Phenon cluster analysis as a method to investigate epidemiological relatedness between sources of Campylobacter jejuni.

AIMS: To develop a method for assessing the relative epidemiological significance of possible infection sources for human campylobacteriosis. METHODS AND RESULTS: Using fluorescent amplified fragment length polymorphism (AFLP), 243 apparently epidemiologically unrelated Campylobacter jejuni isolates were genotyped (77 human, 46 cattle, 49 pet and 71 poultry isolates). In total 136 different phena were identified, of which 48 were clusters grouping at least two isolates. Isolates from different sources were frequently clustered together, underlining the high degree of source mixing and the lack of host specificity of C. jejuni. The phena were classified into different phenon types according to the sources of the isolates they contained. The occurrence of these phenon types was analysed using an area-proportional Euler diagram to describe epidemiological relatedness among C. jejuni isolates. Group separation statistics revealed that 43% of analysed human isolates expressed maximum similarity to other human isolates, 9% to cattle isolates, 21% to pet isolates and 27% to poultry isolates; these results were in accordance with the pattern observed in the phenon cluster analysis. CONCLUSIONS: Based on the grouping of strains into molecular similarity clusters, ecological patterns between sources can be investigated. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach is a new methodological contribution to establish the relative epidemiological significance of concurrent infection sources.

Campylobacter spp. in dogs and cats in Switzerland: risk factor analysis and molecular characterization with AFLP.

A 1-year cross-sectional study was carried out to determine the prevalence, risk factors for carriage, and genetic diversity of Campylobacter spp. in healthy dogs and cats in Switzerland. Veterinary practitioners collected samples from 1268 animals (all ages) presented for vaccination. The prevalence of Campylobacter spp. in 634 dogs and 596 cats that were eligible for the study was 41.2% (confidence interval 95%: 37.3-45.1%) and 41.9% (CI 95%: 37.9-46%), respectively. Risk factors identified for carriage of Campylobacter jejuni were found to be different from risk factors for C. upsaliensis/C. helveticus. Young animals (< or =3 years) had significantly higher odds of carrying C. upsaliensis/C. helveticus than older animals (OR 1.8-3.3), whereas for C. jejuni carriage, the age was not a risk factor. Amplified fragment length polymorphism (AFLP) genotyping revealed heterogeneity among C. jejuni strains and was found to clearly separate C. helveticus from C. upsaliensis. It was shown that cats more often carry C. helveticus with an estimated prevalence of 28.2%, whereas dogs mainly are carrying C. upsaliensis.

Verocytotoxin-producing Escherichia coli in patients with diarrhoea in Switzerland.

The present study was conducted between October 1996 and October 1998 to estimate the frequency of verocytotoxin-producing Escherichia coli among outpatients with diarrhoea in Switzerland. Among 3,041 subjects studied, 16 (0.5%) verocytotoxin-producing Escherichia coli infections were identified. Eleven cases were in infants and children

[The necrotizing enteritis by Clostridium perfringens type C in piglets: II. Molecular epidemiology study]. / Die nekrotisierende Enteritis des Saugferkels durch Clostridium perfringens Typ C: II. Molekularepidemiologische Studie.

Investigations were performed on shedding of C. perfringens in sows from four different pig farms. In two farms where no outbreaks of necrotizing enteritis had been observed, no strains of C. perfringens producing beta-toxin were detected in the faeces of sows. In contrast, C. perfringens strains producing beta-toxin were detected in sows on both farms suffering outbreaks of acute necrotizing enteritis. Strains of C. perfringens producing beta-toxin were invariably positive for the beta 2-toxin gene. However, strains carrying the beta 2-toxin gene only (i.e. negative for beta-toxin) were present in animals on all farms with roughly similar frequencies (mean 28.2% carriers). Some sows carried C. perfringens strains of both toxin genotypes simultaneously. Whereas these data further support the role of betatoxin as a cause of necrotizing enteritis, the role of beta 2-toxin in intestinal disease of piglets remains unclear. To establish the role of faecal shedding vs. environmental contamination as reservoirs of C. perfringens type C, strains were isolated from teats and feedlot trough swabs (toxin genotype beta/beta 2), as well as from fodder (genotype beta 2). However, sows carried this pathogen intermittently and in small numbers. This renders an individual, reliable diagnosis of carrier sows very difficult. Ribotyping of 34 C. perfringens isolates of different toxin genotypes showed five distinct profiles. Different toxin genotypes can belong to the same ribotype, and the same toxin genotype can be present in different ribotypes. Thus, even if a majority (79.4%) of strains investigated in a limited geographic region belonged to ribotype 1, ribotyping offered discrimination of strains beyond toxin typing.

