Relebactam restores susceptibility of resistant Pseudomonas aeruginosa and Enterobacterales and enhances imipenem activity against chromosomal AmpC-producing species: analysis of global SMART 2018-2020.

BACKGROUND: Carbapenem-resistant bacteria are an increasing problem in clinical practice; thus, it is important to identify ß-lactamase inhibitors (e.g., relebactam) that can restore carbapenem susceptibility. We report analyses of relebactam enhancement of imipenem activity against both imipenem-nonsusceptible (NS) and imipenem-susceptible (S) Pseudomonas aeruginosa and Enterobacterales. Gram-negative bacterial isolates were collected for the ongoing Study for Monitoring Antimicrobial Resistance Trends global surveillance program. Clinical and Laboratory Standards Institute-defined broth microdilution minimum inhibitory concentrations (MIC) were used to determine the imipenem and imipenem/relebactam antibacterial susceptibilities of P. aeruginosa and Enterobacterales isolates. RESULTS: Between 2018 and 2020, 36.2% of P. aeruginosa (N = 23,073) and 8.2% of Enterobacterales (N = 91,769) isolates were imipenem-NS. Relebactam restored imipenem susceptibility in 64.1% and 49.4% of imipenem-NS P. aeruginosa and Enterobacterales isolates, respectively. Restoration of susceptibility was largely observed among K. pneumoniae carbapenemase-producing Enterobacterales and carbapenemase-negative P. aeruginosa. Relebactam also caused a lowering of imipenem MIC among imipenem-S P. aeruginosa and Enterobacterales isolates from chromosomal Ambler class C ß-lactamase (AmpC)-producing species. For both imipenem-NS and imipenem-S P. aeruginosa isolates, relebactam reduced the imipenem MIC mode from 16 µg/mL to 1 µg/mL and from 2 µg/mL to 0.5 µg/mL, respectively, compared with imipenem alone. CONCLUSIONS: Relebactam restored imipenem susceptibility among nonsusceptible isolates of P. aeruginosa and Enterobacterales and enhanced imipenem susceptibility among susceptible isolates of P. aeruginosa and isolates from Enterobacterales species that can produce chromosomal AmpC. The reduced imipenem modal MIC values with relebactam may result in a higher probability of target attainment in patients.

In Vitro Activity of Ceftolozane-Tazobactam, Imipenem-Relebactam, Ceftazidime-Avibactam, and Comparators against Pseudomonas aeruginosa Isolates Collected in United States Hospitals According to Results from the SMART Surveillance Program, 2018 to 2020.

Ceftolozane-tazobactam (C/T), imipenem-relebactam (IMR), and ceftazidime-avibactam (CZA) were tested against 2,531 P. aeruginosa strains isolated from patients in the United States from 2018 to 2020 as part of the SMART (Study for Monitoring Antimicrobial Resistance Trends) surveillance program. MICs were determined by CLSI broth microdilution and interpreted using CLSI M100 (2021) breakpoints. Imipenem-, IMR-, or C/T-nonsusceptible isolates were screened for ß-lactamase genes: 96.4% of all isolates and ≥70% of multidrug-resistant (MDR), pan-ß-lactam-nonsusceptible, and difficult-to-treat resistance (DTR) isolates were C/T-susceptible; 52.2% of C/T-nonsusceptible isolates remained susceptible to IMR compared to 38.9% for CZA; and 1.7% of isolates tested were nonsusceptible to both C/T and IMR versus 2.2% of isolates with a C/T-nonsusceptible and CZA-resistant phenotype (a difference of 12 isolates). C/T and IMR modal MICs for pan-ß-lactam-nonsusceptible isolates remained at or below their respective susceptible MIC breakpoints from 2018 to 2020, while the modal MIC for CZA increased 2-fold from 2018 to 2019 and exceeded the CZA-susceptible MIC breakpoint in both 2019 and 2020. Only six of 802 molecularly characterized isolates carried a metallo-ß-lactamase, and two isolates carried a GES carbapenemase. Most P. aeruginosa isolates were C/T-susceptible, including many with MDR, pan-ß-lactam-nonsusceptible, DTR, CZA-resistant, and IMR-nonsusceptible phenotypes. While C/T was the most active antipseudomonal agent, IMR demonstrated greater activity than CZA against isolates nonsusceptible to C/T.

