Antimicrobial resistance in Swiss laying hens, prevalence and risk factors

Antimicrobial resistance is an emerging concern to public health, and food-producing animals are known to be a potential source for transmission of resistant bacteria to humans. As legislation of the European Union requires to ban conventional cages for the housing of laying hens on the one hand, and a high food safety standard for eggs on the other hand, further investigations about the occurrence of antimicrobial resistance in alternative housing types are required. In this study, we determined antimicrobial resistance in indicator bacteria from 396 cloacal swabs from 99 Swiss laying hen farms among four alternative housing types during a cross-sectional study. On each farm, four hens were sampled and exposure to potential risk factors was identified with a questionnaire. The minimal inhibitory concentration was determined using broth microdilution in Escherichia coli (n=371) for 18 antimicrobials and in Enterococcus faecalis (n=138) and Enterococcus faecium (n=153) for 16 antimicrobials. All antimicrobial classes recommended by the European Food Safety Authority for E. coli and enterococci were included in the resistance profile. Sixty per cent of the E. coli isolates were susceptible to all of the considered antimicrobials and 30% were resistant to at least two antimicrobials. In E. faecalis, 33% of the strains were susceptible to all tested antimicrobials and 40% were resistant to two or more antimicrobials, whereas in E. faecium these figures were 14% and 39% respectively. Risk factor analyses were carried out for bacteria species and antimicrobials with a prevalence of resistance between 15% and 85%. In these analyses, none of the considered housing and management factors showed a consistent association with the prevalence of resistance for more than two combinations of bacteria and antimicrobial. Therefore we conclude that the impact of the considered housing and management practices on the egg producing farms on resistance in laying hens is low.

Interaction Between Nitrous Oxide, Sevoflurane, and Opioids: A Response Surface Approach

BACKGROUND:: The interaction of sevoflurane and opioids can be described by response surface modeling using the hierarchical model. We expanded this for combined administration of sevoflurane, opioids, and 66 vol.% nitrous oxide (N2O), using historical data on the motor and hemodynamic responsiveness to incision, the minimal alveolar concentration, and minimal alveolar concentration to block autonomic reflexes to nociceptive stimuli, respectively. METHODS:: Four potential actions of 66 vol.% N2O were postulated: (1) N2O is equivalent to A ng/ml of fentanyl (additive); (2) N2O reduces C50 of fentanyl by factor B; (3) N2O is equivalent to X vol.% of sevoflurane (additive); (4) N2O reduces C50 of sevoflurane by factor Y. These four actions, and all combinations, were fitted on the data using NONMEM (version VI, Icon Development Solutions, Ellicott City, MD), assuming identical interaction parameters (A, B, X, Y) for movement and sympathetic responses. RESULTS:: Sixty-six volume percentage nitrous oxide evokes an additive effect corresponding to 0.27 ng/ml fentanyl (A) with an additive effect corresponding to 0.54 vol.% sevoflurane (X). Parameters B and Y did not improve the fit. CONCLUSION:: The effect of nitrous oxide can be incorporated into the hierarchical interaction model with a simple extension. The model can be used to predict the probability of movement and sympathetic responses during sevoflurane anesthesia taking into account interactions with opioids and 66 vol.% N2O.

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.

Fluoroquinolones in the Treatment of Meningitis

The continuous increase of resistant pathogens causing meningitis has limited the efficacy of standard therapeutic regimens. Due to their excellent activity in vitro and their good penetration into the cerebrospinal fluid (CSF), fluoroquinolones appear promising for the treatment of meningitis caused by gram-negative microorganisms, ie, Neisseria meningitidis and nosocomial gram-negative bacilli. The newer fluoroquinolones (moxifloxacin, gemifloxacin, gatifloxacin, and garenoxacin) have excellent activity against gram-positive microorganisms. Studies in animal models and limited clinical data indicate that they may play a future role in the treatment of pneumococcal meningitis. Analysis of pharmacodynamic parameters suggests that CSF concentrations that produce a C(peak)/minimal bactericidal concentration (MBC) ratio of at least 5 and concentrations above the MBC during the entire dosing interval are a prerequisite for maximal bactericidal activity in meningitis. Of interest, newer fluoroquinolones act synergistically with vancomycin and beta-lactam antibiotics (ceftriaxone, cefotaxime, meropenem) against penicillin-resistant pneumococci in experimental rabbit meningitis, potentially providing a new therapeutic strategy. Clinical trials are needed to further explore the usefulness of quinolones as single agents or in combination with other drugs in the therapy of pneumococcal meningitis.

