Acute Community-Acquired Diarrhea Requiring Hospital Admission in Swiss Children

In order to ascertain the prevalence of agents that cause childhood diarrheal illness, stool specimens of 312 consecutive children with community-acquired diarrhea requiring admission were evaluated. Pathogens were detected in 166 (53%) of the 312 children (≥2 pathogens in 28 children): Rotavirus (n=75), Salmonella spp. (n=37), Campylobacter spp. (n=24), Shigella spp. (n=5), Giardia spp. (n=4), Yersinia spp. (n=2), Aeromonas spp. (n=15), Cryptosporidium (n=15), enteropathogenic Escherichia coli (n=13), enterotoxigenic E. coli (n=7), and enterohemorrhagic E. coli (n=5). In conclusion, acute childhood diarrheal illness pathogens, such as Aeromonas, Cryptosporidium, and diarrheagenic E. coli, account for a large proportion of patients with a microbiologically positive stool specime

Detection of type III secretion genes as a general indicator of bacterial virulence

Type III secretion systems of Gram-negative bacteria are specific export machineries for virulence factors which allow their translocation to eukaryotic cells. Since they correlate with bacterial pathogenicity, their presence is used as a general indicator of bacterial virulence. By comparing the genetic relationship of the major type III secretion systems we found the family of genes encoding the inner-membrane channel proteins represented by the Yersinia enterocolitica lcrD (synonym yscV) and its homologous genes from other species an ideal component for establishing a general detection approach for type III secretion systems. Based on the genes of the lcrD family we developed gene probes for Gram-negative human, animal and plant pathogens. The probes comprise lcrD from Y. enterocolitica, sepA from enteropathogenic Escherichia coli, invA from Salmonella typhimurium, mxiA from Shigella sonnei, as well as hrcV from Erwinia amylovora. In addition we included as a control probe the flhA gene from E. coli K-12 to validate our approach. FlhA is part of the flagellar export apparatus which shows a high degree of similarity with type III secretions systems, but is not involved in pathogenicity. The probes were evaluated by screening a series of pathogenic as well as non-pathogenic bacteria. The probes detected type III secretion in pathogens where such systems were either known or were expected to be present, whereas no positive hybridization signals could be found in non-pathogenic Gram-negative bacteria. Gram-positive bacteria were devoid of known type III secretion systems. No interference due to the genetic similarity between the type III secretion system and the flagellar export apparatus was observed. However, potential type III secretion systems could be detected in bacteria where no such systems have been described yet. The presented approach provides therefore a useful tool for the assessment of the virulence potential of bacterial isolates of human, animal and plant origin. Moreover, it is a powerful means for a first safety assessment of poorly characterized strains intended to be used in biotechnological applications

Cloning and Characterization of Two Bistructural S-Layer-RTX Proteins from Campylobacter rectus

Campylobacter rectus is an important periodontal pathogen in humans. A surface-layer (S-layer) protein and a cytotoxic activity have been characterized and are thought to be its major virulence factors. The cytotoxic activity was suggested to be due to a pore-forming protein toxin belonging to the RTX (repeats in the structural toxins) family. In the present work, two closely related genes, csxA and csxB (for C. rectus S-layer and RTX protein) were cloned from C. rectus and characterized. The Csx proteins appear to be bifunctional and possess two structurally different domains. The N-terminal part shows similarity with S-layer protein, especially SapA and SapB of C. fetus and Crs of C. rectus. The C-terminal part comprising most of CsxA and CsxB is a domain with 48 and 59 glycine-rich canonical nonapeptide repeats, respectively, arranged in three blocks. Purified recombinant Csx peptides bind Ca(2+). These are characteristic traits of RTX toxin proteins. The S-layer and RTX domains of Csx are separated by a proline-rich stretch of 48 amino acids. All C. rectus isolates studied contained copies of either the csxA or csxB gene or both; csx genes were absent from all other Campylobacter and Helicobacter species examined. Serum of a patient with acute gingivitis showed a strong reaction to recombinant Csx protein on immunoblots

