Jenni Antikainen

Antimicrobials Increase Travelers' Risk of Colonization by Extended-Spectrum Betalactamase-Producing Enterobacteriaceae

Colonized travelers contribute to the pandemic spread of resistant intestinal bacteria. This study is the first to show that antimicrobial use during travel predisposes to colonization by intestinal extended-spectrum beta-lactamase-producing Enterobacteriaceae. Travelers refrain from taking unnecessary antibiotics.

Characterization of the Collagen-Binding S-Layer Protein CbsA of Lactobacillus crispatus

The cbsA gene of Lactobacillus crispatus strain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli. The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. The cbsA gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three other Lactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsA where deletions and mutations were introduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA. RNA analysis showed that cbsA, but not cbsB, was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of L. crispatus.

Domains in the S‐layer protein CbsA of Lactobacillus crispatus involved in adherence to collagens, laminin and lipoteichoic acids and in self‐assembly

The protein regions in the S‐layer protein CbsA of Lactobacillus crispatus JCM 5810, needed for binding to collagens and laminin, anchoring to bacterial cell wall, as well as self‐assembly, were mapped by deletion analysis of His‐tagged peptides isolated from Escherichia coli and by heterologous expression on Lactobacillus casei. Mature CbsA is 410 amino acids long, and stepwise genetic truncation at both termini revealed that the region 32–271 carries the infor‐mation for self‐assembly of CbsA into a periodic structure. The lactobacillar S‐layer proteins exhibit sequence variation in their assembly domain, but the border regions 30–34 and 269–274 in CbsA are conserved in valine‐rich short sequences. Short deletions or substitutions at these regions affected the morphology of His‐CbsA polymers, which varied from sheet‐like to cylindrical tubular polymers, and further truncation beyond the DNA encoding residues 32 and 271 leads to a non‐periodic aggregation. The self‐assembly of the truncated peptides, as seen by electron microscopy, was correlated with their behaviour in a cross‐linking study. The shorter peptides not forming a regular polymer were observed by the cross‐linking study and mass spectrometry to form dimers, trimers and tetramers, whereas the other peptides were cross‐linked to large multimers only. Binding of solubilized type I and IV collagens was observed with the His‐CbsA peptides 1–274 and 31–287, but not with the smaller peptides regardless of their ability to form regular polymers. Strain JCM 5810 also adheres to immobilized laminin and, in order to analyse the possible laminin binding by CbsA, cbsA and its fragments were expressed on the surface of L. casei. Expression of the CbsA peptides 1–274, 1–287, 28–287 and 31–287 on L. casei conferred adhesiveness to both laminin and collagen immobilized on glass as well as to laminin‐ and collagen‐containing regions in chicken colon and ileum. The C‐terminal peptides 251–410 and 288–410 bound to L. crispatus JCM 5810 cells from which the S‐layer had been depleted by chemical extraction, whereas no binding was seen with the His‐CbsA peptides 1–250 or 1–269 or to cells with an intact S‐layer. The His‐CbsA peptides 251–410 and 288–410 bound to teichoic acids of several bacterial species. The results show that CbsA is an adhesive complex with an N‐terminal assembly domain exhibiting affinity for pericellular tissue components and a cationic C‐terminal domain binding to negatively charged cell wall components.

pH-Dependent Association of Enolase and Glyceraldehyde-3-Phosphate Dehydrogenase of Lactobacillus crispatus with the Cell Wall and Lipoteichoic Acids

The plasminogen-binding proteins enolase and glyceraldehyde-3-phosphate dehydrogenase of Lactobacillus crispatus were localized on the cell surface at pH 5 but released into the medium at an alkaline pH. These proteins bound to lipoteichoic acids at a pH below their isoelectric point. The results indicate that lactobacilli rapidly modify their surface properties in response to changes in pH.

New 16-plex PCR method for rapid detection of diarrheagenic Escherichia coli directly from stool samples.

