Pietro Antonio Cappuccinelli

Coexistence of predominantly nonculturable rhizobia with diverse, endophytic bacterial taxa within nodules of wild legumes

A previous analysis showed that Gammaproteobacteria could be the sole recoverable bacteria from surface-sterilized nodules of three wild species of Hedysarum. In this study we extended the analysis to eight Mediterranean native, uninoculated legumes never previously investigated regarding their root-nodule microsymbionts. The structural organization of the nodules was studied by light and electron microscopy, and their bacterial occupants were assessed by combined cultural and molecular approaches. On examination of 100 field-collected nodules, culturable isolates of rhizobia were hardly ever found, whereas over 24 other bacterial taxa were isolated from nodules. None of these nonrhizobial isolates could nodulate the original host when reinoculated in gnotobiotic culture. Despite the inability to culture rhizobial endosymbionts from within the nodules using standard culture media, a direct 16S rRNA gene PCR analysis revealed that most of these nodules contained rhizobia as the predominant population. The presence of nodular endophytes colocalized with rhizobia was verified by immunofluorescence microscopy of nodule sections using an Enterobacter-specific antibody. Hypotheses to explain the nonculturability of rhizobia are presented, and pertinent literature on legume endophytes is discussed.

Bacillus cereus keratitis associated with contact lens wear

OBJECTIVE We report the first case of contact lens-related Bacillus cereus keratitis and ulcer associated with B. cereus contamination of the contact lens case. This is also the first study to investigate and establish the genetic identity of an organism isolated from the cornea and contact lens case in a patient with contact lens-associated keratitis. DESIGN Case report. INTERVENTION AND TESTING Conjunctival swabs and corneal scrapings from the left eye were inoculated for culture. The contact lens case was also cultured. Antibiotic susceptibility testing was determined by agar disk diffusion method. Initial treatment with topical ciprofloxacin and fortified tobramycin was given. Genetic analysis of the bacterial isolates was performed using polymerase chain reaction (PCR) with enterobacterial repetitive intergenic consensus primers (ERIC; ERIC-PCR). Susceptibility of B. cereus to heat and contact lens disinfecting solutions containing hydrogen peroxide, hydrogen peroxide-catalase, polyquaternium-1, and polyaminopropyl biguanide (PAPB) was tested. MAIN OUTCOME MEASURES Clinical features, culture results, and antibiotic susceptibility testing were analyzed. The ERIC-PCR amplification products were visualized in ethidium bromide-stained agarose gel. Bacterial growth after exposure to heat and contact lens disinfecting solutions was assessed on blood agar plates. RESULTS B. cereus was grown from the conjunctiva, corneal ulcer, and contact lens case. All isolates were sensitive to gentamicin, tobramycin, ciprofloxacin, clindamycin, and vancomycin. The corneal ulcer gradually healed over the next 6 days. Results of ERIC-PCR showed that the isolates from the cornea and contact lens case were indistinguishable, thus demonstrating the source of infecting organism to be the contaminated contact lens case. Exposure to a temperature of 80 degrees C for 20 minutes and incubation with hydrogen peroxide-catalase, polyquaternium-1, and PAPB for the minimum recommended time failed to kill B. cereus. Only exposure to hydrogen peroxide for 4 hours eradicated the organism. CONCLUSIONS B. cereus should be considered a possible etiologic agent of contact lens-associated keratitis. Heat and many types of contact lens disinfecting solutions may be ineffective in eradicating B. cereus from contaminated contact lens cases. Only prolonged exposure to hydrogen peroxide appeared to be sporicidal to B. cereus in this study.

Reduced microbicidal and anti‐tumour activities of human monocytes after ingestion of Plasmodium falciparum‐infected red blood cells

Oxidatively stressed red blood cells (RBC) and Plasmodium falciparum‐infected RBC (PRBC) are avidly phago‐cytosed by human peripheral monocytes. Following the ingestion of PRBC the monocytes’ ability to phagocytose PRBC and to generate aggressive oxidative compounds is severely impaired. In the present work the microbicidal and anti‐tumour capacities of monocytes fed with diamide‐treated RBC and PRBC harbouring mature (trophozoite) parasites have been investigated. The capacity of the latter, but not of the former, to phagocytose Escherichia coli and Staphylococcus aureus and to kill them, as well as ingested Candida albicans cells intracellularly, was found to be markedly impaired. Monocytes that have ingested PRBC had a significantly reduced cytostatic and cytolytic activities against a lymphoblastic tumour cell line. Monocytesfed with oxidatively stressed RBC had normal or sometimes even greater anti‐tumour activities. Monocytes that have ingested PRBC showed a reduced capability to produce superoxide following stimulation with phorbol ester. Such impairment in monocyte functions may explain the reduced antibacterial and anti‐tumour activities of monocytes in malaria patients, and could be consequential to their ability to resist bacterial infections and to provide means for the control of tumour development in those patients.

