Antibiotic susceptibility patterns and resistance genes of starter cultures and probiotic bacteria used in food

A survey of starter and probiotic cultures was carried out to determine the current antibiotic resistance situation in microbial food additives in Switzerland. Two hundred isolates from 90 different sources were typed by molecular and other methods to belong to the genera Lactobacillus (74 samples), Staphylococcus (33 samples), Bifidobacterium (6 samples), Pediococcus (5 samples), or were categorized as lactococci or streptococci (82 samples). They were screened for phenotypic resistances to 20 antibiotics by the disk diffusion method. Twenty-seven isolates exhibiting resistances that are not an intrinsic feature of the respective genera were further analyzed by microarray hybridization as a tool to trace back phenotypic resistances to specific genetic determinants. Their presence was finally verified by PCR amplification or Southern hybridization. These studies resulted in the detection of the tetracycline resistance gene tet(K) in 5 Staphylococcus isolates used as meat starter cultures, the tetracycline resistance gene tet(W) in the probiotic cultures Bifidobacterium lactis DSM 10140 and Lactobacillus reuteri SD 2112 (residing on a plasmid), and the lincosamide resistance gene lnu(A) (formerly linA) in L. reuteri SD 2112.

Comparison of bacterial plaque samples from titanium implant and tooth surfaces by different methods

Studies have shown similarities in the microflora between titanium implants or tooth sites when samples are taken by gingival crevicular fluid (GCF) sampling methods. The purpose of the present study was to study the microflora from curette and GCF samples using the checkerboard DNA-DNA hybridization method to assess the microflora of patients who had at least one oral osseo-integrated implant and who were otherwise dentate. Plaque samples were taken from tooth/implant surfaces and from sulcular gingival surfaces with curettes, and from gingival fluid using filter papers. A total of 28 subjects (11 females) were enrolled in the study. The mean age of the subjects was 64.1 years (SD+/-4.7). On average, the implants studied had been in function for 3.7 years (SD+/-2.9). The proportion of Streptococcus oralis (P<0.02) and Fusobacterium periodonticum (P<0.02) was significantly higher at tooth sites (curette samples). The GCF samples yielded higher proportions for 28/40 species studies (P-values varying between 0.05 and 0.001). The proportions of Tannerella forsythia (T. forsythensis), and Treponema denticola were both higher in GCF samples (P<0.02 and P<0.05, respectively) than in curette samples (implant sites). The microbial composition in gingival fluid from samples taken at implant sites differed partly from that of curette samples taken from implant surfaces or from sulcular soft tissues, providing higher counts for most bacteria studied at implant surfaces, but with the exception of Porphyromonas gingivalis. A combination of GCF and curette sampling methods might be the most representative sample method.

Real-time quantitative broad-range PCR assay for detection of the 16S rRNA gene followed by sequencing for species identification

Here we determined the analytical sensitivities of broad-range real-time PCR-based assays employing one of three different genomic DNA extraction protocols in combination with one of three different primer pairs targeting the 16S rRNA gene to detect a panel of 22 bacterial species. DNA extraction protocol III, using lysozyme, lysostaphin, and proteinase K, followed by PCR with the primer pair Bak11W/Bak2, giving amplicons of 796 bp in length, showed the best overall sensitivity, detecting DNA of 82% of the strains investigated at concentrations of < or =10(2) CFU in water per reaction. DNA extraction protocols I and II, using less enzyme treatment, combined with other primer pairs giving shorter amplicons of 466 bp and 342 or 346 bp, respectively, were slightly more sensitive for the detection of gram-negative but less sensitive for the detection of gram-positive bacteria. The obstacle of detecting background DNA in blood samples spiked with bacteria was circumvented by introducing a broad-range hybridization probe, and this preserved the minimal detection limits observed in samples devoid of blood. Finally, sequencing of the amplicons generated using the primer pair Bak11W/Bak2 allowed species identification of the detected bacterial DNA. Thus, broad-spectrum PCR targeting the 16S rRNA gene in the quantitative real-time format can achieve an analytical sensitivity of 1 to 10 CFU per reaction in water, avoid detection of background DNA with the introduction of a broad-range probe, and generate amplicons that allow species identification of the detected bacterial DNA by sequencing. These prerequisites are important for its application to blood-containing patient samples.

