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.

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.