Risk factors for hospital readmission following complicated urinary tract infection.

Hospital readmissions following severe infections are a major economic burden on the health care system and have a negative influence on patients' quality of life. Understanding the risk factors for readmission, particularly the extent to which they could be prevented, is of a great importance. In this study we evaluated potentially preventable risk factors for 60-day readmission in patients surviving hospitalization for complicated urinary tract infection (cUTI). This was a multinational, multicentre retrospective cohort study conducted in Europe and the Middle East. Our cohort included survivors of hospitalization due to cUTI during the years 2013-2014. The primary outcome was 60-day readmission following index hospitalization. Patient characteristics that could have influenced readmission: demographics, infection presentation and management, microbiological and clinical data; were collected via computerized medical records from infection onset up to 60 days after hospital discharge. Overall, 742 patients were included. The cohort median age was 68 years (interquartile range, (IQR) 55-80) and 43.3% (321/742) of patients were males. The all-cause 60-day readmission rate was 20.1% (149/742) and more than half were readmitted for infection [57.1%, (80/140)]. Recurrent cUTI was the most frequent cause for readmission [46.4% (65/140)]. Statistically significant risk factors associated with 60-day readmission in multivariable analysis were: older age (odds ratio (OR) 1.02 for an one-year increment, confidence interval (CI) 1.005-1.03), diabetes mellitus (OR 1.63, 95% CI 1.04-2.55), cancer (OR 1.7, 95% CI 1.05-2.77), previous urinary tract infection (UTI) in the last year (OR 1.8, 95% CI: 1.14-2.83), insertion of an indwelling bladder catheter (OR 1.62, 95% CI 1.07-2.45) and insertion of percutaneous nephrostomy (OR 3.68, 95% CI 1.67-8.13). In conclusion, patients surviving hospitalization for cUTI are frequently re-hospitalized, mostly for recurrent urinary infections associated with a medical condition that necessitated urinary interventions. Interventions to avoid re-admissions should target these patients.

Risk factors for enterococcal urinary tract infections: a multinational, retrospective cohort study.

Complicated urinary tract infection (cUTI) is a frequent cause of morbidity. In this multinational retrospective cohort study, we aimed to demonstrate risk factors for enterococcal UTI. Univariate and multivariate analyses of risk factors for enterococcal infection were performed. Among 791 hospitalized patients with cUTI, enterococci accounted for approximately 10% of cases (78/791). Risk factors for enterococcal UTI in multivariable analysis were male gender, age range of 55-75 years, catheter-associated UTI, and urinary retention. This information may assist treating physicians in their decision-making on prescribing empiric anti-enterococcus treatment to hospitalized patients presenting with cUTI and thus improve clinical outcomes.

Clinical outcomes of hospitalised patients with catheter-associated urinary tract infection in countries with a high rate of multidrug-resistance: the COMBACTE-MAGNET RESCUING study.

Background: Although catheter-associated urinary tract infection (CA-UTI) is a major healthcare-related problem worldwide, there is a scarcity of current data from countries with high antimicrobial resistance rates. We aimed to determine the clinical outcomes of patients with CA-UTI compared to those of patients with other sources of complicated urinary tract infection (cUTI), and to assess the impact of antimicrobial resistance. We also aimed to identify the factors influencing 30-day mortality among patients with CA-UTI. Methods: This was a multicentre, multinational retrospective cohort study including hospitalised adults with cUTI between January 2013 and December 2014 in twenty hospitals from eight countries from southern Europe, Turkey and Israel. The primary endpoint was 30-day mortality. The secondary endpoints were length of hospital stay, symptom improvement after 7 days' treatment, symptom recurrence at 30 days and readmission 60 days after hospital discharge. Results: Of the 807 cUTI episodes, 341 (42.2%) were CA-UTIs. The time from catheter insertion to cUTI diagnosis was less than 2 weeks in 44.6% of cases. Overall, 74.5% of cases had hospital or healthcare-acquired CA-UTI. Compared to patients with other cUTI aetiologies, those with CA-UTI had the following characteristics: they were more frequently males, older, admitted for a reason other than cUTI and admitted from a long-term care facility; had higher Charlson's comorbidity index; and more frequently had polymicrobial infections and multidrug-resistant Gram-negative bacteria (MDR-GNB). Patients with CA-UTI also had significantly higher 30-day mortality rates (15.2% vs 6%) and longer hospital stay (median 14 [interquartile range -IQR- 7-27] days vs 8 [IQR 5-14] days) than patients with cUTI of other sources. After adjusting for confounders, CA-UTI was not independently associated with an increased risk of mortality (odds ratio, 1.40; 95% confidence interval, 0.77-2.54), and neither was the presence of MDR-GNB. Conclusions: CA-UTI was the most frequent source of cUTI, affecting mainly frail patients. The mortality of patients with CA-UTI was high, though this was not directly related to the infection.