[Endocarditis prophylaxis with amoxicillin, clindamycin or erythromycin? Serum bacteriostatic and bactericidal effect against Streptococcus viridans]

The effectiveness of antibiotic prophylaxis against endocarditis was assessed by testing the bactericidal and bacteriostatic action in serum of 12 healthy volunteers who had taken the recommended antibiotics according to laid down guidelines. Blood was obtained from these subjects every two hours for 12 hours after oral intake of amoxicillin (3 g as a single dose), clindamycin (600 mg a single dose) or erythromycin (1.5 g plus another 0.5 g after six hours), the serum being tested against three Strep. viridans strains. Two of the three strains were "tolerant", i.e. in relation to the minimal inhibition concentration (MIC) their growth was inhibited, but--in relation to the minimal bactericidal concentration (MBC)--they were not killed. A bacteriostatic effect by amoxicillin and clindamycin was demonstrated by a micromethod against all three strains during the 12-hour period. But erythromycin did not achieve bacteriostasis in all serum samples. A bactericidal effect was demonstrated only in those samples that contained amoxicillin, and then only against the non-tolerant of the three strains. These results support the view that amoxicillin and clindamycin are effective in the prophylaxis against Strep. viridans bacteraemia because of their constant bacteriostatic effect, as measured in serum.

Principles in the treatment of bacterial meningitis

The pathophysiologic aspects of bacterial meningitis impose some specific requirements on successful antimicrobial therapy of this disease. Because infections of the subarachnoid space rapidly produce destruction of the brain tissue, treatment must be instituted as early as possible. In the subarachnoid space, efficient host defense mechanisms are absent, particularly at the start of the infection, and therefore antibiotics have to produce a bactericidal effect to eliminate the microorganisms. As animal studies indicate, only drug concentrations 20- to 100-fold higher than the minimal bactericidal concentration are effective in vivo. Because penetration of antibiotics to the site of infection is limited by the blood-brain barrier, the high cerebrospinal fluid concentrations necessary to kill the bacteria may be difficult to achieve and therapy may be limited by toxicity. Even with optimal antibiotic therapy, the morbidity and mortality remain high, and new therapeutic interventions are necessary and should be aimed at modifying selective components of the inflammatory process.

The postantibiotic effect in the treatment of experimental meningitis caused by Streptococcus pneumoniae in rabbits

The relevance of a postantibiotic effect in the treatment of pneumococcal meningitis was evaluated in a rabbit model. After administration of a single intravenous bolus of ampicillin at various dosages, such an effect was observed in all animals. The duration of this effect in vivo (2.5-18 hr) was consistently longer than that in vitro (1-4.3 hr); however, in rabbits the postantibiotic effect was eliminated by the administration of intravenous plus intracisternal beta-lactamase. In an assessment of the potential therapeutic benefit of the postantibiotic effect, the efficacy to two regimens of treatment with different intervals between doses was compared. One group of animals received ampicillin every 4 hr and another every 12 hr. With sufficiently high doses, drug concentrations in cerebrospinal fluid exceeded the minimal bactericidal concentration for most of the 4-hr interval but for only about one-third of the 12-hr interval. The rate of cure was similar for the two regimens and approximated 100% when peak drug concentrations in cerebrospinal fluid exceeded the minimal bactericidal concentration by at least 10-fold.

Antibacterial activity of beta-lactam antibiotics in experimental meningitis due to Streptococcus pneumoniae

In order to define the characteristics of the antibacterial activity of beta-lactam antibiotics in the treatment of bacterial meningitis, the relationship between cerebrospinal fluid (CSF) drug concentrations and the rate of bacterial killing was investigated for penicillin G and four new cephalosporins in an animal model of meningitis due to Streptococcus pneumoniae. All five drugs showed a significant correlation between increasing drug concentrations in CSF and increasing bactericidal rates. Minimal activity was observed in CSF at drug concentrations of approximately the broth minimal bactericidal concentration (MBC). Maximal activity occurred with CSF concentrations 10-30 times higher. In vitro tests did not reproduce the unique correlation of increasing drug concentrations and killing activity found in vivo. When evaluating new beta-lactam antibiotics for the treatment of bacterial meningitis, it is reasonable to establish a minimum standard of CSF drug concentrations of greater than or equal to 30 times the MBC against the infecting organism.

