Short communication: Staphylococcus aureus isolated from colostrum of dairy heifers represent a closely related group exhibiting highly homogeneous genomic and antimicrobial resistance features.

In heifers, intramammary infections caused by Staphylococcus aureus affect milk production and udder health in the first and subsequent lactations, and can lead to premature culling. Not much is known about Staph. aureus isolated from heifers and it is also unclear whether or not these strains are readily transmitted between heifers and lactating herd mates. In this study, we compared phenotypic characteristics, spa types, and DNA microarray virulence and resistance gene profiles of Staph. aureus isolates obtained from colostrum samples of dairy heifers with isolates obtained from lactating cows. Our objective was to (1) characterize Staph. aureus strains associated with mastitis in heifers and (2) determine relatedness of Staph. aureus strains from heifers and lactating cows to provide data on transmission. We analyzed colostrum samples of 501 heifers and milk samples of 68 lactating cows within the same herd, isolating 48 and 9 Staph. aureus isolates, respectively. Staphylococcus aureus strains from heifers, lactating herd mates, and an unrelated collection of 78 strains from bovine mastitis milk of mature cows were compared. With 1 exception each, characterization of all strains from heifers and lactating cows in the same herd yielded highly similar phenotypic and genotypic results. The strains were Staphaurex latex agglutination test negative (Oxoid AG, Basel, Switzerland) and belonged to agr type II, CC705, and spa types tbl 2645 and t12926. They were susceptible to all antimicrobial agents tested. In contrast, the strains from mature cows in other herds were spread across different clonal complexes, spa types, and SplitsTree clusters (http://www.splitstree.org/), thus displaying a far higher degree of heterogeneity. We conclude that strains isolated from colostrum of heifers and mastitis milk of lactating cows in the same herd feature highly similar phenotypic and genomic characteristics, suggesting persistence of the organism during the first and potentially subsequent lactations or transmission between heifers and mature herd mates.

Efficiency of octenidine dihydrochloride alcohol combination compared to ethanol based skin antiseptics for preoperative skin preparation in dogs.

OBJECTIVE: To quantify the bacterial burden after skin disinfection using an alcohol octenidine dihydrochloride combination (Octenisept®) compared to an 74.1% ethanol 10% 2-propanol combination (Softasept N®). STUDY DESIGN: Prospective randomized clinical trial. MATERIAL & METHODS: 61 dogs undergoing clean or clean-contaminated surgeries (excluding surgeries on the gastrointestinal tract) were randomly assigned to group O (skin disinfection with alcohol and octenidine dihydrochloride after washing with octenidine containing soap) or to control group C (skin disinfection using the ethanol-2-propanol combination after washing with a neutral soap without antiseptic ingredients). Samples were then taken from 8 different locations within the surgical field at four different stages: after clipping, after washing, after disinfection and one hour later. At each stage, two different sampling techniques (wet-dry swab technique (WDS) and contact plates (CP)) were used for quantitative analysis of bacterial counts. RESULTS: WDS detected about 100-fold more bacteria compared to CP sampling in cases with high bacterial burden, but was not accurate enough to detect small numbers. CP sampling was therefore used for comparison of treatment protocols. 30 dogs were assigned to group O and 31 to group C. A relative reduction of 69% in group O and 77 percent in group C was observed after the soap wash. No significant differences were detected between both groups. Washing and disinfection resulted in a reduction of bacterial counts of 99.99% in group O versus 99.7% in group C (p = 0.018). Bacterial reduction one hour after washing and disinfection was significantly higher in group O (99.9%) than in group C (98.5%, p = 0.001). CONCLUSION: Additional octenidine dihydrochloride provided a slightly better decontamination effect after disinfection, particularly one hour after, which means it may only be indicated in longer surgeries. WDS is more sensitive but less specific to detect bacteria on the skin than the CP sampling.

Haematological findings in 158 cows with acute toxic mastitis with a focus on the leukogram.