In Vitro Evaluation of the Effectiveness of the Macrolide Rokitamycin and Chlorpromazine against Acanthamoeba castellanii

ABSTRACT The present study demonstrates the in vitro effectiveness of the macrolide rokitamycin and the phenothiazine compound chlorpromazine against Acanthamoeba castellanii. Growth curve evaluations revealed that both drugs inhibit trophozoite growth in dose- and time-dependent ways. The effects of both drugs when they were used at the MICs at which 100% of isolates are inhibited were amoebistatic, but at higher doses they were amoebicidal as well as cysticidal. Experiments showed that when rokitamycin was associated with chlorpromazine or amphotericin B, rokitamycin enhanced their activities. Furthermore, low doses of rokitamycin and chlorpromazine, alone or in combination, blocked the cytopathic effect of A. castellanii against WKD cells derived from the human cornea. These results may have important significance in the development of new anti-Acanthamoeba compounds.

Mycoplasma hominis and Trichomonas vaginalis symbiosis: multiplicity of infection and transmissibility of M. hominis to human cells

Abstract. We recently reported that most Trichomonas vaginalis isolates cultured in vitro are infected by Mycoplasma hominis. In this work, we have characterized some aspects of the relationships between the two microorganisms. PCR, cultivation, and immunological methods revealed that the number of M. hominis organisms carried by T. vaginalis in culture varied from isolate to isolate, suggesting a specific multiplicity of infection. Moreover, infected T. vaginalis isolates were able to pass bacteria not only to M. hominis-free protozoa, but also to human-derived epithelial cells. The in vitro transmission of the bacterium from T. vaginalis to both uninfected parasite isolates and human epithelial cells suggests a role for T. vaginalis as a carrier of the M. hominis infection in vivo.

ADP and Other Metabolites Released from Acanthamoeba castellanii Lead to Human Monocytic Cell Death through Apoptosis and Stimulate the Secretion of Proinflammatory Cytokines.

ABSTRACT Monocytes/macrophages are thought to be involved in Acanthamoeba infections. The aim of this work was to study whether soluble metabolites (ADP and other compounds) released by Acanthamoeba castellanii trophozoites could induce morphological and biochemical changes in human monocytic cells in vitro. We demonstrate here that ADP constitutively released in the medium by A. castellanii, interacting with specific P2y2 purinoceptors expressed on the monocytic cell membrane, caused a biphasic rise in [Ca2+]i, morphological changes characteristics of cells undergoing apoptosis, caspase-3 activation, and secretion of tumor necrosis factor alpha (TNF-α). The same results were found in monocytes exposed to purified ADP. Cell damage and TNF-α release induced by amoebic ADP were blocked by the P2y2 inhibitor suramin. Other metabolites contained in amoebic cell-free supernatants, with molecular masses of, respectively, >30 kDa and between 30 and 10 kDa, also caused morphological modifications and activation of intracellular caspase-3, characteristics of programmed cell death. Nevertheless, mechanisms by which these molecules trigger cell damage appeared to differ from that of ADP. In addition, other amoebic thermolable metabolites with molecular masses of <10 kDa caused the secretion of interleukin-1β. These findings suggest that pathogenic free-living A. castellanii by release of ADP and other metabolites lead to human monocytic cell death through apoptosis and stimulate the secretion of proinflammatory cytokines.

Seroepidemiology of Trichomonas vaginalis in rural women in Zimbabwe and patterns of association with HIV infection

Serological assays using dried blood spots from 5221 women in rural areas of eastern Zimbabwe were used to assess the epidemiology of Trichomonas vaginalis infection, and its association with HIV. Antibodies to T. vaginalis and to HIV were detected by enzyme immunoassays. Behavioural and demographic data were collected by confidential questionnaires. In total, 516 (9.9%) women were seropositive for T. vaginalis and seroprevalence increased with age among younger women. Divorced, widowed and single women were more likely to be seropositive. After controlling for age, seropositivity was significantly associated with being sexually active, having multiple sex partners, having a partner who had multiple sex partners, and having a new sex partner in the past year. Seropositivity was associated with a recent history of genital discharge. Overall, 208 (40.3%) T. vaginalis-positive samples were also positive for HIV, compared with 1106 (23.5%) T. vaginalis-negative samples (age and sex adjusted OR 2.11, 95% CI 1.74-2.55, P < 0.001). There was increased risk for being HIV-positive amongst T. vaginalis-seropositive women regardless of residence, employment or education. In a logistic regression controlling for common risk factors, the association remained significant. T. vaginalis-seropositive young women with a history of genital discharge were much more likely to be HIV-positive than women who were T. vaginalis-seronegative and had no history of discharge (OR 6.08, 95 % CI 2.95-12.53). Although a causal relationship cannot be assumed, detection and treatment of trichomoniasis may be important in strategies to reduce HIV transmission through sexually transmitted infection control.

