Effects of antibiotic therapy on the gastrointestinal microbiota and the influence of Lactobacillus casei

Figures & data

Table 1. Characterisation of participant groups.

Group	Treatment and/or intervention
AP	Antibiotic treatment and intake of a probiotic drink containing Lactobacillus casei Shirota
A	Antibiotic treatment only
P	Intake of a probiotic drink containing L. casei Shirota
C	Control group

Table 2. Primers and TaqMan^®-probes targeting 16rRNA coding regions of bacteria.

Target organism	Primer/probe	Sequence (5′–3′)	Size (bp)	Conc. [pmol/µL]	References
All bacteria	BAC-338-F	ACT CCT ACG GGA GGC AG	468	10	Yu, Lee, Kim, and Hwang (2005)
	BAC-805-R	GAC TAC CAG GGT ATC TAA TCC		10
	BAC-516-P	(6-FAM)-TGC CAG CAG CCG CGG TAA TAC-(BHQ-1)		2
Clostridium cluster IV (Clostridium leptum)	sg-Clept-F	GCA CAA GCA GTG GAG T	239	4	Matsuki, Watanabe, Fujimoto, Takada, and Tanaka (2004)
	sg-Clept-R	CTT CCT CCG TTT TGT CAA		4
	Clept-Pa	(FAM)-AGG GTT GCG CTC GTT-(BHQ-1)		2	Zwielehner et al. (2009)
All bacteroides	AllBac296f	GAG AGG AAG GTC CCC CAC	106	3	Layton et al. (2006)
	AllBac412r	CGC TAC TTG GCT GGT TCA G		3
	AllBac375Bhqr	(6-FAM)-CCA TTG ACC AAT ATT CCT CAC TGC TGC CT-(BHQ-1)		1
Bifidobacterium spp.	Fwd primer	GCG TGC TTA ACA CAT GCA AGT C	125	3	Penders et al. (2005)
	Rev primer	CAC CCG TTT CCA GGA GCT ATT		3
	Probe	(6-FAM)-TCA CGC ATT ACT CAC CCG TTC GCC-(BHQ-1)		1.5
Clostridium cluster XIVa (Lachnos-piraceae)	195-F	GCA GTG GGG AAT ATT GCA	538	7	Meier, Amann, Ludwig, and Schleifer (1999)
	Ccocc-R	CTT TGA GTT TCA TTC TTG CGA A		7	Matsuki et al. (2004)
	Ccocc-P	(6-FAM)-AAA TGA CGG TAC CTG ACT AA-(BHQ-1)		1.5

Table 3. Primers targeting 16rRNA coding regions of bacteria (SYBR^® Green) and butyryl-CoA CoA transferase genes.

Target organism	Primer	Sequence (5′–3′)	Size (bp)	Conc. [pmol/µL]	References
Clostridium cluster XI	C Cluster XI F	ACG CTA CTT GAG GAG GA	180	3	Song, Liu, and Finegold (2004)
	C Cluster XI R	GAG CCG TAG CCT TTC ACT
Enterobacteriaceae	LPW69	AGC ACC GGC TAA CTC CGT	492–509	3	Woo, Leung, Leung, & Yuen (2000)
	pB-00608 r	GAA GCC ACG CCT CAA GGG CAC AA	834–856	3	Ootsubo et al. (2002)
Lactobacillus spp.	Lac1	AGC AGT SGG GAA TCT TCC A	352–700	4	Walter et al. (2001)
	Lac2	ATT YCA CCG CTA CAC ATG		4

Table 4. Group distribution concerning antibiotic-associated diarrhoea (AAD) and Clostridium difficile infection (CDI).

	A (no intervention)	AP (intervention)
Euroclone^®C. difficile A/B kit	1 (n=15)	0 (n=10)
Clinical study	AAD: 63 (n=338)(18.5%)CDI: 21 (6.2%)	AAD: 17 (n=340) (5%)CDI: 1 (0.3%)
	AAD: RRR 73.2% (p<0.001)
	CDI: RRR 95.3% (p<0.001)

A: antibiotic treatment; AP: antibiotic treatment and intake of a probiotic drink containing Lactobacillus casei Shirota; n: sample size; RRR: relative risk reduction.

Figure 1. 16S rRNA qPCR of (A) total bacteria, (B) Clostridium Cluster IV, (C) Clostridium cluster XI, (D) Bifidobacterium spp., (E) Enterobacteriaceae, (F) Lactobacillus spp.