A rapid 16-plex polymerase chain reaction (PCR) suitable for routine diagnostics of diarrheagenic Escherichia coli (EHEC, EIEC, EAEC, ETEC, and EPEC) was developed, validated with control strains, and tested with 250 diarrhoeal stool samples. The specificity was 100% when tested with 289 control bacterial strains, and the analytical sensitivity of automated DNA extraction directly from stool samples was made by boiling the bacterial culture (104–105 colony forming units/ml). The assay design starting directly from extraction of stool DNA allowed same day analysis without compromising sensitivity and specificity, which makes it superior compared to PCR after culturing the bacteria. The 16-plex PCR method demonstrated high prevalence of diarrheagenic E. coli in stool samples of patients returning from abroad (39.0%) in contrast to the patients with no travel history (8.7%; p < 0.001). The high prevalence of diarrheagenic E. coli suggests that their screening should be part of normal diarrhoea diagnostics, at least in the leading diagnostic laboratories.

Glutamine Synthetase and Glucose-6-Phosphate Isomerase Are Adhesive Moonlighting Proteins of Lactobacillus crispatus Released by Epithelial Cathelicidin LL-37

Glutamine synthetase (GS) and glucose-6-phosphate isomerase (GPI) were identified as novel adhesive moonlighting proteins of Lactobacillus crispatus ST1. Both proteins were bound onto the bacterial surface at acidic pHs, whereas a suspension of the cells to pH 8 caused their release into the buffer, a pattern previously observed with surface-bound enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of L. crispatus. The pH shift was associated with a rapid and transient increase in cell wall permeability, as measured by cell staining with propidium iodide. A gradual increase in the release of the four moonlighting proteins was also observed after the treatment of L. crispatus ST1 cells with increasing concentrations of the antimicrobial cationic peptide LL-37, which kills bacteria by disturbing membrane integrity and was here observed to increase the cell wall permeability of L. crispatus ST1. At pH 4, the fusion proteins His(6)-GS, His(6)-GPI, His(6)-enolase, and His(6)-GAPDH showed localized binding to cell division septa and poles of L. crispatus ST1 cells, whereas no binding to Lactobacillus rhamnosus GG was detected. Strain ST1 showed a pH-dependent adherence to the basement membrane preparation Matrigel. Purified His(6)-GS and His(6)-GPI proteins bound to type I collagen, and His(6)-GS also bound to laminin, and their level of binding was higher at pH 5.5 than at pH 6.5. His(6)-GS also expressed a plasminogen receptor function. The results show the strain-dependent surface association of moonlighting proteins in lactobacilli and that these proteins are released from the L. crispatus surface after cell trauma, under conditions of alkaline stress, or in the presence of the antimicrobial peptide LL-37 produced by human cells.

Identification of a high-molecular-mass Lactobacillus epithelium adhesin (LEA) of Lactobacillus crispatus ST1 that binds to stratified squamous epithelium

Lactobacilli belong to the normal gastrointestinal and genital tract microbiota of human and animal hosts. Adhesion is important for bacterial colonization; however, only a few Lactobacillus adhesins have been identified so far. We studied extracted surface proteins from an adhesive Lactobacillus crispatus strain, ST1, which efficiently colonizes the chicken alimentary tract, for their binding to tissue sections of the chicken crop, and identified a novel high-molecular-mass repetitive surface protein that shows specific binding to stratified squamous epithelium. The adhesin binds to both crop epithelium and epithelial cells from human vagina, and was named Lactobacillus epithelium adhesin (LEA). Expression of LEA is strain-specific among L. crispatus strains and corresponds directly to in vitro bacterial adhesion ability. The partial sequence of the lea gene predicts that the LEA protein carries an N-terminal YSIRK signal sequence and a C-terminal LPxTG anchoring motif, as well as a highly repetitive region harbouring 82 aa long repeats with non-identical sequences that show similarity to Lactobacillus Rib/alpha-like repeats. LEA-mediated epithelial adherence may improve bacterial colonization in the chicken crop and the human vagina, which are the natural environments for L. crispatus.

Comparison of repetitive extragenic palindromic sequence-based PCR with PCR ribotyping and pulsed-field gel electrophoresis in studying the clonality of Clostridium difficile.