A low affinity penicillin-binding protein 2x is required for heteroresistance in Streptococcus pneumoniae

Heteroresistance to penicillin in Streptococcus pneumoniae is the ability of subpopulations to grow at a higher antibiotic concentration than expected from the minimal inhibitory concentration (MIC). This may render conventional resistance testing unreliable and lead to therapeutic failure. We investigated the role of the primary β-lactam resistance determinants, penicillin binding proteins PBP2b and PBP2x and secondary resistance determinant PBP1a in heteroresistance to penicillin. Transformants containing PBP genes from heteroresistant strain Spain(23F)2349 in non-heteroresistant strain R6 background were tested for heteroresistance by population analysis profiling (PAP). We found that pbp2x, but not pbp2b or pbp1a alone, conferred heteroresistance to R6. However, a change of pbp2x expression is not observed and therefore expression does not correlate with an increased proportion of resistant subpopulations. Additional ciaR disruption mutants which have been described to mediate PBP-independent β-lactam resistance revealed no heteroresistant phenotype by PAP.We also showed, that the highly resistant subpopulations (HOM*) of transformants containing low affinity pbp2x undergo an increase in resistance upon selection on penicillin plates which partially reverts after passaging on selection-free medium. Shotgun proteomic analysis showed an upregulation of phosphate ABC transporter subunit proteins pstS, phoU, pstB and pstC in these highly resistant subpopulations.In conclusion, the presence of low affinity pbp2x enables certain pneumococcal colonies to survive in the presence of beta lactams. Upregulation of phosphate ABC transporter genes may represent a reversible adaption to antibiotic stress.

The in vitro impact of toothpaste extracts on cell viability.

Toothpastes contain three main components: detergents, abrasives, and fluoride. Detergents, particularly sodium lauryl sulfate, have been proposed as components that enable toothpastes to produce cytotoxic effects in vitro. However, not all toothpastes contain sodium lauryl sulfate, and almost no studies have found an association between detergents and the in vitro cytotoxicity of toothpastes. The present study examined the in vitro cytotoxicity of nine commercially available toothpastes containing four different detergents. Toothpastes were diluted in serum-free medium, centrifuged, and filter sterilized. The half-lethal concentration of the toothpaste-conditioned medium (TCM) was calculated based on the formation of formazan by gingival fibroblasts, oral squamous cell carcinoma HSC-2 cells, and L929 cells. Cell proliferation was analyzed, and live-dead staining was performed, after exposure of cells to conditioned medium prepared with 1% toothpaste (1% TCM). It was found that toothpastes containing sodium lauryl sulfate and amine fluoride strongly inhibited cell viability with the half-lethal concentration being obtained with conditioned medium prepared with approximately 1% toothpaste (1% TCM). Toothpastes containing cocamidopropyl betaine and Steareth-20 showed higher half-lethal concentration values, with the half-lethal concentration being obtained with conditioned medium prepared with 10% (10% TCM) and 70% (70% TCM) toothpaste, respectively. Proliferation and live-dead data were consistent with the cell-viability analyses. These results demonstrate that the type of detergent in toothpastes can be associated with changes in in vitro cell toxicity.

Significant Differences Characterise the Correlation Coefficients between Biocide and Antibiotic Susceptibility Profiles in Staphylococcus aureus.

There is a growing concern by regulatory authorities for the selection of antibiotic resistance caused by the use of biocidal products. We aimed to complete the detailed information on large surveys by investigating the relationship between biocide and antibiotic susceptibility profiles of a large number of Staphylococcus aureus isolates using four biocides and antibiotics commonly used in clinical practice. The minimal inhibitory concentration (MIC) for most clinically-relevant antibiotics was determined according to the standardized methodology for over 1600 clinical S. aureus isolates and compared to susceptibility profiles of benzalkonium chloride, chlorhexidine, triclosan, and sodium hypochlorite. The relationship between antibiotic and biocide susceptibility profiles was evaluated using non-linear correlations. The main outcome evidenced was an absence of any strong or moderate statistically significant correlation when susceptibilities of either triclosan or sodium hypochlorite were compared for any of the tested antibiotics. On the other hand, correlation coefficients for MICs of benzalkonium chloride and chlorhexidine were calculated above 0.4 for susceptibility to quinolones, beta-lactams, and also macrolides. Our data do not support any selective pressure for association between biocides and antibiotics resistance and furthermore do not allow for a defined risk evaluation for some of the compounds. Importantly, our data clearly indicate that there does not involve any risk of selection for antibiotic resistance for the compounds triclosan and sodium hypochlorite. These data hence infer that biocide selection for antibiotic resistance has had so far a less significant impact than feared.

