Dogs as carriers of virulent and resistant genotypes of Clostridioides difficile.

While previous research on zoonotic transmission of community-acquired Clostridioides difficile infection (CA-CDI) focused on food-producing animals, the present study aimed to investigate whether dogs are carriers of resistant and/or virulent C. difficile strains. Rectal swabs were collected from 323 dogs and 38 C. difficile isolates (11.8%) were obtained. Isolates were characterized by antimicrobial susceptibility testing, whole-genome sequencing (WGS) and a DNA hybridization assay. Multilocus sequence typing (MLST), core genome MLST (cgMLST) and screening for virulence and antimicrobial resistance genes were performed based on WGS. Minimum inhibitory concentrations for erythromycin, clindamycin, tetracycline, vancomycin and metronidazole were determined by E-test. Out of 38 C. difficile isolates, 28 (73.7%) carried genes for toxins. The majority of isolates belonged to MLST sequence types (STs) of clade I and one to clade V. Several isolates belonged to STs previously associated with human CA-CDI. However, cgMLST showed low genetic relatedness between the isolates of this study and C. difficile strains isolated from humans in Austria for which genome sequences were publicly available. Four isolates (10.5%) displayed resistance to three of the tested antimicrobial agents. Isolates exhibited resistance to erythromycin, clindamycin, tetracycline and metronidazole. These phenotypic resistances were supported by the presence of the resistance genes erm(B), cfr(C) and tet(M). All isolates were susceptible to vancomycin. Our results indicate that dogs may carry virulent and antimicrobial-resistant C. difficile strains.

An outbreak of listeriosis linked to turkey meat products in the Czech Republic, 2012-2016.

Since 2012-2016 an increased number of listeriosis cases, especially from one region of the Czech Republic, were observed. Most of them were caused by strains of serotype 1/2a, clonal complex 8, indistinguishable by pulsed-field gel electrophoresis. Twenty-six human cases were reported, including two neonatal cases in twins. Three cases were fatal. The typing of Listeria monocytogenes isolates from food enabled to confirm a turkey meat delicatessen as the vehicle of infection for this local outbreak in the Moravian-Silesian Region. The food strains belonging to identical pulsotype were isolated from ready-to-eat turkey meat products packaged by the same producer between 2012 and 2016. This fact confirms that the described L. monocytogenes outbreak strain probably persisted in the environment of the aforementioned food-processing plant over several years. Whole-genome sequencing confirmed a very close relationship (zero to seven different alleles) between isolates from humans, foods and swabs from the environment of the food-processing plant under investigation.

Fatal Pseudomonas aeruginosa pneumonia in a previously healthy woman was most likely associated with a contaminated hot tub.

Community-acquired pneumonia due to Pseudomonas aeruginosa in previously healthy individuals is a rare disease that is associated with high fatality. On 14 February 2010 a previously healthy 49-year-old woman presented to an emergency room with signs and symptoms of pneumonia, 2 days after returning from a spa holiday in a wellness hotel. Blood cultures and respiratory specimens grew P. aeruginosa. Despite adequate antimicrobial therapy, the patient died of septic multiorgan failure on day nine of hospitalization. On February 26, nine water samples were taken from the hotel facilities used by the patient: In the hot tub sample 37,000 colony-forming units of P. aeruginosa/100 ml were detected. Two of five individual colonies from the primary plate used for this hot tub water sample were found to be genetically closely related to the patient's isolates. Results from PFGE, AFLP and MLST analysis allowed the two lung isolates gained at autopsy and the whirlpool bathtub isolates to be allocated into one cluster. The patient most likely acquired P. aeruginosa from the contaminated water in the hotel's hot tub. The detection of P. aeruginosa in high numbers in a hot tub indicates massive biofilm formation in the bath circulation and severe deficiencies in hygienic maintenance. The increasing popularity of hot tubs in hotels and private homes demands increased awareness about potential health risks associated with deficient hygienic maintenance.

Update: Multinational listeriosis outbreak due to 'Quargel', a sour milk curd cheese, caused by two different L. monocytogenes serotype 1/2a strains, 2009-2010.