BACKGROUND: In cows with acute toxic mastitis (ATM), the leukogram aids in the assessment of the severity of disease. The goal of our study was to compare the leukogram of 158 cows with ATM (cases) and 168 clinically healthy cows (controls). We hypothesised that the leukograms of surviving and non-surviving cows differ and that there are variables of the leukogram with sufficient prognostic potential to be used in the decision to treat or not to treat a cow with ATM. The cows were examined clinically and underwent haematological and biochemical examination of blood and bacteriological culture of milk samples. RESULTS: All cows with ATM had a poor appetite or anorexia, and 34 cows (21.5%) were recumbent. A single quarter was affected in 119 cows (75.3%), two quarters in 37 cows (23.4%) and three quarters in two cows (1.3%). Bacteriological culture showed Gram-negative pathogens in 100 cows (63.3%), Gram-positive in 15 (9.5%) and yeast in 4 (2.5%). The median total leukocyte count of cases was 4300 cells/µL (interquartile range = 2300-8200/µL), which was significantly lower than 8000 cells/µL (6525-9300/µL) in controls. Except for band neutrophils and metamyelocytes, the counts of all components of the leukogram were lower in cases compared with controls. Significantly more cows with ATM had leukopenia (60.1 vs. 4.1%) or leukocytosis (10.1 vs. 3.0%) than controls. Diseased cows had significantly lower segmented neutrophil counts than controls (860 vs. 2598 cells/µL), and 69.5 and 17.3%, respectively, had counts below the reference interval. Cases had increased band (77.3%) and metamyelocyte (25.0%) counts compared with controls (0.6 and 0%, respectively). In diseased cows, eosinopenia occurred in 66.4% (controls, 1.8%), monocytopenia in 40.6% (4.2%) and lymphopenia in 60.2% (1.8%). Twenty-one diseased cows (16.4%) had a regenerative and 57 (44.5%) had a degenerative left shift. The median neutrophil-to-lymphocyte ratio was 0.97 in diseased cows and 0.63 in controls. Toxic changes in neutrophils including cytoplasmic basophilia and vacuolisation were seen in 101 (91.8%) of 110 blood smears of diseased cows. The leukogram of the surviving and non-surviving cows did not differ significantly, and the hypothesis was rejected. CONCLUSIONS: ATM results in severe changes in the leukogram particularly leukopenia, lymphopenia, and degenerative left shift. The hypothesis that the leukograms of surviving and non-surviving cows differ was rejected. The leukogram has not sufficient prognostic potential to be used in the decision to treat or not to treat a cow with ATM.

Whole Genome Sequencing Reveals Biopesticidal Origin of Bacillus thuringiensis in Foods.

Bacillus thuringiensis is a microbial insecticide widely used to control agricultural pests. Although generally regarded as safe, B. thuringiensis is phylogenetically intermingled with the foodborne pathogen B. cereus sensu stricto and has been linked to foodborne outbreaks. Limited data on the pathogenicity potential of B. thuringiensis and the occurrence of biopesticide residues in food compromise a robust consumer risk assessment. In this study, we analyzed whole-genome sequences of 33 B. thuringiensis isolates from biopesticides, food, and human fecal samples linked to outbreaks. All food and outbreak-associated isolates genomically matched (≤ 6 wgSNPs; ≤ 2 cgSNPs) with one of six biopesticide strains, suggesting biopesticide products as their source. Long-read sequencing revealed a more diverse virulence gene profile than previously assumed, including a transposase-mediated disruption of the promoter region of the non-hemolytic enterotoxin gene nhe and a bacteriophage-mediated disruption of the sphingomyelinase gene sph in some biopesticide strains. Furthermore, we provide high-quality genome assemblies of seven widely used B. thuringiensis biopesticide strains, which will facilitate improved microbial source tracking and risk assessment of B. thuringiensis-based biopesticides in the future.

Comparative Genome Analysis and Phenotypic Characterization of Clostridium gasigenes CGAS001 Isolated From Chilled Vacuum-Packed Lamb Meat.

Genomic data for psychrophilic bacteria causing blown pack spoilage (BPS) are limited. This study characterizes the genome of a novel Clostridium gasigenes strain CGAS001 isolated from meat juice sample (MJS) of vacuum-packed lamb meat by comparing it with the type strain C. gasigenes DSM 12272 and five strains representing four other BPS-causing Clostridium sensu stricto species. Phenotypic characteristics of the strain, which include biochemical characteristics, antimicrobial resistance and production of putative polyketide, have been determined. The size of its draft genome is 4.1 Mb with 3,845 coding sequences, 28.7% GC content and 95 RNA genes that include 75 tRNAs, 17 rRNAs, and 3 ncRNAs. Average Nucleotide Identity (ANI) and digital DNA-DNA Hybridization (dDDH) predict that C. gasigenes CGAS001 and DSM 12272 constitute a single species (ANI and dDDH = 98.3% for speciation) but two distinct subspecies (dDDH = 73.3% for subspeciation). The genome is characterized by saccharolytic, lipolytic and proteolytic genes as well as hemolysins and phospholipases, which are consistent with its phenotype. The genome also reveals the ability of C. gasigenes to synthesize polyketides which is demonstrated by the antimicrobial activity of a crude polyketide extract against Listeria monocytogenes and Enterococcus devriesei. The strain is resistant to polymyxin B and streptomycin. The genetic and phenotypic analyses suggest that CGAS001 constitutes a novel subspecies of C. gasigenes adapted to a saprophytic lifestyle and can synthesize narrow spectrum antimicrobial compounds.

