Two cases of food-borne botulism in Finland caused by conserved olives, October 2011.

In October 2011 in Finland, two persons fell ill with symptoms compatible with botulism after having eaten conserved olives stuffed with almonds. One of these two died. Clostridium botulinum type B and its neurotoxin were detected in the implicated olives by PCR and mouse bioassay, respectively. The olives were traced back to an Italian manufacturer and withdrawn from the market. The public and other European countries were informed through media and Europe-wide notifications.

Comparison of phenotypic and genotypic detection of penicillin G resistance of Staphylococcus aureus isolated from bovine intramammary infection.

Resistance of Staphylococcus aureus to penicillin G is common among isolates from bovine mastitis. We determined phenotypic resistance to penicillin G for 151 S. aureus isolates derived from dairy cows with intramammary infection by two methods. The methods were determination of minimum inhibitory concentration (MIC) by a standard agar dilution technique and direct testing of beta-lactamase production using a chromogenic cephalosporin, nitrocefin. The results from these tests were compared with the presence of the beta-lactamase (blaZ) gene in the isolates, which was detected by polymerase chain reaction (PCR). Testing beta-lactamase production with nitrocefin was more predictive for the presence of the blaZ gene than the agar dilution method and the results of the former agreed highly with the presence of the blaZ gene in the isolates. In contrast, the resistance breakpoint generally used in the agar dilution method may be too high for prediction of penicillin resistance in S. aureus isolates with borderline MICs. Using this method, 40% of the isolates possessing the blaZ gene were classified as susceptible; however, majority of these isolates produced beta-lactamase when tested with nitrocefin.

Interlaboratory proficiency testing as a tool for improving performance in laboratories diagnosing bovine mastitis.

The National Veterinary and Food Research Institute (Finland) and the Veterinary Laboratories Agency of the Quality Assurance Unit, Department for Environment, Food and Rural Affairs, United Kingdom (previously the Ministry of Agriculture, Fisheries and Food) organized a proficiency testing program for laboratories analyzing veterinary mastitis samples. Three test samples with lyophilized strains of common aerobic bacteria were sent to the participating laboratories 7 times between 2000 and 2003. The participants returned 98% of the requested data. The overall performance of the laboratories varied from 63 to 93% in different testing rounds. All laboratories diagnosed Staphylococcus aureus and Escherichia coli correctly at every round. Improvement in diagnosing individual bacteria was observed for Staphylococcus epidermidis, Streptococcus dysgalactiae, Enterococcus spp. and Klebsiella spp. The overall performance of the laboratories improved with increased participation. The educational role of the program was important. Laboratories working in the veterinary field should implement a documented quality system covering all functions of the laboratory, as well as a planned quality assurance system.

Antimicrobial resistance in Staphylococcus spp., Escherichia coli and Enterococcus spp. in dogs given antibiotics for chronic dermatological disorders, compared with non-treated control dogs.

The aim of this study was to evaluate antimicrobial resistance in canine staphylococci, Escherichia coli and enterococci, which were isolated from 22 dogs with pyoderma and a history of previous antibiotic treatment, compared to bacterial isolates from 56 non-treated control dogs. Two isolates of each bacterial species per dog were investigated, if detected. Staphylococcal isolates from dogs with pyoderma (35 isolates) were more resistant to sulphatrimethoprim than the isolates from controls (56 isolates) (57% vs. 25%, p < 0.004). Multiresistance in staphylococci was also more common in dogs with pyoderma (29% vs. 9%, p = 0.02). A similar trend among isolates of E. coli was detected (24 and 74 isolates from treated and control dogs, respectively), but the differences were not significant. Resistance for macrolide-lincosamides was approximately 20% among staphylococci in both groups. Resistance to ampicillin among enterococci was 4%-7%. The age of the dogs might have an impact on resistance: multiresistance among staphylococcal isolates from younger dogs (< or = 5 years) was more common than in older dogs (26 years) (24%, vs. 0%, 63 and 27 isolates, respectively, p = 0.02). Staphylococci in younger dogs were more resistant to tetracycline (48% vs. 11%, p < 0.001) and sulphatrimethoprim (48% vs. 15%, p < 0.01) than those in older dogs. In contrast, the isolates of E. coli from older dogs tended to be more resistant, although a significant difference was detected only in resistance to tetracycline (13% vs. 2% of 40 and 50 isolates respecthely, p = 0.04)). The results of this small study indicate that resistance in canine staphylococci in the capital area of Finland is comparable with many other countries in Europe. Resistance in indicator bacteria, E. coli and enterococci, was low.

