Irena Zdovc

Antimicrobial susceptibility of animal and human isolates of Clostridium difficile by broth microdilution.

A total of 188 human (n = 92) and animal (n = 96) isolates of Clostridium difficile of different PCR ribotypes were screened for susceptibility to 30 antimicrobials using broth microdilution. When comparing the prevalence of antimicrobial resistance, the isolates of animal origin were significantly more often resistant to oxacillin, gentamicin and trimethoprim/sulfamethoxazole (P<0.01). The most significant difference between the animal and human populations (P = 0.0006) was found in the level of imipenem resistance, with a prevalence of 53.3 % in isolates of human origin and 28.1 % in isolates of animal origin. Overall, the results show similar MICs for the majority of tested antimicrobials for isolates from human and animal sources, which were collected from the same geographical region and in the same time interval. This supports the hypothesis that C. difficile could be transmissible between human and animal hosts. Resistant isolates have been found in all animal species tested, including food and companion animals, and also among non-toxigenic isolates. The isolates of the most prevalent PCR ribotype 014/020 had low resistance rates for moxifloxacin, erythromycin, rifampicin and daptomycin, but a high resistance rate for imipenem. Multiresistant strains were found in animals and humans, belonging to PCR ribotypes 012, 017, 027, 045, 046, 078 and 150, and also to non-toxigenic strains of PCR ribotypes 010 and SLO 080.

Systemic use of selective iNOS inhibitor 1400W or non-selective NOS inhibitor L-NAME differently affects systemic nitric oxide formation after oral Porphyromonas gingivalis inoculation in mice

OBJECTIVE Nitric oxide synthase (NOS) inhibitors are reported to protect against the local tissue damage in gingivitis and periodontal disease by reducing nitroxidative stress during inflammation, but their systemic effects are not well investigated. DESIGN NOS inhibitors systemic effects were investigated in a murine chronic oral inoculation model using live Porphyromonas gingivalis ATCC 33277 (0.3 ml; 10(9)cfu/ml) or sterile broth (0.3 ml). Organ nitric oxide (NO) and plasma nitrite/nitrate (NOx) were determined in mice treated with non-selective NOS inhibitor l-NAME (50mg/kg/24h i.p.) or selective iNOS inhibitor 1400W (10mg/kg/6h i.p.) for the last 5 days of the experiment. Differences between groups were evaluated by nonparametric Wilcoxons rank-sum one-sided two-sample test and the results compared to those obtained from sham-treated (sterile broth) sham-inoculated animals (water for injection i.p./6h). RESULTS Repeated ingestion of P. gingivalis resulted in generalized production of NO in organs and NOx in plasma, the levels of both typically being reduced in P. gingivalis-inoculated-1400W-treated mice, whilst the use of l-NAME was largerly ineffective, even promoting NO/NOx formation. Application of either inhibitor to sham-inoculated animals enhanced NO/NOx formation, due only in part to the repeated i.p. injections. CONCLUSIONS The systemic use of 1400W or l-NAME differently affects systemic nitric oxide formation in mice orally challenged with P. gingivalis, but the sequelae of such an intervention should be evaluated further.

Chronic ingestion of Porphyromonas gingivalis induces systemic nitric oxide response in mice

INTRODUCTION Porphyromonas gingivalis induces nitric oxide (NO) production in various cells, systemic NO elevation being expected in chronic oral challenge. METHODS Groups of BALB/c mice were inoculated orally with either live P. gingivalis ATCC 33277 or sterile broth on days 0, 2 and 4, with or without later administration of the inducible nitric oxide synthase (iNOS) inhibitor 1400W. Plasma and tissues were harvested on day 42 for assays of tumor necrosis factor-alpha (TNF-alpha), nitrite and nitrate (NOx) and tissue NO, or histology and iNOS immunohistochemistry. RESULTS No signs of gingivitis were observed, but plasma NOx was significantly elevated (P = 0.028) as was TNF-alpha (P = 0.079) in P. gingivalis-inoculated animals compared with controls, NOx being reduced when 1400W was used. NO production in organs showed a similar trend, with significant elevation in liver (P = 0.017) and kidneys (P = 0.027), whereas concomitant treatment of inoculated animals with 1400W caused significant reductions in NO in aorta (P = 0.008) and kidneys (P = 0.046). Sham-inoculated 1400W-treated animals had significantly increased plasma NOx (P = 0.004) and liver NO (P = 0.04). NOx in plasma correlated significantly with NO production in lungs (0.35, P = 0.032) and kidneys (0.47, P = 0.003). Immunohistochemistry demonstrated iNOS activity in many tissues in all groups. CONCLUSION Repeated oral administration of P. gingivalis induced systemic NO and NOx production in mice, probably by activating iNOS as suggested by the response to 1400W.

