostic approaches for identification of Burkholderia cepacia complex, Burkholderia vietnamiensis, Burkholderia multivorans, Burkholderia stabilis, and Burkholderia cepacia genomovars I and III. J Clin Microbiol. 2000;38(9):3165-3173.
B. cenocepacia (Genomovar III-A)	BCRG3A1 BCRG3A2	62	378	Mahenthiralingam et al.²²22 Mahenthiralingam E, Bischof J, Byrne SK, et al. DNA-based diagnostic approaches for identification of Burkholderia cepacia complex, Burkholderia vietnamiensis, Burkholderia multivorans, Burkholderia stabilis, and Burkholderia cepacia genomovars I and III. J Clin Microbiol. 2000;38(9):3165-3173.
B. cenocepacia (Genomovar III-B)	BCRG3B1 BCRG3B2	60	781	Mahenthiralingam et al.²²22 Mahenthiralingam E, Bischof J, Byrne SK, et al. DNA-based diagnostic approaches for identification of Burkholderia cepacia complex, Burkholderia vietnamiensis, Burkholderia multivorans, Burkholderia stabilis, and Burkholderia cepacia genomovars I and III. J Clin Microbiol. 2000;38(9):3165-3173.
B. stabilis (Genomovar IV)	BCRG41 BCRG42	64	647	Mahenthiralingam et al.²²22 Mahenthiralingam E, Bischof J, Byrne SK, et al. DNA-based diagnostic approaches for identification of Burkholderia cepacia complex, Burkholderia vietnamiensis, Burkholderia multivorans, Burkholderia stabilis, and Burkholderia cepacia genomovars I and III. J Clin Microbiol. 2000;38(9):3165-3173.
B. vietnamiensis (Genomovar V)	BCRBV1 BCRBV2	62	378	Mahenthiralingam et al.²²22 Mahenthiralingam E, Bischof J, Byrne SK, et al. DNA-based diagnostic approaches for identification of Burkholderia cepacia complex, Burkholderia vietnamiensis, Burkholderia multivorans, Burkholderia stabilis, and Burkholderia cepacia genomovars I and III. J Clin Microbiol. 2000;38(9):3165-3173.
B. dolosa (Genomovar VI)	G6N BCR1	67	135	Vermis et al.²³23 Vermis K, Coenye T, LiPuma JJ, Mahenthiralingam E, Nelis HJ, Vandamme P. Proposal to accommodate Burkholderia cepacia genomovar VI as Burkholderia dolosa sp. nov. Int J Syst Evol Microbiol. 2004;54(3):689-691.
B. ambifaria (genomovar VII)	BCRGC1 BCRGC2	62	810	Coenye et al.²⁴24 Coenye T, Mahenthiralingam E, Henry D, et al. Burkholderia ambifaria sp. nov., a novel member of the Burkholderia cepacia complex including biocontrol and cystic fibrosis-related isolates. Int J Syst Evol Microbiol. 2001;51(4):1481-1490.
B. anthina (genomovar VIII)	BCRG81 BCRG82	61	473	Vandamme et al.²⁵25 Vandamme P, Henry D, Coenye T, et al. Burkholderia anthina sp. nov. and Burkholderia pyrrocinia, two additional Burkholderia cepacia complex bacteria, may confound results of new molecular diagnostic tools. FEMS Immunol Med Microbiol. 2002;33(2):143-149.
Complexo B. cepacia - Gene recA	BCR1 BCR2	58	1043	Mahenthiralingam et al.²²22 Mahenthiralingam E, Bischof J, Byrne SK, et al. DNA-based diagnostic approaches for identification of Burkholderia cepacia complex, Burkholderia vietnamiensis, Burkholderia multivorans, Burkholderia stabilis, and Burkholderia cepacia genomovars I and III. J Clin Microbiol. 2000;38(9):3165-3173.
A. xylosoxidans	AX-F1 AX-B1	56	163	Liu et al.²⁶26 Liu L, Coenye T, Burns JL, Whitby PW, Stull TL, LiPuma JJ. Ribosomal DNA-directed PCR for identification of Achromobacter (Alcaligenes) xylosoxidans recovered from sputum samples from cystic fibrosis patients. J Clin Microbiol. 2002;40(4):1210-1213.
S. maltophilia	SM1 SM4	58	531	Whitby et al.²⁷27 Whitby PW, Carter KB, Burns JL, Royall JA, LiPuma JJ, Stull TL. Identification and detection of Stenotrophomonas maltophilia by rRNA-directed PCR. J Clin Microbiol. 2000;38(12):4305-4309.
P. sputorum	spuF spur	63	813	Coenye et al.²⁸28 Coenye T, Liu L, Vandamme P, John J. Identification of Pandoraea species by 16S ribosomal DNA-based PCR assays. J Clin Microbiol. 2001;39(12):4452-4455.
Genus Ralstonia	RalGS-F RalGS-R	58	546	Coenye et al.²⁹29 Coenye T, Spilker T, Reik R, Vandamme P, Lipuma JJ. Use of PCR analyses to define the distribution of Ralstonia species recovered from patients with cystic fibrosis. J Clin Microbiol. 2005;43(7):3463-3466.
Genus Cupriavidus	ral2f ral2r	58-61	187	Barrett and Parker³⁰30 Barrett CF, Parker MA. Coexistence of Burkholderia, Cupriavidus, and Rhizobium sp. nodule bacteria on two Mimosa spp. in Costa Rica. Appl Environ Microbiol. 2006;72(2):1198-1206.

