Ciprofloxacin Pharmacokinetics/Pharmacodynamics against Susceptible and Low-Level Resistant Escherichia coli Isolates in an Experimental Ascending Urinary Tract Infection Model in Mice.

The mouse ascending urinary tract infection model was used to study the pharmacokinetic/pharmacodynamic (PKPD) relationships of the effect of ciprofloxacin in subcutaneous treatment for 3 days with varying doses and dosing intervals against a susceptible Escherichia coli strain (MIC, 0.032 mg/liter). Further, a humanized dose of ciprofloxacin was administered for 3 days against three E. coli strains with low-level resistance, i.e., MICs of 0.06, 0.25, and 1 mg/liter, respectively. Against the susceptible isolate, ciprofloxacin was highly effective in clearing the urine with daily doses from 10 mg/kg, but the dosing regimen had to be divided into at least two doses for optimal effect. Ciprofloxacin could not clear the urine or kidneys for the low-level-resistant strains. PKPD correlations with all strains combined showed that for the AUC24/MIC there was a slightly higher correlation with effect in urine and kidneys (R2, 0.71 and 0.69, respectively) than the %T>MIC (R2, 0.41 and 0.61, respectively). Equal correlations for the two PKPD indices were found for reduction of colony counts (CFU) in the bladder tissue, but not even the highest dose of 28 mg/kg × 6 could clear the bladder tissue. In conclusion, ciprofloxacin is highly effective in clearing the urine and kidney tissue for fully susceptible E. coli, while even low-level resistance in E. coli obscures this effect. While the effect of ciprofloxacin is mostly AUC/MIC driven against E. coli infection in the urinary tract, the effect in urine depends on the presence of ciprofloxacin in the urine during most of a 24-h period.

Pharmacokinetics and Pharmacodynamics of Fosfomycin and Its Activity against Extended-Spectrum-ß-Lactamase-, Plasmid-Mediated AmpC-, and Carbapenemase-Producing Escherichia coli in a Murine Urinary Tract Infection Model.

Fosfomycin has become an attractive treatment alternative for urinary tract infections (UTIs) due to increasing multidrug resistance (MDR) in Escherichia coli In this study, we evaluated the pharmacokinetic (PK) and pharmacodynamic (PD) indices of fosfomycin and its in vivo activity in an experimental murine model of ascending UTI. Subcutaneous administration of fosfomycin showed that the mean peak plasma concentrations of fosfomycin were 36, 280, and 750 mg/liter following administration of a single dose of 0.75, 7.5, and 30 mg/mouse, respectively, with an elimination half-life of 28 min, and urine peak concentrations of 1,100, 33,400, and 70,000 mg/liter expected to be sustained above 1 mg/liter (MIC of the test strain, NU14) for 5, 8, and 9.5 h, respectively. The optimal PK/PD indices for reducing urine colony counts (number of CFU per milliliter) were determined to be the area under the concentration-time curve/MIC from 0 to 72 h and the maximum concentration/MIC on the basis of the dose-dependent bloodstream PK and the results of an evaluation of six dosing regimens. With a dosing regimen of 15 mg/mouse twice (every 36 h), fosfomycin significantly reduced the number of CFU per milliliter of all susceptible strains in urine, including clinical MDR strains, except for one clinical strain (P = 0.062). Variable degrees of reduction were observed in the bladder and kidneys. No significant reductions in the number of CFU per milliliter were observed with the resistant strains. In conclusion, fosfomycin shows concentration-dependent in vivo activity, and the results suggest that fosfomycin is an effective alternative to carbapenems in treating MDR E. coli in uncomplicated UTIs. The data on the effectiveness of fosfomycin against the MDR isolates along with the results of PK/PD modeling should facilitate the further development of improved recommendations for its clinical use.

Dissemination and Characteristics of a Novel Plasmid-Encoded Carbapenem-Hydrolyzing Class D ß-Lactamase, OXA-436, Found in Isolates from Four Patients at Six Different Hospitals in Denmark.

