Piero Cappuccinelli

New Cluster of Plasmid-Located Class 1 Integrons in Vibrio cholerae O1 and a dfrA15 Cassette-Containing Integron in Vibrio parahaemolyticus Isolated in Angola

ABSTRACT The resistance profile and its correlation with mobile genetic elements were investigated in 11 Vibrio cholerae O1 and 2 Vibrio parahaemolyticus clinical isolates, as well as in 1 V. cholerae O1 and 1 V. cholerae non-O1 environmental isolate, isolated between 1991 and 1996 in different provinces of Angola. All clinical isolates of V. cholerae O1 were resistant to ampicillin, chloramphenicol, trimethoprim, sulfamethoxazole, and tetracycline. They also contained a large conjugative plasmid (p3iANG) with a set of three class 1 integrons harboring dfrA15, blaP1, and qacH-aadA8 cassettes, which code for resistance to trimethoprim, beta-lactams, quaternary ammonium compounds, and aminoglycosides, clustered in a 19-kb region. Chloramphenicol (cat1), kanamycin (aph), sulfonamide (sul2), and tetracycline (tetG) resistance genes were also carried on the plasmid within the same 19-kb region. A chromosomal integron containing the dfrA15 cassette was also revealed in V. parahaemolyticus strains. SXT integrase genes were present in six V. cholerae isolates but apparently were not associated with known SXT-associated resistance genes. This study indicates that plasmids and integrons contributed mainly to the circulation of multiple-drug resistance determinants in Vibrio strains from Angola.

Environmental Vibrio spp., isolated in Mozambique, contain a polymorphic group of integrative conjugative elements and class 1 integrons

Circulation of mobile genetic elements linked to drug resistance spread was studied in Vibrio strains isolated from surface urban water (river and sea) and shellfish samples in 2002-2003 in Maputo, Mozambique. Class 1 integrons and integrating conjugative elements (ICE) were investigated by PCR and mating experiments in strains of major health interest: 10 Vibrio cholerae, six Vibrio parahaemolyticus, two Vibrio alginolyticus and one Vibrio fluvialis. Resistance to at least two antibiotics (predominantly beta-lactams) was detected in all the strains, with additional resistances to sulfamethoxazole, spectinomycin, streptomycin and/or trimethoprim. Class 1 integrons contributed partially to the expression of drug resistance and were found in five isolates: four V. cholerae (blaP1 cassette, one strain also contained the dfrA15 cassette) and one V. alginolyticus (aadA2 cassette). ICEs, apparently devoid of resistance genes, were found in eight V. cholerae, three V. parahaemolyticus and one V. fluvialis isolates. A wide variability was observed by molecular characterization of ICEs. Five ICEs were included in the SXT/R391 family and seven ICEs were not classified. Our results indicate that the SXT/R391 family and related ICEs comprise a large class of polymorphic genetic elements widely circulating in environmental Vibrio strains in Africa, beside those evidently linked to drug resistance in clinical isolates.

Identification of Trichomonas vaginalis α-Actinin as the Most Common Immunogen Recognized by Sera of Women Exposed to the Parasite

A study on presence of antibodies to Trichomonis vaginalis in serum was done on a group of 500 pregnant, asymptomatic Angolan women. A serologic screening, done by ELISA, revealed that 41% of the women had IgG and IgM against the parasite. Analysis of sera by immunoblotting revealed that 94.4% of sera with anti-T. vaginalis IgG class antibodies were reactive against a common immunogenic protein of 115 kDa. The common immunogen was identified as the protozoan alpha-actinin. All sera recognizing the 115-kDa antigen were reactive against both native and recombinant T. vaginalis alpha-actinin and nonreactive against human alpha-actinin. The findings presented in this work offer a new tool for epidemiologic studies and open new perspectives for vaccination.

