Increased bacterial adherence and biomass in Pseudomonas aeruginosa bacteria exposed to clarithromycin

Long-term low-dose macrolides alter response in patients with chronic sessile Pseudomonas aeruginosa colonization. We examined the effect of clarithromycin on 1) adherence of P. aeruginosa cells and 2) biofilm formation. A suspended-coupon continuous-flow biofilm reactor model was used. Adherent P. aeruginosa bacteria were established for 24 h, immediately followed by a 24-h continuous-flow operation (CFO) phase with serial sampling. In addition, the effect of clarithromycin on adherent biomass was assessed quantitatively using a colorimetric assay. Isolates preexposed to clarithromycin were more adherent to the suspended coupons than nonexposed isolates (P=0.021). After 2 h of CFO, a 1.30+/-0.86 log colony-forming unit (CFU)/cm(2) decrease was observed in controls compared with a 0.08+/-0.55 log CFU/cm(2) decrease in isolates exposed to clarithromycin. Furthermore, a concentration-dependent increase in biofilm biomass was observed with the addition of clarithromycin in a standard mucoid P. aeruginosa strain (1-64 microg/mL, P<0.001) and 44 clinical P. aeruginosa strains (2 or 32 microg/mL, P<0.001). Clarithromycin increased bacterial adherence to the suspended coupons, and increased biomass was observed in isolates treated with clarithromycin.     

Prevalence,Resistance Mechanisms,and Susceptibility of Multidrug-Resistant Bloodstream Isolates of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an important pathogen commonly implicated in nosocomial infections. The occurrence of multidrug-resistant (MDR) P. aeruginosa strains is increasing worldwide and limiting our therapeutic options. The MDR phenotype can be mediated by a variety of resistance mechanisms, and the corresponding relative biofitness is not well established. We examined the prevalence, resistance mechanisms, and susceptibility of MDR P. aeruginosa isolates (resistant to ≥3 classes of antipseudomonal agents [penicillins/cephalosporins, carbapenems, quinolones, and aminoglycosides]) obtained from a large, university-affiliated hospital. Among 235 nonrepeat bloodstream isolates screened between 2005 and 2007, 33 isolates (from 20 unique patients) were found to be MDR (crude prevalence rate, 14%). All isolates were resistant to carbapenems and quinolones, 91% were resistant to penicillins/cephalosporins, and 21% were resistant to the aminoglycosides. By using the first available isolate for each bacteremia episode (n = 18), 13 distinct clones were revealed by repetitive-element-based PCR. Western blotting revealed eight isolates (44%) to have MexB overexpression. Production of a carbapenemase (VIM-2) was found in one isolate, and mutations in gyrA (T83I) and parC (S87L) were commonly found. Growth rates of most MDR isolates were similar to that of the wild type, and two isolates (11%) were found to be hypermutable. All available isolates were susceptible to polymyxin B, and only one isolate was nonsusceptible to colistin (MIC, 3 mg/liter), but all isolates were nonsusceptible to doripenem (MIC, >2 mg/liter). Understanding and continuous monitoring of the prevalence and resistance mechanisms of MDR P. aeruginosa would enable us to formulate rational treatment strategies to combat nosocomial infections.Pseudomonas aeruginosa is an important pathogen commonly implicated in serious nosocomial infections such as pneumonia and sepsis. The occurrence of multidrug-resistant P. aeruginosa strains is increasing worldwide and limiting our therapeutic options. Resistance in P. aeruginosa may be mediated via several distinct mechanisms (3, 13). In multidrug-resistant isolates, the relative contributions of different molecular mechanisms toward phenotypic multidrug resistance are not well established. While there are multiple surveillance studies tracking the resistance of P. aeruginosa to various antimicrobial agents over the years (6, 7), studies reporting trends in concomitant resistance to multiple agents over time are scarce.A hypermutable state is often observed in multidrug-resistant pathogens causing chronic infections in cystic fibrosis patients. It is often related to defects in genes involved in the mismatch repair system (e.g., mutS and mutL). It is also commonly believed that in multidrug-resistant P. aeruginosa isolates, reduced virulence may result due to decreased biofitness. However, recent data suggest otherwise, and multidrug-resistant P. aeruginosa may remain fully pathogenic (8).Many first-line agents are ineffective if used alone against multidrug-resistant P. aeruginosa isolates. Consequently, unconventional agents or those that are less preferred (due to toxicity concerns) may have to be used. The objectives of this study were to examine (i) the prevalence of multidrug-resistant P. aeruginosa, (ii) the mechanism and biofitness cost of multidrug resistance, and (iii) the susceptibilities of multidrug-resistant P. aeruginosa isolates to polymyxin antibiotics and a newer carbapenem (doripenem). These investigations are expected to provide a rationale for effective treatment strategies to combat nosocomial infections.(This study was presented in part at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy-Infectious Diseases Society of America 46th Annual Meeting, Washington, DC, 25 to 28 October 2008 [26].)     

