Antimicrobial resistance gene shuffling and a three-element mobilisation system in the monophasic Salmonella typhimurium strain ST1030.

In this study we describe the genetic elements and the antimicrobial resistance units (RUs) harboured by the Salmonella Typhimurium monophasic variant 1,4,[5],12:i:- strain ST1030. Of the three identified RUs two were chromosomal, RU1 (IS26-blaTEM-1-IS26-strAB-sul2- IS26) and RU2 (IS26-tetR(B)-tetA(B)-ΔIS26), and one, RU3 (a sul3-associated class 1 integron with cassette array dfrA12-orfF-aadA2-cmlA1-aadA1), was embedded in a Tn21-derived element harboured by the conjugative I1 plasmid pST1030-1A. IS26 elements mediated the antimicrobial resistance gene (ARG) shuffling and this gave rise to pST1030-1A derivatives with different sets of ARGs. ST1030 also harboured two ColE1-like plasmids of which one, pST1030-2A, was mobilisable and the target of an intracellular translocation of the Tn21-derived element; the second (pST1030-3) was an orphan mob-associated oriT plasmid co-transferred with pST1030-1A and pST1030-2A. pST1030-2A and pST1030-3 also carried a parA gene and a type III restriction modification system, respectively. Overall analysis of our data reinforces the role played by IS26, Tn21-derived elements and non-conjugative plasmids in the spread of ARGs and supplies the first evidence, at least in Salmonella, for the identification of a natural isolate harbouring a three-element mobilisation system in the same cell.

IS26 mediated antimicrobial resistance gene shuffling from the chromosome to a mosaic conjugative FII plasmid.

In the present study we report the identification of a sul3-associated class 1 integron containing the dfrA12-orfF-aadA2-cmlA1-aadA1-qacH array embedded in a Tn21-derived element that is part of a conjugative FII plasmid named pST1007-1A. The plasmid was identified in the Salmonella Typhimurium strain ST1007, a member of a clinically relevant clonal MDR lineage diffuse in Italy. ST1007 exhibited resistance to ampicillin, chloramphenicol, streptomycin, sulphamethoxazole, tetracycline and trimethoprim encoded by blaTEM-1, cmlA1, (aadA1, aadA2, strAB), (sul2, sul3), tet(B) and dfrA12 genes, respectively. Apart from pST1007-1A, ST1007 also harbours two chromosome-integrated resistance units RU1 (blaTEM-1-sul2-strAB) and RU2 (tet(B)), flanked by IS26 elements. RU1 and RU2 were able to move as translocatable units, respectively TU1 and TU2, and integrate via IS26 mediated recombination into pST1007-1A. A family of conjugative plasmids, harbouring different sets of antimicrobial resistance genes (ARG) was then generated: pST1007-1B (dfrA12-aadA2-cmlA1-aadA1-sul3- tet(B)), pST1007-1C (dfrA12-aadA2-cmlA1-aadA1-sul3-blaTEM-1-sul2-strAB), pST1007-1D (blaTEM-1-sul2-strAB), pST1007-1E (tet(B)) and pST1007-1F (dfrA12-aadA2-cmlA1-aadA1-sul3- tet(B) -blaTEM-1-sul2-strAB). pST1007-1A is also a mosaic plasmid containing two distinct DNA fragments acquired from I1 plasmids through recombination within the repA4, rfsF and repeat-3 sites. This study further highlights the role played by IS26 in intracellular ARGs shuffling. Moreover, attention has been focused on recombination hot spots that might play a key role in generating mosaic plasmids.

A novel group of IncQ1 plasmids conferring multidrug resistance.

The IncQ is a group of non-conjugative but mobilisable plasmids that are found and stably maintained in a wide range of bacteria contributing to the spread of antimicrobial resistance genes and to the insurgence of multidrug resistant bacteria. Here we report the identification, in clinical Salmonella Typhimurium strains, of an IncQ1 plasmid (pNUC) which confers resistance to sulfamethoxazole, streptomycin and tetracycline through the presence of sul2, strAB and tetA genes, respectively. pNUC was detected in five multidrug resistant S. Typhimurium strains collected in Southern Italy from various hospitals and years of isolation. Bioinformatics analyses highlighted the presence of pNUC-like plasmids in pathogenic bacteria of various Enterobacteriaceae genera or species. Taken as a whole, these plasmids constitute a novel group of IncQ1 plasmids that might have originated through recombination events between a tetR-tetA gene cluster (possibly derived from a Tn1721) and a recipient IncQ1 plasmid related to RSF1010. Our findings raise concerns regarding the possible contribution of the newly identified group of IncQ1 plasmids to the spread of tetracycline resistance.