ICEVchInd5 is prevalent in epidemic Vibrio cholerae O1 El Tor strains isolated in India

Integrative conjugative elements (ICEs) of the SXT/R391 family are self-transmissible mobile elements mainly involved in antibiotic resistance spread among γ-Proteobacteria, including Vibrio cholerae. We demonstrated that the recently described ICEVchInd5 is prevailing in V. cholerae O1 clinical strains isolated in Wardha province (Maharashtra, India) from 1994 to 2005. Genetic characterization by ribotyping and multiple-locus SSR analysis proved the same clonal origin for V. cholerae O1 isolates in Wardha province over an 11-year period and was used to assess the correlation between strain and ICE content among ours and different Indian reference strains. In silico analysis showed the existence of at least 3 sibling ICEs of ICEVchInd5 in V. cholerae O1 El Tor reference strains, isolated in the Indian subcontinent after 1992.

New V. cholerae atypical El Tor variant emerged during the 2006 epidemic outbreak in Angola

BackgroundV. cholerae is the etiological agent of cholera, a major public health concern in most developing countries. Virulence of V. cholerae relies on the powerful cholera toxin, encoded by the CTX prophage. The emergence of new pathogenic variants in the recent years has been mostly associated with new CTX prophage rearrangements.ResultsIn this retrospective study, we show that the epidemic V. cholerae O1 El Tor strain responsible for the 2006 outbreak in Angola is clonally and genetically different from El Tor strains circulating in the 1990s in the same area. Strains from 2006 carry ICEVchAng3 of the SXT/R391 family. This ICE is associated with a narrower multidrug resistance profile compared to the one conferred by plasmid p3iANG to strains of the 1990s. The CTX prophage carried by 2006 El Tor strains is characterized by rstRET and ctxBCla alleles organized in a RS1-RS2-Core array on chromosome I. Interestingly, the newly emerging atypical strain belongs to a clade previously known to comprise only clinical isolates from the Indian subcontinent that also contain the same ICE of the SXT/R391 family.ConclusionsOur findings remark the appearance of a novel V. cholerae epidemic variant in Africa with a new CTXΦ arrangement previously described only in the Indian Subcontinent.

High rate of levofloxacin resistance in a background of clarithromycin- and metronidazole-resistant Helicobacter pylori in Vietnam

Antimicrobial resistance in Helicobacter pylori has increased worldwide and has become a major cause of treatment failure in many countries, including Vietnam. It is advisable to perform an antibiogram to provide optimal regimens for H. pylori eradication. This study evaluated the rate of antibiotic resistance to the four commonly used antibiotics against H. pylori at a tertiary care hospital in Central Vietnam and analysed point mutations in genes related to clarithromycin (CLA) and levofloxacin (LFX) resistance. A total of 92 H. pylori strains from gastric biopsy specimens were tested; 42.4% were resistant to CLA (primary, 34.2%; secondary, 73.7%), 41.3% to LFX (primary, 35.6%; secondary, 63.2%), 76.1% to metronidazole (MTZ) and 1.1% to amoxicillin. Multidrug resistance was observed in 56.5% (primary, 50.7%; secondary, 78.9%) of isolates (P<0.05). The rate of resistance to LFX was significantly higher in females than males (P<0.05). Most of the CLA- and LFX-resistant strains harboured resistance-associated mutations, with common positions at A2143G and T2182C in the 23S rRNA gene and at Asn-87 or Asp-91 in GyrA. Minimum inhibitory concentrations (MICs) increased in strains carrying quadruple mutations in their 23S rRNA gene and in strains with Asn-87 GyrA mutation (P<0.05). One high-level LFX-resistant strain (MIC=32mg/L) had new mutations with a combination of N87A, A88N and V65I. High resistance rates to CLA, MTZ and LFX discourage standard and LFX-based triple therapies as first-line treatment in Vietnam.