(A: antibiotic treatment; AP: antibiotic treatment and intake of a probiotic drink containing Lactobacillus casei Shirota; P: intake of a probiotic drink containing L. casei Shirota; C: control group; 1: day 0, 2: day 3, 3: day 5).

Figure 2. PCR-DGGE fingerprinting of 16S rRNA coding regions amplified with primer pair 341GC-518.

(A: antibiotic treatment; AP: antibiotic treatment and intake of a probiotic drink containing Lactobacillus casei Shirota; P: intake of a probiotic drink containing L. casei Shirota; C: control group; 1: day 0, 3: day 5, SL: standard line).

Figure 3. Percentage of bacterial subgroups in relation to the analysed bacteria. In patients under antibiotic treatment, there were more bacteria which cannot be identified by the primers used in this study.

(A: antibiotic treatment; AP: antibiotic treatment and intake of a probiotic drink containing Lactobacillus casei Shirota; P: intake of a probiotic drink containing L. casei Shirota; C: control group; 1: day 0, 2: day 3, 3: day 5).

Figure 4. Abundance of butyryl-CoA CoA transferase genes in group A (antibiotic treatment), AP (antibiotic treatment and intake of a probiotic drink containing Lactobacillus casei Shirota), P (intake of a probiotic drink containing L. casei Shirota) and C (control group) (1: day 0, 2: day 5, 3: day 5). Amplification was done by 16S rRNA qPCR with primer pair BCoATscrF/R.

Yu, Y., Lee, C., Kim, J., & Hwang, S. (2005). Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnology and Bioengineering, 89(6), 670–679.

 PubMed Web of Science ®Google Scholar

Matsuki, T., Watanabe, K., Fujimoto, J., Takada, T., & Tanaka, R. (2004). Use of 16S rRNA gene-targeted group-specific primers for real-time PCR analysis of predominant bacteria in human feces. Applied and Environmental Microbiology, 70(12), 7220–7228.

 PubMed Web of Science ®Google Scholar

Zwielehner, J., Liszt, K., Handschur, M., Lassl, C., Lapin, A., & Haslberger, A.G. (2009). Combined PCR-DGGE fingerprinting and quantitative-PCR indicates shifts in fecal population sizes and diversity of Bacteroides, bifidobacteria and Clostridium cluster IV in institutionalized elderly. Experimental Gerontology, 44(6–7), 440–446.

 PubMed Web of Science ®Google Scholar

Layton, A., Mckay, L., Williams, D., Garrett, V., Gentry, R., & Sayler, G. (2006). Development of Bacteroides 16S rRNA gene TaqMan-based real-time PCR assays for estimation of total, human, and bovine fecal pollution in water. Applied and Environmental Microbiology, 72(6), 4214–4224.

 PubMed Web of Science ®Google Scholar

Penders, J., Vink, C., Driessen, C., London, N., Thijs, C., & Stobberingh, E.E. (2005). Quantification of Bifidobacterium spp., Escherichia coli and Clostridium difficile in faecal samples of breast-fed and formula-fed infants by real-time PCR. FEMS Microbiology Letters, 243(1), 141–147.

 PubMed Web of Science ®Google Scholar

Meier, H., Amann, R., Ludwig, W., & Schleifer, K.H. (1999). Specific oligonucleotide probes for in situ detection of a major group of gram-positive bacteria with low DNA G + C content. Systematic and Applied Microbiology, 22(2), 186–196.

 PubMed Web of Science ®Google Scholar

Song, Y., Liu, C., & Finegold, S.M. (2004). Real-time PCR quantitation of clostridia in feces of autistic children. Applied and Environmental Microbiology, 70(11), 6459–6465.

 PubMed Web of Science ®Google Scholar

Woo, P.C., Leung, P.K., Leung, K.W., & Yuen, K.Y. (2000). Identification by 16S ribosomal RNA gene sequencing of an Enterobacteriaceae species from a bone marrow transplant recipient. Molecular Pathology, 53(4), 211–215.

 PubMedGoogle Scholar

Otsubo, M., Shimizu, T., Tanaka, R., Sawabe, T., Tajima, K., Yoshimizu, M., et al. (2002). Oligonucleotide probe for detecting Enterobacteriaceae by in situ hybridization. Journal of Applied Microbiology, 93(1), 60–68.

 PubMed Web of Science ®Google Scholar

Walter, J., Hertel, C., Tannock, G.W., Lis, C.M., Munro, K., & Hammes, W.P. (2001). Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis. Applied and Environmental Microbiology, 67(6), 2578–2585.

 PubMed Web of Science ®Google Scholar