Risk Factors for Treatment Failure and Mortality Among Hospitalized Patients With Complicated Urinary Tract Infection: A Multicenter Retrospective Cohort Study (RESCUING Study Group).

Background: Complicated urinary tract infections (cUTIs) are responsible for a major share of all antibiotic consumption in hospitals. We aim to describe risk factors for treatment failure and mortality among patients with cUTIs. Methods: A multinational, multicentre retrospective cohort study, conducted in 20 countries in Europe and the Middle East. Data were collected from patients' files on hospitalised patients with a diagnosis of cUTI during 2013-2014. Primary outcome was treatment failure, secondary outcomes included 30 days all-cause mortality,among other outcomes. Multivariable analysis using a logistic model and the hospital as a random variable was performed to identify independent predictors for these outcomes. Results: A total of 981 patients with cUTI were included. Treatment failure was observed in 26.6% (261/981), all cause 30-day mortality rate was 8.7% (85/976), most of these in patients with catheter related UTI (CaUTI). Risk factors for treatment failure in multivariable analysis were ICU admission (OR 5.07, 95% CI 3.18-8.07), septic shock (OR 1.92, 95% CI 0.93-3.98), corticosteroid treatment (OR 1.92, 95% CI 1.12-3.54), bedridden (OR 2.11, 95%CI 1.4-3.18), older age (OR 1.02, 95% CI 1.0071.03-), metastatic cancer (OR 2.89, 95% CI 1.46-5.73) and CaUTI (OR 1.48, 95% CI 1.04-2.11). Management variables, such as inappropriate empirical antibiotic treatment or days to starting antibiotics were not associated with treatment failure or 30-day mortality. More patients with pyelonephritis were given appropriate empirical antibiotic therapy than other CaUTI [110/171; 64.3% vs. 116/270; 43%, p <0.005], nevertheless, this afforded no advantage in treatment failure rates nor mortality in these patients. Conclusions: In patients with cUTI we found no benefit of early appropriate empirical treatment on survival rates or other outcomes. Physicians might consider supportive treatment and watchful waiting in stable patients until the causative pathogen is defined.

Predictive factors for multidrug-resistant gram-negative bacteria among hospitalised patients with complicated urinary tract infections.

Background: Patients with complicated urinary tract infections (cUTIs) frequently receive broad-spectrum antibiotics. We aimed to determine the prevalence and predictive factors of multidrug-resistant gram-negative bacteria in patients with cUTI. Methods: This is a multicenter, retrospective cohort study in south and eastern Europe, Turkey and Israel including consecutive patients with cUTIs hospitalised between January 2013 and December 2014. Multidrug-resistance was defined as non-susceptibility to at least one agent in three or more antimicrobial categories. A mixed-effects logistic regression model was used to determine predictive factors of multidrug-resistant gram-negative bacteria cUTI. Results: From 948 patients and 1074 microbiological isolates, Escherichia coli was the most frequent microorganism (559/1074), showing a 14.5% multidrug-resistance rate. Klebsiella pneumoniae was second (168/1074) and exhibited the highest multidrug-resistance rate (54.2%), followed by Pseudomonas aeruginosa (97/1074) with a 38.1% multidrug-resistance rate. Predictors of multidrug-resistant gram-negative bacteria were male gender (odds ratio [OR], 1.66; 95% confidence interval [CI], 1.20-2.29), acquisition of cUTI in a medical care facility (OR, 2.59; 95%CI, 1.80-3.71), presence of indwelling urinary catheter (OR, 1.44; 95%CI, 0.99-2.10), having had urinary tract infection within the previous year (OR, 1.89; 95%CI, 1.28-2.79) and antibiotic treatment within the previous 30 days (OR, 1.68; 95%CI, 1.13-2.50). Conclusions: The current high rate of multidrug-resistant gram-negative bacteria infections among hospitalised patients with cUTIs in the studied area is alarming. Our predictive model could be useful to avoid inappropriate antibiotic treatment and implement antibiotic stewardship policies that enhance the use of carbapenem-sparing regimens in patients at low risk of multidrug-resistance.