Antimicrobial resistance in bacteria from Swiss veal calves at slaughter

Bacteria with antimicrobial resistance can be transferred from animals to humans and may compromise antimicrobial treatment in case of infection. To determine the antimicrobial resistance situation in bacteria from Swiss veal calves, faecal samples from 500 randomly selected calves originating from 129 farms were collected at four big slaughterhouses. Samples were cultured for Escherichia coli, Enterococcus sp. and Campylobacter sp. and isolated strains were tested for antimicrobial susceptibility to selected antimicrobial agents by the minimal inhibitory concentration technique using the broth microdilution method. From 100 farms, data on farm management, animal husbandry and antimicrobial treatments of the calves were collected by questionnaire. Risk factors associated with antimicrobial resistance were identified by logistic regression. In total, 467 E. coli, 413 Enterococcus sp. and 202 Campylobacter sp. were isolated. Of those, 68.7%, 98.7% and 67.8%, respectively, were resistant to at least one of the tested antimicrobial agents. Resistance was mainly observed to antimicrobials frequently used in farm animals. Prevalence of resistance to antimicrobials important for human treatment was generally low. However, a rather high number of quinupristin/dalfopristin-resistant Enterococcus faecium and ciprofloxacin-resistant Campylobacter sp. were detected. External calf purchase, large finishing groups, feeding of milk by-products and administration of antimicrobials through feed upon arrival of the animals on the farm significantly increased the risk of antimicrobial resistance at farm level. Participation in a quality assurance programme and injection of a macrolide upon arrival of the animals on the farm had a protective effect. The present study showed that veal calves may serve as a reservoir for resistant bacteria. To ensure food safety, veal calves should be included in the national monitoring programme for antimicrobial resistance in farm animals. By improving farm management and calf husbandry the prevalence of resistance may be reduced.

A case of necrotizing fasciitis with septic shock in a cat caused by Acinetobacter baumannii

A 4-year-old, neutered female, domestic shorthair cat admitted to the animal hospital for recurrent constipation presumed to be due to post-traumatic injuries, went into shock with signs including fever and ataxia followed by stupor. On the fifth day of hospitalization, the cat developed severe, diffuse oedema of the ventral abdomen with multifocal to coalescing erythematous areas and small vesicle formation. The results of bacteriological cultures of liver, spleen and kidney specimens led to the diagnosis of Acinetobacter baumannii sepsis. Histopathological findings of skin samples taken during necropsy showed an extensive epidermal and dermal necrosis with septic vasculitis and numerous intralesional gram-negative bacteria. Detection of the bla(OXA-51-like) gene specific for A. baumannii by PCR, performed retrospectively on samples of the deep layers of the skin, confirmed the presence of A. baumannii also in the cutaneous lesions. To our knowledge this is the first report of a necrotizing fasciitis with septic shock in a cat caused by A. baumannii.

[Herd problem: udder health. Retrospective study of farms assessed by the Swiss Bovine Health Service (BHS) from 1999 to 2004]

Data from 59 farms with complaints of udder health problems and insufficient quality of delivered milk that had been assessed by the Swiss Bovine Health Service (BHS) between 1999 and 2004 were retrospectively analysed. Data evaluated included farm characteristics such as farm size, herd size, average milk yield, milking system and housing system, deficits of the milking equipment and the milking practices, and bacteriological results of milk samples from all cows in lactation. The average size of the farms assessed by the BHS was larger than the size of the were evaluated, 42 showed obvious failures which the farm managers could have noticed. Only 5 of the 57 milkers carried out their work according to the generally valid guidelines of the National Mastitis Council. More than 2 basic mistakes were observed in the milking practices of 36 milkers. In 51 farms, mixed infections with several problem bacteria (those present in at least 20 % of the tested cows on a farm) were found. Staphylococcus aureus proved to be the most common problem germ. As the bacteria responsible for the herd problem (the sole problem bacteria detectable on a particular farm) Staphylococcus aureus was detected in 4 farms. The current study revealed that education in the area of milking techniques and milking practices of farmers should be improved in order to reduce the incidence of udder health problems on herd level. Staphylococcus aureus is the most important problem bacteria involved in herds with udder health problems in Switzerland. Staphylococcus aureus might be used in practice as the indicator germ for early recognition of management problems in dairy farms.

