Sharing more than friendship - transmission of NDM-5 ST167 and CTX-M-9 ST69 Escherichia coli between dogs and humans in a family, Finland, 2015.

IntroductionCarbapenemase-producing Enterobacteriaceae (CPE) have rarely been reported in dogs, and never in animals in Finland. However, in April 2015, two meropenem-resistant Escherichia coli were identified from two dogs in one family. Both dogs suffered from chronic otitis externa. Methods: Epidemiological and molecular investigations (pulsed-field gel electrophoresis (PFGE), multilocus sequence typing) were conducted to investigate the source of infection and transmission routes. Results: In both dogs and one family member New Delhi metallo-beta-lactamase (NDM-5)-producing multidrug-resistant ST167 E. coli was found. Whole genome sequencing confirmed that the isolates were identical or only had one or two allelic differences. Additionally, the dogs and humans of the family carried an identical extended-spectrum beta-lactamase (ESBL) CTX-M-group 9 E. coli ST69 strain, indicating interspecies transmission. While the original source remains unclear, human-to-canine transmission is possible. No carbapenems had been administered to the dogs, but exposure to numerous other antimicrobials likely sustained the bacteria and supported its propagation in the canine host. Conclusion: To our knowledge, canine clinical NDM-5 E. coli in Europe, and confirmed CPE transmission between dogs and humans have not been previously reported. The screening of veterinary Enterobacteriaceae isolates for carbapenem resistance is highly recommended.

Antimicrobial resistance in Staphylococcus pseudintermedius and the molecular epidemiology of methicillin-resistant S. pseudintermedius in small animals in Finland.

Objectives: To investigate antimicrobial susceptibility in Staphylococcus pseudintermedius and the occurrence of methicillin-resistant S. pseudintermedius (MRSP), to explore the molecular structure of the MRSP population and to analyse risk factors for MRSP. Methods: Susceptibility data for clinical S. pseudintermedius isolates in 2011-15 were analysed using WHONET. All MRSP isolates in 2010-14 ( n = 362) were typed using PFGE. Representative isolates ( n = 87) of clusters were analysed using MLST and staphylococcal cassette chromosome mec (SCC mec ) typing. Risk factors were analysed using logistic regression. Results: Of the clinical S. pseudintermedius ( n = 1958; 98% from dogs), 14% were MRSP. Resistance to other antimicrobials varied between 12% and 39%. No trends were observed over time. Among clinical specimens (from infection sites) and screening specimens (from potential carriers), respectively, 2.5% (267/10 813) and 9% (211/2434) revealed MRSP. MLST revealed 42 different STs, including 19 new ones. Clonal complexes 71, 45 and 258 were the most common, but the MRSP population diversified over the years. A clinical S. pseudintermedius isolate was more likely to be MRSP if the patient was on antimicrobials at the time of sampling or was male. The presence of MRSP in screening specimens was more likely if the patient was on multiple antimicrobials at the time of sampling. Specimens from private clinics (versus the Veterinary Teaching Hospital of the University of Helsinki) had a higher likelihood of MRSP in both analyses. Conclusions: Resistance to antimicrobials among S. pseudintermedius in Finland is high, emphasizing the importance of infection control measures and susceptibility testing prior to therapy. The diverse MRSP population indicates non-clonal spread.

Characterisation of and risk factors for extended-spectrum ß-lactamase producing Enterobacterales (ESBL-E) in an equine hospital with a special reference to an outbreak caused by Klebsiella pneumoniae ST307:CTX-M-1.