Cost of hospitalised patients due to complicated urinary tract infections: a retrospective observational study in countries with high prevalence of multidrug-resistant Gram-negative bacteria: the COMBACTE-MAGNET, RESCUING study.

OBJECTIVE: Complicated urinary tract infections (cUTIs) impose a high burden on healthcare systems and are a frequent cause of hospitalisation. The aims of this paper are to estimate the cost per episode of patients hospitalised due to cUTI and to explore the factors associated with cUTI-related healthcare costs in eight countries with high prevalence of multidrug resistance (MDR). DESIGN: This is a multinational observational, retrospective study. The mean cost per episode was computed by multiplying the volume of healthcare use for each patient by the unit cost of each item of care and summing across all components. Costs were measured from the hospital perspective. Patient-level regression analyses were used to identify the factors explaining variation in cUTI-related costs. SETTING: The study was conducted in 20 hospitals in eight countries with high prevalence of multidrug resistant Gram-negative bacteria (Bulgaria, Greece, Hungary, Israel, Italy, Romania, Spain and Turkey). PARTICIPANTS: Data were obtained from 644 episodes of patients hospitalised due to cUTI. RESULTS: The mean cost per case was €5700, with considerable variation between countries (largest value €7740 in Turkey; lowest value €4028 in Israel), mainly due to differences in length of hospital stay. Factors associated with higher costs per patient were: type of admission, infection source, infection severity, the Charlson comorbidity index and presence of MDR. CONCLUSIONS: The mean cost per hospitalised case of cUTI was substantial and varied significantly between countries. A better knowledge of the reasons for variations in length of stays could facilitate a better standardised quality of care for patients with cUTI and allow a more efficient allocation of healthcare resources. Urgent admissions, infections due to an indwelling urinary catheterisation, resulting in septic shock or severe sepsis, in patients with comorbidities and presenting MDR were related to a higher cost.

Retrospective observational study to assess the clinical management and outcomes of hospitalised patients with complicated urinary tract infection in countries with high prevalence of multidrug resistant Gram-negative bacteria (RESCUING).

INTRODUCTION: The emergence of multidrug resistant (MDR) Gram-negative bacteria (GNB), including carbapenemase-producing strains, has become a major therapeutic challenge. These MDR isolates are often involved in complicated urinary tract infection (cUTI), and are associated with poor clinical outcomes. The study has been designed to gain insight into the epidemiology, clinical management, outcome and healthcare cost of patients with cUTI, especially in countries with high prevalence of MDR GNB. METHODS AND ANALYSIS: This multinational and multicentre observational, retrospective study will identify cases from 1 January 2013 to 31 December 2014 in order to collect data on patients with cUTI as a cause of hospital admission, and patients who develop cUTI during their hospital stay. The primary end point will be treatment failure defined as the presence of any of the following criteria: (1) signs or symptoms of cUTI present at diagnosis that have not improved by days 5-7 with appropriate antibiotic therapy, (2) new cUTI-related symptoms that have developed within 30 days of diagnosis, (3) urine culture taken within 30 days of diagnosis, either during or after completion of therapy, that grows ≥10(4) colony-forming unit/mL of the original pathogen and (4) death irrespective of cause within 30 days of the cUTI diagnosis. SAMPLE SIZE: 1000 patients afford a power of 0.83 (α=0.05) to detect an absolute difference of 10% in the treatment failure rate between MDR bacteria and other pathogens. This should allow for the introduction of about 20 independent risk factors (or their interaction) in a logistic regression model looking at risk factors for failure. ETHICS AND DISSEMINATION: Approval will be sought from all relevant Research Ethics Committees. Publication of this study will be considered as a joint publication by the participating investigator leads, and will follow the recommendations of the International Committee of Medical Journal Editors (ICMJE). TRIAL REGISTRATION NUMBER: NCT02641015; Pre-results.