Cutaneous listeriosis in a veterinarian with the evidence of zoonotic transmission - a case report.

A case of Listeria monocytogenes skin infection in a man is presented. A 54‐year‐old male veterinary practitioner developed pustular changes on the skin of arms and hands after assisting with the delivery of a stillborn calf. Listeria monocytogenes was isolated from the skin lesions on the arms and from the bovine placenta. Listeria monocytogenes isolates were serotyped and genotyped with pulsed‐field gel electrophoresis (PFGE) to confirm the suspected transmission of the pathogen from animal to human. All isolates were of serotype 4b with identical pulsotype. To the best of our knowledge, this is the first case of cutaneous listeriosis in which the evidence for zoonotic transmission of L. monocytogenes is supported by genotyping methods.

Single gavage with Porphyromonas gingivalis reduces acute systemic nitric oxide response in mice

INTRODUCTION Porphyromonas gingivalis, an important periodontal pathogen, can also induce host responses in distant tissues. P. gingivalis induces nitric oxide (NO) production in immune system cells and non-immune system cells, therefore NO might be involved in an acute systemic host response. METHODS Eighteen female BALB/c mice were perorally inoculated with 10(8) colony-forming units live P. gingivalis ATCC 33277. Plasma nitrite and nitrate (NOx) and NO production in lungs, aorta, heart, liver, spleen, kidneys, and brain were measured at intervals after inoculation and compared with levels in 11 control animals. RESULTS NOx levels were significantly (P = 0.017) lower at 7, 13, and 25 h after P. gingivalis inoculation. A similar trend in NO production occurred in most tested organs, but never reached statistical significance. The correlation between NOx in plasma and NO in liver was positive (Spearman correlation coefficient = 0.81, P = 0.0025) and marginal for kidney (0.58, P = 0.059). CONCLUSION Single peroral inoculation of mice with P. gingivalis reduces the acute systemic NO response. As NO is important for host defense, the reduction of NO levels after exposure is likely to delay the host response, increasing the chances that infection with P. gingivalis will become established.

Antimicrobial Resistance and Molecular Characterization of Extended-Spectrum β-Lactamases and Other Escherichia coli Isolated from Food of Animal Origin and Human Intestinal Isolates

Antibiotics have always appeared miraculous, saving innumerable lives. However, the unwise use of antimicrobial drugs has led to the appearance of resistant bacteria. The purpose of this study was to evaluate antimicrobial resistance in Escherichia coli (n =160) isolated from food of animal origin. The focus was on E. coli -producing extended-spectrum β-lactamases. E. coli was chosen because it is a part of the normal microbiota in mammals and can enter the food chain during slaughtering and food manipulation. Subsequently, its resistance genes can be transferred to pathogenic bacteria and human microbiota. Phenotypic and genotypic analyses of selected antimicrobial resistances were carried out together with a molecular analysis of virulence genes. E. coli isolates from food of animal origin were compared with clinical E. coli strains isolated from the human intestinal tract. Extended-spectrum β-lactamase-producing E. coli isolates were found in 9.4% of food isolates and in 1.8% of intestinal isolates. Phylogenetically, the majority of food (86.3%) and intestinal E. coli (58.1%) isolates were found to belong to the commensal phylogenetic groups A and B1. The distribution of 4 of 14 analyzed virulence factors was similar in the food and intestinal isolates. Strains isolated from food in Slovenia harbored resistance genes and virulence factors, which can constitute a problem for food safety if not handled properly.

Antimicrobial Susceptibility Patterns of Clostridium difficile Isolates from Family Dairy Farms

A significant risk factor for developing Clostridium difficile infection (CDI) in humans and animals is associated with the antimicrobial use. It has often been hypothesized that farm animals could be the source for human infection with Clostridium difficile (CD). In the European Union, family‐run dairy farms are the predominant farming model, which are more interlinked within the community compared to large‐scale intensive dairy or beef farms. Therefore, it is important to investigate antimicrobial susceptibility patterns of CD in such environment. A total of 159 CD isolates from 20 family dairy farms were tested with a customized broth microdilution plate for their antimicrobial resistance. Seventeen antimicrobials were selected (amoxicillin, ceftriaxone, clindamycin, daptomycin, erythromycin, fusidic acid, imipenem, levofloxacin, linezolid, metronidazole, moxifloxacin, oxacillin, rifampicin, tetracycline, tigecycline, trimethoprim/sulfamethoxazole and vancomycin), which are commonly used for treatment of CDI in veterinary and human medicine, or were previously applied in CD epidemiological studies. Antimicrobials, which are used for treatment of CDI in humans (metronidazole, vancomycin, fusidic acid, tigecycline, linezolid) inhibited CD growth in vitro. Most CD isolates were resistant to erythromycin (93.1%), daptomycin (69.2%) and clindamycin (46.5%). High multiple‐resistance was found in CD ribotype 012 (n = 5, 100%), some CD SLO 060 (n = 4, 25%) and one CD 033 (n = 1, 1.1%). High multiple‐resistance in this study was linked with CD ribotypes and not with the origin of CD. The low prevalence of these ribotypes (6.3%; 10/159) indicates that family‐run dairy farms are an unlikely source of CD with multiple‐resistance to antimicrobials.