BACKGROUND: Extended-spectrum ß-lactamase producing Enterobacterales (ESBL-E) are important causative agents for infections in humans and animals. At the Equine Veterinary Teaching Hospital of the University of Helsinki, the first infections caused by ESBL-E were observed at the end of 2011 leading to enhanced infection surveillance. Contact patients were screened for ESBL-E by culturing infection sites and rectal screening. This study was focused on describing the epidemiology and microbiological characteristics of ESBL-E from equine patients of the EVTH during 2011-2014, and analysing putative risk factors for being positive for ESBL-E during an outbreak of Klebsiella pneumoniae ST307. RESULTS: The number of ESBL-E isolations increased through 2012-2013 culminating in an outbreak of multi-drug resistant K. pneumoniae ST307:blaCTX-M-1:blaTEM:blaSHV during 04-08/2013. During 10/2011-05/2014, altogether 139 ESBL-E isolates were found from 96 horses. Of these, 26 were from infection-site specimens and 113 from rectal-screening swabs. A total of 118 ESBL-E isolates from horses were available for further study, the most numerous being K. pneumoniae (n = 44), Escherichia coli (n = 31) and Enterobacter cloacae (n = 31). Hospital environmental specimens (N = 47) yielded six isolates of ESBL-E. Two identical E. cloacae isolates originating from an operating theatre and a recovery room had identical or highly similar PFGE fingerprint profiles as five horse isolates. In the multivariable analysis, mare-foal pairs (OR 4.71, 95% CI 1.57-14.19, P = 0.006), length of hospitalisation (OR 1.62, 95% CI 1.28-2.06, P < 0.001) and passing of a nasogastric tube (OR 2.86, 95% CI 1.03-7.95, P = 0.044) were associated with being positive for ESBL-E during the K. pneumoniae outbreak. CONCLUSIONS: The occurrence of an outbreak caused by a pathogenic ESBL-producing K. pneumoniae ST307 strain highlights the importance of epidemiological surveillance of ESBL-E in veterinary hospitals. Limiting the length of hospitalisation for equine patients may reduce the risk of spread of ESBL-E. It is also important to acknowledge the importance of nasogastric tubing as a potential source of acquiring ESBL-E. As ESBL-E were also found in stomach drench pumps used with nasogastric tubes, veterinary practices should pay close attention to appropriate equipment cleaning procedures and disinfection practices.

Agricultural, socioeconomic and environmental variables as risks for human verotoxigenic Escherichia coli (VTEC) infection in Finland.

BACKGROUND: Verotoxigenic E. coli (VTEC) is the cause of severe gastrointestinal infection especially among infants. Between 10 and 20 cases are reported annually to the National Infectious Disease Register (NIDR) in Finland. The aim of this study was to identify explanatory variables for VTEC infections reported to the NIDR in Finland between 1997 and 2006. We applied a hurdle model, applicable for a dataset with an excess of zeros. METHODS: We enrolled 131 domestically acquired primary cases of VTEC between 1997 and 2006 from routine surveillance data. The isolated strains were characterized by virulence type, serogroup, phage type and pulsed-field gel electrophoresis. By applying a two-part Bayesian hurdle model to infectious disease surveillance data, we were able to create a model in which the covariates were associated with the probability for occurrence of the cases in the logistic regression part and the magnitude of covariate changes in the Poisson regression part if cases do occur. The model also included spatial correlations between neighbouring municipalities. RESULTS: The average annual incidence rate was 4.8 cases per million inhabitants based on the cases as reported to the NIDR. Of the 131 cases, 74 VTEC O157 and 58 non-O157 strains were isolated (one person had dual infections). The number of bulls per human population and the proportion of the population with a higher education were associated with an increased occurrence and incidence of human VTEC infections in 70 (17%) of 416 of Finnish municipalities. In addition, the proportion of fresh water per area, the proportion of cultivated land per area and the proportion of low income households with children were associated with increased incidence of VTEC infections. CONCLUSIONS: With hurdle models we were able to distinguish between risk factors for the occurrence of the disease and the incidence of the disease for data characterised by an excess of zeros. The density of bulls and the proportion of the population with higher education were significant both for occurrence and incidence, while the proportion of fresh water, cultivated land, and the proportion of low income households with children were significant for the incidence of the disease.

Comparison of Streptococcus halichoeri isolates from canine and fur animal infections: biochemical patterns, molecular characteristics and genetic relatedness.

BACKGROUND: Streptococcus halichoeri infections have been reported in grey seals, a European badger, a Stellar sea lion and humans, but its presence in companion and fur animals is unknown. Since 2010, S. halichoeri-like bacteria (SHL) have been isolated from fur animals and dogs in Finland. Our aim was to retrospectively investigate laboratory records for SHL from canine and fur animal infections, characterize the isolates and compare their genetic relatedness in relation to three reference strains: CCUG 48324T, originating from a grey seal, and strains 67100 and 61265, originally isolated from humans. RESULTS: A total of 138 and 36 SHLs from canine and fur animal infections, respectively, were identified in the laboratory records. SHL was commonly associated with skin infections, but rarely as the only species. A set of 49 canine and 23 fur animal SHLs were further characterized. MALDI-TOF confirmed them as being S. halichoeri. The growth characteristics were consistent with the original findings, but isolates were catalase positive. In total, 17 distinct API 20 Strep patterns were recorded among all 75 isolates tested, of which pattern 5563100 was the most common (n = 30). Antimicrobial resistance to erythromycin and clindamycin was common in canine isolates, but rare in fur animal isolates. Three clusters were observed by PFGE, and 16S rRNA sequencing revealed 98.1-100% similarities with the human strains and 98.1-99.5% with the seal strain. A phylogenetic tree of concatenated 16S rRNA and rpoB revealed closely related isolates with two clades. Fifteen canine isolates were identical to the human strains based on concatenated 16S rRNA and rpoB sequencing. CONCLUSIONS: Streptococcus halichoeri appears to be quite a common bacterial species in the skin of dogs and fur animals. The clinical significance of S. halichoeri is uncertain, as it was rarely isolated as a monoculture. No apparent temporal or spatial clustering was detected, but isolates from different sources were genetically very similar. Because many canine isolates were genetically similar to the human reference strains, transmission between dogs and humans may be possible. WGS sequencing of strains from different sources is needed to further investigate the epidemiology and virulence of S. halichoeri.