Molecular epidemiology and genetic linkage of macrolide and aminoglycoside resistance in Staphylococcus intermedius of canine origin.

A collection of 77 Staphylococcus intermedius isolates from dogs and cats in Switzerland was examined for resistance to erythromycin. Resistance profiles for 14 additional antibiotics were compared between erythromycin-resistant and susceptible isolates. A resistance prevalence of 27% for erythromycin was observed in the population under study. Complete correlation between resistance to erythromycin, and to spiramycin, streptomycin, and neomycin was observed. The erythromycin-resistant isolates all had a reduced susceptibility to clindamycin when compared to the erythromycin-susceptible isolates. Both constitutive and inducible resistance phenotypes were observed for clindamycin. Ribotyping showed that macrolide-aminoglycoside resistance was randomly distributed among unrelated strains. This suggests that this particular resistance profile is not related to a single bacterial clone but to the horizontal transfer of resistance gene clusters in S. intermedius populations. The erythromycin-resistant isolates were all carrying erm(B), but not erm(A), erm(C), or msr(A). The erm(B) gene was physically linked to Tn5405-like elements known as resistance determinants for streptomycin, streptothricin, neomycin and kanamycin. Analysis of the region flanking erm(B) showed the presence of two different groups of erm(B)-Tn5405-like elements in the S. intermedius population examined and of elements found in Gram-positive species other than staphylococci. This strongly suggests that erm(B) or the whole erm(B)-Tn5405-like elements in S. intermedius originate from other bacterial species, possibly from enterococci.

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.

[The role of common starlings (Sturnus vulgaris) in the epidemiology of bacterial, potentially human pathogenic, disease agents]. / Die Rolle des Gemeinen Stars (Sturnus vulgaris) in der Epidemiologie bakterieller, potentiell humanpathogener Krankheitserreger.

Since a long time a public garden in Basel is known as a site for overnight accommodation and assembly of starlings. The birds cause an immense faecal contamination of the park and the neighbouring district. A nursery and a primary school are directly affected. To evaluate the health risk coming from the starlings droppings for the population, particularly for the children and to assess the role of starlings in the transmission of diseases to humans and in the epidemiology of human diseases the presence of human bacterial pathogens in the faeces of starlings was determined. Some of the isolated strains were further typed and compared to strains of human origin. C. jejuni, L. monocytogenes and C. psittaci were most often found. The typing of some C. jejuni and L. monocytogenes isolates showed a great variety of geno-, sero- respectively phage types that did not belong to the strains most often found in isolates of human origin. Starlings can harbour human pathogens and therefore a potential risk of infection comes from their droppings. It seems however rather improbable, that these birds present a constant direct source of infection for human beings.

The role of Acinetobacter baumannii as a nosocomial pathogen for dogs and cats in an intensive care unit.

Acinetobacter baumannii is a nosocomial pathogen associated with high morbidity and mortality in humans. Whereas infections with strains of Acinetobacter species have been reported in various situations, the importance of A baumannii as a nosocomial pathogen in veterinary hospitals has not been studied so far. In this retrospective case series, we describe 17 dogs and 2 cats from which A baumannii had been isolated during a 2 1/2-year period. In 7 dogs, A baumannii induced systemic signs of illness, whereas 12 animals showed signs of local infection. In all animals with systemic infection, and in 2 with localized infection, A baumannii contributed to the death of the animal or contributed to euthanasia; the remaining 8 dogs and both cats recovered. Molecular typing of the isolates with restriction polymorphisms of ribosomal DNA provided evidence of nosocomial spread of this pathogen and for the presence of several strains of A baumannii in the hospital environment.

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.

[The seroprevalence of Mycoplasma bovis in lactating cows in Switzerland, particularly in the republic and canton of Jura]. / Etude sur la séroprévalence de Mycoplasma bovis chez la vache laitière en Suisse, en particulier dans la République et Canton du Jura.

