Antibacterial activity of moxifloxacin on bacteria associated with periodontitis within a biofilm

The activity of moxifloxacin was compared with ofloxacin and doxycycline against bacteria associated with periodontitis within a biofilm (single strain and mixed population) in vitro. Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of moxifloxacin, ofloxacin and doxycyline were determined against single strains and mixed populations in a planktonic state. Single-species biofilms of two Porphyromonas gingivalis and two Aggregatibacter actinomycetemcomitans strains and a multi-species biofilm consisting of 12 species were formed for 3 days. The minimal biofilm eradication concentrations (MBECs) were determined after exposing the biofilms to the antibacterials (0.002 - 512 µg ml-1) for 18 h, addition of nutrient broth for 3 days and subsequent subcultivation. Photographs were taken by using confocal laser scanning microscopy and scanning electron microscopy. The MICs and MBCs did not differ between ofloxacin and moxifloxacin against A. actinomycetemcomitans, moxifloxacin was more active than the other tested antibacterials against anaerobes and the mixed population. The single-species biofilms were eradicated by moderate concentrations of the antibacterials, the lowest MBECs were always found for moxifloxacin (2-8 µg ml-1). MBECs against the multi-species biofilms were 128 µg ml-1, >512 µg ml-1 and >512 µg ml-1 for moxifloxacin, ofloxacin and doxycycline, respectively. In summary, moxifloxacin in a topical formulation may have potential as an adjunct to mechanical removal of the biofilms.

Enhanced antibiotic multi-resistance in nasal and faecal bacteria after agricultural use of streptomycin

Streptomycin is used in arboriculture to control fire blight. Using sheep as a model, multidrug-resistant bacteria in mammals were found to be selected after the intentional release of streptomycin into the environment. Escherichia coli and Staphylococcus spp. were isolated from the faeces and nasal cavities, respectively, of sheep grazing on a field sprayed with streptomycin at concentrations used in orchards (test group) and on a field without streptomycin (control group). Before the application of streptomycin, the percentage of streptomycin-resistant E. coli isolates in faeces was 15.8% in the control group and 14.7% in the test group. After the application of streptomycin, the overall number of streptomycin-resistant E. coli isolates was significantly higher in the test group (39.9%) than in the control group (22.3%). Streptomycin-resistant Staphylococcus isolates were only detected after the application of streptomycin. Streptomycin resistance was frequently associated with resistance to sulfamethoxazole, ampicillin, tetracycline and chloramphenicol and less frequently to cefotaxime in E. coli, and to tetracycline, fusidic acid and tiamulin in Staphylococcus spp. This study shows that the application of low concentrations of streptomycin on grass, as occurs during the spraying of orchards, selects for multidrug-resistant nasal and enteric bacterial flora, including extended-spectrum beta-lactamase-producing E. coli.

Thiazolides, a Novel Class of Anti-Infective Drugs, Effective Against Viruses, Bacteria, Intracellular and Extracellular Protozoan Parasites and Proliferating Mammalian Cells

The thiazolide nitazoxanide (2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide; NTZ) is composed of a nitrothiazole- ring and a salicylic acid moiety, which are linked together through an amide bond. NTZ exhibits a broad spectrum of activities against a wide range of helminths, protozoa, enteric bacteria, and viruses infecting animals and humans. Since the first synthesis of the drug, a number of derivatives of NTZ have been produced, which are collectively named thiazolides. These are modified versions of NTZ, which include the replacement of the nitro group with bromo-, chloro-, or other functional groups, and the differential positioning of methyl- and methoxy-groups on the salicylate ring. The presence of a nitro group seems to be the prerequisite for activities against anaerobic or microaerophilic parasites and bacteria. Intracellular parasites and viruses, however, are susceptible to non-nitro-thiazolides with equal or higher effectiveness. Moreover, nitro- and bromo-thiazolides are effective against proliferating mammalian cells. Biochemical and genetic approaches have allowed the identification of respective targets and the molecular basis of resistance formation. Collectively, these studies strongly suggest that NTZ and other thiazolides exhibit multiple mechanisms of action. In microaerophilic bacteria and parasites, the reduction of the nitro group into a toxic intermediate turns out to be the key factor. In proliferating mammalian cells, however, bromo- and nitro-thiazolides trigger apoptosis, which may also explain their activities against intracellular pathogens. The mode of action against helminths may be similar to mammalian cells but has still not been elucidated.