Streptococcus halichoeri: Comparative Genomics of an Emerging Pathogen.

Streptococcus halichoeri is an emerging pathogen with a variety of host species and zoonotic potential. It has been isolated from grey seals and other marine mammals as well as from human infections. Beginning in 2010, two concurrent epidemics were identified in Finland, in fur animals and domestic dogs, respectively. The fur animals suffered from a new disease fur animal epidemic necrotic pyoderma (FENP) and the dogs presented with ear infections with poor treatment response. S. halichoeri was isolated in both studies, albeit among other pathogens, indicating a possible role in the disease etiologies. The aim was to find a possible common origin of the fur animal and dog isolates and study the virulence factors to assess pathogenic potential. Isolates from seal, human, dogs, and fur animals were obtained for comparison. The whole genomes were sequenced from 20 different strains using the Illumina MiSeq platform and annotated using an automatic annotation pipeline RAST. The core and pangenomes were formed by comparing the genomes against each other in an all-against-all comparison. A phylogenetic tree was constructed using the genes of the core genome. Virulence factors were assessed using the Virulence Factor Database (VFDB) concentrating on the previously confirmed streptococcal factors. A core genome was formed which encompassed approximately half of the genes in Streptococcus halichoeri. The resulting core was nearly saturated and would not change significantly by adding more genomes. The remaining genes formed the pangenome which was highly variable and would still evolve after additional genomes. The results highlight the great adaptability of this bacterium possibly explaining the ease at which it switches hosts and environments. Virulence factors were also analyzed and were found primarily in the core genome. They represented many classes and functions, but the largest single category was adhesins which again supports the marine origin of this species.

Description of two Serratia marcescens associated mastitis outbreaks in Finnish dairy farms and a review of literature.

BACKGROUND: Infection with Serratia spp. have been associated with mastitis outbreaks in dairy cattle herds. Environmental contamination or a point source, like a teat dip product, have often been observed to be potential sources of such outbreaks. We describe two Serratia marcescens associated mastitis outbreaks associated with a contaminated teat dip containing a tertiary alkyl amine, n,n-bis (3-aminopropyl) dodecylamine in two dairy cattle farms in Finland. S. marcescens strains isolated from milk and environmental samples were identified by the MALDI-TOF method. RESULTS: Six specimens (n = 19) on Herd 1 and all specimens (n = 9) on Herd 2 were positive for S. marcescens. Positive specimens were from mastitis milk and teat dip liquid and equipment. Bacteria were not isolated from the unopened teat dip canister. The same clone of S. marcescens was isolated from milk samples and teat dip samples within the farms. Pulsed field gel electrophoresis results to the S. marcescens isolates from these two different herds were tested with unweighted pair-group method using arithmetic average clustering analysis. The isolates were not same clone in both herds, because similarity in that test was only 75% when cut-off value to similarity is 85%. CONCLUSIONS: Our investigation showed that the post milking teat dip and/or temporary containers were contaminated with S. marcescens and these were most likely the sources for new mastitis cases. The negative result from the unopened teat dip canister and positive results from refillable containers demonstrated that the product itself was not contaminated with S. marcescens at the production unit, but became contaminated at the farm level.

Improved DNA probe detection of Listeria monocytogenes in enrichment culture after physical-chemical fractionation.