Bovine mastitis in Finland 2001--prevalence, distribution of bacteria, and antimicrobial resistance.

A nationwide survey was conducted in Finland to estimate prevalence of bovine mastitis, distribution of mastitis pathogens, and in vitro antimicrobial susceptibility of different mastitis pathogens. In total, 12,661 quarter milk samples were collected from 3282 dairy cows at 216 farms. These were randomly selected from a database covering all Finnish dairy farms. Quarter milk samples collected by the dairy advisors were submitted for somatic cell counting, bacteriological examination, and testing for antimicrobial susceptibility. If the milk SCC of a cow or of a quarter exceeded 300,000/mL, the cow was defined as having mastitis. The results were compared with those of a previous survey done in 1995. The prevalence of mastitis continued to decrease from 38% in 1995 to 31% in 2001. Compared with the study from 1995, the number of quarters with bacterial growth in 2001 increased significantly from 21.0 to 33.5%. This mainly resulted from increased prevalence of Corynebacterium bovis. Coagulase-negative staphylococci remained the most common bacterial group, comprising almost one-half of the pathogens isolated, whereas the relative number of Staphylococcus aureus isolations decreased from the time of the previous study. According to in vitro antimicrobial susceptibility testing, the enterococci demonstrated the highest level of resistance. Compared with the other Nordic countries, penicillin resistance among the staphylococci was still at a relatively high level in Finland (52.1 and 32.0% for Staphylococcus aureus and coagulase-negative staphylococci, respectively). Streptococci isolated from mastitis were very susceptible to beta-lactam antibiotics, as also found in the previous survey in 1995.

In vitro antimicrobial susceptibility of Escherichia coli isolates from clinical bovine mastitis in Finland and Israel.

Minimal inhibition concentration (MIC) values of 100 Finnish and 100 Israeli Escherichia coli isolated from clinical bovine mastitis were determined for ampicillin, cephalexin, ceftazidime, dihydrostreptomycin, gentamicin, tetracycline, trimethoprim-sulfadiazine, and ciprofloxacin by an agar dilution method. The in vitro antimicrobial susceptibility of the E. coli isolates was high; only 27% showed resistance to one or more tested antimicrobial agents. Fifteen percent of the Israeli isolates and 14% of the Finnish isolates were resistant to tetracycline, 3 and 16% to cephalexin, 10 and 7% to ampicillin, 13 and 9% to dihydrostreptomycin, and 4 and 2% to trimethoprim-sulfadiazine. No gentamicin-, ceftazidime-, or ciprofloxacin-resistant isolates were detected. Eleven percent of all the isolates were resistant to two or more antimicrobial agents. Tetracycline was most often associated with multiresistant patterns. Most of the multiresistant isolates had very high MIC values, whereas most of those that were resistant to only one tested antibiotic had MIC values close to the susceptibility breakpoint. Antimicrobial resistance appeared to pose no problem in E. coli isolated from mastitic milk of both countries. This is probably due to the controlled use of antimicrobial agents in the treatment of dairy herds. Some differences were present in the resistance patterns, which may reflect the different use of antimicrobial agents in these two countries.

Prevalence of campylobacters in chicken flocks during the summer of 1999 in Finland.

