666

Molecular Detection of Bacterial Contamination in Gnotobiotic Rodent UnitsChristopher D. Packey, Ian M. Carroll, Maureen A. Bower, Susan L. Tonkonogy, R.Balfour Sartor

Gnotobiotic rodents facilitate the study of commensal bacteria and their roles in humanphysiology and pathophysiology. To ensure sterility, animals must be screened frequentlyfor contamination by the traditional approach of culturing and Gram staining of feces. Yetmany bacteria are uncultivable, fecal Gram stains can be difficult to interpret, and thesemethods are labor-intensive and time-consuming. Aims: To develop molecular methods ofdetecting contamination in gnotobiotic units. Methods: We collected fresh fecal pellets frommice housed in germ free (GF) isolators (N=8), a contaminated ex-GF isolator, an isolatorcolonized with 2 bacterial species, and a specific pathogen free (SPF) animal room (N=4).DNA from fecal samples was extracted using a phenol/chloroform extraction method com-bined with physical disruption of bacterial cells. Polymerase chain reactions (PCR) werecarried out using templates of fecal DNA and primers that amplify the genes encoding the16S rDNA from all bacterial groups. The numbers of 16S rDNA gene copies in eachDNA sample were then quantified using quantitative Real-Time PCR (qPCR) with the sametemplates and primers and the appropriate set of standards. Finally, the 16S rDNA genewas amplified from fecal DNA from contaminated ex-GF mice, sequenced, and cross-referenced with a computerized data base to identify the bacterial species. Results: PCRyielded amplicons from isolated fecal DNA from, in descending order of intensity, all SPFmice, dual associated mice, and contaminated ex-GF mice, as well as from mice from onepresumed GF isolator. Fecal DNA from mice from the other 7 GF isolators did not yieldamplicons. qPCR confirmed that the level of bacteria in the mouse feces from the questionableisolator, (4.0 X 108 copies 16S rDNA gene/μg feces), was 210-fold greater than the levelsin the other 7 GF isolators, (1.9 X 106 copies/μg, p < 0.0001), and was comparable to thelevel in the known contaminated isolator, (3.0 X 108 copies/μg.) The source of DNA inmouse feces from GF isolators detected by qPCR was dead bacteria in autoclaved food.Upon review, both cultures and Gram stains from the isolator that we determined to becontaminated based on PCR and qPCR results had been deemed indeterminate by gnotobioticfacility staff. Sequencing the 16S rDNA gene revealed the contaminating bacterial speciesin the ex-GF isolator to be Bacillus simplex. Conclusions: We developed molecular methodsto screen for contamination in gnotobiotic units that are more reliable and less time-consuming than current traditional methods and can be used to identify the contaminat-ing species.

667

Temporal Stability of Faecal Microbiota By Inter Spacer rDNA BacterialProfiling (IS-pro)Matthijs E. Grasman, Andries E. Budding, Chris J. Mulder, Paul H. Savelkoul, Adriaan A.van Bodegraven

Introduction Faecal microbiota is considered to be relatively stable in time within healthyindividuals, unlike the markedly fluctuating microbiota in IBD patients, particularly duringflare-up. Since most intestinal bacteria are uncultivable, we developed a rapid, highly reprodu-cible molecular method for high-throughput bacterial profiling: IS-pro. Aims To determineshort-term and long-term stability of faecal microbiota of healthy individuals by means ofIS-pro. Materials and methods Short-term intestinal microbiota variation was assessed withday-to-day faecal samples in one individual over a three-week period. General compositionof diet was different between weekdays and weekends. In addition, eight individuals providedtwo faecal samples with an interval of one month. Long-term variation was assessed in eighthealthy individuals who provided two faecal samples with an interval up to two years.Samples were analysed by use of IS-pro. IS-pro is based on two features of the bacterialgenome: species-specific length of the inter spacer (IS) region between the 16s and 23srDNA and phylum-specific labelled primer sequences. With these specific labelled primers,species can be directly sorted into either Firmicutes/Actinobacteria or Bacteroidetes phylum.By colour and size sorting of amplified fragments specific bacterial profiles are created.Results Short-term: mean similarity of day-to-day bacterial profiles was 86% (79-92%).Substantial variation was observed between pre- and post-weekend profiles (70-74% similar-ity), in particular in species belonging to the Firmicutes phylum (53-59% similarity). Dietseemed to account for this short-term difference. Mean similarity of faecal profiles after onemonth in eight individuals was 71% (48-82%) Long-term: each of eight individuals showeda unique bacterial profile, sharing few species with other individuals. In cluster analysis,faecal profiles were host-specific, even after an interval between samples up to two years.Intra-individual profiles were relatively stable in time with a mean similarity of 70% (48-84%). Conclusion By use of IS-pro, faecal microbiota showed a relatively high day-to-daystability within an individual: 79-92% similarity. Short-term (1 month) and long-term (upto 2 years) stability was equal, both with similarity around 70%. Inter-individual similarity waslow. Diet seems to alter faecal bacterial composition. Short-term and long-term differences,although limited and stable, may thwart analysis of disease-specific changes in intestinal mic-robiota.