[Pancytopenia and hemolysis--diagnosis, differential diagnosis and therapy of pernicious anemia]

Pernicious anemia and Vitamin B12 deficiency have a wide range of symptoms and are a common finding in the elderly. A 73 year old female is admitted to the hospital because of dyspnea, fatigue and loss of appetite and weight. While previous medical history and physical examination are inconspicuous, laboratory findings show severe pancytopenia with macrocytosis, low reticulocyte count and marked signs of hemolysis. A very low serum level of vitamin B12 and chronic atrophic type A gastritis upon endoscopy with presence of parietal cell antibodies in the serum lead to the diagnosis of pernicious anemia. Complete restitution is achieved by parenteral vitamin B12 substitution. Nowadays, severe pernicious anemia is only rarely seen. The differential diagnosis of pancytopenia (with macrocytic anemia) combined with hemolysis and the essential hints to the diagnosis of pernicious anemia are discussed, and thereby practical aspects including therapy actualized.

Lipopolysaccharides from atherosclerosis-associated bacteria antagonize TLR4, induce formation of TLR2/1/CD36 complexes in lipid rafts and trigger TLR2-induced inflammatory responses in human vascular endothelial cells

Infection with bacteria such as Chlamydia pneumonia, Helicobacter pylori or Porphyromonas gingivalis may be triggering the secretion of inflammatory cytokines that leads to atherogenesis. The mechanisms by which the innate immune recognition of these pathogens could lead to atherosclerosis remain unclear. In this study, using human vascular endothelial cells or HEK-293 cells engineered to express pattern-recognition receptors (PRRs), we set out to determine Toll-like receptors (TLRs) and functionally associated PRRs involved in the innate recognition of and response to lipopolysaccharide (LPS) from H. pylori or P. gingivalis. Using siRNA interference or recombinant expression of cooperating PRRs, we show that H. pylori and P. gingivalis LPS-induced cell activation is mediated through TLR2. Human vascular endothelial cell activation was found to be lipid raft-dependent and to require the formation of heterotypic receptor complexes comprising of TLR2, TLR1, CD36 and CD11b/CD18. In addition, we report that LPS from these bacterial strains are able to antagonize TLR4. This antagonistic activity of H. pylori or P. gingivalis LPS, as well as their TLR2 activation capability may be associated with their ability to contribute to atherosclerosis.

Prevention of brain injury by the nonbacteriolytic antibiotic daptomycin in experimental pneumococcal meningitis

Bacteriolytic antibiotics cause the release of bacterial components that augment the host inflammatory response, which in turn contributes to the pathophysiology of brain injury in bacterial meningitis. In the present study, antibiotic therapy with nonbacteriolytic daptomycin was compared with that of bacteriolytic ceftriaxone in experimental pneumococcal meningitis, and the treatments were evaluated for their effects on inflammation and brain injury. Eleven-day-old rats were injected intracisternally with 1.3 x 10(4) +/- 0.5 x 10(4) CFU of Streptococcus pneumoniae serotype 3 and randomized to therapy with ceftriaxone (100 mg/kg of body weight subcutaneously [s.c.]; n = 55) or daptomycin (50 mg/kg s.c.; n = 56) starting at 18 h after infection. The cerebrospinal fluid (CSF) was assessed for bacterial counts, matrix metalloproteinase-9 levels, and tumor necrosis factor alpha levels at different time intervals after infection. Cortical brain damage was evaluated at 40 h after infection. Daptomycin cleared the bacteria more efficiently from the CSF than ceftriaxone within 2 h after the initiation of therapy (log(10) 3.6 +/- 1.0 and log(10) 6.3 +/- 1.4 CFU/ml, respectively; P < 0.02); reduced the inflammatory host reaction, as assessed by the matrix metalloproteinase-9 concentration in CSF 40 h after infection (P < 0.005); and prevented the development of cortical injury (cortical injury present in 0/30 and 7/28 animals, respectively; P < 0.004). Compared to ceftriaxone, daptomycin cleared the bacteria from the CSF more rapidly and caused less CSF inflammation. This combined effect provides an explanation for the observation that daptomycin prevented the development of cortical brain injury in experimental pneumococcal meningitis. Further research is needed to investigate whether nonbacteriolytic antibiotic therapy with daptomycin represents an advantageous alternative over current bacteriolytic antibiotic therapies for the treatment of pneumococcal meningitis.