Bacterial detection in foods by nucleic acid probes is limited by microflora competition during selective enrichment. Probe target concentration by extraction and fractionation of enrichments may diminish this limitation. The 1-h AccuProbe chemiluminescent culture identification test for Listeria monocytogenes was used as a model. Its high detection threshold provides a stringent challenge for evaluating enrichment work-up protocols. Detection of L. monocytogenes, at 1-4 colony-forming units/g food, was not consistently possible in 48 h enrichment cultures using AccuProbe. Concentration by cell sedimentation was occasionally helpful but the volume of co-sedimented food limited concentration to about 10-fold. To improve concentration, enrichment sediments were sonicated or enzymatically lysed to release the probe's target, r-RNA. The RNA was separated from non-RNA material by extraction with phenol and precipitation with ethanol. Enrichments (250 mL) were concentrated 2500-fold, and the limitation was food RNA volume. A strongly competitive Enterococcus faecium food isolate was used to demonstrate the effect of artificial competition on the kit's ability to detect L. monocytogenes in enrichments. High competitor concentrations repressed the level of the target below the detection threshold, but concentration of r-RNA enabled detection of L. monocytogenes. The effectiveness of this enrichment sample work-up was demonstrated with naturally contaminated hummus.

Epidemiology of methicillin resistant Staphylococcus pseudintermedius in guide dogs in Finland.

BACKGROUND: Methicillin resistant Staphylococcus pseudintermedius (MRSP) and Staphylococcus aureus (MRSA) are common multi-drug resistant (MDR) bacteria in dogs. In 2012-2013 three dogs of the Guide Dog School of the Finnish Federation of the Visually Impaired were found to be MRSP positive. Guide dogs have regular contact with each other during their first year of life and prolonged contact when in training. Since dogs are placed in different parts of Finland after training, there is a risk for national spread of MDR bacteria. In this study the prevalence of MRSP and MRSA, as well as the risk factors for MRSP were determined in the Finnish guide dog population. MRSP isolates were investigated using molecular methods and compared to the earlier isolates. RESULTS: Out of 132 tested dogs 4 were MRSP positive thus giving the prevalence estimate of 3% (95% CI: 1-8%) for MRSP in the target population. MRSA was not detected (prevalence estimate 0%, 95% CI: 0-3%). Risk factors associated with MRSP were being a breeding bitch (OR = 8.4; 95% CI: 1.1-64.1, P = 0.012), the number of veterinary visits (OR = 1.23; 95% CI: 1.0-1.5, P = 0.025) and number of antimicrobial courses (OR = 1.63; 95% CI: 1.0-2.55; P = 0.035). Identified MRSP isolates belonged to five different sequence types (ST45, 71, 402, 403 and 404). All ST71 isolates carried SCCmec II-III, while the SCCmec type of the ST45 and ST402 (a single locus variant of ST45) isolates were non-typeable with the method used. CONCLUSIONS: MRSP and MRSA had low prevalence in the studied dog population despite the close contact between dogs, and the MRSP population was heterogenic. Antimicrobial therapy and veterinary visits are risk factors for MRSP even among a small case group.

Application of molecular genetic methods in diagnostics and epidemiology of food-borne bacterial pathogens.

Salmonella enterica, Campylobacter and Yersinia species, Shiga toxin-producing Escherichia coli (STEC), Listeria monocytogenes and Clostridium perfringens are the bacterial pathogens constituting the greatest burden of food-borne disease in Finland. Several molecular genetic methods have been applied to diagnose, discriminate and survey these bacteria. PCR, PCR-RFLP and PFGE are the most widely and successfully used. However, these methods are unable to replace conventional and internationally standardised phenotyping. Electronic database libraries of the different genomic profiles will enable continuous surveillance of infections and detection of possible infection clusters at an early stage. Furthermore, whole-genome sequence data have opened up new insights into epidemiological surveillance. Laboratory-based surveillance performed in a timely manner and exploiting adequate methods, and co-operation at local, national and international levels are among the key elements in preventing food-borne diseases. This paper reviews different applications of molecular genetic methods for investigating enteric bacterial pathogens and gives examples of the methods successfully used in diagnostics and epidemiological studies in Finland.

Tracing shigatoxigenic Escherichia coli O103, O145, and O174 infections from farm residents to cattle.

Severe diarrheal infections caused by Shigatoxigenic Escherichia coli O103:H2:stx(1):eae-epsilon:ehx, O145:H28:stx(1):eae-gamma:ehx (two cases in a family), and O174:H21:stx(2c) in farm residents were traced to cattle. Molecular methods were applied to the isolation and characterization of the strains. The causative strains were also isolated from cattle samples 1 or 4 months later.