The sub-lethal effects and tissue concentration of the human pharmaceutical atenolol in rainbow trout (Oncorhynchus mykiss).

Atenolol is a highly prescribed anti-hypertensive pharmaceutical and a member of the group of β-blockers. It has been detected at concentrations ranging from ng L(-1) to low μg L(-1) in waste and surface waters. The present study aimed to assess the sub-lethal effects of atenolol on rainbow trout (Oncorhynchus mykiss) and to determine its tissue-specific bioconcentration. Juvenile rainbow trout were exposed for 21 and 42 days to three concentration levels of atenolol (1 μg L(-1) - environmentally relevant concentration, 10 μg L(-1), and 1000 μg L(-1)). The fish exposed to 1 μg L(-1) atenolol exhibited a higher lactate content in the blood plasma and a reduced haemoglobin content compared with the control. The results show that exposure to atenolol at concentrations greater than or equal to 10 μg L(-1) significantly reduces both the haematocrit value and the glucose concentration in the blood plasma. The activities of the studied antioxidant enzymes (catalase and superoxide dismutase) were not significantly affected by atenolol exposure, and only the highest tested concentration of atenolol significantly reduced the activity of glutathione reductase. The activities of selected CYP450 enzymes were not affected by atenolol exposure. The histological changes indicate that atenolol has an effect on the vascular system, as evidenced by the observed liver congestion and changes in the pericardium and myocardium. Atenolol was found to have a very low bioconcentration factor (the highest value found was 0.27). The bioconcentration levels followed the order liver>kidney>muscle. The concentration of atenolol in the blood plasma was below the limit of quantification (2.0 ng g(-1)). The bioconcentration factors and the activities of selected CYP450 enzymes suggest that atenolol is not metabolised in the liver and may be excreted unchanged.

Conversion of CD95 (Fas) Type II into Type I signaling by sub-lethal doses of cycloheximide

CD95 (Fas/Apo-1)-mediated apoptosis was shown to occur through two distinct pathways. One involves a direct activation of caspase-3 by large amounts of caspase-8 generated at the DISC (Type I cells). The other is related to the cleavage of Bid by low concentration of caspase-8, leading to the release of cytochrome c from mitochondria and the activation of caspase-3 by the cytochrome c/APAF-1/caspase-9 apoptosome (Type II cells). It is also known that the protein synthesis inhibitor cycloheximide (CHX) sensitizes Type I cells to CD95-mediated apoptosis, but it remains contradictory whether this effect also occurs in Type II cells. Here, we show that sub-lethal doses of CHX render both Type I and Type II cells sensitive to the apoptogenic effect of anti-CD95 antibodies but not to chemotherapeutic drugs. Moreover, Bcl-2-positive Type II cells become strongly sensitive to CD95-mediated apoptosis by the addition of CHX to the cell culture. This is not the result of a restraint of the anti-apoptotic effect of Bcl-2 at the mitochondrial level since CHX-treated Type II cells still retain their resistance to chemotherapeutic drugs. Therefore, CHX treatment is granting the CD95-mediated pathway the ability to bypass the mitochondria requirement to apoptosis, much alike to what is observed in Type I cells.

Postmortem diagnostics using MSCT and MRI of a lethal streptococcus group A infection at infancy: a case report.

Postmortem cross-sectional imaging using multislice computed tomography (MSCT) and magnetic resonance imaging (MRI) was considered as a base for a minimal invasive postmortem investigation in forensic medicine such as within the Virtopsy approach. We present the case of a 3-year-old girl with a lethal streptococcus group A infection and the findings of postmortem imaging in this kind of natural death. Postmortem MSCT and MRI revealed an edematous occlusion of the larynx at the level of the vocal cords, severe pneumonia with atelectatic parts of both upper lobes and complete atelectasis of both lower lobes, purulent fluid-filled right main bronchus, enlargement of cervical lymph nodes and pharyngeal tonsils, and additionally, a remaining glossopharyngeal cyst as well as an ureter fissus of the right kidney. All relevant autopsy findings could be obtained and visualized by postmortem imaging and confirmed by histological and microbiological investigations supporting the idea of a minimal invasive autopsy technique.