In vitro selection of resistance in Escherichia coli and Klebsiella spp. at in vivo fluoroquinolone concentrations

<p>Abstract</p> <p>Background</p> <p>Fluoroquinolones are potent antimicrobial agents used for the treatment of a wide variety of community- and nosocomial- infections. However, resistance to fluoroquinolones in Enterobacteriaceae is increasingly reported. Studies assessing the ability of fluoroquinolones to select for resistance have often used antimicrobial concentrations quite different from those actually acquired at the site of infection. The present study compared the ability to select for resistance of levofloxacin, ciprofloxacin and prulifloxacin at concentrations observed <it>in vivo </it>in twenty strains of <it>Escherichia coli </it>and <it>Klebsiella </it>spp. isolated from patients with respiratory and urinary infections. The frequencies of spontaneous single-step mutations at plasma peak and trough antibiotic concentrations were calculated. Multi-step selection of resistance was evaluated by performing 10 serial cultures on agar plates containing a linear gradient from trough to peak antimicrobial concentrations, followed by 10 subcultures on antibiotic-free agar. <it>E. coli </it>resistant strains selected after multi-step selection were characterized for DNA mutations by sequencing <it>gyrA</it>, <it>gyrB</it>, <it>parC </it>and <it>parE </it>genes.</p> <p>Results</p> <p>Frequencies of mutations for levofloxacin and ciprofloxacin were less than 10^-11at peak concentration, while for prulifloxacin they ranged from <10^-11to 10^-5. The lowest number of resistant mutants after multistep selection was selected by levofloxacin followed by ciprofloxacin and prulifloxacin. Both ciprofloxacin- and prulifloxacin-resistant mutants presented mutations in <it>gyrA </it>and <it>parC</it>, while levofloxacin resistance was found associated only to mutations in <it>gyrA</it>.</p> <p>Conclusions</p> <p>Among the tested fluoroquinolones, levofloxacin was the most capable of limiting the occurrence of resistance.</p

Ability of Lactobacillus kefiri LKF01 (DSM32079) to colonize the intestinal environment and modify the gut microbiota composition of healthy individuals

Abstract Background Probiotics have been observed to positively influence the host's health, but to date few data about the ability of probiotics to modify the gut microbiota composition exist. Aims To evaluate the ability of Lactobacillus kefiri LKF01 DSM32079 (LKEF) to colonize the intestinal environment of healthy subjects and modify the gut microbiota composition. Methods Twenty Italian healthy volunteers were randomized in pre-prandial and post-prandial groups. Changes in the gut microbiota composition were detected by using a Next Generation Sequencing technology (Ion Torrent Personal Genome Machine). Results L. kefiri was recovered in the feces of all volunteers after one month of probiotic administration, while it was detected only in three subjects belonging to the pre-prandial group and in two subjects belonging to the post-prandial group one month after the end of probiotic consumption. After one month of probiotic oral intake we observed a reduction of Bilophila, Butyricicomonas, Flavonifractor, Oscillibacter and Prevotella. Interestingly, after the end of probiotic administration Bacteroides, Barnesiella, Butyricicomonas, Clostridium, Haemophilus, Oscillibacter, Salmonella, Streptococcus, Subdoligranolum, and Veillonella were significantly reduced if compared to baseline samples. Conclusion L. kefiri LKF01 showed a strong ability to modulate the gut microbiota composition, leading to a significant reduction of several bacterial genera directly involved in the onset of pro-inflammatory response and gastrointestinal diseases

Gender and Age-Dependent Etiology of Community-Acquired Urinary Tract Infections

Urinary tract infections (UTIs) are among the most frequent community-acquired infections worldwide. Escherichia coli is the most common UTI pathogen although underlying host factors such as patients' age and gender may influence prevalence of causative agents. In this study, 61 273 consecutive urine samples received over a 22-month period from outpatients clinics of an urban area of north Italy underwent microbiological culture with subsequent bacterial identification and antimicrobial susceptibility testing of positive samples. A total of 13 820 uropathogens were isolated and their prevalence analyzed according to patient's gender and age group. Overall Escherichia coli accounted for 67.6% of all isolates, followed by Klebsiella pneumoniae (8.8%), Enterococcus faecalis (6.3%), Proteus mirabilis (5.2%), and Pseudomonas aeruginosa (2.5%). Data stratification according to both age and gender showed E. coli isolation rates to be lower in both males aged ≥60 years (52.2%), E. faecalis and P. aeruginosa being more prevalent in this group (11.6% and 7.8%, resp.), as well as in those aged ≤14 years (51.3%) in whom P. mirabilis prevalence was found to be as high as 21.2%. Streptococcus agalactiae overall prevalence was found to be 2.3% although it was shown to occur most frequently in women aged between 15 and 59 years (4.1%). Susceptibility of E. coli to oral antimicrobial agents was demonstrated to be as follows: fosfomycin (72.9%), trimethoprim/sulfamethoxazole (72.9%), ciprofloxacin (76.8%), ampicillin (48.0%), and amoxicillin/clavulanate (77.5%). In conclusion, both patients' age and gender are significant factors in determining UTIs etiology; they can increase accuracy in defining the causative uropathogen as well as providing useful guidance to empiric treatment