Detection of Psychrophilic Clostridium spp. Causing "Blown Pack" Spoilage in Meat Juice Samples from Chilled Vacuum-Packed Beef and Lamb Meat Imported from Different Countries to Switzerland.

"Blown pack" spoilage (BPS) of chilled vacuum-packed meat is mainly caused by anaerobic and psychrophilic Clostridium spp., including C. estertheticum, C. gasigenes, C. frigoriphilum, and C. frigidicarnis. Recently, its occurrence has been reported in several countries, especially in internationally traded meat. Therefore, this study aimed at detecting the occurrence of psychrophilic Clostridium spp. causing BPS in meat juice samples (MJS) from chilled vacuum-packed beef and lamb meat imported from other countries to Switzerland. One hundred fifty-four MJS (n = 78 from beef; n = 76 from lamb meat) were screened for psychrophilic Clostridium spp. by quantitative PCR, whereby MJS with a crossing point PCR cycle value <35 and >35 were considered positive and negative, respectively. Psychrophilic Clostridium spp. were detected in 10 MJS, of which 2 were from beef and 8 were from lamb meat. The two beef MJS originated from Spain and Lithuania, whereas the lamb MJS originated from New Zealand (six) and Australia (two). This is the first report of psychrophilic Clostridium spp. in MJS from chilled vacuum-packed beef and lamb meat imported from other countries to Switzerland and provides further evidence that the risk of BPS in lamb meat is higher than in beef.

Growth Potential of Listeria monocytogenes in Three Different Salmon Products.

Cold smoked salmon and sushi salmon have been implicated in outbreaks of listeriosis. We performed challenge tests and a durability study with Listeria monocytogenes on different salmon products to determine the growth potential of this important food-borne pathogen. Data from the challenge test showed a significant growth potential of L. monocytogenes on all of the tested salmon products, with faster growth in sushi salmon than in cold smoked salmon. In identical products that were naturally contaminated at low levels, the durability study did not confirm a high growth potential, possibly due to interactions with competing microflora. The injection of sodium lactate (NaL) at a high concentration (30%) into cold smoked salmon significantly reduced the growth potential of L. monocytogenes. In addition to good manufacturing practices, the injection of higher concentrations of NaL may therefore be a useful additional hurdle to prevent growth of L. monocytogenes to high numbers in the tested salmon products.

Draft Genome Sequences of Two Phylogenetically Distinct Clostridium gasigenes Strains, CM001 and CM004, Isolated from Chilled Vacuum-Packed Meat.

We present the draft genome sequences of Clostridium gasigenes strains CM001 and CM004. The genomes are 4,147,089 and 4,191,074 bp with GC contents of 28.7% and 28.8%, respectively. Although both strains belong to the same species, whole-genome sequence-based analyses reveal that the strains are phylogenetically distinct.

Staphylococcus aureus Population Structure and Genomic Profiles in Asymptomatic Carriers in Switzerland.

Staphylococcus aureus is a leading cause for clinical infections and food intoxications, causing over 100,000 yearly cases of bacteremia in the United States and 434 food-borne outbreaks in the European Union. Approximately 30% of the population permanently carry S. aureus asymptomatically in their nasal cavity. The risk of infection and transmission to food items or the environment is higher in individuals that are nasally colonized. In addition, S. aureus can acquire various antimicrobial resistances leading to therapeutic failure, additional medical costs, and fatalities. Methicillin-resistant S. aureus (MRSA) cause a considerable burden of disease in humans and animals. MRSA carriage has been associated with animal and in particular livestock contact. Extensive current data on the virulence gene profiles, as well as data on antimicrobial resistance determinants is crucial in developing effective strategies to mitigate the burden of disease. To this end, we screened the anterior nares of 160 test subjects (87 pupils and 73 members of farmer families) in Switzerland for S. aureus carriage. A total of 73 S. aureus isolates were obtained. Factors such as exposure to farm or companion animals and personal medical history were recorded using a questionnaire. Using a DNA microarray, isolates were assigned to clonal complexes (CCs), and virulence and resistance gene profiles were determined. The collected strains were assigned to 20 CCs, among others CC1, CC7, CC8, CC15, CC30, CC45, CC97, and CC398. Two MRSA strains and one multiresistant isolate carrying genes blaZ/I/R, InuA, aadD, tetK, and fosB were isolated from farmers with intensive exposure to animals. Strains carrying pvl, causing severe skin lesions and necrotizing pneumonia, as well as tetracycline, erythromycin, and kanamycin resistance genes were found in individuals that had taken antibiotics during the last year. A variety of superantigenic toxin genes was detected, including among others, the toxic shock syndrome toxin (tst1), and various enterotoxins (sea, sec, sel, and the egc cluster). Contact to chickens was identified as a significant factor contributing to S. aureus colonization.