We previously reported an outbreak of listeriosis in Austria and Germany due to consumption of Quargel cheese. It comprised 14 cases (including five fatalities) infected by a serotype 1/2a Listeria monocytogenes (clone 1), with onset of illness from June 2009 to January 2010. A second strain of L. monocytogenes serotype 1/2a (clone 2) spread by this product could be linked to further 13 cases in Austria (two fatal), six in Germany (one fatal) and one case in the Czech Republic, with onset of disease from December 2009 to end of February 2010.

Listeriosis outbreak caused by acid curd cheese Quargel , Austria and Germany 2009.

We report an outbreak of listeriosis in Austria and Germany due to the consumption of Quargel cheese produced by an Austrian manufacturer. At the time of writing this report, the outbreak was known to account for 14 outbreak cases in 2009, including four cases with lethal outcome. On 23 January 2010, the cheese product was voluntarily withdrawn from the market.

An outbreak of multidrug-resistant tuberculosis among refugees in Austria, 2005-2006.

SETTING: In 2005-2006, the Austrian reference laboratory for tuberculosis (TB) identified multidrug-resistant (MDR) isolates from four cases of TB showing genotypes indistinguishable from each other. OBJECTIVE: To clarify the chain of transmission of this MDR-TB strain. DESIGN: An epidemiological case series investigation by reviewing TB notification reports and hospital discharge letters. RESULTS: The 38-year-old primary case of the MDR-TB cluster had initially been identified as a case of non-MDR pulmonary TB in June 2004, 7 months after being detained for illegal immigration. In March 2005, he was lost to follow-up for 4 months. In June 2005, he presented with pulmonary and laryngeal TB due to MDR-TB. After discharge, the case was again lost to follow-up until April 2006, when he was readmitted with recurrent MDR-TB. A three-case cluster of pulmonary MDR-TB sharing the same strain as the primary case was detected in April 2006: the index case's 5-month-old daughter and a 25-year-old friend with a 6-month-old son. CONCLUSION: As MDR-TB has originated in the human immunodeficiency virus seronegative community in Austria, there is a clear need to implement national guidelines for the management of drug-resistant TB in Austria.

Meticillin-resistant Staphylococcus aureus: occurrence of a new spa type in two acute care hospitals in Austria.

Typing multiply-resistant bacteria using molecular techniques is high priority for national health authorities. Routine typing of meticillin-resistant Staphylococcus aureus (MRSA) was initiated in Austria 2005 and was performed by sequence analysis of the variable X region of protein A gene (spa), characterisation of the mec gene (SCCmec) and testing for Panton-Valentine leukocidin (PVL), enterotoxins, toxic shock syndrome toxin and the epidermolytic toxin genes. Ten different spa types, including newly identified t2023, were found among 66 clinical MRSA isolates originating from two neighbouring hospitals under the same management. Spa type t2023 was initially isolated in December 2005 from hospital A, where it became the dominant spa type during 2006 (nine of 16 isolates). The occurrence of type t2023 in hospital B remained a unique event and could be epidemiologically linked to a patient transferred from hospital A. Spa type t2023 is very similar to spa type t001. An isolate of spa type t001 from hospital A showed an enterotoxin gene pattern, multilocus sequence type (MLST) and SmaI macrorestriction PFGE pattern indistinguishable from that of t2023. Epidemiological differences suggested that infection control measures can prevent MRSA cross-transmission. Hospital B had a more stringent MRSA isolation policy, a higher nurse:patient ratio and provided more resources for infection control than hospital A.

Outbreak of acute gastroenteritis in an Austrian boarding school, September 2006.

An outbreak of acute gastroenteritis occurred in September 2006 in a boarding school in eastern Austria. Of 113 cases, 101 were hospitalised. In order to identify the outbreak source, a retrospective cohort study on the group at risk was performed, including 222 pupils and 30 staff members. Food exposure in the canteen of the school was identified as the most relevant common link among the cases in the case series investigation. Although the preliminary microbiological investigation made Norovirus infections possible, an in-depth descriptive epidemiological investigation later pointed to food intoxication rather than a viral infection as the cause of the outbreak. The analytical epidemiological investigation implicated boiled rice and chicken wings served in the canteen as the most likely source of the outbreak. Staphylococcus aureus was identified as the causative agent. Further molecular characterisation revealed that the predominant S. aureus type in this outbreak was a new spa type, t2046. The same spa type was isolated from stool specimens of the majority of the cases investigated, from samples of the incriminated boiled rice, and also from a swab of a palmar skin lesion of one of the healthy kitchen workers, who is therefore the most likely source of contamination. This outbreak underlines again the importance of compliance with the basic guidelines for kitchen hygiene.