SINGLE ORAL INOCULATION WITH ESCHERICHIA COLI (ATCC 25922) STIMULATES GENERALISED PRODUCTION OF NITRIC OXIDE IN MICE

Nitric oxide (NO) production was investigated in the lungs, thoracic aorta, heart, liver, spleen, kidneys and brain of mice inoculated orally with Escherichia coli ATCC 25922. Detection of NO was performed by electron paramagnetic resonance (EPR) using diethyldithiocarbamate (DETC) spin trap. Nitric oxide synthase (NOS) inhibitors [nonselective: L-NAME and inducible NOS (iNOS) selective: 1400W] were used to determine the source of NO. Spin-trap only and untreated mice were included as controls. Within 2.5 hours (h) of a single oral inoculation with E. coli half of the animals had increased NO levels in all investigated organs. Thereafter the signals dropped before increasing again to reach maximal median values by 25 h in all organs of all inoculated mice. The most intense response occurred in livers, followed by aorta and lungs. Early (2.5 h) inhibition of the signal was achieved using both NOS inhibitors. L-NAME was also effective at 25 h, while 1400W-treated mice had increased NO levels beyond 7 h. The generalised increase in NO production in the short and longer term indicates a host response to E. coli administered by the oral route of infection.

Determination of N-acylhomoserine lactones of Pseudomonas aeruginosa in clinical samples from dogs with otitis externa

BackgroundBacterial intercellular communication, called quorum sensing, takes place via the production and collective response to signal molecules. In Gram-negative bacteria, like Pseudomonas aeruginosa, these signaling molecules are N-acylhomoserine lactones (AHLs). P. aeruginosa is a common cause of inflammation of the ear canal (otitis externa) in dogs. It employs quorum sensing to coordinate the expression of host tissue-damaging factors, which are largely responsible for its virulence. The treatment of P. aeruginosa-associated otitis is challenging due to a high intrinsic resistance of P. aeruginosa to several antibiotics.Attenuation of quorum sensing signals to inhibit bacterial virulence is a novel strategy for the treatment of resistant bacterial pathogens, including P. aeruginosa. Therefore, it is important to recognize and define quorum sensing signal molecules in clinical samples. To date, there are no reports on determination of AHLs in the veterinary clinical samples. The purpose of this study was to validate an analytical procedure for determination of the concentration of AHLs in the ear rinses from dogs with P. aeruginosa-associated otitis externa.Samples were obtained with rinsing the ear canals with physiological saline solution. For validation, samples from healthy dogs were spiked with none or different known amounts of the selected AHLs. With the validated procedure, AHLs were analyzed in the samples taken in weekly intervals from two dogs, receiving a standard treatment for P. aeruginosa-associated otitis externa.ResultsValidation proved that the procedure enables quantification of AHLs in non-clinical and clinical samples. In addition, a time dependent reduction of AHL concentration was detected for the treated dogs.ConclusionsOur results indicate that liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is superior in detecting AHLs compared to other chromatographic techniques. This is the first report on determination of AHLs in the clinical samples of veterinary importance. The analytical procedure described in this paper is capable of supporting novel antimicrobial strategies, which target quorum sensing.

Detection of Bacillus anthracis spores in postal and environmental samples in cases of suspected bioterrorism

From October 2001 to the end of 2004, a series of postal samples containing material suspected of containing Bacillus anthracis spores were examined. Environmental samples from dwelling places of some addressees and laboratory areas, including equipment, were also examined. Classical culture methods for isolation and identification were slightly modified and conformed to environmental samples. The most common bacteria isolated from postal samples were different Bacillus spp. (B. cereus, B. licheniformis, B. subtilis, B. mycoides). In cases of isolation of nonmotile and nonhaemolytic Bacillus spp., morphologically resembling B. anthracis, identification was supplemented by molecular method and mouse bioassay. All the samples resulted negative to Bacillus anthracis but in several cases an extended procedure was necessary to confirm the negative result.