Bacterial Vaginosis-Associated Bacteria and Uterine Fibroids: A Nested Case-Control Study.

BACKGROUND: Reproductive tract infections are hypothesized to influence uterine fibroid development, yet few studies have investigated the common condition of bacterial vaginosis (BV). The literature is currently limited to data using self-report of BV. METHODS: We conducted a nested case-control study of 200 women (100 cases and 100 controls) from a large study of 23- to 35-year-old African American women, 1310 of whom were fibroid-free and prospectively followed up for 5 years to identify incident fibroids with standardized ultrasound examinations. We used quantitative polymerase chain reaction, an objective molecular method, to assess 9 BV-associated and 4 Lactobacillus species from vaginal swab specimens. We used hierarchical logistic regression to compute odds ratios and 95% confidence intervals to examine associations between bacterial species (both individually and grouped as (1) "optimal" Lactobacillus and (2) BV-associated species) with fibroid incidence and number. We also examined vaginal imbalance (quantitatively more BV-associated bacteria than optimal Lactobacilli). RESULTS: Contrary to our hypothesis, we found no increase in fibroid incidence or number among women with more BV-associated bacteria. High imbalance (only BV-associated bacteria, no optimal Lactobacillus bacteria) was actually inversely associated with fibroid incidence (odds ratio, 0.38; 95% confidence interval, 0.17-0.81). CONCLUSIONS: This is the first study of ultrasound-detected incident fibroids and molecular vaginal bacterial assessment. We found no evidence that BV-associated bacteria increase the risk of fibroid incidence or number.

Mycoplasma genitalium and Bacterial Vaginosis-Associated Bacteria in a Non-Clinic-Based Sample of African American Women.

BACKGROUND: Mycoplasma genitalium is associated with adverse reproductive problems. However, prevalence estimates from studies that screen women not seeking care are rare. Studies have reported co-occurrence of M. genitalium with bacterial vaginosis (BV), but no prior study of specific BV-associated bacteria has been conducted in African Americans whose reproductive tract infection burden is high. METHODS: Using quantitative polymerase chain reaction, we screened vaginal swabs for M. genitalium, 9 BV-associated bacteria, and 4 Lactobacillus species from 200 participants drawn from a cohort of African Americans 23 to 35 years old. Sexual history, herpes serostatus, and Nugent score had been assessed. Prevalence of M. genitalium was computed. The associations of other vaginal bacteria with M. genitalium were examined with binomial regression. RESULTS: M. genitalium prevalence was 18%. Detection and quantity of 2 BV-associated bacteria were significantly associated with a higher prevalence of M. genitalium (Leptotrichia/Sneathia: detection prevalence ratio (PR) of 2.9 [95% confidence interval {CI}, 1.1-7.7] and quantity PR of 1.2 [95% CI, 1.0-1.3]; Megasphaera phylotype 1: detection PR of 2.2 [95% CI, 1.2-4.2] and quantity PR of 1.1 [95% CI, 1.0-1.2]). Increased quantity of L. iners was also positively associated with M. genitalium (PR, 1.3 [95% CI, 1.0-1.8]). Nugent ≥7, herpes serostatus, and lifetime number of sex partners were not associated with M. genitalium. CONCLUSIONS: Specific BV-associated microbes and L. iners were associated with M. genitalium, but Nugent ≥7 was not. Studies are needed to confirm a high prevalence of M. genitalium in African Americans and to understand its interactions with other vaginal bacteria.

Treatment of Clostridium difficile Infection with a Small-Molecule Inhibitor of Toxin UDP-Glucose Hydrolysis Activity.