Phenotypic and genotypic identification of streptococci and related bacteria isolated from bovine intramammary infections.

BACKGROUND Streptococcus spp. and other Gram-positive, catalase-negative cocci (PNC) form a large group of microorganisms which can be found in the milk of cows with intramammary infection. The most frequently observed PNC mastitis pathogens (major pathogens) are Streptococcus uberis, Strep. dysgalactiae, and Strep. agalactiae. The remaining PNC include a few minor pathogens and a large nonpathogenic group. Improved methods are needed for the accurate identification and differentiation of PNC. A total of 151 PNC were collected from cows with intramammary infection and conclusively identified by 16S rRNA sequencing as reference method. Nine phenotypic microbiological tests (alpha-hemolysis, CAMP reaction, esculin hydrolysis, growth on kanamycin esculin azide agar and on sodium chloride agar, inulin fermentation, hippurate hydrolysis, leucine aminopeptidase and pyrrolidonyl peptidase activity), multiplex PCR for the three major pathogens (target genes for Strep. uberis, Strep. dysgalactiae and Strep. agalactiae: pauA, 16S rRNA, and sklA3, respectively), and mass spectroscopy using the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS) were evaluated for the diagnosis and discrimination of the three clinically most relevant PNC. RESULTS The probability that a strain of Strep. uberis, Strep. dysgalactiae and Strep. agalactiae was correctly identified by combining the results of the 9 phenotypic tests was 92%, 90%, and 100%, respectively. Applying the multiplex PCR, all strains of the three major pathogens were correctly identified and no false positive results occurred. Correct identification was observed for all strains of Strep. uberis and Strep. agalactiae using MALDI-TOF MS. In the case of Strep. dysgalactiae, some variability was observed at the subspecies level, but all strains were allocated to one single cluster. CONCLUSIONS The results of the present study show that reliable identification of the clinically most relevant PNC (Strep. uberis, Strep. agalactiae and Strep. dysgalactiae) can be obtained by use of a combination of colony morphology, hemolysis type and catalase reaction, and a multiplex PCR with specific primers restricted to these 3 pathogens. The MALDI-TOF MS is a fast method that shows promising results, although identification of Strep. dysgalactiae at the subspecies level is not yet satisfactory.

Identification of the Causative Bacteria in Musculoskeletal Infections Using 16s rDNA - Denaturing Gradient Gel Electrophoresis Analysis