In order to determine the prevalence of campylobacter positive broiler flocks in Finland, every flock from all three major slaughterhouses was studied during the period from 1 May to 30 September 1999. Caecal samples were taken in the slaughterhouses from five birds per flock. A total of 1132 broiler flocks were tested and 33 (2.9%) of those were campylobacter positive. Thirty-one isolates were C. jejuni and two isolates were C. coli. Isolates were serotyped for heat-stable antigens (Penner) and genotyped with pulsed-field gel electrophoresis (PFGE). The most common serotypes were serotypes 6, 7, 12 and 4-complex. Together with SmaI and KpnI patterns there were 18 different PFGE genotypes. Simultaneous monitoring of chicken flocks and typing of the isolates produced data which can be used to study the epidemiology of campylobacters in chicken as well as their role in human infections.

Use of phenotyping and genotyping to verify transmission of Escherichia coli O157:H7 from dairy farms.

A total of 80 human infections by Escherichia coli O157:H7 were documented in Finland in 1997 and 1998. Most were sporadic and their sources undetermined. Five cases not associated with one another, one of which led to secondary transmission within a family, could be traced to five different dairy farms. These five case patients (age range 2-17 years, median age 3 years) were hospitalised with bloody diarrhoea; two of them developed haemolytic uraemic syndrome. All nine human isolates obtained were sorbitol negative, carried the verocytotoxin 2 and eae genes, and produced verocytotoxin and enterohaemolysin. The phage and pulsed-field gel electrophoresis types of the human and bovine isolates from the corresponding farms were indistinguishable. The cattle (20-70 animals per farm) were monitored for up to 2 years after the human cases. The proportion of cattle excreting the type that caused the human infections varied from 3.2 to 66.7% when sampled soon after the human cases, and from 0.0 to 5.3% about a year or so later. On most of the farms, the animals excreted the pathogen intermittently. On one farm, Escherichia coli O157 isolates with other characteristics were also occasionally isolated. Although the infections were traced back to the farms, it could not be established whether the source was unpasteurised milk or direct or indirect contact with cattle. The results of this study emphasise the need for special recommendations for children visiting or living on a farm to prevent these infections.

Occurrence of Escherichia coli O157 in Finnish cattle.

Bovine faecal samples were collected during June-December 1997 at 14 major abattoirs slaughtering cattle in Finland. Escherichia coli O157 was isolated from 19 of the 1448 samples (1.31%) after enrichment and immunomagnetic separation (IMS). The positive faecal isolates originated from 16 farms and eight abattoirs. The occurrence of E. coli O157 was highest in July (8/204; 3.92%) and September (6/244; 2.46%). No E. coli O157 was detected in November and December, nor from the faecal samples from the northernmost region where cattle density is low. All of the isolates carried the eae gene and showed the enterohaemolytic phenotype. All except one were motile and had the flagella antigen H7. Seventeen of the isolates were positive for stx(2) gene and one carried both the stx(1) and stx(2) genes. Of the 17 isolates with stx genes, 16 were verocytotoxin-positive in a reversed passive latex agglutination test after polymyxin extraction but only eight without extraction. The isolates belonged to 10 different pulsed-field gel electrophoresis (PFGE) patterns. The most common PFGE pattern (1.42) was detected in eight isolates (42.1%). Four PFGE patterns (1.1; 1.6; 1.12; 1.14) were identical with those isolated from humans in Finland, suggesting that at least some human E. coli O157 infections may be of bovine origin.

Exposure of Listeria monocytogenes within an epidemic caused by butter in Finland.