Characterization of the polymyxin B resistome of Pseudomonas aeruginosa.

Multidrug resistance in Pseudomonas aeruginosa is increasingly becoming a threat for human health. Indeed, some strains are resistant to almost all currently available antibiotics, leaving very limited choices for antimicrobial therapy. In many such cases, polymyxins are the only available option, although as their utilization increases so does the isolation of resistant strains. In this study, we screened a comprehensive PA14 mutant library to identify genes involved in changes of susceptibility to polymyxin B in P. aeruginosa. Surprisingly, our screening revealed that the polymyxin B resistome of this microorganism is fairly small. Thus, only one resistant mutant and 17 different susceptibility/intrinsic resistance determinants were identified. Among the susceptible mutants, a significant number carried transposon insertions in lipopolysaccharide (LPS)-related genes. LPS analysis revealed that four of these mutants (galU, lptC, wapR, and ssg) had an altered banding profile in SDS-polyacrylamide gels and Western blots, with three of them exhibiting LPS core truncation and lack of O-antigen decoration. Further characterization of these four mutants showed that their increased susceptibility to polymyxin B was partly due to increased basal outer membrane permeability. Additionally, these mutants also lacked the aminoarabinose-substituted lipid A species observed in the wild type upon growth in low magnesium. Overall, our results emphasize the importance of LPS integrity and lipid A modification in resistance to polymyxins in P. aeruginosa, highlighting the relevance of characterizing the genes that affect biosynthesis of cell surface structures in this pathogen to follow the evolution of peptide resistance in the clinic.

The two-component system CprRS senses cationic peptides and triggers adaptive resistance in Pseudomonas aeruginosa independently of ParRS.

Cationic antimicrobial peptides pass across the outer membrane by interacting with negatively charged lipopolysaccharide (LPS), leading to outer membrane permeabilization in a process termed self-promoted uptake. Resistance can be mediated by the addition of positively charged arabinosamine through the action of the arnBCADTEF operon. We recently described a series of two-component regulators that lead to the activation of the arn operon after recognizing environmental signals, including low-Mg(2+) (PhoPQ, PmrAB) or cationic (ParRS) peptides. However, some peptides did not activate the arn operon through ParRS. Here, we report the identification of a new two-component system, CprRS, which, upon exposure to a wide range of antimicrobial peptides, triggered the expression of the LPS modification operon. Thus, mutations in the cprRS operon blocked the induction of the arn operon in response to several antimicrobial peptides independently of ParRS but did not affect the response to low Mg(2+). Distinct patterns of arn induction were identified. Thus, the responses to polymyxins were abrogated by either parR or cprR mutations, while responses to other peptides, including indolicidin, showed differential dependency on the CprRS and ParRS systems in a concentration-dependent manner. It was further demonstrated that, following exposure to inducing antimicrobial peptides, cprRS mutants did not become adaptively resistant to polymyxins as was observed for wild-type cells. Our microarray studies demonstrated that the CprRS system controlled a quite modest regulon, indicating that it was quite specific to adaptive peptide resistance. These findings provide greater insight into the complex regulation of LPS modification in Pseudomonas aeruginosa, which involves the participation of at least 4 two-component systems.

The intrinsic resistome of Pseudomonas aeruginosa to ß-lactams.

