Quantification of Intestinal Lactobacillus Species in Children with Functional Constipation by Quantitative Real-Time PCR

Figures & data

Table 1 List of Primers Used in This Study

Specificity	Primers	Sequence (5ʹ→3ʹ)	Annealing T (°C)	Amplicon Length (bp)	Target Gene	Genome Copy Number	Reference
L. casei	LcasF LcasR	TTTGAGGGGACGACCCTCAAGCA CGCCGACAAGCTATGAATTCACTTG	55	339	16S-23S spacer region	3	Our study
L. paracasei	PC2a CPRrev	GGATTGGGTTTTGCGTGATGGTCGC TGCATTTCCCCGCTTTCATGACT	68	261	mutL	1	7
L. rhamnosus	LrhamF LrhamR	GGACAGGTAGAAAGTCAAACGA GCTGACCGTAAACGCAATCTTAG	65	186	mutL	1	8
L. plantarum	Lp-F Lp-R	AAAATCATGCGTGCGGGTAC ATGTTGCGTTGGCTTCGTCT	55	210	pyrG	1	9
L. reuteri	Lreu-1 Lreu-4	CAGACAATCTTTGATTGTTTAG GCTTGTTGGTTTGGGCTCTTC	60	305	16S-23S spacer region	6	10
L. acidophilus	LacidoF LacidoR	TGCAAAGTGGTAGCGTAAGC CCTTTCCCTCACGGTACTG	47	207	16S-23S spacer region	4	11
L. fermentum	Lfer-3 Lfer-4	ACTAACTTGACTGATCTACGA TTCACTGCTCAAGTAATCATC	50	191	16S-23S spacer region	5	11

Table 2 Specificity of the L. Casei Primer Pair Used in This Study

Strain	Origin	Specificity
		Conventional PCR	Quantitative PCR
Lactobacillus casei	ATCC 39392	+	+
Lactobacillus paracasei	ATCC 25598	–	–
Lactobacillus rhamnosus	PTCC 1637	–	–
Lactobacillus plantarum	PTCC 1058	–	–
Lactobacillus reuteri	PTCC 1655	–	–
Lactobacillus fermentum	PTCC 1638	–	–
Lactobacillus acidophilus	PTCC 1643	–	–
Bifidobacterium bifidum	PTCC 1644	–	–
Bifidobacterium breve	ATCC 15700	–	–
Bifidobacterium longum	ATCC 15707	–	–
Bifidobacterium animalis	PTCC 1631	–	–
Escherichia coli	PTCC 1330	–	–
Salmonella typhi	PTCC 1609	–	–
Salmonella enterica	PTCC 1709	–	–
Shigella flexneri	PTCC 1865	–	–
Listeria monocytogenes	PTCC 1783	–	–
Enterobacter aerogenes	PTCC 1221	–	–
Clostridium difficile	ATCC 9689	–	–
Staphylococcus aureus	PTCC 1189	–	–
Bacillus cereus	PTCC 1015	–	–
Enterococcus faecalis	PTCC 1778	–	–
Pseudomonas aeruginosa	PTCC 1074	–	–

Table 3 Demographic and Clinical Characteristics of the Subjects

Parameters	Controls (40) N (%)	Constipated Children (40) N (%)	Total N (%)	P-value
Sex				0.370*
Female	19 (47.5)	23 (57.5)	42(52.5)
Male	21 (52.5)	17 (42.5)	38(47.5)
Age (months)^◊	8.1±3.2	8.1±3.1		0.950^£
Abdominal pain				<0.001^€
Yes	3(7.5)	36(90)	39(48.8)
No	37(92.5)	4(10)	41(51.3)
History of Hard Bowel Movement
Yes	-	28(70)	28(70)
No	-	12(30)	12(30)
Fecal Incontinence
Yes	-	29(72.5)	29(72.5)
No	-	11(27.5)	11(27.5)
History of Fecal Retention
Yes	-	35(87.5)	35(87.5)
No	-	5(12.5)	5(12.5)
Duration of constipation (months)	-	7.4 ± 2.8
Family history of constipation				<0.001*
Yes	14(35)	32(80)	46(57.5)
No	26(65)	8(20)	34(42.5)
Painful Defecation
Yes	-	37(92.5)	37(92.5)
No	-	3(7.5)	3(7.5)

Notes: ^◊Mean ± standard deviation. *Pearson’s chi-square test. ^£Mann–Whitney test. ^€Fisher’s exact test.

