Gram Stain-Specific-Probe-Based Real-Time PCR for Diagnosis and Discrimination of Bacterial Neonatal Sepsis

Yi-Dong Wu; Li-Hua Chen; Xiu-Jing Wu; Shi-Qiang Shang; Jin-Tu Lou; Li-Zhong Du; Zheng-Yan Zhao

doi:10.1128/JCM.02237-07

DOI: 10.1128/JCM.02237-07

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FIG. 1.
Comparison of the standard curves of the Gram stain-specific probes from serial dilutions of bacteria. (A) Standard curve of G⁺ probe for S. aureus. (B) Standard curve of G⁻ probe for E. coli.

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TABLE 1.

Representative bacterial species detected by GSPBRT-PCR using a pair of specific probes

Genus	Species	GSPBRT-PCR C_T^a		GenBank accession no.
Genus	Species	G⁺ probe	G⁻ probe	GenBank accession no.
Gram-positive bacteria
Bacillus	B. subtilis	16.28 ± 1.08		AB065370
Enterococcus	E. avium	17.67 ± 0.24		AJ301825
	E. faecalis	19.26 ± 1.28		AJ276460
	E. faecium	16.37 ± 0.08		AJ874342
Listeria	L. monocytogenes	17.10 ± 0.24		AY946290
Staphylococcus	S. aureus	16.97 ± 0.45		X68417
	S. auricularis	20.47 ± 0.77		D83358
	S. capitis	16.32 ± 0.15		AY030321
	S. cohnii	19.21 ± 1.32		AJ717378
	S. epidermidis	21.06 ± 0.19		L37605
	S. haemolyticus	20.17 ± 0.97		L37600
	S. hominis	22.45 ± 0.97		AY030318
	S. hyicus	22.70 ± 0.41		D83368
	S. lentus	18.24 ± 0.28		D83370
	S. saprophyticus	21.49 ± 0.46		D83371
	S. schleiferi	24.45 ± 0.37		D83372
	S. simulans	16.56 ± 0.26		D83373
	S. warneri	18.62 ± 0.73		L37603
	S. xylosus	19.01 ± 1.49		D83374
Streptococcus	S. agalactiae	17.24 ± 0.28		AB023574
	S. mitis	23.45 ± 0.72		AY005045
	S. mutans	22.21 ± 0.42		AF139603
	S. pneumoniae	21.87 ± 0.45		AF003930
	S. pyogenes	17.12 ± 0.54		AB023575
	S. sanguis	19.10 ± 1.58		AF003928
Gram-negative bacteria
Citrobacter	C. braakii		20.45 ± 0.56	AF025368
	C. freundii		20.78 ± 0.45	AM184281
Edwardsiella	E. tarda		16.79 ± 0.48	DQ233654
Enterobacter	E. aerogenes		14.85 ± 0.76	AJ251468
	E. cloacae		21.81 ± 0.82	AJ251469
Escherichia	E. coli		16.35 ± 0.36	AF233451
Haemophilus	H. influenzae		24.38 ± 0.38	AF224306
	H. haemolyticus		23.33 ± 0.46	M75045
Klebsiella	K. oxytoca		19.11 ± 0.66	EF127829
	K. pneumoniae		18.05 ± 0.38	AF130981
Neisseria	N. meningitidis		22.18 ± 0.34	AF059671
Pasteurella	P. haemolytica		19.78 ± 0.56	M75080
	P. multocida		20.55 ± 0.48	DQ228979
	P. trehalosi		21.30 ± 0.38	DQ841185
Proteus	P. mirabilis		16.52 ± 0.44	AF008582
	P. penneri		17.45 ± 1.07	AJ634474
	P. vulgaris		20.22 ± 0.32	AJ301683
Providencia	P. alcalifaciens		22.22 ± 0.45	AJ301684
	P. rettgeri		21.38 ± 0.52	AM040492
	P. stuartii		23.67 ± 0.56	AM040491
Pseudomonas	P. aeruginosa		22.75 ± 0.41	AF094720
Salmonella	S. enterica serovar Paratyphi		23.06 ± 0.40	X80682
	S. enterica serovar Typhi		23.06 ± 0.88	U88545
Serratia	S. marcescens		21.48 ± 0.34	EF035134
	S. plymuthica		22.20 ± 0.50	EF064206
Shigella	S. dysenteriae		20.74 ± 0.55	X96966
Yersinia	Y. enterocolitica		18.55 ± 0.50	AF366378
	Y. pestis		17.35 ± 0.78	AF366383

↵ a Mean ± standard deviation of three independent experiments.