Pseudomonas aeruginosa is a relevant opportunistic pathogen particularly problematic due to its low intrinsic susceptibility to antibiotics. Intrinsic resistance has been traditionally attributed to the low permeability of cellular envelopes together with the presence of chromosomally-encoded detoxification systems such as multidrug efflux pumps or antibiotic inactivating enzymes. However, some recently published articles indicate that several other elements can contribute to the phenotype of intrinsic resistance of bacterial pathogens. In a recently published article, we explored the chromosomally-encoded determinants that contribute to the phenotype of susceptibility of P. aeruginosa to ceftazidime, imipenem and carbapenem. Using a comprehensive library of transposon-tagged insertion mutants, we found 37 loci in the chromosome of P. aeruginosa that contributed to its intrinsic resistance, because mutants in these loci were more susceptible to antibiotics than their parental strain. 41 further loci could potentially be involved in the acquisition of resistance, because mutants in these loci were less susceptible than their wild-type counterpart. These results indicate that the intrinsic resistome of P. aeruginosa involves several elements, belonging to different functional families and cannot be considered as a specific mechanism of adaptation to the recent usage of antibiotics as therapeutic agents. In the current article, we summarize the findings of the paper and discuss their implications for understanding the evolution of antibiotic resistance and for defining novel targets for the search of new antimicrobials. Finally, the validity of recent theories on the mechanisms of action of antibiotics is discussed taken into consideration the results of our paper and other recently published works on the mechanisms of intrinsic resistance to antibiotics of P. aeruginosa.

Genetic determinants involved in the susceptibility of Pseudomonas aeruginosa to beta-lactam antibiotics.

The resistome of P. aeruginosa for three ß-lactam antibiotics, namely, ceftazidime, imipenem, and meropenem, was deciphered by screening a comprehensive PA14 mutant library for mutants with increased or reduced susceptibility to these antimicrobials. Confirmation of the phenotypes of all selected mutants was performed by Etest. Of the total of 78 confirmed mutants, 41 demonstrated a reduced susceptibility phenotype and 37 a supersusceptibility (i.e., altered intrinsic resistance) phenotype, with 6 mutants demonstrating a mixed phenotype, depending on the antibiotic. Only three mutants demonstrated reduced (PA0908) or increased (glnK and ftsK) susceptibility to all three antibiotics. Overall, the mutant profiles of susceptibility suggested distinct mechanisms of action and resistance for the three antibiotics despite their similar structures. More detailed analysis indicated important roles for novel and known ß-lactamase regulatory genes, for genes with likely involvement in barrier function, and for a range of regulators of alginate biosynthesis.

Adaptive resistance to the "last hope" antibiotics polymyxin B and colistin in Pseudomonas aeruginosa is mediated by the novel two-component regulatory system ParR-ParS.

As multidrug resistance increases alarmingly, polymyxin B and colistin are increasingly being used in the clinic to treat serious Pseudomonas aeruginosa infections. In this opportunistic pathogen, subinhibitory levels of polymyxins and certain antimicrobial peptides induce resistance toward higher, otherwise lethal, levels of these antimicrobial agents. It is known that the modification of lipid A of lipopolysaccharide (LPS) is a key component of this adaptive peptide resistance, but to date, the regulatory mechanism underlying peptide regulation in P. aeruginosa has remained elusive. The PhoP-PhoQ and PmrA-PmrB two-component systems, which control this modification under low-Mg2+ conditions, do not appear to play a major role in peptide-mediated adaptive resistance, unlike in Salmonella where PhoQ is a peptide sensor. Here we describe the identification and characterization of a novel P. aeruginosa two-component regulator affecting polymyxin-adaptive resistance, ParR-ParS (PA1799-PA1798). This system was required for activation of the arnBCADTEF LPS modification operon in the presence of subinhibitory concentrations of polymyxin, colistin, or the bovine peptide indolicidin, leading to increased resistance to various polycationic antibiotics, including aminoglycosides. This study highlights the complexity of the regulatory network controlling resistance to cationic antibiotics and host peptides in P. aeruginosa, which has major relevance in the development and deployment of cationic antimicrobials.

Swarming of Pseudomonas aeruginosa is controlled by a broad spectrum of transcriptional regulators, including MetR.