Data on the levels of bacteria and the amounts of food consumed in food-borne outbreaks provides an excellent opportunity to study the effects of exposure to Listeria monocytogenes. Between June 1998 and April 1999, an outbreak caused by L. monocytogenes serotype 3a in butter occurred in Finland. The majority of the cases were immunocompromised and hospitalized at the Helsinki University Central Hospital (HUCH), where 7-g butter packages produced by a dairy plant were used as the only butter brand. The butter had also been sold to 10 other central hospitals as well as to the retail market. Based on the data on hospital stay, butter consumption and the qualitative and quantitative analyses of L. monocytogenes in butter, the attack rates and exposure were estimated. Incubation studies on the naturally contaminated small butter packages showed that the levels found in the packages at the time of detection of the outbreak could reliably be used for these estimations. However, the levels of L. monocytogenes in 500-g packages increased. The attack rate among HUCH patients varied from 70 to 117 cases per 1000 patients at risk, depending on which estimate of the contamination level of butter (100-60%) was used. The highest single dose (7.7 x 10(4) CFU in one meal) could have been sufficient to cause the listeriosis cases at HUCH. However, this data also supports another hypothesis, according to which these listeriosis cases were caused by a prolonged daily consumption of contaminated butter during the hospital stay. The estimated daily dose, based on the hospital kitchen data or the highest detected level in a wholesale sample (11,000 CFU/g), would have varied from 1.4 x 10(1) to 2.2 x 10(3) CFU/day or from 2.2 x 10(4) to 3.1 x 10(5) CFU/day, respectively. The choice of the hypothesis has a crucial impact on the interpretation of this data for the dose-response estimations as well as for the discussion on Food Safety Objectives. Due to the susceptibility of hospital patients, special care must be taken in order to avoid even low levels of L. monocytogenes in food served.

An outbreak of Listeria monocytogenes serotype 3a infections from butter in Finland.

In February 1999, an outbreak of listeriosis caused by Listeria monocytogenes serotype 3a occurred in Finland. All isolates were identical. The outbreak strain was first isolated in 1997 in dairy butter. This dairy began delivery to a tertiary care hospital (TCH) in June 1998. From June 1998 to April 1999, 25 case patients were identified (20 with sepsis, 4 with meningitis, and 1 with abscess; 6 patients died). Patients with the outbreak strain were more likely to have been admitted to the TCH than were patients with other strains of L. monocytogenes (60% vs. 8%; odds ratio, 17.3; 95% confidence interval, 2.8-136.8). Case patients admitted to the TCH had been hospitalized longer before cultures tested positive than had matched controls (median, 31 vs. 10 days; P=.008). An investigation found the outbreak strain in packaged butter served at the TCH and at the source dairy. Recall of the product ended the outbreak.

Toxigenic strains of Bacillus licheniformis related to food poisoning.

Toxin-producing isolates of Bacillus licheniformis were obtained from foods involved in food poisoning incidents, from raw milk, and from industrially produced baby food. The toxin detection method, based on the inhibition of boar spermatozoan motility, has been shown previously to be a sensitive assay for the emetic toxin of Bacillus cereus, cereulide. Cell extracts of the toxigenic B. licheniformis isolates inhibited sperm motility, damaged cell membrane integrity, depleted cellular ATP, and swelled the acrosome, but no mitochondrial damage was observed. The responsible agent from the B. licheniformis isolates was partially purified. It showed physicochemical properties similar to those of cereulide, despite having very different biological activity. The toxic agent was nonproteinaceous; soluble in 50 and 100% methanol; and insensitive to heat, protease, and acid or alkali and of a molecular mass smaller than 10,000 g mol(-1). The toxic B. licheniformis isolates inhibited growth of Corynebacterium renale DSM 20688(T), but not all inhibitory isolates were sperm toxic. The food poisoning-related isolates were beta-hemolytic, grew anaerobically and at 55 degrees C but not at 10 degrees C, and were nondistinguishable from the type strain of B. licheniformis, DSM 13(T), by a broad spectrum of biochemical tests. Ribotyping revealed more diversity; the toxin producers were divided among four ribotypes when cut with PvuII and among six when cut with EcoRI, but many of the ribotypes also contained nontoxigenic isolates. When ribotyped with PvuII, most toxin-producing isolates shared bands at 2.8 +/- 0.2, 4.9 +/- 0.3, and 11.7 +/- 0.5 or 13.1 +/- 0.8 kb.

Occurrence and typing of Listeria monocytogenes strains in retail vacuum-packed fish products and in a production plant.