Origin of Vibrio cholerae in Haiti

As pointed out in the December, 2010, editorial, the ongoing cholera outbreak in Haiti placed this diarrhoeal infectious disease at the forefront of the global public health agenda. As of Dec 3, 2010, WHO reported 121 518 cases, and 2591 deaths associated with cholera infection. Since Haiti was not previously aff ected by cholera during the current seventh pandemic its population is more susceptible to Vibrio cholerae infection. The epidemic strain responsible for the outbreak was identifi ed as V cholerae O1 biotype El Tor, resistant to co-trimoxazole (trimethoprim– sulfamethoxazole), furazolidone, sulfafurazole, streptomycin, and nalidixic acid. We analysed the genome of three clonal isolates sequenced recently by the US Centers for Disease Control and Prevention (AELH00000000, AELI00000000, and AELJ00000000). The Haitian strains contain an integrative conjugative element (ICE) of the SXT/R391 family, a major drugresistance-spreading vector in bacteria, which is 99% identical to ICEVchInd5. This ICE, which confers resistance to co-trimoxazole, sulfafurazole, and streptomycin, was originally identifi ed in strains of V cholerae isolated in India, which are also resistant to nalidixic acid, and clonally belong to the most prevalent epidemic clade in the Indian subcontinent, represented by the reference strain CIRS101. The Haitian clone carries a genotype 7 ctxB gene coding for the cholera toxin subunit B. This genotype was described only in an altered El Tor V cholerae variant isolated during the harsh cholera epidemic in Orissa, India, in 2007. Whole-genome alignment and comparative genomic analysis of the Haitian strains, with the representative V cholerae O1 variants from Central America and Indian subcontinent, confi rmed that the Haitian strain is strictly phylogenetically related to CIRS101 from India. This strain is one of the highly virulent Indian V cholerae O1 that are gradually spreading all over the world; it is not surprising that this strain easily took advantage of the susceptibility of the Haitian people to the disease, and the poor sanitation caused by the earthquake in Haiti.

Phenotypic features and molecular characterization of plasmids in Salmonella abortusovis

Summary: The phenotypic and genotypic characteristics of 69 wild-type strains of Salmonella abortusovis from Sardinia and other Italian regions, representing four different epidemic outbreaks, were studied. Biochemical profiles, pathogenicity factors, resistance to antibacterial drugs and genomic organization were investigated. None of the strains tested showed any haemagglutination with different types of red blood cells, and electron microscopy revealed no fimbriae. Adhesivity to epithelial cells was present in all strains tested. Only 16% of the strains were resistant to streptomycin, and no other drug resistances were found. The restriction patterns of chromosomal DNA did not show heterogeneity and a high-molecular-mass (50-67 kb) plasmid was found in all strains. Restriction analysis of plasmid DNA from strains from different geographical areas, collected over a period of 30 years, showed a HindIII restriction profile characterized by two patterns, one with three fragments, stable and common to all strains, and a second polymorphic pattern with five fragments. The stable pattern included a 3·7 kb HindIII fragment that hybridized with a S. typhimurium probe containing a virulence region including the spvC and spvD genes. These studies allowed us to outline a genetic correlation among S. abortusovis isolated from different outbreaks.

Immunofluorescence of microtubular structures during the cell cycle of Dictyostelium discoideum

SUMMARY: Microtubules and microtubule-organizing centres of Dictyostelium discoideum were investigated using indirect immunofluorescence with antibodies against a microtubule-associated protein from a rabbit preimmune serum. The microtubule system showed a cell cycle dependent variation. The cytoplasmic microtubule network present in interphase disappeared in mitosis, in contrast to the astral fibres and central spindle fibres which remained evident. In both cell cycle stages microtubules radiated from microtubule-organizing centres which corresponded to the nucleus-associated organelles in interphase and the spindle pole bodies in mitosis.

Differentiation without mitosis in Dictyostelium discoideum.

Dictyoselium discoideum Ax-2 amoebae incubated in the presence of the microtubule inhibitor nocodazole, irreversibly lost their ability to multiply. Nocodazole-treated cells remained viable and RNA and protein synthesis continued for at least 48 h. When nocodazole-treated amoebae were allowed to develop on Millipore filters or on agar slides they differentiated with some delay when compared with controls. These results show that mitosis, naturally present during the developmental cycle of Dictyostelium discoideum Ax-2, is not indispensible for differentiation.