Clostridium difficile infection (CDI) is the leading cause of hospital-acquired infectious diarrhea, with significant morbidity, mortality, and associated health care costs. The major risk factor for CDI is antimicrobial therapy, which disrupts the normal gut microbiota and allows C. difficile to flourish. Treatment of CDI with antimicrobials is generally effective in the short term, but recurrent infections are frequent and problematic, indicating that improved treatment options are necessary. Symptoms of disease are largely due to two homologous toxins, TcdA and TcdB, which are glucosyltransferases that inhibit host Rho GTPases. As the normal gut microbiota is an important component of resistance to CDI, our goal was to develop an effective nonantimicrobial therapy. Here, we report a highly potent small-molecule inhibitor (VB-82252) of TcdA and TcdB. This compound inhibits the UDP-glucose hydrolysis activity of TcdB and protects cells from intoxication after challenge with either toxin. Oral dosing of the inhibitor prevented inflammation in a murine intrarectal toxin challenge model. In a murine model of recurrent CDI, the inhibitor reduced weight loss and gut inflammation during acute disease and did not cause the recurrent disease that was observed with vancomycin treatment. Lastly, the inhibitor demonstrated efficacy similar to that of vancomycin in a hamster disease model. Overall, these results demonstrate that small-molecule inhibition of C. difficile toxin UDP-glucose hydrolysis activity is a promising nonantimicrobial approach to the treatment of CDI.

Trichomonas vaginalis is most frequently detected in women at the age of peri-/premenopause: an unusual pattern for a sexually transmitted pathogen.

BACKGROUND: Trichomonas vaginalis is the most common nonviral sexually transmitted infection. However, because it is not a reportable disease in the United States, there is limited information on the age of infected individuals and their geographic distribution. OBJECTIVE: The purpose of this study was to evaluate the detection rates of T vaginalis infection compared with Chlamydia trachomatis by age and state in a commercial laboratory setting. STUDY DESIGN: Quantitative real-time polymerase chain reactions were used to detect the presence of T vaginalis and C trachomatis in cervicovaginal samples that were obtained during gynecologic examinations. A total of 1,554,966 and 1,999,077 samples from females 10-79 years old were analyzed retrospectively for the presence of T vaginalis and C trachomatis, respectively. RESULTS: The highest detection rate of an infection with T vaginalis was ages 47-53 years. For C trachomatis, the highest detection rate was ages 14-20 years. T vaginalis detection rate distribution by age shows a bimodal pattern with first peak at ages 21-22 years (4.0-4.1%) and a higher second peak at ages 48-51 years (5.4-5.8%). C trachomatis prevalence distribution by age shows a maximum peak of 8.6% at age 17 years and a rapid decline thereafter. In general, the detection rates of both pathogens were higher in the southeast and in states along the Mississippi River Valley than in other parts of the country. A nucleotide polymorphism associated with T vaginalis metronidazole resistance (ntr6TVK80STOP) was not associated with age and was found most frequently in specimens from New Mexico and Vermont. CONCLUSIONS: The detection rate of T vaginalis does not appear to decrease with age as observed for C trachomatis and reaches maximum rates in women 48-51 years old. The geographic distribution of T vaginalis appears to be broadly similar to that of other sexually transmitted diseases. The ntr6TVK80STOP polymorphism did not have a specific association with age or geography.

Synthesis and SAR studies of novel benzodiazepinedione-based inhibitors of Clostridium difficile (C. difficile) toxin B (TcdB).

Synthesis and structure-activity relationships (SAR) of a novel series of benzodiazepinedione-based inhibitors of Clostridium difficile toxin B (TcdB) are described. Compounds demonstrating low nanomolar affinity for TcdB, and which possess improved stability in mouse plasma vs. earlier compounds from this series, have been identified. Optimized compound 11d demonstrates a good pharmacokinetic (PK) profile in mouse and hamster and is efficacious in a hamster survival model of Clostridium difficile infection.

Identification and initial optimization of inhibitors of Clostridium difficile (C. difficile) toxin B (TcdB).

The discovery, synthesis and preliminary structure-activity relationship (SAR) of a novel class of inhibitors of Clostridium difficile (C. difficile) toxin B (TcdB) is described. A high throughput screening (HTS) campaign resulted in the identification of moderately active screening hits 1-5 the most potent of which was compound 1 (IC50 = 0.77 µM). In silico docking of an early analog offered suggestions for structural modification which resulted in the design and synthesis of highly potent analogs 13j(IC50 = 1 nM) and 13 l(IC50 = 7 nM) which were chosen as leads for further optimization.

Development and Validation of a Highly Accurate Quantitative Real-Time PCR Assay for Diagnosis of Bacterial Vaginosis.