CD4(+)IL-13(+) cells in peripheral blood well correlates with the severity of atopic dermatitis in children

In atopic dermatitis (AD) a Th1/Th2 imbalance has been reported, and interleukin (IL)-13 seems to play a pivotal role in the inflammatory network. We tried to assess the correlation between the immunological marker CD4(+)IL-13(+) and the clinical phase of extrinsic AD in children

Isolation of biofilm producing microorganisms isolated from urinary indwelling catheter in geriatric hospitalized patients

Introduction: In nature, bacterial cells can exist in two different states: individual (planktonic) cells, or biofilm. In biofilms, bacterial cells are adherent to a surface and contained in an extracellular matrix mainly composed of polysaccharides and proteins.The cells in biofilms differ from planktonic counterparts for a different pattern of gene expression and increased resistance to antibiotics.This means that biofilms in hospital can cause persistent infections, due to the immunocompromised state of the patient and from a previous infection or exposure to the antibiotic. The aim of our work was to: 1) to assess the presence of microorganisms in institutionalized geriatric patients carrying indwelling catheter 2) assess the ability of gram-negative microorganisms to produce biofilm. Methods: 150 samples from urine of patients with indwelling urinary catheter hospitalized at ASP Pio Albergo Trivulzio were studied. Adhesion of bacteria was tested using LB medium diluted 1:4 and staining the biofilm cells with crystal violet. Results: 120 samples (80%) tested positive for the presence of microorganisms; in 96 samples only one microrganism was detected, while 24 were contaminated with 2 or more bacterial species. 88% of isolates testing positive to a single species consisted of Gram negative microorganisms: E. coli (48%), P. mirabilis (16%), P. aeruginosa (13%), K. pneumoniae (5%), P. stuartii (2%), C. freundii (1%), E. aerogenes (1%), E. cloacae (1%), K. oxytica (1%), M. morganii (1%).Adhesion assays show that all strains of P. aeruginosa, K. pneumoniae, K. oxytoca, E. cloacae and C. freundii isolates produced biofilm. In contrast, strains of E. aerogenes and M. morganii did not produce any biofilm. 26% of E. coli strains were able to produce biofilm, as well as 93% of P. mirabilis and 50% of P. stuartii.Overall, 55% of Gram negative microrganisms isolated were capable of producing biofilms. Conclusion: The use of a nutritionally poor medium (LB diluted 1:4) and a synthetic substrate define an experimental model in vitro that plays in a relatively faithful to what may occur in vivo. The fact that biofilms are known to be more tolerant to antibiotics and the high proportion of biofilm-forming bacteria isolated from patients carrying indwelling underlines the need for novel antimicrobial agents with higher potency on bacterial biofilms than the ones currently used in therapy

The cientificWorldJOURNAL Research Article Gender and Age-Dependent Etiology of Community-Acquired Urinary Tract Infections