Bacteria colonizing root nodules of wild legumes exhibit virulence-associated properties of mammalian pathogens

Bacteria not proficient in nitrogen fixing symbiosis were proven able to invade root nodules of three wild legumes of the genus Hedysarum in Algeria and to be multiplying in these in place of the natural rhizobium symbionts. The involved species featured taxa known as human pathogens including: Enterobacter cloacae, Enterobacter kobei, Escherichia vulneris, Pantoea agglomerans and Leclercia adecarboxylata. A direct screening of the phenotypic determinants of virulence using human cultured cells tested positive for the traits of cytotoxicity, vital stain exclusion and adhesion to epithelia. Antibiogram analyses revealed also a complex pattern of multiple antibiotic resistances. The data suggest that legume root nodules can be a site of survival and of active multiplication for populations of mammalian pathogens, which could thus alternate between the target animal and a number of neutral plant hosts. The worldwide distribution of as yet uninvestigated legumes raises the concern that these represent a general niche that could enhance the hazards posed by microorganisms of clinical nature.

Diversity among human non-typhoidal salmonellae isolates from Zimbabwe

BACKGROUND Non-typhoidal Salmonella infections are an important public health problem in sub-Saharan Africa, especially among children and HIV-seropositive patients in whom they may cause invasive disease. METHODS In order to better understand the epidemiology of Salmonella infections in southern Africa we typed, using serotyping, phage typing and multilocus sequence typing, 167 non-typhoidal Salmonella strains isolated from human clinical specimens during 1995-2000. RESULTS The most common serovars were Salmonella Typhimurium DT56/ST313, Salmonella Enteritidis PT4 and Salmonella Isangi ST216. Isolates of Salmonella Isangi showed a multidrug-resistant phenotype that was resistant to extended-spectrum cephalosporins. Twelve new sequence types and six new serotypes of Salmonella were identified. CONCLUSIONS Given the diversity detected in the study it seems likely that many new variants of S. enterica are extant in Zimbabwe and by implication across sub-Saharan Africa. We have demonstrated the presence in Zimbabwe of a multidrug-resistant strain of the serovar Salmonella Isangi and demonstrated the diversity of Salmonella circulating in one sub-Saharan African country. Further studies on the characteristics of Salmonella Isangi isolates from Zimbabwe, including plasmid typing and genotyping, are essential if effective control of the spread of this potential pathogen in sub-Saharan Africa is to be achieved.

Acanthamoeba castellanii (Genotype T4) Stimulates the Production of Interleukin-10 as well as Pro-inflammatory Cytokines in THP-1 Cells, Human Peripheral Blood Mononuclear Cells and Human Monocyte-Derived Macrophages

ABSTRACT Free-living amoebae of the genus Acanthamoeba can cause severe and chronic infections in humans, mainly localized in immune privileged sites, such as the brain and the eye. Monocytes/macrophages are thought to be involved in Acanthamoeba infections, but little is known about how these facultative parasites influence their functions. The aim of this work was to investigate the effects of Acanthamoeba on human monocytes/macrophages during the early phase of infection. Here, THP-1 cells, primary human monocytes isolated from peripheral blood, and human monocyte-derived macrophages were either coincubated with trophozoites of a clinical isolate of Acanthamoeba (genotype T4) or stimulated with amoeba-derived cell-free conditioned medium. Production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-6 [IL-6], and IL-12), anti-inflammatory cytokine (IL-10), and chemokine (IL-8) was evaluated at specific hours poststimulation (ranging from 1.5 h to 23 h). We showed that both Acanthamoeba trophozoites and soluble amoebic products induce an early anti-inflammatory monocyte-macrophage phenotype, characterized by significant production of IL-10; furthermore, challenge with either trophozoites or their soluble metabolites stimulate both proinflammatory cytokines and chemokine production, suggesting that this protozoan infection results from the early induction of coexisting, opposed immune responses. Results reported in this paper confirm that the production of proinflammatory cytokines and chemokines by monocytes and macrophages can play a role in the development of the inflammatory response during Acanthamoeba infections. Furthermore, we demonstrate for the first time that Acanthamoeba stimulates IL-10 production in human innate immune cells, which might both promote the immune evasion of Acanthamoeba and limit the induced inflammatory response.