Bacterial vaginosis (BV) is the most common gynecological infection in the United States. Diagnosis based on Amsel's criteria can be challenging and can be aided by laboratory-based testing. A standard method for diagnosis in research studies is enumeration of bacterial morphotypes of a Gram-stained vaginal smear (i.e., Nugent scoring). However, this technique is subjective, requires specialized training, and is not widely available. Therefore, a highly accurate molecular assay for the diagnosis of BV would be of great utility. We analyzed 385 vaginal specimens collected prospectively from subjects who were evaluated for BV by clinical signs and Nugent scoring. We analyzed quantitative real-time PCR (qPCR) assays on DNA extracted from these specimens to quantify nine organisms associated with vaginal health or disease:Gardnerella vaginalis,Atopobium vaginae, BV-associated bacteria 2 (BVAB2, an uncultured member of the orderClostridiales),Megasphaeraphylotype 1 or 2,Lactobacillus iners,Lactobacillus crispatus,Lactobacillus gasseri, andLactobacillus jensenii We generated a logistic regression model that identifiedG. vaginalis,A. vaginae, andMegasphaeraphylotypes 1 and 2 as the organisms for which quantification provided the most accurate diagnosis of symptomatic BV, as defined by Amsel's criteria and Nugent scoring, with 92% sensitivity, 95% specificity, 94% positive predictive value, and 94% negative predictive value. The inclusion ofLactobacillusspp. did not contribute sufficiently to the quantitative model for symptomatic BV detection. This molecular assay is a highly accurate laboratory tool to assist in the diagnosis of symptomatic BV.

An ecoclimatic framework for evaluating the resilience of vegetation to water deficit.

The surge in global efforts to understand the causes and consequences of drought on forest ecosystems has tended to focus on specific impacts such as mortality. We propose an ecoclimatic framework that takes a broader view of the ecological relevance of water deficits, linking elements of exposure and resilience to cumulative impacts on a range of ecosystem processes. This ecoclimatic framework is underpinned by two hypotheses: (i) exposure to water deficit can be represented probabilistically and used to estimate exposure thresholds across different vegetation types or ecosystems; and (ii) the cumulative impact of a series of water deficit events is defined by attributes governing the resistance and recovery of the affected processes. We present case studies comprising Pinus edulis and Eucalyptus globulus, tree species with contrasting ecological strategies, which demonstrate how links between exposure and resilience can be examined within our proposed framework. These examples reveal how climatic thresholds can be defined along a continuum of vegetation functional responses to water deficit regimes. The strength of this framework lies in identifying climatic thresholds on vegetation function in the absence of more complete mechanistic understanding, thereby guiding the formulation, application and benchmarking of more detailed modelling.

Trichomonas vaginalis metronidazole resistance is associated with single nucleotide polymorphisms in the nitroreductase genes ntr4Tv and ntr6Tv.

Metronidazole resistance in the sexually transmitted parasite Trichomonas vaginalis is a problematic public health issue. We have identified single nucleotide polymorphisms (SNPs) in two nitroreductase genes (ntr4Tv and ntr6Tv) associated with resistance. These SNPs were associated with one of two distinct T. vaginalis populations identified by multilocus sequence typing, yet one SNP (ntr6Tv A238T), which results in a premature stop codon, was associated with resistance independent of population structure and may be of diagnostic value.

Cervical and vaginal flora specimens are highly concordant with respect to bacterial vaginosis-associated organisms and commensal Lactobacillus species in women of reproductive age.

Matched vaginal and cervical specimens from 96 subjects were analyzed by quantitative PCR for the presence and concentration of bacterial vaginosis-associated microbes and commensal Lactobacillus spp. Detection of these microbes was 92% concordant, indicating that microbial floras at these body sites are generally similar.

Detecting DNA methylation of the BCL2, CDKN2A and NID2 genes in urine using a nested methylation specific polymerase chain reaction assay to predict bladder cancer.