Surface antigens and potential virulence factors from parasites detected by comparative genomics of perfect amino acid repeats

ABSTRACT: BACKGROUND: Many parasitic organisms, eukaryotes as well as bacteria, possess surface antigens with amino acid repeats. Making up the interface between host and pathogen such repetitive proteins may be virulence factors involved in immune evasion or cytoadherence. They find immunological applications in serodiagnostics and vaccine development. Here we use proteins which contain perfect repeats as a basis for comparative genomics between parasitic and free-living organisms. RESULTS: We have developed Reptile http://reptile.unibe.ch, a program for proteome-wide probabilistic description of perfect repeats in proteins. Parasite proteomes exhibited a large variance regarding the proportion of repeat-containing proteins. Interestingly, there was a good correlation between the percentage of highly repetitive proteins and mean protein length in parasite proteomes, but not at all in the proteomes of free-living eukaryotes. Reptile combined with programs for the prediction of transmembrane domains and GPI-anchoring resulted in an effective tool for in silico identification of potential surface antigens and virulence factors from parasites. CONCLUSION: Systemic surveys for perfect amino acid repeats allowed basic comparisons between free-living and parasitic organisms that were directly applicable to predict proteins of serological and parasitological importance. An on-line tool is available at http://genomics.unibe.ch/dora.

Use of ampicillin-sulbactam for treatment of experimental meningitis caused by a beta-lactamase-producing strain of Escherichia coli K-1

We evaluated the pharmacokinetics and therapeutic efficacy of ampicillin combined with sulbactam in a rabbit model of meningitis due to a beta-lactamase-producing strain of Escherichia coli K-1. Ceftriaxone was used as a comparison drug. The MIC and MBC were 32 and greater than 64 micrograms/ml (ampicillin), greater than 256 and greater than 256 micrograms/ml (sulbactam), 2.0 and 4.0 micrograms/ml (ampicillin-sulbactam [2:1 ratio, ampicillin concentration]) and 0.125 and 0.25 micrograms/ml (ceftriaxone). All antibiotics were given by intravenous bolus injection in a number of dosing regimens. Ampicillin and sulbactam achieved high concentrations in cerebrospinal fluid (CSF) with higher dose regimens, but only moderate bactericidal activity compared with that of ceftriaxone was obtained. CSF bacterial titers were reduced by 0.6 +/- 0.3 log10 CFU/ml/h with the highest ampicillin-sulbactam dose used (500 and 500 mg/kg of body weight, two doses). This was similar to the bactericidal activity achieved by low-dose ceftriaxone (10 mg/kg), while a higher ceftriaxone dose (100 mg/kg) produced a significant increase in bactericidal activity (1.1 +/- 0.4 log10 CFU/ml/h). It appears that ampicillin-sulbactam, despite favorable CSF pharmacokinetics in animals with meningitis, may be of limited value in the treatment of difficult-to-treat beta-lactamase-producing bacteria, against which the combination shows only moderate in vitro activity.

Pefloxacin therapy for experimental endocarditis caused by methicillin-susceptible or methicillin-resistant strains of Staphylococcus aureus

The therapeutic efficacy of pefloxacin in experimental endocarditis caused by methicillin-susceptible or methicillin-resistant Staphylococcus aureus was evaluated. In rabbits infected with a methicillin-susceptible strain, 4 days of pefloxacin therapy significantly reduced both the number of bacteria per gram of vegetation and the mortality rate compared with untreated controls, and pefloxacin was equivalent to cephalothin. Pefloxacin was also as effective as vancomycin in reducing vegetation titers and mortality rate in animals with endocarditis caused by a methicillin-resistant strain. These results suggest that pefloxacin may be an effective agent in the therapy of serious infections caused by either methicillin-susceptible or -resistant strains of S. aureus.