668

Regulation of Urease Membrane Recruitment and Activation By theCytoplasmic Histidine Kinase, HP0244 of Helicobacter pylori: A RapidResponse to AcidityGeorge Sachs, Yi Wen, Jing Feng, Siddharth Singh, Elizabeth A. Marcus, David R. Scott

Background: The neutralophile, H. pylori, is the only known pathogen colonizing thenormal human stomach. It uses unique acid acclimation rather than acid tolerant/resistancemechanisms. Important for this acclimation is very high UreA/UreB apoenzyme content andurea access to urease via the H+-gated urea channel, UreI. Since the pH of its gastric habitatcan fall rapidly from pH ~5.0 to pH < 3.0 (Scott et al PNAS 2007), a rapid reaction to highacidity is needed. Deletion of the cytoplasmic histidine kinase, HP0244, abolishes survivalat pH 2.5 with 10 mM urea in contrast to wildtype (Wen et al J Bact 2008) suggesting that

A-103 AGA Abstracts

this kinase might regulate acid responses. Aim: To identify the rapid response of ureaseapoenzyme regulated by HP0244 and its functional consequence. Methods: Wildtype andHP0244 mutants were exposed to pH 4.5 for 30 min and membrane recruitment of ureaseapoenzyme and accessory genes and urease activity were compared in a highly purifiedmembrane preparation. Since the addition of 50mM NH4Cl results in, first, influx of NH3

producing alkalization and then NH4+ with re-acidification, the role of UreI in transport of

NH3 and NH4+ was determined by the effect of 50mM NH4Cl on pHin and membrane

potential using BCECF-AM and DiSC3(5) fluorimetry in wildtype and ureI deletion mutants.Protein synthesis was measured by 35S-methionine incorporation. Results: There is UreIdependent recruitment of UreA, UreB and UreE to the membrane at pH 4.5out independentof protein synthesis since it is not affected by chloramphenicol. There is a ~ twofold increasein membrane urease activity and equivalent depletion of cytosolic urease. Deletion of HP0244abolished recruitment. Membrane potential collapsed at pH 2.5 in HP0244 deletion mutantswith 10mM urea but not in the wildtype, accounting for the loss of survival. UreI is ableto transport NH3 as evidenced by the decreased rate of alkalization following NH4Cl additionin the presence of methylamine that competes with NH3. UreI also transports NH4

+ sincecytoplasmic re-acidification and membrane repolarization after NH4Cl addition are abolishedin ureI deletion mutants. Conclusions: UreI-dependent membrane recruitment and activa-tion of urease is regulated by the histidine kinase, HP0244. Formation of this pH-dependentcomplex allows direct access of urea to urease via UreI and efflux of urease-generatedNH3 and NH4

+ through UreI. This enables rapid periplasmic neutralization and consistentcytoplasmic pH homeostasis. Functioning of this complex appears essential for survival atthe pH of the site of H. pylori habitation. HP0244 is a therefore new target for eradication.

