Plasmid-mediated fluoroquinolone resistance determinants in Escherichia coli from community uncomplicated urinary tract infection in an area of high prevalence of quinolone resistance.

In Italy fluoroquinolones (FQs) are extensively prescribed in empirical therapy of uncomplicated urinary tract infection (UTI) despite recommendations in national guidelines and widespread antibiotic resistance in community. To survey the dissemination of plasmid-mediated quinolone resistance in a peak area of FQs consumption, E. coli strains from 154 community and 41 local hospital patients were collected; low level ciprofloxacin resistance qnrA, qnrB, qnrS, and aac(6)'-Ib-cr genes were screened by PCR and patterns of transferable resistances were determined. Clinical ciprofloxacin resistance in hospital doubled community value, while overall rates of FQ resistance genes were similar (31.6% and 27.8%). Prevalence of aac(6')-Ib-cr gene was 11% in outpatients (21%, inpatients) and risk of harbouring this variant was significantly associated with gentamicin resistance; linkage to ceftazidime resistance was significant (P=0.001) and six out of eight strains produced CTX-M-15 and TEM-1 beta lactamases. In transconjugants, the unique pattern ampicillin/kanamycin-gentamicin/ ESBL + was associated with aac(6')-Ib-cr gene presence and with an increase of ciprofloxacin MIC value. Data highlight the need to monitor the resistance risk factors in the local community to provide clinicians with well-grounded guidelines for UTI therapy.

Necrotic cell death in human amniotic cells infected by Listeria monocytogenes.

Listeria monocytogenes can cause a placental-foetal infection that results in spontaneous abortion, premature labour, stillbirth, or neonatal sepsis and meningitis. Bacteria cross the maternofoetal barrier at the villous syncytiotrophoblast level and subsequently spread from the placenta to the fetus. L. monocytogenes is able to induce different kinds of death in a variety of cells. Murine hepatocytes, murine T and human B lymphocytes, and murine dendritic cells die by apoptosis, whereas bacterial infection of murine and human macrophages leads mainly to necrotic cell death. As we previously described the efficient infection and growth of L. monocytogenes in a human amniotic cell line, we investigated the fate of these cells in order to analyse the mode of cell death. Our results provide biochemical and morphological evidence of necrotic death induced by L. monocytogenes infection.

Correlation between liver fibrosis and inflammation in patients transplanted for HCV liver disease.

Hepatitis C virus (HCV) re-infection after liver transplantation (LT) is characterized by an accelerated disease progression in recent years with unclear mechanisms. We evaluate the relationship between progression of liver fibrosis and histological necro-inflammation in HCV recipients, according to age of transplant. Fifty-five patients transplanted (1993-2002) for HCV liver disease, were included in the study. Recipients were retrospectively stratified in three different age of transplant, of 40 months each: group 1) from January 1993 to May 1996; group 2) from June 1996 to august 1999; group 3) from September 1999 to December 2002. Grading (necro-inflammation) and staging (fibrosis) scores were evaluated in liver biopsies at 1, 2 and 3 years from LT (Ishak classification). For all age of transplant the main factor associated with fibrosis progression, was grading score (p < 0.05). However mean staging score for each point of grading increased from 0.3 +/- 0.2 in older LT to 0.7 +/- 0.5 in newer ones (p = 0.01). In conclusion in HCV-LT patients (1) liver fibrosis is strictly associated to histological necro-inflammation; (2) the proportion of this relationship has been changing in recent years since newer LT patients, show an increased amount of fibrosis in comparison with the older ones, for similar grading score.

Virulence traits in Escherichia coli strains isolated from outpatients with urinary tract infections.

This study aims to characterize phenotypic and genotypic virulence traits in Escherichia coli strains, isolated from outpatients with urinary tract infections, comparing with those obtained from inpatients. Information on the pathogenic behavior of the uropathogenic strains was obtained by monitoring different biological properties, such as autoagglutination, hemagglutination, adhesiveness to and invasion of human bladder (HT1376) cells, biofilm formation, phylogenetic grouping, and virulence-related genes. The results show similar behavior in the two groups concerning autoagglutination, hemagglutination, and biofilm formation. None of the strains examined was invasive. However, in strains from outpatients there was an increased adhesion to HT1376 cells compared with clinical strains, a significant higher presence of genes codifying for adhesins and cell protection factors, and a lower proportion of strains belonging to B1 group. These findings add further information on the pathogenic traits of community E. coli, since strains isolated from the outpatients' group were differently "armed" in comparison with those of clinical cases, and more suitable to infect healthy individuals.