Musculoskeletal infections are infections of the bone and surrounding tissues. They are currently diagnosed based on culture analysis, which is the gold standard for pathogen identification. However, these clinical laboratory methods are frequently inadequate for the identification of the causative agents, because a large percentage (25-50%) of confirmed musculoskeletal infections are false negatives in which no pathogen is identified in culture. My data supports these results. The goal of this project was to use PCR amplification of a portion of the 16S rRNA gene to test an alternative approach for the identification of these pathogens and to assess the diversity of the bacteria involved. The advantages of this alternative method are that it should increase sample sensitivity and the speed of detection. In addition, bacteria that are non-culturable or in low abundance can be detected using this molecular technique. However, a complication of this approach is that the majority of musculoskeletal infections are polymicrobial, which prohibits direct identification from the infected tissue by DNA sequencing of the initial 16S rDNA amplification products. One way to solve this problem is to use denaturing gradient gel electrophoresis (DGGE) to separate the PCR products before DNA sequencing. Denaturing gradient gel electrophoresis (DGGE) separates DNA molecules based on their melting point, which is determined by their DNA sequence. This analytical technique allows a mixture of PCR products of the same length that electrophoreses through agarose gels as one band, to be separated into different bands and then used for DNA sequence analysis. In this way, the DGGE allows for the identification of individual bacterial species in polymicrobial-infected tissue, which is critical for improving clinical outcomes. By combining the 16S rDNA amplification and the DGGE techniques together, an alternative approach for identification has been used. The 16S rRNA gene PCR-DGGE method includes several critical steps: DNA extraction from tissue biopsies, amplification of the bacterial DNA, PCR product separation by DGGE, amplification of the gel-extracted DNA, and DNA sequencing and analysis. Each step of the method was optimized to increase its sensitivity and for rapid detection of the bacteria present in human tissue samples. The limit of detection for the DNA extraction from tissue was at least 20 Staphylococcus aureus cells and the limit of detection for PCR was at least 0.05 pg of template DNA. The conditions for DGGE electrophoreses were optimized by using a double gradient of acrylamide (6 – 10%) and denaturant (30-70%), which increased the separation between distinct PCR products. The use of GelRed (Biotium) improved the DNA visualization in the DGGE gel. To recover the DNA from the DGGE gels the gel slices were excised, shredded in a bead beater, and the DNA was allowed to diffuse into sterile water overnight. The use of primers containing specific linkers allowed the entire amplified PCR product to be sequenced and then analyzed. The optimized 16S rRNA gene PCR-DGGE method was used to analyze 50 tissue biopsy samples chosen randomly from our collection. The results were compared to those of the Memorial Hermann Hospital Clinical Microbiology Laboratory for the same samples. The molecular method was congruent for 10 of the 17 (59%) culture negative tissue samples. In 7 of the 17 (41%) culture negative the molecular method identified a bacterium. The molecular method was congruent with the culture identification for 7 of the 33 (21%) positive cultured tissue samples. However, in 8 of the 33 (24%) the molecular method identified more organisms. In 13 of the 15 (87%) polymicrobial cultured tissue samples the molecular method identified at least one organism that was also identified by culture techniques. Overall, the DGGE analysis of 16S rDNA is an effective method to identify bacteria not identified by culture analysis.

Suppression of peripheral blood mononuclear cell proliferation by a periodontopathic bacteria {\it Actinobacillus actinomycetemcomitans\/}

Actinobacillus actinomycetemcomitans (Aa) is a gram-negative coccobacillus implicated as a major pathogen in juvenile periodontitis. The immunosuppressive activity of a sonic extract (designated 100SN) derived from Aa was investigated. 100SN suppressed spontaneous proliferation as well as proliferative response to the mitogens, PHA and PWM, of human peripheral blood mononuclear cells (PBMC). 100SN-induced suppression of PHA-stimulated proliferation was heat-sensitive, inactivated by pronase and trypsin, dose-dependent and non-cytotoxic. There were no significant changes in the CD4$\sp+$ or CD8$\sp+$ subsets of PBMC after 7-day incubation with 100SN. There was a trend toward increased levels of the CD4$\sp+$CD45R$\sp{\rm hi}$CDw29$\sp{\rm lo}$ (naive cells, associated with suppressor-inducer activity) and CD4$\sp+$CDw29$\sp{\rm hi}$CD45R$\sp{\rm lo}$ (memory cells, associated with helper-inducer activity) subsets. The target of 100SN appeared to be the non-adherent cells and suppression by 100SN could not be reversed by indomethacin (IDM), the cyclo-oxygenase inhibitor of prostaglandin (PG) synthesis. The mechanism of 100SN-induced suppression was studied in terms of inhibition involving IL-2-regulated T cell proliferation and the results point to the possibility that suppression occurred subsequent to IL-2 receptor binding.^ The suppressive activity observed could occur through multiple mechanisms including cell-cell; contact or release of soluble factors. Supernatants derived from 7-day cultures of PBMC and 100SN (designated CSN-A) were able to suppress proliferative response of PBMC to PHA without affecting cell viability. Analysis of CSN-A showed that it contained PGE2 and soluble IL-2 receptors. Suppression by CSN-A could be partially overcome by either IDM or exogenous IL-2. Significant suppression was also maintained when both IDM and exogenous IL-2 were added at the same time. These findings suggest that PGE2 and soluble IL-2 receptors contribute to the suppression observed but other suppressive cytokine(s) may be involved. Collectively, the data indicate that a factor derived from oral bacteria associated with juvenile periodontitis have profound effects on cellular immune responses, and that these effects may be partially mediated by secondary factors produced by the host in response to the bacteria. ^