Evaluation of a rapid test for the diagnosis of pneumococcal pneumonia.

We evaluated the usefulness of a rapid immunochromatographic pneumococcal urinary antigen test (UAT) for the diagnosis of pneumonia over a period of five years. The UAT was positive in 32 (2.3%) urine samples obtained from 1414 patients. In 46 of these 1414 patients results of UAT and/or sputum/pleural fluid culture and/or blood culture and/or procalcitonin levels were available and therefore the study was concentrated on these patients. A concordance between UAT positivity and the presence of Streptococcus pneumoniae in the sputum was observed in only 4 of 46 (8.7%) patients for which both urine and sputum samples were analyzed. A discordant result (UAT positive and absence of S. pneumoniae in sputum samples) was recorded in 8 of 46 (17.4 %) patients. UAT negative results with sputum culture positive for S. pneumoniae were recorded in 28.3% of patients. In 20 patients, UAT tested positive but sputum culture was not performed. A concordance between UAT positivity and the isolation of S. pneumoniae from blood was seen in 2 of 46 patients whereas a discordant result (UAT positive and blood culture negative) was seen in 12 (26.1%) patients. A concordance between the UAT and high levels (≥2ng/ml) of procalcitonin was observed in 4 out of 46 patients, whereas a positive UAT result and a procalcitonin negative result were observed in 2 patients. In our experience the UAT allows the detection of the etiological agent of pneumonia, and also when sputum and/or blood cultures are negative for S. pneumoniae, when the clinical picture is suggestive of alveolar pneumonia.

Relation of C. pneumoniae antibodies to inflammation and atherosclerosclerosis in dialysis patients.

AIM: the aim of this study was to investigate the association between the presence of antibodies to C. pneumoniae, some markers of inflammation and the presence of preclinical atherosclerotic lesions in hemodyalisis (HD) patients treated with different dialytic membranes. METHODS: C. pneumoniae antibodies were measured by microimmunofluorescence in blood samples of 68 chronic HD patients and in 120 healthy blood donors. Intima-media thickness (IMT) of carotid and of femoral arteries, eco-color doppler of sovraortic trunk and lower limb vessels were evaluated. Plasma levels of C-reactive protein (CRP) and terminal complement (C) complex, C5b-9, were measured. RESULTS: HD patients treated by cellulosic membranes have significantly higher plasma levels of C5b-9 and of CRP compared to those treated by synthetic membranes. A significantly higher prevalence of IgG antibodies to C. pneumoniae and also at higher titre was observed in HD patients in comparison to the controls (66% vs. 28%). The carotid artery mean wall thickness was significantly lower in C. pneumoniae seronegative patients than C. pneumoniae seropositive patients. Similar results were obtained for limb arteries. The use of cellulosic membranes, but not synthetic membranes, was associated with higher carotid IMT and this was independent of the C. pneumoniae serology status. CONCLUSION: in addition to known risk factors, the type of dialytic membrane used may contribute to the progression of atherosclerosis lesions in HD patients. Our data strengthen the evidences that C. pneumoniae infection under high inflammatory status might be a further risk factor for progression of atherosclerosis in HD patients, particularly in those treated with cellulosic membranes.

Acanthamoeba T4 and T15 genotypes associated with keratitis infections in Italy.

Thus far there is little data available concerning Acanthamoeba associated amoebic keratitis (AK) from Italy. In order to understand the incidence of Acanthamoeba in patients with ocular infections and to characterize the isolates at the molecular level, ocular specimens and contact lenses or lens case solutions from 140 patients were analysed by culture and by an 18S rRNA (Rns) gene-based PCR method. Nineteen (13.6%) patients showed Acanthamoeba culture positive samples. Eleven out of the 14 genetically characterized isolates were assigned to the T4 genotype. Three isolates, two of them from patients with keratitis responding to specific anti-Acanthamoeba therapy, were identified as belonging to the T15 genotype. This finding represents the first association between the T15 genotype and human amoebic keratitis. PCR amplification of the 18S ribosomal DNA proved to be a sensitive method, potentially able to detect Acanthamoeba without the need of long culture incubation, and thus considerably useful for clinical applications.