PURPOSE: Detection of methylated DNA has been shown to be a good biomarker for bladder cancer. Bladder cancer has the highest recurrence rate of any cancer and, as such, patients are regularly monitored using invasive diagnostic techniques. As urine is easily attainable, bladder cancer is an optimal cancer to detect using DNA methylation. DNA methylation is highly specific in cancer detection. However, it is difficult to detect because of the limited amount of DNA present in the urine of patients with bladder cancer. Therefore, an improved, sensitive and noninvasive diagnostic test is needed. MATERIALS AND METHODS: We developed a highly specific and sensitive nested methylation specific polymerase chain reaction assay to detect the presence of bladder cancer in small volumes of patient urine. The genes assayed for DNA methylation are BCL2, CDKN2A and NID2. The regions surrounding the DNA methylation sites were amplified in a methylation independent first round polymerase chain reaction and the amplification product from the first polymerase chain reaction was used in a real-time methylation specific polymerase chain reaction. Urine samples were collected from patients receiving treatment at Wolfson Medical Center in Holon, Israel. RESULTS: In a pilot clinical study using patient urine samples we were able to differentiate bladder cancer from other urogenital malignancies and nonmalignant conditions with a sensitivity of 80.9% and a specificity of 86.4%. CONCLUSIONS: We developed a novel methylation specific polymerase chain reaction assay for the detection and monitoring of bladder cancer using DNA extracted from patient urine. The assay may also be combined with other diagnostic tests to improve accuracy.

Population pharmacokinetic/pharmacodynamic assessment of imipenem/cilastatin/relebactam in patients with hospital-acquired/ventilator-associated bacterial pneumonia.

In the phase III RESTORE-IMI 2 study (ClinicalTrials.gov: NCT02493764), the combination antibacterial agent imipenem/cilastatin/relebactam (IMI/REL) demonstrated noninferiority to piperacillin/tazobactam for the end points of all-cause mortality at day 28 and favorable clinical response at the early follow-up visit in adult participants with gram-negative hospital-acquired bacterial pneumonia/ventilator-associated bacterial pneumonia (HABP/VABP). Existing population pharmacokinetic models for imipenem (IPM) and REL were updated using data from patients with HABP/VABP from RESTORE-IMI 2. Creatinine clearance (CrCl), body weight, infection type, and ventilation status were significant covariates in the updated model. The following simulations were performed to calculate the pharmacokinetic/pharmacodynamic joint probability of target attainment among patients with HABP/VABP and varying degrees of renal function: augmented renal clearance (CrCl ≥150 ml/min), normal renal function (CrCl ≥90 to <150 ml/min), renal impairment (mild, CrCl ≥60 to <90 ml/min; moderate, CrCl ≥30 to <60 ml/min; or severe, CrCl ≥15 to <30 ml/min), and end-stage renal disease (CrCl <15 ml/min). At the recommended IMI/REL dosing regimens across renal categories, greater than 90% of patients in all renal function groups were predicted to achieve joint pharmacokinetic/pharmacodynamic targets at a minimum inhibitory concentration breakpoint of ≤2 µg/ml, regardless of ventilation status. This modeling and simulation analysis supports use of the recommended IMI/REL dosing regimens, adjusted based on renal function, in patients with HABP/VABP.

Compartmentalization of gene expression during Bacillus subtilis spore formation.

Gene expression in members of the family Bacillaceae becomes compartmentalized after the distinctive, asymmetrically located sporulation division. It involves complete compartmentalization of the activities of sporulation-specific sigma factors, sigma(F) in the prespore and then sigma(E) in the mother cell, and then later, following engulfment, sigma(G) in the prespore and then sigma(K) in the mother cell. The coupling of the activation of sigma(F) to septation and sigma(G) to engulfment is clear; the mechanisms are not. The sigma factors provide the bare framework of compartment-specific gene expression. Within each sigma regulon are several temporal classes of genes, and for key regulators, timing is critical. There are also complex intercompartmental regulatory signals. The determinants for sigma(F) regulation are assembled before septation, but activation follows septation. Reversal of the anti-sigma(F) activity of SpoIIAB is critical. Only the origin-proximal 30% of a chromosome is present in the prespore when first formed; it takes approximately 15 min for the rest to be transferred. This transient genetic asymmetry is important for prespore-specific sigma(F) activation. Activation of sigma(E) requires sigma(F) activity and occurs by cleavage of a prosequence. It must occur rapidly to prevent the formation of a second septum. sigma(G) is formed only in the prespore. SpoIIAB can block sigma(G) activity, but SpoIIAB control does not explain why sigma(G) is activated only after engulfment. There is mother cell-specific excision of an insertion element in sigK and sigma(E)-directed transcription of sigK, which encodes pro-sigma(K). Activation requires removal of the prosequence following a sigma(G)-directed signal from the prespore.

Uropathogenic Escherichia coli dominantly suppress the innate immune response of bladder epithelial cells by a lipopolysaccharide- and Toll-like receptor 4-independent pathway.

