In vitro activity studies of doripenem and two other carbapenems tested against Pseudomonas aeruginosa and other non-fermentative bacilli.

Over the past decade, an increasing prevalence of infections caused by non-fermenting Gram-negative bacteria has been reported in many countries. Among these bacteria, Pseudomonas aeruginosa and Acinetobacter baumannii have been associated with high mortality and treatment failures. Treatment options for multidrug-resistant P. aeruginosa and A. baumannii infections are limited to carbapenems in most cases. The mechanisms of carbapenem resistance have been identified in P. aeruginosa and other Gram-negative non-fermenters, including enzyme production, overexpression of efflux pumps, porin deficiencies, and target- site alterations. This article reviews the in vitro activity of doripenem and compares it with that of imipenem and meropenem against a large collection of non-fermenting Gram-negative bacilli, obtained in worldwide surveillance studies between 2000 and 2010. A detailed examination of the available data demonstrate that doripenem has more potent in vitro antibacterial activity against P. aeruginosa and Acinetobacter species compared to other carbapenems. Furthermore, doripenem has a limited ability to select for carbapenem-resistant mutants in vitro.

Specific detection of Arcobacter spp. in estuarine waters of Southern Italy by PCR and fluorescent in situ hybridization.

AIM: To evaluate the reliability of culture-independent methods in comparison with culture-dependent ones for the detection of Arcobacter spp. in estuarine waters of Southern Italy. METHODS AND RESULTS: PCR and fluorescent in situ hybridization (FISH) procedures were used to detect arcobacters directly in water samples and after enrichment cultures. The samples totally were positive by molecular methods (PCR and FISH) but only 75% were culture positive, confirming the limitation of these latter to detect Arcobacter spp. in natural samples. Culturable arcobacters were retrieved in all times except in July, and isolated species were ascribed only to Arcobacter cryaerophilus. CONCLUSIONS: Culturable and nonculturable forms of Arcobacter in the estuarine environment were present. PCR assays were more sensitive than traditional culture in detecting Arcobacter butzleri and A. cryaerophilus. FISH comparatively to PCR technique may provide information about cell morphology and viability of single cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Our investigation indicates the existence of an environmental reservoir of potential pathogenic arcobacters in an estuarine Italian area, which may survive under a viable but not culturable state.

Molecular detection of Bartonella henselae and Bartonella clarridgeiae in clinical samples of pet cats from Southern Italy.

Bartonella henselae is considered an emerging pathogen of veterinary and medical interest that can be occasionally transmitted to humans. Cats are considered to be the only reservoir host for B. henselae. In this study, we used a nested-PCR assay to investigate the prevalence of B.henselae and Bartonella clarridgeiae DNA in peripheral blood samples, fine needle lymph node aspirate specimens and oral swabs from 85 cats in order to develop an easy diagnostic strategy for the selection of infection-free cats that are being considered as pets, especially for immunocompromised patients. Overall, molecular analysis showed that 71 cats (83.5%) tested PCR positive for the presence of B. henselae DNA. PCR amplification of DNA B. henselae produced positive products from lymph node aspirate specimens (62/85; 72.9%) similar to those obtained from blood samples (60/85; 70.6%) and higher than those from oral swabs (51/85; 60%) of cats. No PCR product was obtained for B. clarridgeiae. The simultaneous analysis of three different clinical samples in our study increased the diagnostic possibilities for B. henselae infection in the examined cats from 60-72.9% to 83.5%. Lymph node aspirates were found to be the most effective clinical samples for the detection of B. henselae and blood samples were the next best. Oral swab samples were used in this study with good results when considered in combination with blood and/or lymph node aspiration. The use of nested-PCR assay on these three clinical samples may enhance the diagnostic sensitivity for bartonellosis in cats irrespective of the clinical status of animals.

Pet cats as carriers of Arcobacter spp. in Southern Italy.