Figure 1 Representative standard curves obtained by plotting the average Ct values against the estimated log10 CFU/PCR for L. casei (A), L paracasei (B), L rhamnosus (C), L fermentum (D), L acidophilus (E), L plantarum (F) and L. reuteri (G).

Table 4 Intra-Assay Repeatability, Inter-Assay Reproducibility and Detection Limits of qPCR

Target	Quantities (log10 CFU/gram)	Intra-Assay Repeatability (Ct)			Inter-Assay Reproducibility (Ct)			Detection Limit (log10 CFU/g feces)
		Mean	SD	CV %	Mean	SD	CV %
L. plantarum	4.75	19.57	0.06	0.31	19.55	0.02	0.10	3.86
L. paracasei	4.36	22.49	0.04	0.18	22.49	0.02	0.09	3.75
L. rhamnosus	4.31	22.65	0.05	0.22	22.61	0.02	0.09	3.77
L. reuteri	4.63	22.89	0.02	0.09	22.90	0.02	0.09	2.84
L. casei	4.39	22.47	0.02	0.09	22.48	0.02	0.09	3.80
L. fermentum	4.67	21.44	0.04	0.19	21.45	0.02	0.09	3.00
L. acidophilus	4.21	22.92	0.05	0.22	22.94	0.02	0.09	3.43

Abbreviations: SD, standard deviation; CV, coefficients of variation.

Figure 2 Quantity of seven Lactobacillus species in the feces of constipated children and healthy controls. Statistical significance of observed differences in the amount of Lactobacillus species between both constipated and healthy groups was measured by the Mann–Whitney U-test. Bars represent standard errors. P < 0.05 was marked with one asterisk (∗), and P < 0.2 with two asterisks (∗∗). One-way ANOVA testing shows the significant differences in quantity (log₁₀ CFU/gram) between Lactobacillus species.

Table 5 Prevalence of Lactobacillus Species, Detected by Quantitative PCR

Species	Prevalence n (%) by qPCR
	Non Constipated N (%)	Constipated N (%)	Total N (%)
L. plantarum	10 (12.5)	10 (12.5)	20 (25)
L. paracasei	13 (16.2)	11 (13.7)	24 (30)
L. rhamnosus	6 (7.5)	5 (6.2)	11 (13.7)
L. reuteri	3 (3.7)	4 (5)	7 (8.7)
L. casei	2 (2.5)	3 (3.7)	5 (6.2)
L. fermentum	3 (3.7)	3 (3.7)	6 (7.5)
L. acidophilus	3 (3.7)	4 (5)	7 (8.7)

Table 6 The Quantity of Fecal Lactobacilli in Constipated Children and Healthy Controls

Species	Healthy Controls					Constipated Children					*P-value
	Quantities (log CFU/gram) Max Min		Range	Mean	SD	Quantities (log CFU/gram) Max Min		Range	Mean	SD
L. palntarum	6.39	5.48	0.92	6.01	0.33	4.85	4.31	0.54	4.55	0.19	<0.0001
L. paracasei	6.42	5.49	0.92	6.07	0.30	5.12	3.99	1.12	4.45	0.34	<0.0001
L. rhamnosus	6.39	5.96	0.43	6.25	0.15	4.93	4.50	0.43	4.71	0.17	<0.0043
L. reuteri	6.98	6.82	0.16	6.91	0.08	3.17	2.84	0.98	2.61	0.48	<0.0001
L. fermentum	7.02	6.90	0.11	6.95	0.06	3.58	3.27	0.30	3.46	0.16	<0.0001
L. casei	6.56	6.54	0.01	6.55	0.01	3.88	3.62	0.26	3.77	0.13	<0.2
L. acidophilus	6.51	6.25	0.25	6.35	0.13	3.58	3.40	0.18	3.49	0.09	<0.0001

Notes: *Values of P <0.05 were significantly different.

Abbreviation: SD, standard deviation.