Clostridium difficile infection is most often induced by antibiotic treatment. Recently, morbidity and mortality resulting especially from C. difficile PCR ribotype 027 have increased significantly. In addition, more severe disease has been associated with C. difficile PCR ribotype 078 strains. Thus, reliable typing methods for epidemic control are needed. In the present study, we compared an automated repetitive extragenic palindromic sequence-based PCR (rep-PCR) method (DiversiLab; Bacterial Barcodes, Inc., Athens, GA, USA) to PCR ribotyping and pulsed-field gel electrophoresis (PFGE) typing using 205 isolates of C. difficile (including 24 previously characterized isolates). Among the 181 clinical isolates, a total of 31 different PCR ribotypes, 38 different PFGE types and subtypes and 28 different rep-PCR types were found. Six major rep-PCR groups (DL1-DL6) harboured 86% of the clinical isolates. All isolates belonging to PCR ribotypes 027 and 001 clustered in their own rep-PCR groups, enabling us to screen out the hypervirulent ribotype 027 strain. Within the PCR ribotype 001, four subgroups were found using rep-PCR. Overall, in 75% (135/181) of the isolates, the classification attributed following rep-PCR and PCR ribotyping was comparable. In conclusion, the automated rep-PCR-based typing method represents an option for first-line molecular typing in local clinical microbiology laboratories. The method was easy to use as well as rapid, requiring less hands-on time than PCR ribotyping or PFGE typing. The conventional PCR ribotyping or PFGE, however, are needed for confirmatory molecular epidemiology. In addition, more epidemiology-oriented studies are needed to examine the discriminatory power of automated rep-PCR with isolates collected from a larger geographical area and during a longer period of time.

Novel Microbiological and Spatial Statistical Methods to Improve Strength of Epidemiological Evidence in a Community-Wide Waterborne Outbreak

Failures in the drinking water distribution system cause gastrointestinal outbreaks with multiple pathogens. A water distribution pipe breakage caused a community-wide waterborne outbreak in Vuorela, Finland, July 2012. We investigated this outbreak with advanced epidemiological and microbiological methods. A total of 473/2931 inhabitants (16%) responded to a web-based questionnaire. Water and patient samples were subjected to analysis of multiple microbial targets, molecular typing and microbial community analysis. Spatial analysis on the water distribution network was done and we applied a spatial logistic regression model. The course of the illness was mild. Drinking untreated tap water from the defined outbreak area was significantly associated with illness (RR 5.6, 95% CI 1.9–16.4) increasing in a dose response manner. The closer a person lived to the water distribution breakage point, the higher the risk of becoming ill. Sapovirus, enterovirus, single Campylobacter jejuni and EHEC O157:H7 findings as well as virulence genes for EPEC, EAEC and EHEC pathogroups were detected by molecular or culture methods from the faecal samples of the patients. EPEC, EAEC and EHEC virulence genes and faecal indicator bacteria were also detected in water samples. Microbial community sequencing of contaminated tap water revealed abundance of Arcobacter species. The polyphasic approach improved the understanding of the source of the infections, and aided to define the extent and magnitude of this outbreak.

High number of diarrhoeal co-infections in travellers to Benin, West Africa

BackgroundTravellers’ diarrhoea (TD) is the most frequent health problem among travellers to the tropics. Using routine techniques, the aetiology mostly remains unresolved, whereas modern molecular methods enable reducing the number of equivocal cases considerably. While many studies address the aetiology of TD in Asian, Central American and North African tourist resorts, only few focus on Western Africa.MethodsStool samples from 45 travellers travelling in Benin, West Africa, were analyzed by a new multiplex qPCR assay for Salmonella, Yersinia, Campylobacter, Vibrio cholerae, Shigella or enteroinvasive (EIEC), enterohaemorrhagic (EHEC), enterotoxigenic (ETEC), enteroaggregative (EAEC), and enteropathogenic Escherichia coli (EPEC).ResultsAll 18 pre-travel samples proved negative for bacterial pathogens. Of the 39/45 (87%) travellers having had TD, EPEC was detected in post-travel samples in 30 (77%) cases, EAEC in 23 (59%), ETEC in 22 (56%), Shigella or EIEC in 7 (18%), EHEC in two (5%), and Salmonella in one (3%). In 31(79%) of the TD cases two or more bacterial pathogens were identified. Two (8%) samples remained negative: both patients had taken antimicrobials for TD.ConclusionsEPEC, EAEC and ETEC were the most common findings. 79% of the cases had a co-infection. As modern diagnostics reveals in most patients a multitude of pathogens, the role of each pathogen should be re-evaluated.