AIMS: To evaluate the presence of Arcobacter spp. in different biological samples from domestic cats in Southern Italy by using a species-specific PCR assay and thus to elucidate their potential significance as sources of human infection. METHODS AND RESULTS: We investigated the prevalence of Arcobacter DNA in oral swabs, in peripheral blood samples and fine needle lymph node aspirate specimens from 85 cats of which 17 were clinically healthy and 68 had clinical signs of oral disease or lymphadenomegaly. Overall, molecular analysis has shown that Arcobacter-specific DNA was found in 78.8% (67 of 85) of all the cats. In the 67 Arcobacter-positive cats, 66 (77.6%) and 29 (34.1%) were found positive for Arcobacter butzleri and Arcobacter cryaerophilus, respectively. None of the examined samples gave a PCR product for Arcobacter skirrowii. CONCLUSIONS: This study demonstrates that pet cats commonly carry Arcobacter in the oral cavity. According to the clinical data, the Arcobacter detection results showed no significant difference between cats with oral pathology and those suffering from other different pathologies. SIGNIFICANCE AND IMPACT OF THE STUDY: Pet cats harbour Arcobacter spp. and may play a role in their dissemination in the domestic habitat. The high prevalence in a limited number of cat samples in this study may be of significance.

Induction and resuscitation of viable nonculturable Arcobacter butzleri cells.

Two strains of Arcobacter butzleri, ATCC 49616 and an environmental isolate, became nonculturable in seawater microcosms at 4 degrees C by 20 days and at room temperature by 14 days. Nonculturable cells were viable for up to 270 days of incubation in microcosms. Resuscitation of A. butzleri cells from microcosms at both temperatures was achieved 9 days after nutrient addition.

New and investigational triazole agents for the treatment of invasive fungal infections.

The incidence of invasive fungal infections (IFIs) caused by both common and uncommon opportunistic fungi is increasing along with emerging fungal resistance. Since traditional agents (amphotericin B, fluconazole, itraconazole) are limited by an inadequate spectrum of activity, drug resistance or toxicity, there is a great interest in the development of new antifungal agents for treatment of IFIs in high-risk populations. In recent years a number of systemic antifungal drugs have become available and options for treatment of IFIs have expanded. A new generation of triazole agents (voriconazole, posaconazole, isavuconazole, ravuconazole and albaconazole), with a broad spectrum of activity and sufficient improvements in potency to overcome resistance have emerged and represent an alternative to conventional antifungals for the prevention and treatment of IFIs. This article focuses on the microbiology, pharmacology, clinical efficacy and safety of the new antifungal triazole generation.

Occurrence of Helicobacter pylori DNA in the coastal environment of southern Italy (Straits of Messina).

AIMS: The occurrence of Helicobacter pylori in the coastal zone of the Straits of Messina (Italy) as free-living and associated with plankton was studied. METHODS AND RESULTS: Monthly sampling of seawater and plankton was carried out from April 2002 to March, 2003. All environmental samples analysed by cultural method, did not show the presence of H. pylori. The DNA extracted from all environmental samples was tested by PCR by using primers for H. pylori 16S rRNA, ureA and cagA. 16S rRNA PCR yielded amplified products of 522-bp in 15 of 36 (41.7%) of the environmental samples. By using the ureA primers to amplify the urea signal sequences, the predicted PCR products of 491-bp were obtained from eight (22.2%) of 36 environmental samples. PCR with cagA primers yielded amplified products of 349-bp in DNA extracted of seven of 36 (19.4%) of the environmental samples. When 16S rRNA, ureA and cagA amplified gene sequences were aligned with H. pylori 26695 and J99 genome sequences, we obtained a percentage of alignment over 90%. CONCLUSIONS: The detection of H. pylori genes in marine samples allows us to consider the marine environment a possible reservoir for this pathogenic bacterium. SIGNIFICANCE AND IMPACT OF THE STUDY: The direct detection of H. pylori genes may be relevant in order to consider the marine environment as significant reservoir for this bacterium.

Distribution of potentially pathogenic bacteria as free living and plankton associated in a marine coastal zone.