Suitability of partial 16S ribosomal RNA gene sequence analysis for the identification of dangerous bacterial pathogens.

AIMS: In a bioterrorism event a rapid tool is needed to identify relevant dangerous bacteria. The aim of the study was to assess the usefulness of partial 16S rRNA gene sequence analysis and the suitability of diverse databases for identifying dangerous bacterial pathogens. METHODS AND RESULTS: For rapid identification purposes a 500-bp fragment of the 16S rRNA gene of 28 isolates comprising Bacillus anthracis, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Yersinia pestis, and eight genus-related and unrelated control strains was amplified and sequenced. The obtained sequence data were submitted to three public and two commercial sequence databases for species identification. The most frequent reason for incorrect identification was the lack of the respective 16S rRNA gene sequences in the database. CONCLUSIONS: Sequence analysis of a 500-bp 16S rDNA fragment allows the rapid identification of dangerous bacterial species. However, for discrimination of closely related species sequencing of the entire 16S rRNA gene, additional sequencing of the 23S rRNA gene or sequencing of the 16S-23S rRNA intergenic spacer is essential. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides comprehensive information on the suitability of partial 16S rDNA analysis and diverse databases for rapid and accurate identification of dangerous bacterial pathogens.

Characterization of Erwinia amylovora strains from different host plants using repetitive-sequences PCR analysis, and restriction fragment length polymorphism and short-sequence DNA repeats of plasmid pEA29.

AIMS: The three main aims of the study were the assessment of the genetic relationship between a deviating Erwinia amylovora strain isolated from Amelanchier sp. (Maloideae) grown in Canada and other strains from Maloideae and Rosoideae, the investigation of the variability of the PstI fragment of the pEA29 plasmid using restriction fragment length polymorphism (RFLP) analysis and the determination of the number of short-sequence DNA repeats (SSR) by DNA sequence analysis in representative strains. METHODS AND RESULTS: Ninety-three strains obtained from 12 plant genera and different geographical locations were examined by repetitive-sequences PCR using Enterobacterial Repetitive Intergenic Consensus, BOX and Repetitive Extragenic Palindromic primer sets. Upon the unweighted pair group method with arithmetic mean analysis, a deviating strain from Amelanchier sp. was analysed using amplified ribosomal DNA restriction analysis (ARDRA) analysis and the sequencing of the 16S rDNA gene. This strain showed 99% similarity to other E. amylovora strains in the 16S gene and the same banding pattern with ARDRA. The RFLP analysis of pEA29 plasmid using MspI and Sau3A restriction enzymes showed a higher variability than that previously observed and no clear-cut grouping of the strains was possible. The number of SSR units reiterated two to 12 times. The strains obtained from pear orchards showing for the first time symptoms of fire blight had a low number of SSR units. CONCLUSIONS: The strains from Maloideae exhibit a wider genetic variability than previously thought. The RFLP analysis of a fragment of the pEA29 plasmid would not seem a reliable method for typing E. amylovora strains. A low number of SSR units was observed with first epidemics of fire blight. SIGNIFICANCE AND IMPACT OF THE STUDY: The current detection techniques are mainly based on the genetic similarities observed within the strains from the cultivated tree-fruit crops. For a more reliable detection of the fire blight pathogen also in wild and ornamentals Rosaceous plants the genetic features of deviating E. amylovora strains have to be studied in detail.

Enumeration of probiotic bacilli spores in animal feed: interlaboratory study.