Helicobacter pylori seroprevalence in selected groups of Albanian volunteers.

BACKGROUND: Albania is a Mediterranean, South-East European developing country where epidemiological data on infectious diseases are scarce. In this study, the seroprevalence of Helicobacter pylori infection in 1,088 Albanian healthy volunteers (472 females followed-up to a prenatal clinic, 173 recruits, 443 health care workers) was evaluated. MATERIALS AND METHODS: Sera were tested for immunoglobulin-G (IgG) antibodies against H. pylori using a quantitative enzyme immunosorbent assay. RESULTS: The overall H. pylori seroprevalence was 70.7%. The H. pylori seroprevalence increased by age, from 60.4% in individuals younger than 20 years to 81% among those > or = 50 years of age with a significant trend of increase by age. The overall seroprevalence was 73.9% for females and 59.5% for males. In addition the seroprevalence was 55.3% for people living in rural areas and 72.3% for people living in urban areas. No significant differences were found according to level of education except for individuals with elementary level of education. Nurses and hospital auxiliaries have significantly higher H. pylori seroprevalence when compared to other health care workers (physicians and office workers). When each variable (age, gender, area of residence, occupation, and education level) was adjusted for the confounding effect of the other variables by stepwise logistic analysis, we observed that age greater than 40 years and female gender remain the only variables independently associated with the presence of H. pylori IgG antibodies. CONCLUSIONS: H. pylori is highly prevalent among the Albanian population. Improving living conditions, education in hygiene, and the supply of running water are measures to prevent the transmission of H. pylori infection and other infections spread by the fecal-oral route in Albania.

Amebic liver abscess: report of three cases.

Amebic abscess is a common manifestation of extraintestinal amebiasis and it is associated with relatively high morbidity and mortality. We present three cases seen in Bari, Southern Italy, one of which was autochthonous and the other two were not. Diagnosis was performed by elevated antibody titre for E. histolytica through immunofluorescence assay and positive antigen determination by ELISA in stools and in abscess aspirate. Fever often accompanied by chills, abdominal pain, weight loss and hepatomegaly were present. Laboratory findings also revealed leukocytosis with neutrophilia. Pleural effusion was observed in two patients. In all our patients multiple abscesses were observed. All the patients were treated with metronidazole and two of them also underwent the aspiration of the amoebic abscess. In all of them there was improvement of the clinical picture, as demonstrated by computerized tomography.

Campylobacter fetus bacteremia in an immunocompromised patient: case report and review of the literature.

A 33-year-old woman underwent a liver transplantation and splenectomy in 1985 and had followed immunosuppressive therapy until 1995. Afterwards a non-Hodgkin lymphoma was diagnosed and chemotherapy was started. In January 2000, because of suspect transplantation rejection she was treated with steroid and immunosuppressive therapy. Fever occurred after two months and Cytomegalovirus (CMV) infection was diagnosed. Ganciclovir was started with clinical remission. In November 2000 fever recurred without clinical symptoms. Lymphoma recurrence was excluded and CMV was detected by PCR in several biological fluids. Blood cultures were positive for a bacterium that was identified as Campylobacter fetus. The patient was successfully treated with intravenous ciprofloxacin. For persistent CMV viremia therapy with gancyclovir was stopped and foscarnet was used (60mg/Kg/tid i.v. for two weeks). Bacteremia due to C. fetus is rare, occurring mainly in immunocompromised patients. In our patient the immunosuppressive therapy, chemotherapy for lymphoma and CMV infection had made the patient susceptible to bacteremia with this infrequently found bacterium. The clinical microbiologist should be aware of this infection in immunocompromised hosts.

The prolongation of triple therapy for Helicobacter pylori does not allow reaching therapeutic outcome of sequential scheme: a prospective, randomised study.