AIMS: To determine the abundance of faecal and nonfaecal bacteria related to human and animal health, as free living or associated with small (>64 microm) and large (>200 microm) plankton, samples were collected monthly from the coastal zone at Messina (Italy). METHODS AND RESULTS: Different enrichment and selective cultural methods were used to determine the abundance of bacteria in sea water and plankton. The bacteria were more frequently isolated from water and large plankton than from small plankton. Vibrio and Aeromonas spp. showed different distribution patterns in water and plankton. Faecal indicators were always present in water and the large size class plankton samples. Enterococci associated with large plankton were more abundant than E. coli in the winter. Vibrio species distributions were different in water and plankton samples. Among arcobacters only A. butzleri was isolated from water and plankton samples. Campylobacter spp. was always absent in small plankton and more frequent in large plankton than in water. CONCLUSIONS: The colonization of zooplankton by potentially pathogenic bacteria is a widespread phenomenon. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of potentially pathogenic bacteria in sea water and associated with plankton can have ecological and epidemiological implications.

Detection of Arcobacter spp. in the coastal environment of the Mediterranean Sea.

The occurrence of Arcobacter spp. was studied in seawater and plankton samples collected from the Straits of Messina, Italy, during an annual period of observation by using cultural and molecular techniques. A PCR assay with three pairs of primers targeting the 16S and 23S rRNA genes was used for detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii in cultures and environmental samples. Only one of the Arcobacter species, A. butzleri, was isolated from seawater and plankton samples. With some samples the A. butzleri PCR assay gave amplified products when cultures were negative. A. cryaerophilus and A. skirrowii were never detected by culture on selective agar plates; they were detected only by PCR performed directly with environmental samples. Collectively, our data suggest that culturable and nonculturable forms of Arcobacter are present in marine environments. The assay was useful for detecting Arcobacter spp. both as free forms and intimately associated with plankton. This is the first report showing both direct isolation of A. butzleri and the presence of nonculturable Arcobacter spp. in the coastal environment of the Mediterranean Sea.

In vitro susceptibility of Arcobacter butzleri and Arcobacter cryaerophilus to different antimicrobial agents.

Seventeen strains of Arcobacter butzleri and thirteen of Arcobacter cryaerophilus, were tested for their antimicrobial susceptibility to 26 antimicrobial agents. Among beta-lactams agents in this study, imipenem was the most active agent against both A. butzleri and A. cryaerophilus isolates with MIC(90) values of 2 and 4 mg/l, respectively. The most active cephalosporin tested was cefepime, although it was more active against A. butzleri (MIC(90) 8 mg/l) than A. cryaerophilus (MIC(90) 64 mg/l). Levofloxacin, marbofloxacin, enrofloxacin and ciprofloxacin were the best-performing fluoroquinolones against these species. Of the aminoglycosides, amikacin was the most active agent against both A. butzleri and A. cryaerophilus strains with MIC(90) values of 64 and 16 mg/l, respectively. All isolates showed high levels of resistance to penicillins, macrolides, chloramphenicol, trimethoprim and vancomycin.

Correlation between Helicobacter pylori infection and IL-18 mRNA expression in human gastric biopsy specimens.

Our data indicate that H. pylori infection is associated with active interleukin-18 production in patients with chronic gastritis. Different cell types appear to be involved in this activity and may play a role in the development of immunopathologic damage.

Cytokine induction in murine bladder tissue by type 1 fimbriated Escherichia coli.

The local cytokine response to uropathogenic phenotype Escherichia coli KBC211 infection exhibits characteristics of both TH1 and TH2 profiles. Interleukin (IL)-6, MIP-2, IL-12, IL-18, and tumor necrosis factor-alpha (TNF-alpha) are expressed, but IL-4, IL-5, and IL-10 are also present at low levels. This is clearly a complex response that should be explored more fully. The relative contributions of the bladder epithelium and other cells of the bladder wall should also be determined. Epithelial cytokine responses may be considerable, and because these cells are the first to encounter the pathogen, they will be of great importance in the immune response to pathogenic E. coli.

p53 and anti-p53 antibodies as possible markers of a switch towards a neoplastic phenotype in patients infected by Helicobacter pylori.

Helicobacter pylori is a definite carcinogen whose mechanism of action is still unknown. The aim of this work was (1) to determine the presence of p53 protein and related antibodies in patients affected by various gastric pathologies and chronically infected with H. pylori, and (2) to try to discover a test to be used as a marker of a possible switch towards a neoplastic phenotype.