Draft Genome Sequence of Streptococcus parasuis 4253, the First Available for the Species.

Here, we report the draft genome sequence of Streptococcus parasuis strain 4253. This is the first publicly available genome sequence of a S. parasuis strain.

Molecular types, virulence profiles and antimicrobial resistance of Escherichia coli causing bovine mastitis.

BACKGROUND: Escherichia coli is an important aetiological agent of bovine mastitis worldwide. METHODS: In this study, 82 E. coli from bovine mastitis milk samples from 49 farms were analysed for their genetic diversity using phylogenetic grouping and multilocus sequence typing. The isolates were examined by PCR for a selection of virulence factors (VFs). Antimicrobial susceptibility profiles were assessed using the disk diffusion method. RESULTS: The most prevalent phylogroups were group B1 (41.5 per cent of the isolates) and group A (30.5 per cent). A variety of 35 different sequence types (STs) were identified, including ST1125 (11 per cent), ST58 (9.8 per cent), ST10 (8.5 per cent) and ST88 (7.3 per cent). Aggregate VF scores (the number of unique VFs detected for each isolate) ranged from 1 to 3 for 63.4 per cent of the isolates and were at least 4 for 12.2 per cent. For 24.4 per cent of the isolates, the score was 0. The three most frequent VFs were traT, fyuA and iutA. The majority (72 per cent) of the isolates harboured traT. The majority (68.3 per cent) of the isolates were fully susceptible to all antimicrobials tested, with 22 per cent resistant to ampicillin and 14.6 per cent to tetracycline. Resistance rates were low for gentamicin (3.7 per cent), amoxicillin/clavulanic acid (2.4 per cent) and ceftiofur (1.2 per cent), respectively. CONCLUSION: Among the study's sample population, E. coli strains were genotypically diverse, even in cows from the same farm, although some STs occurred more frequently than others. Susceptibility to clinically relevant compounds remained high.

Sequence Types and Antimicrobial Resistance Profiles of Streptococcus uberis Isolated From Bovine Mastitis.

Bovine mastitis is one of the most common diseases among dairy cows and causes high economic losses in dairy industries worldwide. Streptococcus uberis is one of the most frequently identified pathogens causing the disease. In this study, 153 S. uberis strains isolated from mastitis milk samples were analyzed for their genetic diversity using multi locus sequence typing (MLST). Moreover, antibiotic susceptibility testing was performed using a microdilution assay and 11 antimicrobial agents including penicillin, which is the first line agent for treatment of bovine mastitis in Switzerland. MLST was successful for 152 (99.3%) of the strains. Overall, 103 different sequence types (STs) were determined, including 91 novel STs. S. uberis belonging to clonal complex (CC) 5 represented 47 (30.7%) of the mastitis cases. Two (1.3%) of the strains belonged to CC86 and one (0.7%) to CC143. The population structure identified in this work suggests that environmental transmission is the predominant route of infection in herds in Switzerland. Antimicrobial susceptibility testing determined a resistance rate of 11.8% for pirlimycin and elevated MIC90-values for marbofloxacin as well as for erythromycin. This study highlights the importance of genetic characterization of S. uberis and the need for veterinary breakpoints for surveillance of antimicrobial resistance in S. uberis.

Microbiological quality and presence of foodborne pathogens in raw milk cheeses and raw meat products marketed at farm level in Switzerland.