BACKGROUND AND AIM: One-week triple therapy for Helicobacter pylori revealed, during these last few years, a decrease in the eradication rate, so that the prolongation of its duration has been proposed. A sequential scheme recently showed very satisfactory results. We performed a prospective randomised study with the aim of either evaluating whether the triple therapy prolongation may improve its effectiveness and comparing its outcome with that of sequential regimen. PATIENTS AND METHODS: Three hundred and forty-two H. pylori positive patients completed the study. They were randomised to receive one of the following treatments: (i) a 7-day triple therapy comprising of rabeprazole (20 mg, b.i.d.) plus clarithromycin (500 mg, b.i.d.) and amoxycillin (1 g, b.i.d.); (ii) a 10-day triple therapy comprising the same scheme; (iii) a 10-day sequential regimen comprising of rabeprazole (20 mg, b.i.d.) plus amoxycillin (1 g, b.i.d.) for 5 days followed by rabeprazole (20 mg, b.i.d.) plus clarithromycin (500 mg, b.i.d.) and tinidazole (500 mg, b.i.d.) for the next 5 days. Therapeutic results were expressed using both intention-to-treat and per protocol analyses with 95% confidence intervals. A model of multivariate logistic regression analysis was performed using therapeutic outcome as a dependent variable and including endoscopic finding, smoking habit, age and sex as candidates for the model. RESULTS: Sequential regimen showed a significant gain in the eradication rate as compared to the 7-day (P < 0.0001) and the 10-day (P < 0.01) triple therapies, respectively. Overall eradication was lower in smokers than in non-smokers, but the difference remained significant only in the 7-day triple therapy (P < 0.01). Additionally, the overall eradication was higher in peptic ulcer than dyspepsia (P < 0.01), even if this difference was significant only for both triple therapies. CONCLUSIONS: Seven-day triple therapy achieves disappointing eradication rates in dyspeptics and smokers. Prolonging triple therapy to 10 days does not significantly improve the eradication rate. The novel 10-day sequential regimen is more effective and equally tolerated than the 10-day triple therapy.

Chlamydia pneumoniae in community-acquired pneumonia: seven years of experience.

OBJECTIVES: To determine the prevalence of Chlamydia pneumoniae in community-acquired pneumonia during a period of seven years. METHODS: Serum samples from 311 patients with pneumonia were evaluated using microimmunofluorescence assay to detect C. pneumoniae -specific IgG and IgM antibodies. RESULTS: Thirty nine patients (12.5%) complied with the diagnostic criteria of acute C. pneumoniae infection (a four-fold rise in the titer of IgG antibody, or a single IgG titer > or = 1:512, or a single IgM titer > or = 1:16). All patients were diagnosed as having pneumonia. Co-infection with other respiratory tract pathogens was found in four patients. CONCLUSIONS: C. pneumoniae is an important cause of pneumonia also in our area. Pneumonia due to this bacterium occurs in the cold months and in early spring; in addition we have observed periods of increased incidence of one years duration and periods of low incidence lasting one-two years. Therapy with macrolides and levofloxacin was effective in all patients with C. pneumoniae infection.

Comparison of two microimmunofluorescence tests for detecting antibodies to Chlamydia pneumoniae.

Two microimmunofluorescence (MIF) tests were compared for detection of antibodies to Chlamydia pneumoniae: the microimmunofluorescence of Washington University and the microimmunofluorescence of Chlamydia Serofia. Concordant positive results at the same dilution were observed for IgG in 37.33% of sera tested and concordant negative results were found in 44%. Variations of one fold dilution were observed in 36 sera. Extensive variations (2-3 two-fold dilutions) in the numeric titer values were observed in 20 serum samples with titers of antibody generally higher in the Chlamydia Serofia MIF than in the Washington MIF, resulting in a diagnosis of current infection in three patients. IgM were found with both methods only in one patient. The discrepancies observed may be due to several factors including the different TWAR strain used as antigen in the two tests and the dilution of the FITC-labelled conjugated anti-human IgG. We think that MIF serology may also be influenced by the type of response of the host that may depend on the "local strain" of C. pneumoniae that may express different antigens or in different amounts in comparison with the strains used by the commercial kit.

Protease-activated receptor 1 and plasminogen activator inhibitor 1 expression in chronic allograft nephropathy: the role of coagulation and fibrinolysis in renal graft fibrosis.