Invasive pathway of Listeria ivanovii in human amnion-derived WISH cells.

Among Listeria genus, only two species, Listeria ivanovii and Listeria monocytogenes, are pathogenic. L. ivanovii is almost only associated with infections in animals, mainly sheep and cattle, and has rarely been associated with human infections, whereas L. monocytogenes causes severe illnesses in both humans and animals. To further investigate the pathogenetic features of L. ivanovii in humans, we undertook a study in which the intracellular behaviour of this pathogen was analysed in WISH cells, a cell line derived from human amniotic tissue, and compared to that of L. monocytogenes. Using microbiological, biochemical, and ultrastructural approaches, we demonstrate that L. ivanovii can adhere to and invade human amniotic cells, lyse the phagosomal membrane, polymerize host cell actin, and spread from cell to cell more efficiently than L. monocytogenes. However, although L. ivanovii is capable of specifically infecting and replicating in human amnion cells, its survival in cytoplasm is limited compared to that of L. monocytogenes.

Extended double-dosage HBV vaccination after liver transplantation is ineffective, in the absence of lamivudine and prior wash-out of human Hepatitis B immunoglobulins.

BACKGROUND: The recommended prophylaxis against hepatitis B virus recurrence after liver transplantation based on hepatitis B immunoglobulins and lamivudine is highly expensive. A recent study reported a significant anti-HBs (antibodies against hepatitis B surface antigen) response after a reinforced vaccination against hepatitis B virus, a result not confirmed in a study from our group. Concomitant lamivudine treatment and the achievement of complete washout of anti-hepatitis B-specific immunoglobulin prior to vaccination in our study could explain the contradiction. AIMS: To test the efficacy of a reinforced anti-hepatitis B virus vaccination schedule without lamivudine and without previous anti-hepatitis B-specific immunoglobulin washout. METHODS: A double reinforced course of S-recombinant hepatitis B virus vaccination was given to seven male patients who were transplanted for hepatitis B virus-related cirrhosis. Vaccination consisted of two cycles of three intramuscular double doses (40 microg), given at month 0, 1, 2, and 3, 4, 5, respectively. The first dose was given 2 weeks after stopping lamivudine and the intravenous administration of anti-HBs immunoglobulins. The latter was continued throughout the study and follow-up period to maintain an anti-HBs titre >100 IU/L. RESULTS: At the end of both the first and the second vaccination cycle none of the patients developed an anti-HBs titre greater than the basal anti-HBs titre. CONCLUSION: These data confirm and expand our previous data on the lack of effectiveness of conventional recombinant hepatitis B virus vaccination in liver transplant recipients.

Apoptotic death of Listeria monocytogenes-infected human macrophages induced by lactoferricin B, a bovine lactoferrin-derived peptide.

Listeria monocytogenes, an intracellular facultative food-borne pathogen, was reported to induce apoptosis in vitro and in vivo in a variety of cell types with the exception of murine macrophages. These cells represent the predominant compartment of bacterial multiplication and die as a result of necrosis. In this study we showed that human non-activated and IFN-gamma-activated macrophagic-like (THP-1) cells infected with L. monocytogenes, mainly die by necrosis rather than by an apoptotic process. Two natural products derived from bovine milk, lactoferrin and its derivative peptide lactoferricin B, are capable of regulating the fate of infected human macrophages. Bovine lactoferrin treatment of macrophages protects them from L. monocytogenes-induced death whereas lactoferricin B, its derivative peptide, determines a shifting of the equilibrium from necrosis to apoptosis.

Listeria monocytogenes infection of Caco-2 cells: role of growth temperature.