Activity and postantibiotic effect of marbofloxacin, enrofloxacin, difloxacin and ciprofloxacin against feline Bordetella bronchiseptica isolates.

The minimum inhibitory concentration (MIC) and postantibiotic effect (PAE) of marbofloxacin, enrofloxacin, difloxacin and ciprofloxacin were evaluated in vitro against 43 feline-source Bordetella bronchiseptica strains. All strains tested were susceptible to marbofloxacin and enrofloxacin (MIC90 0.5mg/l), while 93 and 84% of the strains were susceptible, respectively, to ciprofloxacin and difloxacin with MIC(90) values of, respectively, 1 and 8mg/l. The PAE was studied in 10 strains by exposure of bacteria to marbofloxacin, enrofloxacin, difloxacin and ciprofloxacin at 5 and 10 times minimum inhibitory concentration (MIC) for 1 and 2h. Regrowth was determined by measuring the viable counts after drug removal by a 10(3) dilution procedure. PAEs increased as a function of concentration and exposure time. The mean duration of PAEs varied between 1.1 and 8.2h, showing the following order: marbofloxacin>enrofloxacin>ciprofloxacin>difloxacin. These data are encouraging since fluoroquinolones have a possible role in the clinical treatment of B. bronchiseptica infections, and the strong PAE caused by quinolones may contribute to the in vivo efficacy of these drugs.

Antimicrobial activity and postantibiotic effect of flurithromycin against Helicobacter pylori strains.

The minimal inhibitory concentrations (MIC) of flurithromycin on 49 clinical isolates of Helicobacter pylori was investigated. The MICs were determined using an agar dilution technique. Flurithromycin inhibited the growth of H. pylori strains with MIC(50) and MIC(90) values of 0.156 and 0.625 mg/l, respectively. The postantibiotic effects (PAE) were studied on ten strains, by exposure of the bacteria to flurithromycin at five and ten times MIC for 1 or 2 h. Regrowth was determined by measuring the viable counts after drug removal by a 10(3) dilution procedure. All PAEs increased as a function of concentration and time of exposure. The mean duration of PAEs varied between 1.5 and 6 h. These data are encouraging since macrolides play a key role in the clinical treatment of H. pylori infections, and the strong PAE caused by flurithromycin may contribute to the in vivo efficacy of this drug.

Synergic activities of streptococcal pyrogenic exotoxin A and lipoteichoic acid in cytokine induction.

The present study was carried out to gain insight into the mechanisms involved in the pathogenesis of streptococcal toxic shock syndrome (TSS) and other acute invasive diseases caused by Streptococcus pyogenes (GAS). Specifically, since both whole bacteria and their soluble products are often present in the blood in these conditions, we sought to detect possible synergic activities of somatic and extracellular products in inducing mediators release. For this purpose, whole blood cultures from healthy donors were incubated with different concentrations of streptococcal pyrogenic exotoxin A (SpeA), which is considered a major molecular effector of TSS, heat-killed GAS and cell-wall components such as lipoteichoic acid (LTA) and soluble peptidoglican (sPGN). Significant levels of TNF-alpha, IL-1 alpha and IFN-gamma were found in supernatants from cultures incubated with each of the four inducers alone. Whole GAS and both cell-wall components were more effective (p < 0.05) than SpeA in inducing cytokine release. Whole GAS, at weight basis, was a more potent inducer than LTA and sPGN and LTA, at weight basis, was a more potent inducer than sPGN. In order to verify possible additive or synergic effects of exotoxic and parietal compounds in inducing cytokine release, whole blood cells were incubated with mixtures of SpeA and LTA at different molecular ratio. TNF-alpha, IL-1 alpha and IFN-gamma levels in supernatants were significantly (p < 0.05) higher in supernatants of cultures stimulated simultaneously with the two components than those of cultures stimulated with a single agent. Moreover, these levels were significantly higher than the sum of cytokine levels induced by single components. This study shows that parietal compounds can act in synergy with exotoxins in inducing the release of cytokines, which appear to be the major mediators of TSS.