Pseudomonas aeruginosa exhibits swarming motility on semisolid surfaces (0.5 to 0.7% agar). Swarming is a more than just a form of locomotion and represents a complex adaptation resulting in changes in virulence gene expression and antibiotic resistance. In this study, we used a comprehensive P. aeruginosa PA14 transposon mutant library to investigate how the complex swarming adaptation process is regulated. A total of 233 P. aeruginosa PA14 transposon mutants were verified to have alterations in swarming motility. The swarming-associated genes functioned not only in flagellar or type IV pilus biosynthesis but also in processes as diverse as transport, secretion, and metabolism. Thirty-three swarming-deficient and two hyperswarming mutants had transposon insertions in transcriptional regulator genes, including genes encoding two-component sensors and response regulators; 27 of these insertions were newly identified. Of the 25 regulatory mutants whose swarming motility was highly impaired (79 to 97%), only 1 (a PA1458 mutant) had a major defect in swimming, suggesting that this regulator might influence flagellar synthesis or function. Twitching motility, which requires type IV pili, was strongly affected in only two regulatory mutants (pilH and PA2571 mutants) and was moderately affected in three other mutants (algR, ntrB, and nosR mutants). Microarray analyses were performed to compare the gene expression profile of a swarming-deficient PA3587 mutant to that of the wild-type PA14 strain under swarming conditions. PA3587 showed 63% homology to metR, which encodes a regulator of methionine biosynthesis in Escherichia coli. The observed dysregulation in the metR mutant of nine different genes required for swarming motility provided a possible explanation for the swarming-deficient phenotype of this mutant.

Novel genetic determinants of low-level aminoglycoside resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa strains isolated from patients with persistent lung infections and cystic fibrosis have been found to gradually develop aminoglycoside resistance over time. The aim of this study was to identify potential contributors to low-level aminoglycoside resistance, which may cause such graduated increases in resistance. The Harvard P. aeruginosa PA14 nonredundant library, consisting of approximately 5,800 mutants, was screened for twofold or greater increases in tobramycin resistance. Mutants carrying mutations in a total of 135 unique genes were identified and confirmed to have reduced susceptibility to tobramycin. Many of these genes were involved predominantly in energy metabolism; however, most of these mutants did not exhibit growth defects under the conditions tested, although some exhibited the small-colony phenotype and/or defects in growth under anaerobic conditions. Lipopolysaccharide mutants were also identified, and it was found that tobramycin had a reduced ability to permeabilize the outer membranes of these mutants. The results of this study emphasize the complexity of the interactions that tobramycin may have within the bacterial cell and introduce a large number of novel genes which may play a role in tobramycin resistance.

Complex ciprofloxacin resistome revealed by screening a Pseudomonas aeruginosa mutant library for altered susceptibility.

Pseudomonas aeruginosa offers substantial therapeutic challenges due to its high intrinsic resistance to many antibiotics and its propensity to develop mutational and/or adaptive resistance. The PA14 comprehensive mutant library was screened for mutants exhibiting either two- to eightfold increased susceptibilities (revealing genes involved in intrinsic resistance) or decreased susceptibilities (mutational resistance) to the fluoroquinolone ciprofloxacin. Thirty-five and 79 mutants with increased and decreased susceptibilities, respectively, were identified, as confirmed by broth dilution.

Mutator genes giving rise to decreased antibiotic susceptibility in Pseudomonas aeruginosa.

Screening of the PA14 genomic transposon mutant library for resistance to ceftazidime, tobramycin, and ciprofloxacin led to the discovery of several mutants that appeared in more than one screen. Testing of the frequency of mutation to ciprofloxacin resistance revealed previously known mutator genes, including mutS and mutL, as well as mutators that have not yet been described for P. aeruginosa, including PA3958 and RadA (PA4609).

Induction by cationic antimicrobial peptides and involvement in intrinsic polymyxin and antimicrobial peptide resistance, biofilm formation, and swarming motility of PsrA in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important opportunistic pathogen that causes infections that can be extremely difficult to treat due to its high intrinsic antibiotic resistance and broad repertoire of virulence factors, both of which are highly regulated. It is demonstrated here that the psrA gene, encoding a transcriptional regulator, was upregulated in response to subinhibitory concentrations of cationic antimicrobial peptides. Compared to the wild type and the complemented mutant, a P. aeruginosa PAO1 psrA::Tn5 mutant displayed intrinsic supersusceptibility to polymyxin B, a last-resort antimicrobial used against multidrug-resistant infections, and the bovine neutrophil antimicrobial peptide indolicidin; this supersusceptibility phenotype correlated with increased outer membrane permeabilization by these agents. The psrA mutant was also defective in simple biofilm formation, rapid attachment, and swarming motility, all of which could be complemented by the cloned psrA gene. The role of PsrA in global gene regulation was studied by comparing the psrA mutant to the wild type by microarray analysis, demonstrating that 178 genes were up- or downregulated >or=2-fold (P

Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances.