The diversity of OXA-48-like carbapenemases is continually expanding. In this study, we describe the dissemination and characteristics of a novel carbapenem-hydrolyzing class D ß-lactamase (CHDL) named OXA-436. In total, six OXA-436-producing Enterobacteriaceae isolates, including Enterobacter asburiae (n = 3), Citrobacter freundii (n = 2), and Klebsiella pneumoniae (n = 1), were identified in four patients in the period between September 2013 and April 2015. All three species of OXA-436-producing Enterobacteriaceae were found in one patient. The amino acid sequence of OXA-436 showed 90.4 to 92.8% identity to the amino acid sequences of other acquired OXA-48-like variants. Expression of OXA-436 in Escherichia coli and kinetic analysis of purified OXA-436 revealed an activity profile similar to that of OXA-48 and OXA-181, with activity against penicillins, including temocillin; limited or no activity against extended-spectrum cephalosporins; and activity against carbapenems. The blaOXA-436 gene was located on a conjugative ∼314-kb IncHI2/IncHI2A plasmid belonging to plasmid multilocus sequence typing sequence type 1 in a region surrounded by chromosomal genes previously identified to be adjacent to blaOXA genes in Shewanella spp. In conclusion, OXA-436 is a novel CHDL with functional properties similar to those of OXA-48-like CHDLs. The described geographical spread among different Enterobacteriaceae and the plasmid location of blaOXA-436 illustrate its potential for further dissemination.

Development of contractile properties in the fetal porcine urinary bladder.

BackgroundIn early fetal life, the bladder is merely a conduit allowing urine to pass through freely into the amniotic cavity. As the striated external urethral sphincter evolves, the bladder acquires its reservoir and voiding functions. We characterized the myogenic and neurogenic contractions of the normal fetal porcine bladder from midterm until close to full-term gestation.MethodsContractile responses were measured in vitro using bladder strips from fetuses at 60 (N=23) and 100 days (N=21) of gestation. Spontaneous activity, and the responses to potassium chloride (KCl) solution, electrical field stimulation (EFS), and receptor activation were recorded. The smooth muscle content was evaluated histologically.ResultsHistological studies revealed that the fractional content of smooth muscle doubled between the two time points, and passive tension was adjusted to take that into account. Spontaneous activity was regular at 60 days, changing toward an irregular pattern at 100 days. Contractile force elicited by KCl and carbachol increased significantly with gestational age, while contractions to the purinergic agonist, α-ß-methylene adenosine 5'-triphosphate did not. The responses to EFS were almost completely blocked by atropine.ConclusionSpontaneous myogenic contractions become irregular and contractile responses to muscarinic receptor stimulation increase during gestation, as the bladder reservoir and voiding functions develop.

Use of WGS data for investigation of a long-term NDM-1-producing Citrobacter freundii outbreak and secondary in vivo spread of blaNDM-1 to Escherichia coli, Klebsiella pneumoniae and Klebsiella oxytoca.