Bacteria causing bacteremia in pediatric cancer patients presenting with febrile neutropenia – species distribution and susceptibility patterns

PURPOSE Infections are a major cause of morbidity and mortality in pediatric cancer patients. The aim of this study was to establish the microbiological spectrum and the susceptibility patterns of bacteremia-causing bacteria in pediatric cancer patients with febrile neutropenia in relation to the use of prophylactic and empirical antibiotics. METHODS We analyzed positive blood cultures of pediatric cancer patients presenting with febrile neutropenia between 2004 and 2011 in Groningen and Amsterdam (the Netherlands) and in Bern (Switzerland), using different antibiotic prophylactic and empirical regimens. RESULTS A total of 156 patients with 202 bacteremias, due to 248 bacteria species, were enrolled. The majority (73%) of bacteremias were caused by Gram-positive bacteria. Gram-negative bacteria, especially Pseudomonas aeruginosa, were observed significantly more often in Bern, where no fluoroquinolone prophylaxis was used. Ciprofloxacin-resistant bacteria were cultured more often from patients who did receive ciprofloxacin prophylaxis, compared to the patients who did not (57 versus 11%, p = 0.044). CONCLUSIONS Gram-positive bacteria predominated in this study. We showed that the use of prophylactic antibiotics in pediatric cancer patients was associated with increased resistance rates, which needs further study. The strategy for empiric antimicrobial therapy for febrile neutropenia should be adapted to local antibiotic resistance patterns.

Conjugal Transfer of the Tn916-like Transposon TnFO1 from Enterococcus faecalis Isolated from Cheese to Other Gram-positive Bacteria

A Tn916-like transposon (TnFO1) was found in the multiple antibiotic resistant Enterococcus faecalis strain FO1 isolated from a raw milk cheese. In this strain, the tetracycline determinant was localized by DNA-DNA hybridization with a tetM nucleotide probe on the chromosome and on a 30-kb plasmid. The transposon TnFO1 was identified and characterized by DNA-DNA hybridization experiments with the five internal HincII fragments of Tn916. The tetracycline resistance determinant was identified by its complete nucleotide sequence as TetM. Transposon TnFO1 was also detected in its circular form by DNA-DNA hybridization and PCR amplification. Both ends including the joining region of the closed circular transposon TnFO1 were sequenced. TnFO1 could be transferred by conjugation from Enterococcus faecalis into Enterococcus faecalis, Lactococcus lactis subsp. lactis biovar. diacetylactis, Listeria innocua, Leuconostoc mesenteroides and Staphylococcus aureus, and from Lactococcus lactis subsp. lactis biovar. diacetylactis into Listeria innocua. Pulsed-field electrophoresis of genomic DNA from E. faecalis FO1 transconjugants showed that transposon TnFO1 integrated at different sites.

Role of milk and meat products as vehicles for antibiotic-resistant bacteria

This subject is reviewed under the following headings: Microbial contamination of raw meat and raw milk; Antibiotic resistance of food-borne pathogens; Antibiotic resistance of commensal and potentially pathogenic bacteria as a new threat in food microbiology; Antibiotic-resistant staphylococci in fermented meat and [in] milk products; Antibiotic-resistant Enterococcus sp. in fermented meat and [in] milk products; Enterococci in farm animals and meat; Enterococci in fermented food; Molecular characterization of resistance of food-borne enterococci; and Further ecological and epidemiological considerations of resistant live bacteria in food. It is concluded that further research is needed, particularly into the possible transfer of the resistance of bacteria consumed in meat or milk products to the indigenous bacteria of the human consumer.