Genetic diversity among clonal lineages within Escherichia coli O157:H7 stepwise evolutionary model.

Escherichia coli O157:H7 variants were examined for trait mutations and by molecular subtyping to better define clonal complexes postulated on the O157:H7 evolution model. Strains of beta-glucuronidase-positive, sorbitol-negative O157:H7 isolated in United States and Japan were identical to A5 clonal strain and shared sequence type (ST)-65 by multilocus sequence typing (MLST); thus, they belong in A5. However, these strains exhibited pulsed-field gel electrophoresis (PFGE) profile differences that suggested genomic divergence between populations. Sorbitol-fermenting O157 (SFO157) strains from Finland, Scotland, and Germany were identical to A4 clonal strain and belong in A4. Some SFO157 strains, isolated years apart and from different countries, had identical PFGE profiles, suggesting a common origin. Despite similarities, some Finnish and Scottish and all of the German strains have ST-75 ("German clone"), whereas others have ST-76, a new variant ("Scottish clone"). MLST of strains in other clonal complexes also discriminated strains thought to be identical and showed that genetic differences will further distinguish clonal populations into subclones.

Clinical Escherichia coli strains carrying stx genes: stx variants and stx-positive virulence profiles.

Altogether, 173 Shiga toxin-producing Escherichia coli (STEC) serotype O157 (n = 111) and non-O157 (n = 62) isolates from 170 subjects were screened by PCR-restriction fragment length polymorphism for eight different stx genes. The results were compiled according to serotypes, phage types of O157, production of Stx toxin and enterohemolysin, and the presence of eae. The stx genes occurred in 11 combinations; the most common were stx(2) with stx(2c) (42%), stx(2) alone (21%), and stx(1) alone (16%). Of the O157 strains, 64% carried stx(2) with stx(2c) versus 2% of the non-O157 strains (P < 0.001). In the non-O157 strains, the prevailing gene was stx(1) (99% versus 1% in O157 strains; P < 0.001). In addition, one strain (O Rough:H4:stx(2c)) which has not previously been described as associated with hemolytic-uremic syndrome (HUS) was found. Ten stx-positive virulence profiles were responsible for 71% of all STEC infections. Of these profiles, five accounted for 71% of the 21 strains isolated from 20 patients with HUS or thrombotic thrombocytopenic purpura (TTP). The strains having the virulence profile that caused mainly HUS or TTP or bloody diarrhea produced Stx with titers of >/=1:128 (90%) more commonly than did other strains (51%; P < 0.001). These strains were also more commonly enterohemolytic (98% versus 68% for other strains; P < 0.001) and possessed the eae gene (100%) more commonly than did other strains (74%; P < 0.001). A particular virulence profile, O157:H7:PT2:stx(2):stx(2c):eae:Ehly, was significantly more frequently associated with HUS and bloody diarrhea than were other profiles (P = 0.02) and also caused the deaths of two children. In this study, the risk factors for severe symptoms were an age of <5 years and infection by the strain of O157:H7:PT2 mentioned above.

Serology and genetics of the flagellar antigen of Escherichia coli O157:H7a,7c.

Among Escherichia coli strains of the O55:H7 serovar, which is considered the ancestor of Shiga toxin-producing E. coli (STEC) O157:H7, two subtypes, H7a,7b and H7a,7c (briefly, H7a,b and H7a,c, respectively), of the H7 flagellar antigen have been described previously [J. Wright and R. Villanueva, J. Hyg. (Camb.) 51:39-48, 1953; Y. A. Ratiner and V. A. Sinelnikova, Zh. Microbiol. Epidemiol. Immunobiol. 3:111-116, 1969). We have now studied 13 STEC O157:H7 strains and 1 O55:H7 strain that were epidemiologically unrelated, that originated from six countries on two continents, and that had different profiles when analyzed by multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, and PCR for stx and eae. They were all found to possess the H7a,c flagellar antigen. Serum cross-absorption assays confirmed that their H antigens were indistinguishable from each other and from that of E. coli O55:H7a,c but differed from the standard H7a,b antigen of E. coli H test strain U5/41. It was shown by phage-mediated transduction that the flagellin genes for these two H-antigen subserotypes were alleles of the E. coli fliC locus. On the basis of the serological data obtained in this study and the molecular characteristics of E. coli fliC(H7) alleles recently published, it is inferred that H7a,c and H7a,b are the main serological subtypes of the group of E. coli H7 flagellins.