Primers	Genus/species	Targets	Proposed modifications^a a According to identification carried out with the reference strains and clinical isolates. AT, annealing temperature; NMT, no modification to previously proposed temperature.
	Literature	The present study
BCR1/BCR2	Bcc bacteria	Bcc bacteria	AT 55 ºC and 56 ºC
AX-F1/AX-B1	A. xylodoxidans	Achromobacter sp.	NMT
SM-1/SM4	S. maltophilia	S. maltophilia	NMT
spuF/spuR	P. sputorum	Pandoraea sp. + Ralstonia sp.	NMT
RalGS-F/RalGS-R	Ralstonia sp.	Ralstonia sp. + Cupriavidus sp.	NMT
ral2f/ral2r	Cupriavidus sp.	Cupriavidus sp. + Ralstonia sp. + Pandoraea sp. + Achromobacter sp.	AT 62 ºC

Bacteria tested	Genus/species Primers
	Bcc BCR1/BCR2	P. sputorum spuF/spuR	Ralstonia sp. RalGS-F/RalGS-R	A. xylosoxidans AX-F1/AX-B1	S. maltophilia SM-1/SM4	Cupriavidus sp. ral2f/ral2r
Reference strains
A. denitrificans LMG 1231	Negative	Negative	Negative	Positive	Negative	Positive ^a a Unexpected results. Gen., genomovar.
A. piechaudii LMG 1873	Negative	Negative	Negative	Positive	Negative	Positive ^a a Unexpected results. Gen., genomovar.
A. xylosoxidans LMG 1863	Negative	Negative	Negative	Positive	Negative	Positive ^a a Unexpected results. Gen., genomovar.
A. dolens LMG	Negative	Negative	Negative	Positive	Negative	Positive ^a a Unexpected results. Gen., genomovar.
A. insuavis	Negative	Negative	Negative	Positive	Negative	Positive ^a a Unexpected results. Gen., genomovar.
A. mucicolens	Negative	Negative	Negative	Positive	Negative	Positive ^a a Unexpected results. Gen., genomovar.
A. ruhlandii	Negative	Negative	Negative	Positive	Negative	Positive ^a a Unexpected results. Gen., genomovar.
C. gillardi LMG 5886	Negative	Negative	Positive ^a a Unexpected results. Gen., genomovar.	Negative	Negative	Positive
P. norimbergensis LMG 18379	Negative	Positive	Negative	Negative	Negative	Positive ^a a Unexpected results. Gen., genomovar.
P. pnomenusa LMG 18087	Negative	Positive	Negative	Negative	Negative	Positive ^a a Unexpected results. Gen., genomovar.
P. pulmonicula LMG 18106	Negative	Positive	Negative	Negative	Negative	Positive ^a a Unexpected results. Gen., genomovar.
P. apista LMG 16407	Negative	Positive	Negative	Negative	Negative	Positive ^a a Unexpected results. Gen., genomovar.
R. picketti LMG 5942	Negative	Positive ^a a Unexpected results. Gen., genomovar.	Positive	Negative	Negative	Positive ^a a Unexpected results. Gen., genomovar.
S. maltophilia LMG 958	Negative	Negative	Negative	Negative	Positive	Negative