Sporadic cases of infection with Mycoplasma bovis have been observed in Switzerland since 1983. However, five severe outbreaks of endemic mastitis in a geographically distinct, small region (Canton du Jura) during the period 1995 to 1997 prompted the present investigation on the seroprevalence of infection with M. bovis among milking cows in Switzerland. A commercially prepared indirect enzyme immunoassay was used. Among a stratified random sample of 118 herds of milking cows in Switzerland, at least one positive animal was detected in 56 (47%) of the herds, whereas 6.1% of the 1816 individual animals tested positive. An epidemiological study was performed in the Canton du Jura region among 51 herds in order to assess the importance of management factors in the spread of M. bovis infection. The herd-level prevalence was 78%, and the seroprevalence at the level of the 1354 individual animals tested was 13.4%. A multivariate analysis of possible risk factors showed purchase of animals to be the only variable significantly associated with serological status of the herd with an "odds ratio" of 10.8.

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 Ca2+. 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.

Detection of RTX toxin genes in gram-negative bacteria with a set of specific probes.

The family of RTX (RTX representing repeats in the structural toxin) toxins is composed of several protein toxins with a characteristic nonapeptide glycine-rich repeat motif. Most of its members were shown to have cytolytic activity. By comparing the genetic relationships of the RTX toxin genes we established a set of 10 gene probes to be used for screening as-yet-unknown RTX toxin genes in bacterial species. The probes include parts of apxIA, apxIIA, and apxIIIA from Actinobacillus pleuropneumoniae, cyaA from Bordetella pertusis, frpA from Neisseria meningitidis, prtC from Erwinia chrysanthemi, hlyA and elyA from Escherichia coli, aaltA from Actinobacillus actinomycetemcomitans and lktA from Pasteurella haemolytica. A panel of pathogenic and nonpathogenic gram-negative bacteria were investigated for the presence of RTX toxin genes. The probes detected all known genes for RTX toxins. Moreover, we found potential RTX toxin genes in several pathogenic bacterial species for which no such toxins are known yet. This indicates that RTX or RTX-like toxins are widely distributed among pathogenic gram-negative bacteria. The probes generated by PCR and the hybridization method were optimized to allow broad-range screening for RTX toxin genes in one step. This included the binding of unlabelled probes to a nylon filter and subsequent hybridization of the filter with labelled genomic DNA of the strain to be tested. The method constitutes a powerful tool for the assessment of the potential pathogenicity of poorly characterized strains intended to be used in biotechnological applications. Moreover, it is useful for the detection of already-known or new RTX toxin genes in bacteria of medical importance.

Detection system for Escherichia coli-specific virulence genes: absence of virulence determinants in B and C strains.

We describe a rational approach to simultaneously test Escherichia coli strains for the presence of known virulence genes in a reverse dot blot procedure. Specific segments of virulence genes of E. coli designed to have similar hybridization parameters were subcloned on plasmids and subsequently amplified by PCR as unlabeled probes in amounts sufficient to be bound to nylon membranes. Various pathogenic isolates and laboratory strains of E. coli were probed for the presence of virulence genes by labeling the genomic DNA of these strains with digoxigenin and then hybridizing them to the prepared nylon membranes. These hybridization results demonstrated that besides the E. coli K-12 safety strain derivatives, E. coli B and C strains are also devoid of genes encoding any of the investigated virulence factors. In contrast, pathogenic E. coli control strains, used to evaluate the method, showed typical hybridization patterns. The described probes and their easy application on a single filter were shown to provide a useful tool for the safety assessment of E. coli strains to be used as hosts in biotechnological processes. This approach might also be used for the identification and characterization of clinically significant E. coli isolates from human and animal species.

Risk factors for sporadic salmonellosis in Switzerland.