Urinary tract infections are a major source of morbidity among women, with the majority caused by uropathogenic Escherichia coli. Our objective was to test if uropathogenic E. coli suppress the innate immune response of bladder epithelial cells. We found that bladder epithelial cells secrete interleukin-6 and interleukin-8 in response to non-pathogenic E. coli, whereas they failed to do so in response to uropathogenic E. coli. Uropathogenic E. coli prevented interleukin-6 secretion in response to non-pathogenic E. coli and a panel of Toll-like receptor agonists, as well as to interleukin-1beta, but not to tumor necrosis factor alpha. These results indicate that receptors with a Toll/interleukin-1 receptor domain are specifically targeted, and that suppression is not a consequence of toxicity. One candidate for mediating immune suppression is bacterial lipopolysaccharide. However, lipopolysaccharide isolated from either uropathogenic or non-pathogenic E. coli stimulated interleukin-6 secretion to similar levels. In addition, uropathogenic E. coli did not stimulate interleukin-6 secretion from cells expressing a dominant negative Toll-like receptor 4, and prevented cells lacking Toll-like receptor 4 from secreting interleukin-6 in response to synthetic lipoprotein. We conclude that uropathogenic E. coli suppress the innate immune response through a pathway partially independent of lipopolysaccharide and Toll-like receptor 4.

Long-term dynamics of a fragmented rainforest mammal assemblage.

Habitat fragmentation is a severe threat to tropical biotas, but its long-term effects are poorly understood. We evaluated longer-term changes in the abundance of larger (>1 kg) mammals in fragmented and intact rainforest and in riparian "corridors" in tropical Queensland, with data from 190 spotlighting surveys conducted in 1986-1987 and 2006-2007. In 1986-1987 when most fragments were already 20-50 years old, mammal assemblages differed markedly between fragmented and intact forest. Most vulnerable were lemuroid ringtail possums (Hemibelideus lemuroides), followed by Lumholtz's tree-kangaroos (Dendrolagus lumholtzi) and Herbert River ringtail possums (Pseudocheirus herbertensis). Further changes were evident 20 years later. Mammal species richness fell significantly in fragments, and the abundances of 4 species, coppery brushtail possums (Trichosurus vulpecula johnstoni), green ringtail possums (Pseudochirops archeri), red-legged pademelons (Thylogale stigmatica), and tree-kangaroos, declined significantly. The most surprising finding was that the lemuroid ringtail, a strict rainforest specialist, apparently recolonized one fragment, despite a 99.98% decrease in abundance in fragments and corridors. A combination of factors, including long-term fragmentation effects, shifts in the surrounding matrix vegetation, and recurring cyclone disturbances, appear to underlie these dynamic changes in mammal assemblages.

Sporulation of Bacillus subtilis.

Differentiation of vegetative Bacillus subtilis into heat resistant spores is initiated by the activation of the key transcription regulator Spo0A through the phosphorelay. Subsequent events depend on the cell compartment-specific action of a series of RNA polymerase sigma factors. Analysis of genes in the Spo0A regulon has helped delineate the mechanisms of axial chromatin formation and asymmetric division. There have been considerable advances in our understanding of critical controls that act to regulate the phosphorelay and to activate the sigma factors.

Identification of intrinsically metronidazole-resistant clades of Gardnerella vaginalis.

Gardnerella vaginalis is associated with bacterial vaginosis (BV), the most common cause of vaginal discharge. Metronidazole is a front-line therapy for BV, and treatment failure and recurrent disease are common problems. Whole-genome sequencing studies have revealed that G. vaginalis has a population structure that consists of 4 clades: clades 1 and 3 are associated with BV, whereas clades 2 and 4 are not. To determine if metronidazole susceptibility is associated with population structure, we analyzed 87 clinical isolates and found that metronidazole resistance (MIC ≥32 µg/mL) was highly associated with clade (P<0.0001), as 14/14 clade 3 isolates (100%) and 22/22 clade 4 isolates (100%) exhibited resistance, compared to only 16/37 clade 1 isolates (35%) and 1/14 clade 2 isolates (7.1%). The identification of intrinsically metronidazole-resistant G. vaginalis clades will facilitate future studies on the relationship between metronidazole resistance and BV treatment failure.