B. cepacia 1254	Positive	Negative	Negative	Negative	Negative	Negative
B. multivorans 788	Positive	Negative	Negative	Negative	Negative	Negative
B. cenocepacia LMG 21462	Positive	Negative	Negative	Negative	Negative	Negative
B. cenocepacia 818 Gen. IIIA	Positive	Negative	Negative	Negative	Negative	Negative
B. cenocepacia 17604 Gen. IIIA	Positive	Negative	Negative	Negative	Negative	Negative
B. cenocepacia 842 Gen. IIIB	Positive	Negative	Negative	Negative	Negative	Negative
B. cenocepacia 805 Gen. IIIB	Positive	Negative	Negative	Negative	Negative	Negative
B. stabilis 790	Positive	Negative	Negative	Negative	Negative	Negative
B. stabilis 825	Positive	Negative	Negative	Negative	Negative	Negative
B. vietnamiensis LMG 10929	Positive	Negative	Negative	Negative	Negative	Negative
B. vietnamiensis 1109	Positive	Negative	Negative	Negative	Negative	Negative
B. dolosa LMG-18943	Positive	Negative	Negative	Negative	Negative	Negative
B. ambifaria LMG 19182	Positive	Negative	Negative	Negative	Negative	Negative
B. ambifaria ATCC-53266	Positive	Negative	Negative	Negative	Negative	Negative
B. ambifaria AMMD	Positive	Negative	Negative	Negative	Negative	Negative
B. anthina LMG-20980	Positive	Negative	Negative	Negative	Negative	Negative
B. anthina LMG-16670	Positive	Negative	Negative	Negative	Negative	Negative
B. pyrrocinia ATCC-39227	Positive	Negative	Negative	Negative	Negative	Negative
B. pyrrocinia LMG-14191	Positive	Negative	Negative	Negative	Negative	Negative
Isolates
Bcc (n = 91)	Positive	Negative	Negative	Negative	Negative	Negative
Achromobacter sp. (n = 85)	Negative	Negative	Negative	Positive	Negative	Positive ^a a Unexpected results. Gen., genomovar.
S. maltophilia (n = 12)	Negative	Negative	Negative	Negative	Positive	Negative
Ralstonia sp. (n = 10)	Negative	Positive ^a a Unexpected results. Gen., genomovar.	Positive	Negative	Negative	Positive ^a a Unexpected results. Gen., genomovar.
Pandoraea sp. (n = 2)	Negative	Positive	Negative	Negative	Negative	Positive ^a a Unexpected results. Gen., genomovar.
Cupriavidus sp. (n = 1)	Negative	Negative	Positive ^a a Unexpected results. Gen., genomovar.	Negative	Negative	Positive

[1] *Corresponding author at: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Avenida do Café, s/n^o, Ribeirão Preto, SP, Brazil. E-mail: aldarini@usp.br (A.L. Darini).

Brasil

Brasil

A practical molecular identification of nonfermenting Gram-negative bacteria from cystic fibrosis

Abstract