Fourteen out of 17 laboratories completed an interlaboratory study comparing 2 pretreatment protocols of feed samples containing authorized probiotic bacilli spores. Both methods used tryptone soy agar for enumeration. Pretreatment A involved preparation of a suspension of the feed sample in 50% ethanol. For pretreatment B, the sample was suspended in peptone salt solution and heated at 80 degrees C for 10 min. Each laboratory analyzed 12 samples (6 per pretreatment), which represented duplicates of a high (10(9) colony-forming units [CFU]/g) and low (10(5) CFU/g) level of bacilli spores or a blank that contained vegetative probiotic bacteria only. For pretreatment A, the repeatability relative standard deviation (RSD(r)) was 2.9% for the low level and 2.5% for the high. The reproducibility relative standard deviation (RSDR) values were 7.8 and 5.9%, respectively. Pretreatment B revealed RSD(r) values of 1.1 and 1.0%, and RSDR values of 5.8 and 3.4%, respectively. The heat treatment (pretreatment B) of feed samples had better precision data, resulted in higher viable bacilli counts, and was more effective in deactivating vegetative background flora. It is therefore recommended for adoption for official control purposes and for CEN and ISO standards.

Chromosomal instability of fanconi anemia cells is not the consequence of a defective repair activity of the ribosomal protein S3.

Fanconi anemia (FA) is an autosomal recessive chromosomal breakage disorder characterized by developmental defects, hypersensitivity toward oxygen and DNA crosslinking agents, and susceptibility to cancer. An increased level of reactive oxygen intermediates and an increased level of 8-oxoguanine in FA cells point to a defective oxygen metabolism. Recent investigations showed that FA cells from several complementation groups have a reduced capacity to repair oxidatively damaged DNA. One major enzyme involved in the repair of oxidative DNA lesions is the ribosomal protein S3. Previous reports implied a role for the ribosomal protein S3 in DNA repair in FA cells. However, a more detailed analysis of the ribosomal protein S3 in FA cells from complementation groups A-E could not confirm this. DNA analysis and Western blot analysis did not show significant differences in ribosomal protein S3 between FA cells and cells from healthy individuals. Furthermore, even the overexpression of the ribosomal protein S3 did not reduce the chromosomal instability of FA cells.

Involvement of the Fanconi anemia protein FA-C in repair processes of oxidative DNA damages.

Fanconi anemia (FA) is an autosomal recessive disorder characterized by skeletal abnormalities, pancytopenia and a marked predisposition to cancer. FA cells exhibit chromosomal instability and hypersensitivity towards oxygen and cross-linking agents such as diepoxybutane and mitomycin C. An increased level of reactive oxygen intermediates and an elevation of 8-oxoguanine in FA cells point to a defective oxygen metabolism in FA cells. We investigated the repair activity of oxidatively damaged DNA in lymphoblastoid cells from FA patients of complementation groups A-E. The repair activity for oxidatively damaged DNA was significantly reduced in lymphoblastoid cell lines of complementation groups B-E. Complementation of the FA-C cell line with the wild type FA-C gene restored the repair activity to normal. This indicates that the FA-C protein participates in the repair of oxidatively damaged DNA.

Overexpression of thioredoxin in Fanconi anemia fibroblasts prevents the cytotoxic and DNA damaging effect of mitomycin C and diepoxybutane.

Adult T cell leukemia derived factor (ADF)/thioredoxin (Trx) is known to be an important intracellular antioxidant involved in a number of redox reactions such as ribonucleotide reductase (RNR) as well as of tyrosinase. Since RNR is a key enzyme of nucleotide metabolism and DNA synthesis, a reduced Trx level would result in reduced enzymatic activity and cause DNA damage. Furthermore, Trx is considered to be an effective regulator of redox sensitive gene expression. The role of Trx in nucleotide metabolism and gene expression may be an explanation for increased chromosomal instability as well as hypersensitivity towards oxygen, ROI and ROI generating agents. The activity of tyrosinase, the key enzyme of melanin biosynthesis, is influenced by the thioredoxin level and by superoxide radicals. Low thioredoxin levels and high superoxide concentrations activate tyrosinase causing hyperpigmentation of the skin. In addition to the observed high superoxide concentration in Fanconi anemia (FA) patients, a low thioredoxin level might be responsible for the hyperpigmentation (café-au-lait spots) in this disease. We observed that overexpression of the thioredoxin cDNA in FA fibroblasts completely abolished the DNA damaging effects of mitomycin C and diepoxybutane and inhibited the constitutive activity of the nuclear factor kappaB (NF-kappaB) in SV40 transformed FA fibroblasts. However, spontaneous chromosomal breakage was not affected.