BACKGROUND: Chronic allograft nephropathy (CAN), the major cause of renal graft failure, frequently displays extensive interstitial fibrin deposition. Little is known in regard to the cause of the altered coagulation/fibrinolysis balance and its relevance in the pathogenesis of CAN. Thrombin, present within the fibrin clots, can interact with a specific receptor, protease-activated receptor 1 (PAR-1), and modulate a variety of cell functions. On the other hand, the derangement of the fibrinolytic system may directly affect extracellular matrix (ECM) degradation. METHODS: In the present study, we investigated, by in situ hybridization, PAR-1 gene expression and the mRNA levels for tissue factor and plasminogen activator inhibitor 1 (PAI-1), two key regulatory molecules of coagulation and fibrinolysis, in 16 CAN biopsies and in 10 normal human kidney grafts. The thrombin-induced transforming growth factor beta (TGF-beta) gene and protein expression in proximal tubular cells (PTC) was investigated by Northern blotting and ELISA, respectively. RESULTS: Fibrin deposits, absent in normal grafts, were observed in the interstitial space and arterial wall of CAN. Tissue factor gene expression was not increased either at the vascular or at the interstitial level in CAN. On the contrary, PAI-1 gene expression, barely detectable in control tissue, was strikingly increased in CAN, with a distribution resembling the pattern of fibrin deposition. Note that PAI-1 gene expression was directly correlated with the degree of interstitial fibrosis. In addition, fibrin deposits were strictly associated with a marked increase of PAR-1 gene expression in endothelial cells and PTC. The tubular expression of PAR-1 was significantly higher in Banff grade II-III than in grade I. In vitro, incubation of PTC with thrombin caused a significant up-regulation of TGF-beta gene expression, followed by an increased TGF-beta release into the supernatant. Interestingly, urine from CAN patients contained significantly higher levels of TGF-beta. CONCLUSIONS: Fibrin deposits in CAN may result from the increased expression of PAI-1 and the subsequent inhibition of fibrinolysis. The reduced fibrinolysis may cause, in turn, a decreased ECM turnover. Finally, thrombin, preserved in the active form within the fibrin clots, may interact with PAR-1 highly expressed on PTC and induce an up-regulation of ECM deposition in a TGF-beta-dependent manner.

Helicobacter heilmannii gastritis: a histological and immunohistochemical trait.

AIM: Biopsies of the gastric antrum were reviewed over a period of 10 years to determine the prevalence of Helicobacter heilmannii in symptomatic subjects from this geographical area and to relate its presence to distinctive histopathological and immunohistochemical features. METHODS: Biopsies from 7926 symptomatic patients were reviewed. Ten serial sections were stained with haematoxylin and eosin for conventional histology. Another 10 sections were stained with the Gram method for spiral bacteria. When H heilmannii was suspected, 10 additional serial sections were stained with methylene blue to obtain homogeneous colouring. An equal number of sections from patients affected by isolated H heilmannii or H pylori gastritis were analysed by immunohistochemistry to evaluate lymphoid aggregate/mucosal lymphocyte clonality (CD20 and CD3) and tumour necrosis factor alpha (TNF-alpha) in stromal cells. RESULTS: The prevalence of H heilmannii was 0.1% (eight of 7926), whereas H pylori was present in 60.7% of patients (4813 of 7926). In two of the eight H heilmannii positive patients both helicobacters were found. In all subjects infected by H heilmannii only, distinctive histology (lymphocyte exudation into gastric foveolae) was seen. Lymphoid aggregates, chronic mucosal inflammation with patchy activity, and the absence of epithelial mucus depletion were regular features of H heilmannii gastritis. Immunohistochemistry did not reveal different lymphocyte clonal patterns between H pylori and H heilmannii gastritis: CD20 positive cells were predominant in the centre of aggregates and mucosal infiltrates, whereas CD3 positive cells were prevalent at the periphery of follicles. Only H pylori gastritis showed a significant increase in TNF-alpha positive stromal cells. CONCLUSION: These data suggest that an unusual lymphocyte reaction, with the tendency to invade the foveolar lumen, is a distinctive histopathological aspect of H heilmannii chronic gastritis, although further studies in a larger series are necessary to confirm this fact. Nevertheless, lymphocyte clones do not differ qualitatively from those found in H pylori infection. Moreover, compared with H heilmannii, H pylori provokes a more intense release of TNF-alpha, suggesting that different inflammatory responses exist to these two organisms.

Chlamydia trachomatis and Mycobacterium tuberculosis lung infection in an HIV-positive homosexual man.