One hundred and ten samples of ready-to-eat, vacuum-packed, smoked and cold-salted fish products were collected from retail outlets in southern Finland during 1996 for examination of the occurrence and level of Listeria monocytogenes. The samples originated from 12 producers. Positive samples with levels exceeding 100 CFU/g were encountered mainly in one of the producers (no. 8). Therefore, 200 samples from the plant and the products of this producer were studied during August-September 1996 and May-September 1997, as well as 55 samples from the six fish farms providing raw material fish to this plant, during September 1997-January 1998. The isolates were characterised by serotyping and pulsed-field gel electrophoresis (PFGE). L. monocytogenes was isolated in 20% (22/110) of the samples from the retail market, originating from 6 producers. Ten of these positive samples contained L. monocytogenes at > 100 CFU/g (maximum 1.37 X 10(4) CFU/g). Seventeen percent (5/30) of cold-smoked and 50% (16/32) of cold-salted rainbow trout samples were contaminated. Only one hot-smoked fish product (2%) was found to be positive by enrichment. Nineteen (86%) of the strains isolated from the retail samples belonged to serovar 1/2a and three (14%) to serovar 4b. In further studies the production line of plant no. 8 was found to be contaminated. All of isolates from up until autumn, 1997 both the products and the production plant were serovar 1/2a; thereafter one strain of 4b and one of 1/2 (H-antigen untypeable) were isolated from the plant. The samples from raw material fish were all negative for L. monocytogenes. The samples from retail market fell into seven PFGE types. Five and nine PFGE types, respectively, were found from the products and the plant of producer no. 8. PFGE type A was detected from the retail products of four producers and was also dominant among the isolates from production plant no. 8. PFGE type A was the only one found repeatedly from skinning, salting and slicing units as well as from products throughout the whole period. PFGE proved to be a powerful tool for studying contamination points and routes in the production plant. The measures based on hazard analysis critical control points (HACCP) program resulted in L. monocytogenes negative samples at production plant no. 8 from the beginning of January 1998.

Bovine mastitis in Finland in 1988 and 1995--changes in prevalence and antimicrobial resistance.

Two surveys were carried out (during 1988 and 1995) to estimate the prevalence of bovine mastitis in Finland. In 1988, 17,111 quarter milk samples were obtained from 4495 cows, and in 1995 the corresponding figures were 10,410 and 2648. Antimicrobial susceptibility of mastitis pathogens was studied. Prevalence of mastitis on cow basis decreased from 47.8% in 1988 to 37.8% in 1995. Staphylococci was the largest group of pathogens isolated. The proportion of Staphylococcus aureus decreased and that of coagulase-negative staphylococci (CNS) increased. The proportion of strains resistant to at least one antibacterial drug increased with regard to S. aureus from 36.9% in 1988, to 63.6% in 1995 and with CNS from 26.6% to 49.7%. Most of the increase in antibacterial resistance was due to a higher number of beta-lactamase producing strains. Multiresistance also increased, but it was proportional to the overall increase in resistance. All the predominant mastitis streptococci were susceptible to beta-lactams tested.

Bovine clinical mastitis due to coagulase-negative staphylococci and their susceptibility to antimicrobials.

A total of 168 coagulase-negative staphylococci (CNS) strains were isolated from milk samples taken from cows with clinical mastitis. The samples were collected between January 1990 and August 1992 from cows in the veterinary surveillance area of the Ambulatory Clinic, College of Veterinary Medicine, Hautjärvi, Finland. In 100 cases the effect of antibiotic treatment was evaluated 3-4 weeks after initial sampling. Clinical symptoms of the animals were recorded, and the inflammatory status of their udders was evaluated using the CMT test and assessing milk NAGase activity. CNS mastitis was most common in young cows during early lactation. Staphylococcus hyicus, S. simulans and S. epidermidis were the most frequently isolated CNS. Clinical symptoms were most severe with S. hyicus. Cure rates for CNS induced mastitis were high.

Antibacterial susceptibility of bovine-mastitis pathogens tested directly in milk from infected quarters.