Urinary tract infections (UTIs) are among the most frequent community-acquired infections worldwide. Escherichia coli is the most common UTI pathogen although underlying host factors such as patients&apos; age and gender may influence prevalence of causative agents. In this study, 61 273 consecutive urine samples received over a 22-month period from outpatients clinics of an urban area of north Italy underwent microbiological culture with subsequent bacterial identification and antimicrobial susceptibility testing of positive samples. A total of 13 820 uropathogens were isolated and their prevalence analyzed according to patient&apos;s gender and age group. Overall Escherichia coli accounted for 67.6% of all isolates, followed by Klebsiella pneumoniae (8.8%), Enterococcus faecalis (6.3%), Proteus mirabilis (5.2%), and Pseudomonas aeruginosa (2.5%). Data stratification according to both age and gender showed E. coli isolation rates to be lower in both males aged ≥60 years (52.2%), E. faecalis and P. aeruginosa being more prevalent in this group (11.6% and 7.8%, resp.), as well as in those aged ≤14 years (51.3%) in whom P. mirabilis prevalence was found to be as high as 21.2%. Streptococcus agalactiae overall prevalence was found to be 2.3% although it was shown to occur most frequently in women aged between 15 and 59 years (4.1%). Susceptibility of E. coli to oral antimicrobial agents was demonstrated to be as follows: fosfomycin (72.9%), trimethoprim/sulfamethoxazole (72.9%), ciprofloxacin (76.8%), ampicillin (48.0%), and amoxicillin/clavulanate (77.5%). In conclusion, both patients&apos; age and gender are significant factors in determining UTIs etiology; they can increase accuracy in defining the causative uropathogen as well as providing useful guidance to empiric treatment. Background Urinary tract infections (UTIs) are among the most frequent bacterial infections worldwide Although women, particularly those aged 16-64 years, are significantly more likely to experience UTIs than men Microbial etiology of UTIs has been regarded as well established, with E. coli being the causative pathogen in 50-80% of cases Methods A retrospective study was performed at the Bacteriological Laboratory of the &quot;Centro Diagnostico Italiano&quot; (CDI), based in Milan (Italy), on all bacterial strains isolated from consecutive urine samples received from outpatients clinics of a high-populated urban area of North Italy, between March 2008 and December 2009. Urine samples, accompanied by microbiology request forms, were delivered either directly to the CDI laboratory or through 7 collaborating laboratories. All sample processing and patients&apos; data collection were carried out centrally by the CDI laboratory. CDI Laboratory follows Internal Quality Control procedures and participates to an External Program for Quality Assessment with positive evaluations. As part of the routine procedure, patients received indications to avoid antimicrobials assumption during the previous 7 days and instructions on urine sampling (including cleaning of the genital area prior to midstream specimen collection) and its transport to the laboratories (within 2 hours of collection). Specimens from collaborating laboratories were transported in Vacutainer tubes containing boric acid at 1-2% as preservative. All samples were plated as soon as possible and no later than 24 hours on Chromagar Orientation-BD plates and incubated for 18-24 hours at 37 • C. Criterion for defining significant bacteriuria (positive samples) was the presence of ≥10 5 colony-forming units (CFU)/mL of urine The statistical analysis was performed by the SAS System version 9.2. The difference between females and males in the frequency of positive samples of each agent was analyzed by the chi-square test. The frequency of the positive samples of each agent as a function of gender and age groups (≤14; 15-29; 30-59; ≥60 years) was analyzed by the logistic regression, including in the model the terms gender, age, and the gender by age interaction. Results A total of 61 273 urine cultures were performed over a 22-month period; of these 13 820 (22.6%) were found to be positive for bacterial infection. Nearly 80% of all isolates were from women (female to male ratio (F/M) = 3.8) and 58% from subjects aged 60 years or more. Cumulatively, the two younger age groups accounted for 11.2% of total isolates. Female to male ratio was highest in age group 15-29 years (F/M = 13.5) and lowest in the youngest age group (F/M = 1.4). Overall the most frequently encountered pathogen was Escherichia coli (67.6%), followed by Klebsiella pneumoniae (8.8%), Enterococcus faecalis (6.3%), Proteus mirabilis (5.2%), Pseudomonas aeruginosa (2.5%), and Streptococcus agalactiae (2.3%) Frequency of isolation of all six main species was found to be statistically different between females and males ( in females, whereas E. faecalis, P. mirabilis, and P. aeruginosa were more common in men. All the six most prevalent bacterial species, with the exception of K. pneumoniae, revealed statistically significant differences in isolation rates within the four chosen age groups Data stratification according to both gender and age showed that differences in frequency of isolation between females and males of E. faecalis and S. agalactiae were not consistent across all age groups Susceptibility to antimicrobials of main isolated uropathogens is shown in K. pneumoniae susceptibility to quinolones (93.0%-95.3%) and to TMP-SMX (89.8%) was higher in comparison to E. coli while fosfomycin activity resulted to be lower (81.0%). E. faecalis susceptibility to ampicillin and fosfomycin was high (96.1% and 100%, resp.), superior with respect to susceptibility to quinolones (71.9%-82.3%). S. agalactiae susceptibility to levofloxacin was found to be 91.1% (data not shown). Discussion As urinary tract infection is a very common disease, its diagnosis and treatment have important implications for patients&apos; health, development of antibiotic resistance, and health care costs The present retrospective study describes the distribution and antimicrobial susceptibility of bacterial species isolated from a large number of urinary samples collected over a 22-month period, as part of routine analyses, from unselected community patients (male and female of any age and clinical condition) living in a urban area in the north of Italy. The high number of available isolates allowed to stratify data according to patients&apos; gender and age and so to evaluate the association of such variables to UTI etiology. As expected E. coli was the most frequently encountered species in our study. Percentage of E. coli isolation (67.6%) well compares with those reported from other outpatients surveys conducted in north (64.6%) Other frequent isolates found in this study included K. pneumoniae, P. mirabilis, and E. faecalis, all having been reported to be highly prevalent species in UTIs Our study, however, showed that prevalence of urinary pathogens following data stratification was not consistent across all age groups further divided by gender. Escherichia coli, for example, was found to be less prevalent in the youngest and oldest male subjects (51.3% and 52.2%, resp.) and more frequent in female patients aged 15 years or older 4 The Scientific World Journal (approximately 71%), Proteus mirabilis prevalence was found to be highest (21.2%) in young males aged ≤14 years, whilst S. agalactiae was mostly found in women aged between 15 and 59 years (approximately 4.0%). Kiffer et al. [21] conducted a study comparable to ours, in terms of patient&apos;s population (both males and females of any age), number of isolates The Scientific World Journal 5 (0-13 years), accounting for 22.1% of isolates (15.6% in our study) with a females to males ratio of 0.45 (0.55 in our study). Already in 1972, Bergström [22] and, more recently, Modarres and Oskoii P. mirabilis has been described to be present in the preputial sac of boys, having been isolated in 22.6% of uncircumcised males of up to 14 years of age Susceptibility of uropathogenic bacteria to antimicrobials agents is also known to vary among countries and over time In our survey, 72.9% of E. coli isolates were susceptible to TMP/SMX. Susceptibility to oral antimicrobials of P. mirabilis strains isolated in our study, was generally lower than that reported both in Italy and other countries. Susceptibility to ciprofloxacin and TMP/SMX of P. mirabilis isolates in our study was of 62.9% and 51.5%, respectively, as compared to rates demonstrated by other authors ranging from 75.5% to 97.9% for ciprofloxacin and from 52.0% to 84.9% for TMP-SMX In conclusion, besides providing further data on the etiology of community-acquired UTIs and antimicrobial susceptibility of uropathogens in Italy, our results confirm that stratification of isolates from unselected patients on the basis of age and gender can improve the assessment of causative pathogens, providing guidance for empiric treatment and interesting clues to the understanding of UTIs etiopathology. In particular, P. mirabilis prevalence was found to be high both in boys (21.2%) and girls (11.8%) suggesting, as previously reporte