A case-control study was conducted to identify determinants for the acquisition of sporadic Salmonella infection in Switzerland. Over a one-year period (1993), 223 case-control pairs were enrolled in the study and risk factors were assessed by means of self-administered questionnaires. Three-quarters of the isolates were identified as Salmonella enteritidis, most of which (80%) belonged to phage type 4. There were distinct differences in risk factors between infections with Salmonella enteritidis and those with other Salmonella serovars. In both groups recent travel abroad was positively associated with illness. This association was more pronounced for infections with non-enteritidis salmonellae [odds ratio (OR) 39.5; 95% confidence interval (CI) 6.6-236.8, compared to Salmonella enteritidis 4.0; 95% CI 1.8-9.1]. Among the presumably imported infections, Salmonella enteritidis was acquired mostly in other European countries, while other serovars were acquired mostly in countries outside of Europe. Eating food containing raw or undercooked eggs during the three days before the onset of illness increased the risk of infection with Salmonella enteritidis. Desserts made with raw eggs (OR 4.6; 95% CI 2.0-10.6) were more strongly associated with disease than consumption of soft-boiled eggs (OR 2.1; 95% CI 1.2-3.7), suggesting that the risk of infection was dependent on the extent to which eggs were cooked. Egg consumption was not associated with infections with serovars other than Salmonella enteritidis; however, the intake of medications other than antacids was found to be a risk factor (OR 3.5; 95% CI 1.1-11.4).

Evolutionary origin of a monophasic Salmonella serovar, 9,12:l,v:-, revealed by IS200 profiles and restriction fragment polymorphisms of the fljB gene.

The emergence in several countries of the monophasic serogroup D1 serovar Salmonella 9,12:l,v:- provided the opportunity to study its evolutionary origin. According to current models, such a variant serovar could have arisen by horizontal transfer of a new flagellar gene to a preexisting monophasic Salmonella strain or, alternatively, by the loss of the phase 2 flagellar gene of an originally biphasic Salmonella strain. Five known serovars of Salmonella, S. panama, S. kapemba, S. goettingen, S. zaiman, and S. mendoza, could have been possible ancestors of the new variant. The profiles of the insertion element IS200, which has been shown to provide phylogenetic markers for serogroup D1 salmonellae, were analyzed in relation to the restriction fragment length polymorphisms of the phase 2 flagellar gene. Together they provide unequivocal evidence that Salmonella 9,12:l,v:- arose from a strain of S. goettingen. Analysis of the flj operon of the variant indicated that loss of phase 2 flagellar antigen expression occurred through deletion of the hin gene and adjacent DNA, thereby blocking the phase 2 flagellar gene in the off position.

Sequential development of resistance to fluoroquinolones and erythromycin in an isolate of Campylobacter jejuni.

A severe episode of Campylobacter jejuni gastroenteritis in a patient with HIV infection was treated with ciprofloxacin and, because of therapeutic failure, subsequently with roxithromycin. After treatment, C. jejuni was again isolated from feces and shown to be resistant to both drugs. We present molecular evidence of the sequential development of both types of resistance in the patient isolate. To our knowledge, this is the first case with documented evidence showing sequential emergence of resistance to fluoroquinolones and erythromycin in a strain of C. jejuni during treatment.

Outbreak of Salmonella dublin infection in France, November - December 1995.

On 20 December 1995, the National Network of Public Health (Reseau National de Sante Publique - RNSP) was notified by the Salmonella and Shigella National Reference Centre (Centre National de Reference - CNR) that a greater than expected number of human i

Association between clinical presentation, biogroups and virulence attributes of Yersinia enterocolitica strains in human diarrhoeal disease.

Traditionally the enteric pathogen Yersinia enterocolitica has been differentiated into biogroups. Despite being considered as non-pathogenic, biogroup 1A isolates have constituted a sizeable fraction of strains from patients with gastroenteritis in many reports. To establish a potential clinical significance for biogroup 1A isolates of Y. enterocolitica, clinical disease in patients with gastroenteritis excreting such isolates was compared with symptoms among patients found infected with pathogenic biogroups. Clinical data and isolates of 66 patients from whom Y. enterocolitica had been isolated by direct plating were available for study. There was an association between patient age below 3 years and infection with 'pathogenic' Y. enterocolitica. The severity of gastroenteritis and other symptoms, however, did not depend on the biogroup, or the presence of the virulence plasmid in the yersinia strain isolated from the patients. Strains belonging to biogroup 1A of Y. enterocolitica showed two clusters of ribotypes, one of which encompassed most isolates recovered from humans, the other being associated with environmental isolates. This might indicate the existence of human-adapted and potentially pathogenic strains among biogroup 1A of Y. enterocolitica.