The aim of the present study was to evaluate the role of temperature in the virulence of Listeria monocytogenes, a Gram-positive facultative intracellular food-borne pathogen. The capacity of bacteria grown at 37, 25 and 4 degrees C to develop haemolytic activity, to enter the Caco-2 enterocyte-like cell line and to multiply intracellularly was investigated. We demonstrated that L. monocytogenes penetration was not significantly influenced by the growth temperature of cultures and that bacteria grown at low temperature were capable of synthesizing internalin and, during the infection process, of restoring the haemolytic phenotype which is normally lacking in the extracellular environment at 4 and 25 degrees C. It can be concluded that L. monocytogenes, frequently present in numerous environmental sources and also in refrigerated food products, produces at low temperature, the virulence factors necessary to invade intestinal cells.

Lactoferrin inhibits herpes simplex virus type 1 adsorption to Vero cells.

This paper describes the ability of human and bovine lactoferrins (HLf; BLf), iron-binding proteins belonging to the non-immune defense system, to interfere with herpes simplex virus type 1 (HSV-1) infection. Since lactoferrins are known to bind to heparan sulphate proteoglycans and to low density lipoprotein receptor, which in turn act as binding sites for the initial interaction of HSV-1 with host cells, we tested the effect of these proteins on HSV-1 multiplication in Vero cells. Both HLf and BLf are found to be potent inhibitors of HSV-1 infection, the concentrations required to inhibit the vital cytopathic effect in Vero cells by 50% being 1.41 microM and 0.12 microM, respectively. HLf and BLf exerted their activity through the inhibition of adsorption of virions to the cells independently of their iron withholding property showing similar activity in the apo- and iron-saturated form. The binding of [35S]methionine-labelled HSV-1 particles to Vero cells was strongly inhibited when BLf was added during the attachment step. BLf interacts with both Vero cell surfaces and HSV-1 particles, suggesting that the hindrance of cellular receptors and/or of viral attachment proteins may be involved in its antiviral mechanism.

Modulation of actA gene expression in Listeria monocytogenes by iron.

This study analysed the invasiveness of Listeria monocytogenes into enterocyte-like Caco-2 cells in which iron depletion was achieved by picolinic acid treatment. Both entry and intracellular multiplication varied depending on the endogenous iron content of bacterial and eukaryotic cells. The behaviour within enterocytes was correlated with a 10-fold increased transcription of the actA gene observed in bacterial cells grown under conditions of iron stress.

The effects of inhibitors of vacuolar acidification on the release of Listeria monocytogenes from phagosomes of Caco-2 cells.

To evaluate the role of the acidic pH of phagosomes on the invasive ability and fate of Listeria monocytogenes within host cells, entry and replication of this gram-positive bacterium in a human enterocyte-like cell line (Caco-2) were investigated by a combination of biochemical and ultrastructural approaches. The effects of inhibitors of vacuolar acidification--the lipophilic weak base ammonium chloride, the carboxylic ionophore monensin and the vacuolar proton ATPase inhibitor bafilomycin A1--on the bacterial invasion pathway were analysed. These agents, which raise the intracellular vesicle acidic pH of living cells by different mechanisms, affected L. monocytogenes replication in Caco-2 cells. Bacteria internalised by bafilomycin-treated cells were unable to escape from phagosomes, as demonstrated by electronmicroscopy. The results provide evidence that low pH is required for efficient intracellular growth of L. monocytogenes.

The effect of iron on the invasiveness of Escherichia coli carrying the inv gene of Yersinia pseudotuberculosis.

The effect of growth in iron-excess or iron-limitation conditions on the invasiveness for HeLa cells of Escherichia coli HB101 carrying plasmid pRI203 which bears the invasion gene of Yersinia pseudotuberculosis was examined. Iron-limitation reduced adhesion and the number of organisms internalised by HeLa cells by about 100-fold. The reduced adhesion of iron-starved bacteria correlated with reduced hydrophobicity and the reduced invasiveness appeared to depend on the plasmid copy number, which was 3.5-fold less than in bacteria grown in iron excess.

Production of a monoclonal antibody directed against the high-affinity nerve growth factor receptor.

The high-affinity nerve growth factor receptor corresponds to the tyrosine protein kinase encoded by the proto-oncogene trkA. Different findings suggest that nerve growth factor (NGF) can be operative in the growth modulation of tumor cell lines possessing high-affinity binding sites for this molecule. Using as immunizing material the SKNBE neuroblastoma cell line transfected with proto-trkA we produced a monoclonal antibody (MAb) able to recognize the high-affinity nerve growth factor receptor. The selected MAb, designated MGR12, is directed against an epitope present on the extracellular domain of the receptor since it showed reactivity on living trkA-expressing cells and was able to immunoprecipitate the proto-trkA molecule. The MGR12 MAb is directed against a non-functional epitope since it neither inhibited NGF binding nor induced receptor internalization. This new reagent appears to be an appropriate tool for analyzing the expression of high-affinity nerve growth factor receptor in tumors of different origin and for elucidating its involvement in tumor progression.