Impact of Multidrug-Resistant Pseudomonas aeruginosa Bacteremia on Patient Outcomes

Trends of rising rates of resistance in Pseudomonas aeruginosa make selection of appropriate empirical therapy increasingly difficult, but whether multidrug-resistant (MDR) P. aeruginosa is associated with worse clinical outcomes is not well established. The objective of this study was to determine the impact of MDR (resistance to three or more classes of antipseudomonal agents) P. aeruginosa bacteremia on patient outcomes. We performed a retrospective cohort study of adult patients with P. aeruginosa bacteremia from 2005 to 2008. Patients were identified by the microbiology laboratory database, and pertinent clinical data were collected. Logistic regression was used to explore independent risk factors for 30-day mortality. Classification and regression tree analysis was used to determine threshold breakpoints for continuous variables. Kaplan-Meier survival analysis was used to compare time to mortality, after normalization of the patients'' underlying risks by propensity scoring. A total of 109 bacteremia episodes were identified; 25 episodes (22.9%) were caused by MDR P. aeruginosa. Patients with MDR P. aeruginosa bacteremia were more likely to receive inappropriate empirical therapy (44.0% and 6.0%, respectively; P < 0.001) and had longer prior hospital stays (32.6 ± 37.3 and 14.4 ± 43.6 days, respectively; P = 0.046). Multivariate regression revealed that 30-day mortality was associated with multidrug resistance (odds ratio [OR], 6.8; 95% confidence interval [CI], 1.9 to 24.0), immunosuppression (OR, 5.0; 95% CI, 1.4 to 17.5), and an APACHE II score of ≥22 (OR, 29.0; 95% CI, 5.0 to 168.2). Time to mortality was also shorter in the MDR cohort (P = 0.011). Multidrug resistance is a significant risk factor for 30-day mortality in patients with P. aeruginosa bacteremia; efforts to curb the spread of MDR P. aeruginosa could be beneficial.Bacterial bloodstream infections are serious medical events with life-threatening consequences and heavy impacts on health care costs through increased disease acuteness and lengths of hospital stay. Over the past decade, the numbers of bloodstream infections caused by Gram-negative bacteria have risen sharply. Among the various Gram-negative bacteria, Pseudomonas aeruginosa is known to be associated with resistance to practically all known antibiotics and is particularly problematic (3). In 2004, P. aeruginosa was placed seventh in prevalence among the causative pathogens in bloodstream infections but was second only to Candida species in bloodstream infection-related mortality. The crude mortality rate exceeded 38%, and the intensive care unit-related mortality was nearly 50% (20).Patients with P. aeruginosa bacteremia are often medically complicated. Up to 90% of these patients have preexisting severe underlying diseases or conditions, such as malignancy, diabetes, renal failure, heart failure, cirrhosis, or other immune system-impairing disease states, or have undergone a solid organ transplant (19). Recent studies have indicated that the initial site of infection, surgery, and pneumonia are important independent predictors of mortality in patients with P. aeruginosa bacteremia, while neutropenia, the presence of a central line, or the urinary tract as the initial site of infection were not highly associated with mortality (10, 19). The rapid course of the infection, combined with a rising tendency toward resistance to multiple agents typically used in empirical treatment, makes P. aeruginosa bacteremia particularly difficult to treat successfully (10).Paramount to the effective treatment of P. aeruginosa bacteremia is the obstacle of multidrug resistance. Recent studies demonstrated the importance of appropriate empirical antibiotic therapy in achieving favorable patient outcomes (10, 14). It can be reasoned that the probability of providing appropriate empirical therapy is lower with multidrug-resistant isolates. Furthermore, there may be only limited therapeutic options for multidrug-resistant isolates, and a less preferred (e.g., more toxic) agent(s) would have to be used as a last resort. It has been proposed that as bacteria gather mutations sequentially resulting in multidrug resistance, biofitness could be lost and the bacteria would become less virulent (11). However, there are also data to suggest that multidrug-resistant P. aeruginosa may remain fully pathogenic (7). The influence of multidrug resistance on the outcomes of patients with P. aeruginosa bloodstream infections remains controversial. The purpose of the present study was to examine the impact of multidrug resistance on outcomes in patients with P. aeruginosa bloodstream infections.     