Pathogenesis of bacterial meningitis: contributions by experimental models in rabbits

Rabbits models of bacterial meningitis have contributed substantially to our understanding of the disease, although the technical characteristics of these models only allow the study of specific aspects of the disease. Bacterial multiplication in the subarachnoidal space is not substantially influenced by host defense mechanisms, mainly because of the lack of sufficient amounts of specific antibodies and functional complement in infected CSF. The multiplying bacteria induce profound changes in the blood-brain barrier, an influx of serum proteins into the CSF and the invasion of polymorphonuclear leukocytes at the site of the infection. The presence of polymorphonuclear leukocytes in CSF not only appears to be of limited value in combating the infection, but also seems to produce deleterious effects on the central nervous system. Components of the leukocytes, such as unsaturated fatty acids, arachidonic metabolites and free oxygen radicals, may contribute to the profound hydrodynamic, structural and metabolic changes that are currently under study in experimental models of the disease. A better understanding of the pathophysiology of bacterial meningitis may allow us to design more effective therapeutic strategies and improve the outcome of this disease.

One-year bacterial colonization patterns of Staphylococcus aureus and other bacteria at implants and adjacent teeth

AIMS: (i) To assess the pattern of early bacterial colonization on titanium oral implants after installation, at 12 weeks and at 12 months, (ii) to compare the microbiota at submucosal implant sites and adjacent subgingival tooth sites and (iii) to assess whether or not early colonization was predictive of 12-month colonization patterns. MATERIAL AND METHODS: Submucosal/subgingival plaque samples from 17 titanium oral implants and adjacent teeth were analyzed by checkerboard DNA-DNA hybridization 30 min, 12 weeks and 12 months after implant installation. RESULTS: At 12 months, none of the inserted implants had been lost or presented with signs of peri-implantitis. The distribution of sites at implants and teeth with bleeding on probing varied between 2% and 11%. Probing pocket depths < or =3 mm were found at 75% of implant sites. At 12 months, the sum of the bacterial counts of 40 species was statistically significantly higher at tooth compared with implant sites (mean difference: 34.4 x 10(5), 95% confidence interval -0.4 to 69.4, P<0.05). At 12 months, higher individual bacterial counts at tooth sites were found for 7/40 species compared with implant sites. Detection or lack of detection of Staphylococcus aureus at implant sites at 12 weeks resulted in the highest positive (e.g. 80%) and negative (e.g. 90%) predictive values, respectively. Between 12 weeks and 12 months, the prevalence of Tannerella forsythia increased statistically significantly at implant sites (P<0.05). Lack of detection of Porphyromonas gingivalis at 12 weeks yielded a negative predictive value of 93.1% of this microorganism being undetectable at implant sites at 12 months. CONCLUSIONS: Within the limits of this study, the findings showed (i) a few differences in the prevalence of bacterial species between implant and adjacent tooth sites at 12 months and (ii) high positive and negative predictive values for selected bacterial species.

Staphylococcus aureus and other bacteria in untreated periodontitis

Whether the subgingival microbiota differ between individuals with chronic and those with aggressive periodontitis, and whether smoking influences bacterial composition, is controversial. We hypothesized that the subgingival microbiota do not differ between sites in individuals with chronic or aggressive periodontitis, or by smoking status. Bacterial counts and proportional distributions were assessed in 84 individuals with chronic periodontitis and 22 with aggressive periodontitis. No differences in probing pocket depth by periodontal status were found (mean, 0.11 mm; 95% CI, 0.6 to 0.8, p = 0.74). Including Staphylococcus aureus, Parvimonas micra, and Prevotella intermedia, 7/40 species were found at higher levels in those with aggressive periodontitis (p < 0.001). Smokers had higher counts of Tannerella forsythia (p < 0.01). The prevalence of S. aureus in non-smokers with aggressive periodontitis was 60.5%. The null hypothesis was rejected, in that P. intermedia, S. aureus, and S. mutans were robust in diagnosing sites in individuals with aggressive periodontitis. S. aureus, S. sanguinis, and T. forsythia differentiated smoking status.