The aim of broth and agar dilution methods is to determine the lowest concentration of the assayed antimicrobial agent (minimal inhibitory concentration, MIC) that, under defined test conditions, inhibits the visible growth of the bacterium being investigated. MIC values are used to determine susceptibilities of bacteria to drugs and also to evaluate the activity of new antimicrobial agents. Agar dilution involves the incorporation of different concentrations of the antimicrobial substance into a nutrient agar medium followed by the application of a standardized number of cells to the surface of the agar plate. For broth dilution, often determined in 96-well microtiter plate format, bacteria are inoculated into a liquid growth medium in the presence of different concentrations of an antimicrobial agent. Growth is assessed after incubation for a defined period of time (16-20 h) and the MIC value is read. This protocol applies only to aerobic bacteria and can be completed in 3 d.

Development of resistance in wild-type and hypermutable Pseudomonas aeruginosa strains exposed to clinical pharmacokinetic profiles of meropenem and ceftazidime simulated in vitro.

In this study we investigated the interplay of antibiotic pharmacokinetic profiles and the development of mutation-mediated resistance in wild-type and hypermutable Pseudomonas aeruginosa strains. We used in vitro models simulating profiles of the commonly used therapeutic drugs meropenem and ceftazidime, two agents with high levels of antipseudomonal activity said to have different potentials for stimulating resistance development. During ceftazidime treatment of the wild-type strain (PAO1), fully resistant mutants overproducing AmpC were selected rapidly and they completely replaced wild-type cells in the population. During treatment with meropenem, mutants of PAO1 were not selected as rapidly and showed only intermediate resistance due to the loss of OprD. These mutants also replaced the parent strain in the population. During the treatment of the mutator P. aeruginosa strain with meropenem, the slowly selected mutants did not accumulate several resistance mechanisms but only lost OprD and did not completely replace the parent strain in the population. Our results indicate that the commonly used dosing regimens for meropenem and ceftazidime cannot avoid the selection of mutants of wild-type and hypermutable P. aeruginosa strains. For the treatment outcome, including the prevention of resistance development, it would be beneficial for the antibiotic concentration to remain above the mutant prevention concentration for a longer period of time than it does in present regimens.

Detection of extended-spectrum beta-lactamases among Enterobacteriaceae by use of semiautomated microbiology systems and manual detection procedures.

Three commercially available microbiology identification and susceptibility testing systems were compared with regard to their ability to detect extended-spectrum beta-lactamase (ESBL) production in Enterobacteriaceae, i.e., the Phoenix Automated Microbiology System (BD Diagnostic Systems, Sparks, MD), the VITEK 2 System (bioMérieux, Marcy l'Etoile, France), and the MicroScan WalkAway-96 System (Dade Behring, Inc., West Sacramento, CA), using routine testing panels. One hundred fifty putative ESBL producers were distributed blindly to three participating laboratories. Conventional phenotypic confirmatory tests such as the disk approximation method, the CLSI double-disk synergy test, and the Etest ESBL were also evaluated. Biochemical and molecular characterization of beta-lactamases performed at an independent laboratory was used as the reference method. One hundred forty-seven isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Serratia marcescens, Proteus mirabilis, Proteus vulgaris, and Morganella morganii were investigated. Of these isolates, 85 were identified as ESBL producers by the reference method. The remaining isolates were identified as non-ESBL producers; they were either hyperproducers of their chromosomal AmpC, Koxy, or SHV enzymes or lacked any detectable beta-lactamase activity. The system with the highest sensitivity for the detection of ESBLs was the Phoenix (99%), followed by the VITEK 2 (86%) and the MicroScan (84%); however, specificity was more variable, ranging from 52% (Phoenix) to 78% (VITEK 2). The performance of the semiautomated systems differed widely with the species investigated. The sensitivities of the conventional test methods ranged from 93 to 94%. The double-disk synergy test showed the highest specificity and positive predictive value among all test methods, i.e., 97% and 98%, respectively.