TABLE 2.

Primers and probes used in this study

Primer or probe	Sequence (5′→3′)^a	T_m^b (°C)	Position
Forward primer (P967F)	CAACGCGAAGAACCTTACC	59.2	967-985
Reverse primer (P1194R)	ACGTCATCCCCACCTTCC	59.0	1194-1177
Gram-positive probe	5′-FAM-ACGACAACCATGCACCACCTG-TAMRA-3′	69.2	1076-1056^c
Gram-negative probe	5′-HEX-ACGACAGCCATGCAGCACCT-TAMRA-3′	68.0	1065-1045^d

↵ a FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine; HEX, 6-carboxyhexachlorofluorescein.
↵ b T _ms (melting temperatures) were calculated with Primer Express software (Applied Biosystems).
↵ c Numbered according to the 16S rRNA gene of S. aureus (GenBank accession number X68417 ).
↵ d Numbered according to the 16S rRNA gene of E. coli (GenBank accession number AF233451 ).

TABLE 3.
Overall results obtained by GSPBRT-PCR compared to blood culture^a
GSPBRT-PCR result Blood culture result Total
Positive Negative
Positive 34 16 50
Negative 0 550 550
Total 34 566 600
↵ a McNemar's test; P = 0.00003.

TABLE 4.

Summary of 34 culture-positive samples and the corresponding GSPBRT-PCR results

Culture identification	No. of samples	GSPBRT-PCR result
Culture identification	No. of samples	Gram^+/−	Ranged C_T value	Avg C_T value
CoNS	20	+	27.02-34.65	32.12
S. aureus	3	+	26.17-31.78	29.79
E. coli	2	−	27.89-32.56	30.23
K. pneumoniae	2	−	29.46-33.16	31.31
C. freundii	1	−	31.37	31.37
S. agalactiae	1	+	31.50	31.50
E. avium	1	+	28.21	28.21
E. faecium	1	+	29.80	29.80
Corynebacterium sp.	1	+	33.21	33.21
A. lwoffii	1	−	33.56	33.56
S. paucimobilis	1	−	34.00	34.00

TABLE 5.

PCR-positive/culture-negative samples analyzed by sequence and clinical diagnosis

Case no.	C_T	No. of CFU	Gram^+/−	Sequence	Clinical symptom	CRP (mg/liter)^b
1	27.31	6.30 × 10³	−	H. influenzae	Sepsis	30
2	28.51	3.80 × 10³	+	S. pneumoniae	Pneumonia	8
3	30.74	8.30 × 10²	+	S. pneumoniae	Pneumonia	24
4	30.84	7.76 × 10²	+	S. haemolyticus	Hyperbilirubinemia	8
5^a	31.03	6.76 × 10²	+	L. monocyogenes	Sepsis	68
6	31.18	6.17 × 10²	+	S. agalactiae	Sepsis	15
7	31.56	4.72 × 10²	+	S. epidermidis	Cyanosis	53
8	32.65	2.25 × 10²	+	Equivocal	Sepsis	4
9^a	32.65	2.25 × 10²	+	Equivocal	Sepsis	102
10^a	32.70	2.18 × 10²	+	Equivocal	Pneumonia	14
11	32.82	2.01 × 10²	+	Failed	Premature infant	6
12	33.30	1.44 × 10²	+	Failed	Pneumonia	2
13^a	33.24	8.32 × 10¹	−	Failed	Sepsis	28
14	34.21	7.76 × 10¹	+	Failed	Sepsis	34
15	34.43	6.70 × 10¹	+	Failed	Sepsis	45
16^a	35.00	2.23 × 10¹	−	Failed	Blood in stool	134

↵ a Patient was pretreated with antibiotics.
↵ b CRP, complement-reactive protein. The detection limit was 1 mg/liter, and a serum value of >8 mg/liter was defined as abnormally elevated.