669

H. pylori Peptide HP(2-20) Promotes Healing of Injured Gastric Mucosa ByInteracting with Formyl-Peptyde ReceptorsMarco Romano, Nella Prevete, Francesca W. Rossi, Felice Rivellese, Fiamma Salerno,Gabriele Delfino, Bianca Liccardo, Stefania Staibano, Giuseppe D'Argenio, Gianni Marone,Amato De Paulis

Background & Aims: Peptide Hp(2-20) derived from the N-terminal sequence of Helicobacterpylori (H. pylori) ribosomal protein L1 possesses a broad-spectrum of anti-microbial activity.In addition to its antimicrobial action, Hp(2-20), through the interaction with formyl-peptidereceptors (FPRs), exerts several immunomodulatory properties in eukaryotic cells. It hasrecently been shown that activation of FPRs facilitates intestinal epithelial cell restitution.We studied whether Hp(2-20) accelerated healing of injured gastric mucosa and assessedthe mechanism(s) underlying any such effect. Methods: 1) Gastric epithelial cell proliferationwas assessed by flow cytometry and cell migration was determined by chemotaxis assayusing a modified Boyden chamber technique, in two gastric cell lines, i.e MKN28 and AGScells; 2) mRNA expression was assessed by RTPCR; 3) protein expression was determinedby Western blot analysis; 4) the effect of Hp(2-20) on mucosal healing was assessed in ratsfollowing indomethacin-induced injury both macroscopically and microscopically. Results:1) FPR-like receptors are expressed in both cell lines, both at the mRNA and protein level;2) Hp(2-20) significantly stimulates migration and proliferation of gastric epithelial cells; 3)this effect is specifically mediated by the interaction with FPRL1 and FPRL2; 4) Hp(2-20)causes activation of FPRs-related downstream signalling pathways inducing phosphorylationof MAPK, Akt, and STAT3; 5) Hp(2-20) upregulates mRNA expression and stimulatessecretion of VEGF; 5) Hp(2-20) significantly accelerates healing of rat gastric mucosa follow-ing injury brought about by indomethacin causing an approximately 40% reduction ingastric mucosal injury compared with control, both at the macroscopic and microscopiclevel. Conclusions: 1) FPRs are expressed in human gastric epithelial cells; 2) H. pylori-derived peptide Hp(2-20) stimulates gastric epithelial cell migration, proliferation and VEGFexpression through interaction with FPRL1 and FPRL2 and activates FPRs-related down-stream signalling pathways; 3) oral delivery of Hp(2-20) significantly improves gastricmucosal healing following damage brought about by indomethacin in the rat. This studyprovides further evidence to the complexity of the relationship between H. pylori and itshost and opens possible scenarios where a bacterial product may prove of use in the therapyand/or prevention of exogenous injury to the human stomach.

670

CagA SHP2 Binding in Natural Infections By Western and East AsianHelicobacter pylori StrainsHiroaki Ogiwara, Mitsushige Sugimoto, Ellen J. Beswick, Victor E. Reyes, David Y.Graham, Yoshio Yamaoka

Background and aims: Helicobacter pylori CagA protein tyrosine-phosphorylation site is atEPIYA motifs within the 3' repeat region of the gene. These repeat regions have been classifiedbased on sequences surrounding EPIYA motifs into types A, B, C, and D. EPIYA-A and -Bare conserved in both Western and East Asian strains; EPIYA-C is specific for Western strainsand EPIYA-D is specific for East Asian strains. CagA transfection experiments reported thatEast Asian type CagA (e.g., ABD) exhibited stronger SHP-2 binding activity than Westerntype (e.g., ABC, ABCC). This difference was related to structural differences between EPIYA-C and -D motifs and it was hypothesized that SHP-2 binding activity might relate to thehigh incidence of gastric cancer in East Asia compared to Western countries. We investigatedCagA-SHP-2 binding during natural infection experiments using live Western and East AsianH. pylori. Methods: 27 East Asian and 26 Western H. pylori with various numbers of EPIYAmotifs were co-cultured with gastric epithelial AGS cells. CagA levels in the cell lysates weremeasured by immunoblot and phospho-CagA levels and CagA-SHP2 binding by immunopre-cipitation. Results: Western and East Asian strains produced similar amounts of CagAprotein in AGS cells irrespective of the number of EPIYA motifs. The amount of H. pyloriattached to the AGS cells was also equal irrespective of the origin of strains and the numberof EPIYA motifs. In contrast, significantly higher total CagA protein levels translocated intoAGS cells from Western strains compared to East Asian strains. Phospho-CagA and SHP-2bound CagA levels showed similar patterns to total CagA protein levels and the levelsdirectly correlated with the number of EPIYA-C motifs among Western strains. Importantly,translocated CagA protein levels were significantly higher in ABC than in ABD type H. pylori

AG

AA

bst

ract

s