Antibacterial susceptibilities of bovine-mastitis pathogens were analysed directly in 57 mastitic milk samples without inoculation with exogenous organisms. Aseptically collected milk was mixed with serial dilutions of antibacterials and the growth was observed using 2,3,5-triphenyltetrazolium chloride (TTC) reduction the following day. The results were compared with those obtained by using calibrated bacterial inocula in turbidimetric minimum-inhibitory-concentration (MIC) determination in broth cultures, and in TTC-broth culture-test and TTC-normal milk-test. The results of different methods all correlated positively when the entire data was used. However, taking the direct test in mastitic milk as the 'true' result, the total discrepancies varied from 34.7% to 48.8%. Antibacterial activities of the trimethoprim-sulphadoxine combination, and of spiramycin and ampicillin, decreased significantly when nutrient broth was replaced by milk as the test medium. The efficacy of trimethoprim-sulphadoxine as an antibacterial agent was also dependent on the source of milk.

Effect of abrasion of teat orifice epithelium on development of bovine staphylococcal mastitis.

The predisposing effect of teat damage on mastitis caused by staphylococci and the pathogenicity of Staphylococcus aureus, Staphylococcus hyicus, and Staphylococcus epidermidis were investigated with an experimental model. The study included three experiments in which the teat canal orifice of 5 cows was slightly abraded. Experimental and control quarters were challenged with a staphylococcal suspension, and the status of the quarters was monitored. Virulence of the staphylococcal strains was studied using a protein-binding test with 125I-labeled proteins (fibronectin, fibrinogen, vitronectin, collagen type I and II, and IgG). Abrasion on the teat orifice epithelium was a predisposing factor for staphylococcal infections. Teat canal infection or colonization developed in 93% of experimental quarters and in 53% of control quarters; IMI developed in 73% of experimental quarters, but in none of the control quarters. Quarter IMI developed more consistently when the contaminating agent was S. aureus. Staphylococcus hyicus was very effective in causing teat canal infections, but S. epidermidis appeared to be less infectious. The S. aureus strain had strong binding sites for most of the proteins tested. The S. hyicus and S. epidermidis strains showed no binding, or only very weak binding, which correlated with lower infection rates.

Association af changes in the bacterial ecology of bovine mastitis with changes in the use of milking machines and antibacterial drugs.

The results of mastitis bacteriology made by the National Veterinary and Food Research Institute in Finland during the past 50 years (1.15 million samples) are viewed in relation to simultaneous changes in dairy cow management. Although intensive preventive measures have been applied for decades, the prevalence of bovine mastitis has not decreased. Instead, pathogenic bacteria are becoming progressively less susceptible to the available therapy. In part this must be due to the emergence of antibiotic resistant strains of bacteria, but it seems that the bacterial spectrum has also changed. The incidence of Streptococcus agalactiae (group B streptococci) has decreased, while in contrast, the incidence of staphylococci, initially Staphylococcus aureus and later coagulase-negative staphylococci, has increased. Results suggest that external pressure, like changes in animal husbandry, including antimicrobial treatments and introduction of modern milking machines, act as selective forces on the bacterial species which cause bovine mastitis.

Growth characteristics of Staphylococcus aureus and Escherichia coli in whey from sequentially infected milk.

The growth of Staphylococcus aureus and Escherichia coli was followed in bovine whey samples which had been prepared from milk previously incubated with cultures of S. aureus or E. coli. Staphylococcal strains were divided into 2 groups according to their ability to form compact or diffuse colonies on serum soft agar, which is related to the absence or presence of capsule respectively. The growth of compact staphylococci was dependent on the bulk tank milk used whereas diffuse colony forming staphylococci grew equally well in all bulk milk, also in all inoculated milk. The growth of E. coli was markedly enhanced in whey samples prepared from milk preincubated with staphylococci. However, clear growth inhibition was seen with E. coli and S. aureus strains when grown in whey prepared from milk preincubated with E. coli. Results indicate that the growth promotion of pathogens due to compositional changes in milk are of importance during the course of infection because the growth pattern on staphylococci is dependent on these compositional changes. The growth-inhibitory effects caused by E. coli may explain difficulties in isolating this organism.