This study investigated the microbiological quality and presence of bacterial foodborne pathogens in 51 raw milk cheeses (mainly semihard and hard cheese) and 53 raw meat products (cured meat products and sausages) marketed at farm level. With regard to Enterobacteriaceae, Escherichia (E.) coli, and coagulase-positive staphylococci (CPS), the examined products were generally of a good microbiological quality. Enterobacteriaceae were found in seven cheeses (1.0×102 - 8.8×104 CFU/g) and one sausage (2.0×102 CFU/g). Three of these cheeses were also positive for E. coli. CPS results were comparable for cheeses (5.9%; 1.0-6.0×102 CFU/g) and meat products (3.8%; 1.0-2.0×102 CFU/g). On the other hand, such raw products may harbor potential health hazards as Listeria (L.) monocytogenes, Shiga toxin-producing E. coli (STEC), and staphylococcal enterotoxin (SE)-producing Staphylococcus (S.) aureus. L. monocytogenes were found in one sausage and the isolate belonged to the serotype 1/2c. The two STEC isolates harbored stx1a (cheese) or stx2e (sausage), but both lacked eae and did not belong to the top five-serogroups. Of the five S. aureus isolates, the three cheese isolates belonged to the clonal complex (CC) 8, CC22, and CC705, the two sausage isolates belonged to CC7, and all isolates harbored genes for SEs. Thus, to avoid contaminations and to prevent foodborne pathogens from entering the food chain, strict compliance with good hygiene practices during milk and cheese production or meat production is of central importance.

Complete and Assembled Genome Sequence of Salmonella enterica subsp. enterica Serotype Senftenberg N17-509, a Strain Lacking Salmonella Pathogen Island 1.

The genome of Salmonella enterica subsp. enterica serotype Senftenberg N17-509, a strain isolated from desiccated coconut, was sequenced using single-molecule real-time sequencing. It consists of a 5.1-Mbp chromosome and a 29-kb linear plasmid.

Effect of a commercial steam-vacuuming treatment implemented after slaughtering for the decontamination of cattle carcasses.

To assess the antimicrobial effect of a commercial steam-vacuuming system newly implemented after slaughtering, 105 cattle carcasses were examined for total viable counts (TVC) at four different areas. Before steam vacuuming, mean TVC of the excision samples were comparable at the perineal area and brisket (3.0-3.1 log CFU cm-2) or the hind leg and shoulder (2.6-2.7 log CFU cm-2). Steam vacuuming reduced mean TVC by 0.9, 0.7, 0.6, and 0.4 log CFU cm-2 at the perineal area, hind leg, shoulder, and brisket, respectively. With regard to the distribution of counts, steam vacuuming increased the proportion of TVC results <3.0 log CFU cm-2 from 74.8% (62.9-87.6% at carcass areas) to 86.7% (71.4-97.1% at carcass areas). Thus, steam vacuuming after slaughtering might be useful for the reduction of contamination in designated carcass areas, but the effect must not be overestimated and decontamination treatments always must be seen part of an integral food safety system.

Detection of Mycobacterium avium subspecies paratuberculosis specific IS900 insertion sequences in bulk-tank milk samples obtained from different regions throughout Switzerland.

BACKGROUND: Since Mycobacterium avium subspecies paratuberculosis (MAP) was isolated from intestinal tissue of a human patient suffering Crohn's disease, a controversial discussion exists whether MAP have a role in the etiology of Crohn's disease or not. Raw milk may be a potential vehicle for the transmission of MAP to human population. In a previous paper, we have demonstrated that MAP are found in raw milk samples obtained from a defined region in Switzerland. The aim of this work is to collect data about the prevalence of MAP specific IS900 insertion sequence in bulk-tank milk samples in different regions of Switzerland. Furthermore, we examined eventual correlation between the presence of MAP and the somatic cell counts, the total colony counts and the presence of Enterobacteriaceae. RESULTS: 273 (19.7%) of the 1384 examined bulk-tank milk samples tested IS900 PCR-positive. The prevalence, however, in the different regions of Switzerland shows significant differences and ranged from 1.7% to 49.2%. Furthermore, there were no statistically significant (p >> 0.05) differences between the somatic cell counts and the total colony counts of PCR-positive and PCR-negative milk samples. Enterobacteriaceae occur as often in IS900 PCR-positive as in PCR-negative milk samples. CONCLUSION: This is the first study, which investigates the prevalence of MAP in bulk-tank milk samples all over Switzerland and infers the herd-level prevalence of MAP infection in dairy herds. The prevalence of 19.7% IS900 PCR-positive bulk-milk samples shows a wide distribution of subclinical MAP-infections in dairy stock in Switzerland. MAP can therefore often be transmitted to humans by raw milk consumption.