Mathematical Modeling To Characterize the Inoculum Effect

Killing by beta-lactams is well known to be reduced against a dense bacterial population, commonly known as the inoculum effect. However, the underlying mechanism of this phenomenon is not well understood. We proposed a semimechanistic mathematical model to account for the reduced in vitro killing observed. Time-kill studies were performed with 4 baseline inocula (ranging from approximately 1 × 10⁵ to 1 × 10⁸ CFU/ml) of Escherichia coli ATCC 25922 (MIC, 2 mg/liter). Constant but escalating piperacillin concentrations used ranged from 0.25× to 256× MIC. Serial samples were taken over 24 h to quantify viable bacterial burden, and all the killing profiles were mathematically modeled. The inoculum effect was attributed to a reduction of effective drug concentration available for bacterial killing, which was expressed as a function of the baseline inoculum. Biomasses associated with different inocula were examined using a colorimetric method. Despite identical drug-pathogen combinations, the baseline inoculum had a significant impact on bacterial killing. Our proposed mathematical model was unbiased and reasonable in capturing all 28 killing profiles collectively (r² = 0.88). Biomass was found to be significantly more after 24 h with a baseline inoculum of 1 × 10⁸ CFU/ml, compared to one where the initial inoculum was 1 × 10⁵ CFU/ml (P = 0.002). Our results corroborated previous observations that in vitro killing by piperacillin was significantly reduced against a dense bacterial inoculum. This phenomenon can be reasonably captured by our proposed mathematical model, and it may improve prediction of bacterial response to various drug exposures in future investigations.Escherichia coli is part of the human gastrointestinal flora and a common pathogen implicated in intra-abdominal infections such as perforated appendicitis and peritonitis. Beta-lactams are often the empirical drug of choice for the management of severe intra-abdominal infections, in view of their spectrum of activity and safety profile. In intra-abdominal infections, a heavy bacterial burden is commonly encountered and the clinical utility of the beta-lactams may be limited by the inoculum effect. This phenomenon is believed to be due to the physiologic state of the bacterial cells, or preferential expression of different penicillin-binding proteins, rendering the bacteria less susceptible to beta-lactams (17). Biofilm production and quorum sensing may also be involved.We and others have previously developed mathematical models to capture the dynamic relationship between a heterogeneous microbial population and constant drug concentrations (10, 13, 15, 16, 21, 24). Our models were further refined to predict the microbial response to multiple antimicrobial agent dosing regimens (fluctuating drug concentration over time) efficiently (14, 22). Such a modeling approach could be used as a decision-support tool for dosing regimen design, and it may be used at different stages of drug development. It is often observed that killing could be more pronounced against a lower bacterial burden than with a higher bacterial burden (1, 3, 18). Capturing this phenomenon in a mathematical model would require a killing function dependent on bacterial burden. However, one common assumption in most modeling approaches is that the killing function is independent of the bacterial burden (4, 11, 19). This modeling assumption is often restrictive and is useful only when there is not a very significant change in bacterial susceptibility to antimicrobials, as the bacterial burden changes. In other cases, it may be misleading in making accurate predictions about the bacterial behaviors. For example, an experiment with a higher bacterial burden may suggest a dosing exposure requirement to suppress regrowth, but this may overestimate the requirement to suppress regrowth at lower bacterial burdens. In order to capture the overall picture satisfactorily, we need a more robust model the conclusions of which can be extended over a wide range of bacterial burdens.Therefore, the objective of this study was to extend our mathematical modeling approach proposed previously, to further account for the reduced in vitro killing observed. For illustrative purposes, a standard wild-type E. coli strain with various baseline inocula was used in this investigation.     

A Smartphone-Based Intervention for Anxiety and Depression in Racially and Ethnically Diverse Adults (EASE): Protocol for a Randomized Controlled Trial