OBJECTIVES: An outbreak of NDM-1-producing Citrobacter freundii and possible secondary in vivo spread of blaNDM-1 to other Enterobacteriaceae were investigated. METHODS: From October 2012 to March 2015, meropenem-resistant Enterobacteriaceae were detected in 45 samples from seven patients at Aalborg University Hospital, Aalborg, Denmark. In silico resistance genes, Inc plasmid types and STs (MLST) were obtained from WGS data from 24 meropenem-resistant isolates (13 C. freundii, 6 Klebsiella pneumoniae, 4 Escherichia coli and 1 Klebsiella oxytoca) and 1 meropenem-susceptible K. oxytoca. The sequences of the meropenem-resistant C. freundii isolates were compared by phylogenetic analyses. In vitro susceptibility to 21 antimicrobial agents was tested. Furthermore, in vitro conjugation and plasmid characterization was performed. RESULTS: From the seven patients, 13 highly clonal ST18 NDM-1-producing C. freundii were isolated. The ST18 NDM-1-producing C. freundii isolates were only susceptible to tetracycline, tigecycline, colistin and fosfomycin (except for the C. freundii isolates from Patient 2 and Patient 7, which were additionally resistant to tetracycline). The E. coli and K. pneumoniae from different patients belonged to different STs, indicating in vivo transfer of blaNDM-1 in the individual patients. This was further supported by in vitro conjugation and detection of a 154 kb IncA/C2 plasmid with blaNDM-1. Patient screenings failed to reveal any additional cases. None of the patients had a history of recent travel abroad and the source of the blaNDM-1 plasmid was unknown. CONCLUSIONS: To our knowledge, this is the first report of an NDM-1-producing C. freundii outbreak and secondary in vivo spread of an IncA/C2 plasmid with blaNDM-1 to other Enterobacteriaceae.

Investigation of a possible outbreak of carbapenem-resistant Acinetobacter baumannii in Odense, Denmark using PFGE, MLST and whole-genome-based SNPs.

OBJECTIVES: The objectives were to study a possible outbreak of carbapenem-resistant Acinetobacter baumannii by comparing three different typing methods (PFGE, MLST and whole-genome SNPs) and to compare the resistance gene profiles of the isolates. METHODS: From December 2012 to October 2013, eight carbapenem-resistant A. baumannii were detected at Odense University Hospital, Odense, Denmark. These isolates were typed by PFGE, with ApaI and SmaI, respectively, and subjected to WGS. The WGS data were used for in silico extraction of MLST types using two different schemes, resistance genes and SNPs, to which 31 publicly available A. baumannii genomes were added. RESULTS: Using ApaI, the eight isolates had four different PFGE profiles, which were further differentiated using SmaI, separating one of the profiles into two distinct PFGE types. Five ST2 (Pasteur MLST) OXA-23-producing isolates, two ST1 OXA-72-producing isolates and one ST158 OXA-23-producing isolate were detected. The five ST2 isolates were subdivided into ST195, ST208 and ST218 using the Oxford MLST scheme. The phylogenetic analysis based on the core genome showed that six of the eight Danish A. baumannii isolates were located in three distinct clusters. The two remaining isolates did not cluster with other Danish or international isolates included in the study. Isolates that clustered using PFGE, Oxford MLST and phylogenetic analysis also shared similar resistance gene profiles. CONCLUSIONS: The SNP profile, Oxford MLST, PFGE and resistance gene profiles clearly indicated spread of three different A. baumannii strains.

Multiple hospital outbreaks of vanA Enterococcus faecium in Denmark, 2012-13, investigated by WGS, MLST and PFGE.

OBJECTIVES: In Denmark, the incidence of vancomycin-resistant Enterococcus faecium (VREfm) has increased since 2012. The aim of this study was to investigate the epidemiology and clonal relatedness of VREfm isolates in Danish hospitals in 2012-13 using WGS. The second aim was to evaluate if WGS-based typing could replace PFGE for typing of VREfm. METHODS: A population-based study was conducted including all VREfm isolates submitted for national surveillance from January 2012 to April 2013. All isolates were investigated by WGS, MLST and PFGE. RESULTS: One-hundred and thirty-two isolates were included. The majority of the isolates were from clinical samples (77%). Gastroenterology/abdominal surgery (29%) and ICUs (29%) were the predominant departments with VREfm. Genomics revealed a polyclonal structure of the VREfm outbreak. Seven subgroups of 3-44 genetically closely related isolates (separated by <17 SNPs) were identified using WGS. Direct or indirect transmission of VREfm between patients and intra- and inter-regional spreading clones was observed. We identified 10 STs. PFGE identified four major clusters (13-43 isolates) and seven minor clusters (two to three isolates). The results from the typing methods were highly concordant. However, WGS-based typing had the highest discriminatory power. CONCLUSIONS: This study emphasizes the importance of infection control measures to limit transmission of VREfm between patients. However, the diversity of the VREfm isolates points to the fact that other important factors may also affect the VREfm increase in Denmark. Finally, WGS is suitable for typing of VREfm and has replaced PFGE for typing of VREfm in Denmark.