The role of oxygen metabolism for the pathological phenotype of Fanconi anemia.

The molecular defect of the hereditary disease Fanconi anemia (FA) remains unknown. The two theoretical possibilities are (1) an impaired DNA crosslink-repair system or (2) a disturbed oxygen metabolism either by overproduction of reactive oxygen intermediates (ROI) or by diminished detoxification of ROI. In order to gain further insight into the molecular mechanism of this disease, we have determined the repair capacity of FA cells challenged by crosslinking agents and have analyzed diverse biological systems that are involved in oxygen metabolism. We have tested normal and FA cells for oxygen consumption and for the activity of the antioxidant phospholipid-hydroperoxide-glutathione-peroxidase (PHGPx). FA cells show a reduced oxygen consumption and an increased PHGPx activity. Since spontaneous and induced chromosomal instability is a main cellular feature of FA, we have analyzed the redox state of cells and the effect of cytochrome P-450 (Cyt P-450) inhibitors and inducers on chromosomal breaks and micronuclei production. Our results indicate that Cyt P-450 enzymes, especially Cyt P-450 1A2, play a crucial role in radical metabolism in FA cells. Furthermore, we have determined NF-kappa B activity in untransformed cells and in SV40-transformed cells by gel shift experiments. NF-kappa B is a multiunit transcription factor that is known to be induced by ROI and that activates the expression of various genes involved in cellular responses to stress. NF-kappa B is constitutively induced in SV40-transformed FA cells probably as a consequence of an increased ROI level. Our results suggest that enzymatic defects in oxygen metabolism mediate the FA phenotype via impaired reactivity with ROI. Cyt P-450 1A2 appears to be a good candidate for the defective enzyme, even though no differences have been measured in the activity of this enzyme in FA and control fibroblasts in pilot experiments.

Werner syndrome: characterization of mutations in the WRN gene in an affected family.

Affected and unaffected members of a Caucasian family with Werner syndrome were analyzed for mutations in the recently described Werner syndrome (WRN) gene and for their relevance to phenotypic expression of chromosomal instability and x-ray hypersensitivity. Two distinct molecular alterations were documented in the family. Analysis of the genomic DNA revealed a single-base exchange from A to T at an intron-exon boundary in the otherwise strongly conserved 5' donor splice site. Consequently, exon 30 is spliced together with the intron. The ensuing structure could be confirmed by the presence and calculated size of the resulting RNA fragments. The patients, all compound heterozygotes, had a 1-bp deletion in the first third of the coding sequence in the other allele. The genotypes of the family members for these mutations were determined and consequences for the cellular phenotype of the otherwise unaffected heterozygotes are documented.

Activity of ternary gelatinase A-TIMP-2-matrix metallo-proteinase complexes.

The progelatinase A-TIMP-2 complex behaves like a Janus. Like TIMP (tissue inhibitor of metalloproteinases) it inhibits active matrix metalloproteinases, and activation with 4-aminophenylmercury acetate leads to a gelatinolytic activity. This activity, however, amounts only to less than 10% of that of free gelatinase A not complexed with TIMP-2. When the progelatinase A-TIMP-2 complex inhibits an active matrix metalloproteinase, a ternary complex is generated. After activation with 4-aminophenylmercury acetate this ternary complex displays a more than tenfold proteolytic activity compared to activated gelatinase A-TIMP-2 complex, thus reaching the activity of free gelatinase A. The activity of the ternary complex is nearly independent from the bound matrix metalloproteinase. When the progelatinase A-TIMP-2 complex is activated at first with 4-aminophenylmercury acetate the generation of the ternary complex is made impossible and not such a significant enhancement of activity is observed. These results suggest that gelatinase A-TIMP-2 complex may be a matrix metalloproteinase of the 'second step': It starts its proteolytic attack after it has switched off the activity of other matrix metalloproteinases.