BackgroundClear health disparities have emerged in the rates of COVID-19 exposure, hospitalization, and death among Black, Hispanic, and American Indian (BHAI) individuals, relative to non-Hispanic White (NHW) individuals. BHAI populations have been disproportionately affected by lower behavioral health access and heightened negative mental health outcomes during the pandemic.ObjectiveThis project directly addresses health disparities in access to behavioral health care during the COVID-19 pandemic among BHAI populations via an adaptation of the established, initially validated, low-cost, mobile app Easing Anxiety Sensitivity for Everyone (EASE) among individuals with symptoms of elevated anxiety or depression or both.MethodsThe EASE trial is a 2-arm, prospective, randomized, blinded-assessor study with intention-to-treat analysis. Participants (N=800; n=200, 25%, Black; n=200, 25%, Hispanic; n=200, 25%, American Indian; and n=200, 25%, NHW) are randomized to receive either EASE or an active comparison condition for anxiety and depression. Participants compete an online prescreener, an enrollment call to provide informed consent, a baseline survey, a 6-month intervention period, and 3- and 6-month postbaseline assessments. Select participants also complete a 3- and 6-month postbaseline qualitative interview via phone or an online platform (eg, Zoom). Participants complete 2 scheduled daily ecological momentary assessments (EMAs) during the 6-month study period. These twice-daily EMAs guide a just-in-time approach to immediate, personalized behavioral health care.ResultsOutcomes include reductions in anxiety and depressive symptoms and functional impairment at 3 and 6 months postrandomization. We also will examine putative mechanisms (eg, anxiety sensitivity [AS] and COVID-19–specific stress and fear) of the intervention effects. Further, as treatment effects may differ across sociocultural factors, perceived discrimination, social support, and socioeconomic status (SES) will be evaluated as potential moderators of treatment effects on the primary outcomes. Process evaluation using data collected during the study, as well as individual interviews with participants, will complement quantitative data.ConclusionsData from this efficacy trial will determine whether EASE successfully improves symptoms of anxiety and depression and whether these improvements outperform an active comparison control app. If successful, findings from this study have the potential to decrease anxiety and depression symptoms among vulnerable populations determined to be most at risk of exacerbated, long-lasting negative health sequelae. Data from this study may be used to support an implementation and dissemination trial of EASE within real-world behavioral health and social service settings.Trial RegistrationClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT05074693","term_id":"NCT05074693"}}NCT05074693; https://clinicaltrials.gov/ct2/show/{"type":"clinical-trial","attrs":{"text":"NCT05074693","term_id":"NCT05074693"}}NCT05074693International Registered Report Identifier (IRRID)DERR1-10.2196/40713     

Development of parvalbumin immunoreactive neurons in normal and intracranially transplanted retinas in the rat

Summary Retinas from embryonic day 14 Sprague-Dawley rats were transplanted to the midbrain or cerebral cortex of newborn (P0) rats of which the right eye was enucleated at the time of transplantation. Parvalbumin immunoreactive (PV-I) neurons were studied in the developing retinal transplants, and in the remaining retina of the host, as well as in normal retinas. PV-I neurons were identifiable in retinas of normal and host rats from postnatal day 5 (P5) onward, with the PV-I somata primarily in the inner half of the inner nuclear layer and in the ganglion cell layer. An adult-like distribution of PV-I neurons was attained at P35, as judged by cell packing density, intensity of immunostaining, laminar distribution and soma size of subpopulations of PV-I cells. A similar time course of development and distribution of PV-I somata was observed in the retinal transplants, except for some minor differences such as a slight delay in PV-I cells achieving their final distribution. These findings provide evidence that PV-I neurons can survive, differentiate and mature according to pre-determined programmes intrinsic to the retinal tissue following transplantation to a new and foreign environment.Visiting research fellow on leave from the Neurosciences Research Institute, Guangzhou Medical College, People's Republic of China     

Topographic distribution of callosal neurons and terminals in the cerebral cortex of the cat

Summary This investigation had four goals: First, to study the general topography of the corpus callosum (CC) of the cat. Second, to study the columnar organization of CC terminals and map their banding pattern in the cortex. Third, to examine the relation between CC neuron density and the presence of CC terminal columns. Fourth, to determine whether CC and anterior commissure (AC) neuron distributions are intermixed. Eight adult cats were subjected to partial commissurotomies, and then to large injections of horseradish peroxidase to one cerebral hemisphere. Processing with tetramethyl benzidine revealed retrogradely labelled cells and anterogradely labelled terminals in the cortex of the uninjected hemisphere. The distributions of these cells and terminals were examined by light microscopy and analyzed by computer microscopic methods. The genu of the CC interconnects frontal portions of the cortex, the body interconnects mostly dorsal portions of the cortex, while the splenium interconnects the temporal and occipital cortices. Reconstructions of the CC terminal columns reveal intricate banding patterns in several non-primary areas of the cortex. CC cell density is greater within than outside the terminal columns. CC and AC neurons intermix in the infragranular lavers of the neocortex.