Association of kidney disease measures with risk of renal function worsening in patients with type 1 diabetes

Background: Albuminuria has been classically considered a marker of kidney damage progression in diabetic patients and it is routinely assessed to monitor kidney function. However, the role of a mild GFR reduction on the development of stage 653 CKD has been less explored in type 1 diabetes mellitus (T1DM) patients. Aim of the present study was to evaluate the prognostic role of kidney disease measures, namely albuminuria and reduced GFR, on the development of stage 653 CKD in a large cohort of patients affected by T1DM. Methods: A total of 4284 patients affected by T1DM followed-up at 76 diabetes centers participating to the Italian Association of Clinical Diabetologists (Associazione Medici Diabetologi, AMD) initiative constitutes the study population. Urinary albumin excretion (ACR) and estimated GFR (eGFR) were retrieved and analyzed. The incidence of stage 653 CKD (eGFR &lt; 60 mL/min/1.73 m2) or eGFR reduction &gt; 30% from baseline was evaluated. Results: The mean estimated GFR was 98 \ub1 17 mL/min/1.73m2 and the proportion of patients with albuminuria was 15.3% (n = 654) at baseline. About 8% (n = 337) of patients developed one of the two renal endpoints during the 4-year follow-up period. Age, albuminuria (micro or macro) and baseline eGFR &lt; 90 ml/min/m2 were independent risk factors for stage 653 CKD and renal function worsening. When compared to patients with eGFR &gt; 90 ml/min/1.73m2 and normoalbuminuria, those with albuminuria at baseline had a 1.69 greater risk of reaching stage 3 CKD, while patients with mild eGFR reduction (i.e. eGFR between 90 and 60 mL/min/1.73 m2) show a 3.81 greater risk that rose to 8.24 for those patients with albuminuria and mild eGFR reduction at baseline. Conclusions: Albuminuria and eGFR reduction represent independent risk factors for incident stage 653 CKD in T1DM patients. The simultaneous occurrence of reduced eGFR and albuminuria have a synergistic effect on renal function worsening

Emerging Circulating Biomarkers in Atherosclerosis: From Molecular Mechanisms to Therapeutic Strategies

Atherosclerosis is a long-term damaging process, and its progression leads to cardiovascular system injury [...