Role of urinary cathelicidin LL-37 and human ß-defensin 1 in uncomplicated Escherichia coli urinary tract infections.

Cathelicidin (LL-37) and human ß-defensin 1 (hBD-1) are important components of the innate defense in the urinary tract. The aim of this study was to characterize whether these peptides are important for developing uncomplicated Escherichia coli urinary tract infections (UTIs). This was investigated by comparing urinary peptide levels of UTI patients during and after infection to those of controls, as well as characterizing the fecal flora of participants with respect to susceptibility to LL-37 and in vivo virulence. Forty-seven UTI patients and 50 controls who had never had a UTI were included. Participants were otherwise healthy, premenopausal, adult women. LL-37 MIC levels were compared for fecal E. coli clones from patients and controls and were also compared based on phylotypes (A, B1, B2, and D). In vivo virulence was investigated in the murine UTI model by use of selected fecal isolates from patients and controls. On average, UTI patients had significantly more LL-37 in urine during infection than postinfection, and patient LL-37 levels postinfection were significantly lower than those of controls. hBD-1 showed similar urine levels for UTI patients and controls. Fecal E. coli isolates from controls had higher LL-37 susceptibility than fecal and UTI E. coli isolates from UTI patients. In vivo studies showed a high level of virulence of fecal E. coli isolates from both patients and controls and showed no difference in virulence correlated with the LL-37 MIC level. The results indicate that the concentration of LL-37 in the urinary tract and low susceptibility to LL-37 may increase the likelihood of UTI in a complex interplay between host and pathogen attributes.

Pharmacokinetic and pharmacodynamic evaluation of nitrofurantoin against Escherichia coli in a murine urinary tract infection model.

The antimicrobial agent nitrofurantoin is becoming increasingly important for treatment of urinary tract infections (UTIs) due to widespread occurrence of multidrug-resistant Escherichia coli. Despite many years of use, little data on nitrofurantoin pharmacokinetics (PK) or -dynamics (PD) exist. The objective of this study was to (i) evaluate the pharmacokinetics of nitrofurantoin in a mouse model and (ii) use that data to design an in vivo dose fractionation study in an experimental model of UTI with E. coli for determination of the most predictive PK/PD index. Nitrofurantoin concentrations in urine were approximately 100-fold larger than concentrations in plasma after oral administration of 5, 10, and 20 mg/kg nitrofurantoin. The area under the curve over the minimum inhibitory concentration (AUC/MIC) was weakly correlated to bacterial reduction in urine (r2 = 0.24), while no such correlation was found for the time that nitrofurantoin stayed above the MIC (T > MIC). Increasing size of single-dose treatment was significantly correlated to eradication of bacteria in the urine, while this was not apparent when the same doses were divided in 2 or 3 doses 8 or 12 h apart. In conclusion, the results indicate that nitrofurantoin activity against E. coli in urine is driven by AUC/MIC.

Reconstructive urologi.

This review summarises the current reconstructive urological procedures seeking to optimise urinary tract function. This includes nephrectomy to avoid complications in non-functioning kidneys and reconstruction of uretero-pelvic junction stenosis. Re-implantation of the ureters is indicated in case of reflux or stenosis. The technique depends on the defect and ranges from re-implantation to transplantation of the kidney into the pelvis. Intestine is used for bladder augmentation or to create a new reservoir. Urethral reconstruction is used for complicated strictures, while penile reconstruction includes insertion of implants and straightening procedures.