Heterogeneity of virulence-related properties in Listeria monocytogenes strains isolated from patients with haematological malignancies.

Listeria monocytogenes is an intracellular foodborne pathogen of humans and animals for which there are indications of virulence differences among strains. Various virulence properties related to different phases of infection process were investigated in L. monocytogenes strains isolated from patients affected by haematological malignancies. In these isolates, besides to the clinical history, we analysed the haemolysin production, the survival to acidic pH, the ability to enter and proliferate in human intestinal-like and human macrophagic-like cells, as well as the allelic polymorphism of the actA gene involved intracellular movement. A general heterogeneity in the virulence properties was detected which did not appear correlated with the clinical outcome of listeriosis but more probably was influenced by the status of the immune defence of the host.

Invasiveness of Shigella flexneri in poliovirus infected HT-29 cells.

In this paper we report the effect in HT-29 cells of mixed infections with poliovirus type 1 and Shigella flexneri serotype 5 strain M90T and derivative strains differing in adhesive as well as invasive properties. HT-29 epithelial intestinal cells derived from a human colon adenocarcinoma are a good model for coinfection studies because they are susceptible both to poliovirus replication and to S. flexneri invasion. The results show that 48 h after infection by poliovirus the invasiveness of wild-type S. flexneri M90T and of its adherent and invasive derivative M90T (pIL22) is increased. However, the alterations produced in HT-29 cells at the later stage of poliovirus infection are not sufficient to promote the internalization of simply non invasive, or non invasive but adherent, isogenic derivative bacterial strains.

Infectious agents in tissues from spontaneous abortions in the first trimester of pregnancy.

Some evidence suggests that intrauterine infection plays a major role in the pathogenesis of early pregnancy loss, but the implication and prevalence of microrganisms in the aetiology of spontaneous abortion during the first trimester of pregnancy has not yet been well established. In this study, we analysed the tissues relative to the product of conception from abortions during the first trimester (51 spontaneous abortions and 56 voluntary pregnancy interruptions) in women attending the Gynecological Sciences Perinatology and Puericulture Department of "Policlinico Umberto I". Specimens were investigated by cultural methods for the presence of yeasts, gram positive, gram negative bacteria, and genital mycoplasma. By molecular diagnostic procedures, DNA sequences of Chlamydia trachomatis, herpes simplex viruses, adenovirus, human papillomaviruses and human polyomaviruses BK and JC were searched. None of these agents could be found in voluntary pregnancy interruption samples, with the exception of 3.6% of specimens positive for adenovirus, whereas spontaneous abortion tissues were positive for at least one microrganism by 31.5%. Data analysis showed the occurrence of both monomicrobial and polymicrobial infections.

[Modern views on the physiopathologic and clinical aspects of pre-eclampsia]. / Moderne vedute sulla fisiopatologia e clinica della preeclampsia.

After an introduction on the hypertensive disease of pregnancy, the paper reviews recent studies on the etiological, pathophysiological and clinical aspects of preeclampsia.

Effect of enterovirus infection on susceptibility of HeLa cells to Shigella flexneri invasivity.

Invasiveness of Shigella flexneri M90T in HeLa cells was significantly increased when cells were preinfected with poliovirus 1, coxsackievirus B3 and echovirus 6. This effect was dependent on the dose of virus used, evident at early stages of viral infection and lasted hours before the appearance of a cytopathic effect. An increase of bacterial invasion ability was also noticed when HeLa cells were incubated with UV-inactivated enteroviruses. This enhancing effect obtained with both viable and UV-inactivated enteroviruses was not observed when in coinfection experiments HN555, a mutant of S. flexneri M90T which lacked invasive properties, was used. The data presented here suggest that the early steps of enterovirus infection induce some alterations of HeLa cells which are responsible for the enhancing of the invasiveness of S. flexneri M90T, but not sufficient to promote internalization of a non-invasive strain.