Genetic relatedness within the genus Campylobacter inferred from rpoB sequences

The genus Campylobacter comprises 17 species, some of which are important animal and human pathogens. To gain more insight into the genetic relatedness of this genus and to improve the molecular tools available for diagnosis, a universal sequencing approach was established for the gene encoding the beta-subunit of RNA polymerase (rpoB) for the genus Campylobacter. A total of 59 strains, including the type strains of currently recognized species as well as field isolates, were investigated in the study. A primer set specific for Campylobacter species enabled straightforward amplification and sequencing of a 530 bp fragment of the rpoB gene. The 16S rRNA gene sequences of all of the strains were determined in parallel. A good congruence was obtained between 16S rRNA and rpoB gene sequence-based trees within the genus Campylobacter. The branching of the rpoB tree was similar to that of the 16S rRNA gene tree, even though a few discrepancies were observed for certain species. The resolution of the rpoB gene within the genus Campylobacter was generally much higher than that of the 16S rRNA gene sequence, resulting in a clear separation of most species and even some subspecies. The universally applicable amplification and sequencing approach for partial rpoB gene sequence determination provides a powerful tool for DNA sequence-based discrimination of Campylobacter species

Genotypes and antibiotic resistance of canine Campylobacter jejuni isolates

Campylobacter jejuni is the most important cause of bacterial gastroenteritis in humans. It is a commensal in many wild and domestic animals, including dogs. Whereas genotypes of human and chicken C. jejuni isolates have been described in some detail, only little information on canine C. jejuni genotypes is available. To gain more information on genotypes of canine C. jejuni and their zoonotic potential, isolates from routine diagnostics of diarrheic dogs as well as isolates of a prevalence study in non-diarrheic dogs were analyzed. Prevalence of thermophilic Campylobacter among non-diarrheic dogs was 6.3% for C. jejuni, 5.9% for Campylobacter upsaliensis and 0.7% for Campylobacter coli. The C. jejuni isolates were genotyped by multi locus sequence typing (MLST) and flaB typing. Resistance to macrolides and quinolones was genetically determined in parallel. Within the 134 genotyped C. jejuni isolates 57 different sequence types (ST) were found. Five STs were previously unrecognized. The most common STs were ST-48 (11.2%), ST-45 (10.5%) and ST-21 (6.0%). Whereas no macrolide resistance was found, 28 isolates (20.9%) were resistant to quinolones. ST-45 was significantly more prevalent in diarrheic than in non-diarrheic dogs. Within the common time frame of isolation 94% of the canine isolates had a ST that was also found in human clinical isolates. In conclusion, prevalence of C. jejuni in Swiss dogs is low but there is a large genetic overlap between dog and human isolates. Given the close contact between human and dogs, the latter should not be ignored as a potential source of human campylobacteriosis

Acute Community-Acquired Diarrhea Requiring Hospital Admission in Swiss Children

In order to ascertain the prevalence of agents that cause childhood diarrheal illness, stool specimens of 312 consecutive children with community-acquired diarrhea requiring admission were evaluated. Pathogens were detected in 166 (53%) of the 312 children (≥2 pathogens in 28 children): Rotavirus (n=75), Salmonella spp. (n=37), Campylobacter spp. (n=24), Shigella spp. (n=5), Giardia spp. (n=4), Yersinia spp. (n=2), Aeromonas spp. (n=15), Cryptosporidium (n=15), enteropathogenic Escherichia coli (n=13), enterotoxigenic E. coli (n=7), and enterohemorrhagic E. coli (n=5). In conclusion, acute childhood diarrheal illness pathogens, such as Aeromonas, Cryptosporidium, and diarrheagenic E. coli, account for a large proportion of patients with a microbiologically positive stool specimen

Molecular Identification and Epidemiological Tracing of Pasteurella multocida Meningitis in a Baby

We report a case of Pasteurella multocida meningitis in a 1-month-old baby exposed to close contact with two dogs and a cat but without any known history of injury by these animals. 16S rRNA gene sequencing of the isolate from the baby allowed identification at the subspecies level and pointed to the cat as a possible source of infection. Molecular typing of Pasteurella isolates from the animals, from the baby, and from unrelated animals clearly confirmed that the cat harbored the same P. multocida subsp. septica strain on its tonsils as the one isolated from the cerebrospinal fluid of the baby. This case stresses the necessity of informing susceptible hosts at risk of contracting zoonotic agents about some basic hygiene rules when keeping pets. In addition, this study illustrates the usefulness of molecular methods for identification and epidemiological tracing of Pasteurella isolates