A 31-year-old homosexual man, who was human immunodeficiency virus (HIV)-positive was admitted for fever and cough. Chest computed tomography (CT) revealed the presence of diffuse interstitial reticular nodulation, and brain nuclear magnetic resonance imaging showed the presence of nodular frontal lesions. Microscopic examination of sputum and other body fluids showed the presence of acid-fast bacilli and culture-only growth Mycobacterium tuberculosis. Serology for respiratory tract pathogens was negative except for Chlamydia. An antibody titer in the immunoglobulin G (IgG) class of 1:64 for Chlamydia pneumoniae and, unexpectedly, an antibody titer of 1:1024 for C. trachomatis were found. The patient was successfully treated with antituberculosis agents, and clarithromycin, for presumptive chlamydial infection.

The effect of azidothymidine on germ tube formation in Candida albicans.

Candida albicans have a marked propensity to cause infections in AIDS patients. A virulent trait of C. albicans is the yeast-hypha transition (Y-->H) which is influenced, in vitro and in vivo, by several factors. Since azidothymidine (AZT) is used in HIV-positive patients, the effect, in vitro, of different concentrations of AZT on C. albicans Y-->H transition was evaluated. C. albicans isolated from HIV-negative and HIV-positive patients were used and strains of C. tropicalis isolated from HIV-positive patients were also tested. AZT concentrations from 0.01 microg/ml to 10 microg/ml did not have any influence on the Y-->H transition, whereas 100 microg/ml AZT significantly inhibited the germ tube formation. AZT did not influence the formation of pseudohyphae in C. tropicalis. It is suggested that C. albicans infection observed in HIV-positive patients was not influenced by AZT therapy, because at currently used dosages, the Y-->H transition was not expected to increase.

MCP-1 and EGF renal expression and urine excretion in human congenital obstructive nephropathy.

BACKGROUND: Obstructive nephropathy is characterized at the histologic level by tubular atrophy and interstitial monocyte infiltration. The molecular mechanisms underlying these histologic changes are still poorly defined. Epidermal growth factor (EGF) produced by tubular cells seems to play a pivotal role in the modulation of tubular cell growth, while monocyte chemotactic peptide-1 (MCP-1) is a powerful and specific chemotactic and activating factor for monocytes. METHODS: Twenty-four patients with congenital ureteropelvic junction obstruction [UPJO; 10 with recurrent urinary tract infection (UTI) and 10 with no UTI] and 15 healthy children were studied. Diagnosis was made by renal ultrasound, intravenous pielography, and MAG3 scan. Urinary samples were collected before and after surgery. In 10 patients, urine was also collected directly from the affected pelvis at the time of surgery. Urinary EGF and MCP-1 levels were measured by enzyme-linked immunosorbent assay. MCP-1 and EGF gene expression were evaluated by in situ hybridization in 15 biopsies from congenital UPJO and in 10 normal kidneys. RESULTS: In normal kidneys, there was a high expression of EGF mRNA, whereas MCP-1 mRNA was undetectable. MCP-1 gene expression was strikingly increased at the tubulointerstitial level in UPJO biopsies compared with controls and was directly correlated with the extent of monocyte infiltration. In addition, UPJO kidney sections showed a marked reduction in EGF gene expression that was directly correlated with the degree of tubular damage. EGF urine concentration was significantly reduced in UPJO when compared with control and directly correlated with its renal gene expression. On the other hand, the MCP-1 urine concentration was strikingly increased in UPJO patients. It is noteworthy that a significant and inverse correlation was observed between the MCP-1 concentration in the urine collected from the obstructed pelvis and the MAG3 clearance of the obstructed kidney (r = -0.76). The presence of recurrent UTI was associated with a significantly higher MCP-1 excretion and a slight reduction in EGF urine concentration. The surgical correction of UPJO was followed by an improvement of renal function together with a significant reduction in MCP-1 excretion and a marked increase in EGF urine concentrations. Interestingly, EGF urine concentration measured before surgery was significantly correlated with the difference between the MAG3 clearance of the obstructed kidney before and after surgery. CONCLUSIONS: MCP-1 and EGF seem to be involved in the pathogenesis of tubulointerstitial damage in congenital obstructive nephropathy, and their urine excretion may represent a powerful prognostic marker in this form of renal disease.