Apoptosis of hippocampal neurons in organotypic slice culture models: direct effect of bacteria revisited

Neurons of the hippocampal dentate gyrus selectively undergo programmed cell death in patients suffering from bacterial meningitis and in experimental models of pneumococcal meningitis in infant rats. In the present study, a membrane-based organotypic slice culture system of rat hippocampus was used to test whether this selective vulnerability of neurons of the dentate gyrus could be reproduced in vitro. Apoptosis was assessed by nuclear morphology (condensed and fragmented nuclei), by immunochemistry for active caspase-3 and deltaC-APP, and by proteolytic caspase-3 activity. Co-incubation of the cultures with live pneumococci did not induce neuronal apoptosis unless cultures were kept in partially nutrient-deprived medium. Complete nutrient deprivation alone and staurosporine independently induced significant apoptosis, the latter in a dose-response way. In all experimental settings, apoptosis occurred preferentially in the dentate gyrus. Our data demonstrate that factors released by pneumococci per se failed to induce significant apoptosis in vitro. Thus, these factors appear to contribute to a multifactorial pathway, which ultimately leads to neuronal apoptosis in bacterial meningitis.

Intestinal bacteria induce TSLP to promote mutualistic T-cell responses

Thymic stromal lymphopoietin (TSLP) is constitutively expressed in the intestine and is known to regulate inflammation in models of colitis. We show that steady-state TSLP expression requires intestinal bacteria and has an important role in limiting the expansion of colonic T helper type 17 (Th17) cells. Inappropriate expansion of the colonic Th17 cells occurred in response to an entirely benign intestinal microbiota, as determined following the colonization of germ-free C57BL/6 or TSLPR(-/-) mice with the altered Schaedler flora (ASF). TSLP-TSLPR (TSLP receptor) interactions also promoted the expansion of colonic Helios(-)Foxp3(+) regulatory T cells, necessary for the control of inappropriate Th17 responses following ASF bacterial colonization. In summary, these data reveal an important role for TSLP-TSLPR signaling in promoting steady-state mutualistic T-cell responses following intestinal bacterial colonization.

Detection of RTX toxin genes in gram-negative bacteria with a set of specific probes

The family of RTX (RTX representing repeats in the structural toxin) toxins is composed of several protein toxins with a characteristic nonapeptide glycine-rich repeat motif. Most of its members were shown to have cytolytic activity. By comparing the genetic relationships of the RTX toxin genes we established a set of 10 gene probes to be used for screening as-yet-unknown RTX toxin genes in bacterial species. The probes include parts of apxIA, apxIIA, and apxIIIA from Actinobacillus pleuropneumoniae, cyaA from Bordetella pertusis, frpA from Neisseria meningitidis, prtC from Erwinia chrysanthemi, hlyA and elyA from Escherichia coli, aaltA from Actinobacillus actinomycetemcomitans and lktA from Pasteurella haemolytica. A panel of pathogenic and nonpathogenic gram-negative bacteria were investigated for the presence of RTX toxin genes. The probes detected all known genes for RTX toxins. Moreover, we found potential RTX toxin genes in several pathogenic bacterial species for which no such toxins are known yet. This indicates that RTX or RTX-like toxins are widely distributed among pathogenic gram-negative bacteria. The probes generated by PCR and the hybridization method were optimized to allow broad-range screening for RTX toxin genes in one step. This included the binding of unlabelled probes to a nylon filter and subsequent hybridization of the filter with labelled genomic DNA of the strain to be tested. The method constitutes a powerful tool for the assessment of the potential pathogenicity of poorly characterized strains intended to be used in biotechnological applications. Moreover, it is useful for the detection of already-known or new RTX toxin genes in bacteria of medical importance.