Phenotypic changes associated with Colistin resistance due to Lipopolysaccharide loss in Acinetobacter baumannii.

Acinetobacter baumannii can acquire resistance to colistin via complete loss of lipopolysaccharide (LPS) biosynthesis due to mutations in the lpxA, lpxC and lpxD genes. However, although colistin is increasingly being used for the treatment of multidrug resistant infections, very few A. baumannii clinical isolates develop colistin resistance through loss of LPS biosynthesis. This may suggest that LPS loss affects virulence traits that play a role in the transmission and pathogenesis of A. baumannii. In this study we characterize multiple virulence phenotypes of colistin resistant, LPS-deficient derivatives of the ATCC 19606 strain and five multidrug resistant clinical isolates and their colistin resistant, LPS-deficient derivatives. Our results indicate that LPS loss results in growth defects compared to the parental strain in vitro both in laboratory media and human serum (competition indices of 0.58 and 7.0 × 10-7, respectively) and reduced ability to grow and disseminate in vivo (competition index 6.7 × 10-8). Infection with the LPS-deficient strain resulted in lower serum levels of pro-inflammatory cytokines TNF-α and IL-6 compared to the parent strain, and was less virulent in a mouse model of disseminated sepsis. LPS loss also significantly affected biofilm production, surface motility, growth under iron limitation and susceptibility to multiple disinfectants used in the clinical setting. These results demonstrate that LPS loss has a significant effect on multiple virulence traits, and may provide insight into the low incidence of colistin resistant strains lacking LPS that have been reported in the clinical setting.

Lipopolysaccharide loss produces partial colistin dependence and collateral sensitivity to azithromycin, rifampicin and vancomycin in Acinetobacter baumannii.

Treatment options for multidrug-resistant (MDR) strains of Acinetobacter baumannii that acquire resistance to colistin are limited. Acinetobacter baumannii can become highly resistant to colistin through complete loss of lipopolysaccharide (LPS) owing to mutations in the genes encoding the first three enzymes involved in lipid A biosynthesis (lpxA, lpxC and lpxD). The objective of this study was to characterise the susceptibility to 15 clinically relevant antibiotics and 6 antimicrobial peptides (AMPs) of MDR A. baumannii clinical isolates that acquired colistin resistance due to mutations in lpxA, lpxC and lpxD as well as their colistin-susceptible counterparts. A dramatic increase in antibiotic susceptibility (≥16-fold increase) was observed upon LPS loss for azithromycin, rifampicin and vancomycin, whereas a moderate increase in susceptibility was seen for amikacin, ceftazidime, imipenem, cefepime and meropenem. Importantly, concentrations ranging from 8 mg/L to 32 mg/L of the six AMPs were able to reduce bacterial viability by ≥3 log10 in growth curve assays. We also demonstrate that colistin resistance results in partial colistin dependence for growth in LPS-deficient strains containing mutations in lpxA, lpxC and lpxD, but not when colistin resistance occurs via LPS modification due to mutations in the PmrA/B two-component system. The results of this study indicate that loss of LPS expression results in collateral sensitivity to azithromycin, rifampicin and vancomycin, and that the six AMPs tested retain activity against LPS-deficient strains, indicating that these antibiotics may be viable treatment options for infections caused by these strains.

Characterization of protective extracellular membrane-derived vesicles produced by Streptococcus pneumoniae.

Extracellular vesicles are produced by many pathogenic microorganisms and have varied functions that include secretion and release of microbial factors, which contribute to virulence. Very little is known about vesicle production by Gram-positive bacteria, as well as their biogenesis and release mechanisms. In this work, we demonstrate the active production of vesicles by Streptococcus pneumoniae from the plasma membrane, rather than being a product from cell lysis. We biochemically characterized them by proteomics and fatty acid analysis, showing that these vesicles and the plasma membrane resemble in essential aspects, but have some differences: vesicles are more enriched in lipoproteins and short-chain fatty acids. We also demonstrate that these vesicles act as carriers of surface proteins and virulence factors. They are also highly immunoreactive against human sera and induce immune responses that protect against infection. Overall, this work provides insights into the biology of this important Gram-positive human pathogen and the role of extracellular vesicles in clinical applications. BIOLOGICAL SIGNIFICANCE: Pneumococcus is one of the leading causes of bacterial pneumonia worldwide in children and the elderly, being responsible for high morbidity and mortality rates in developing countries. The augment of pneumococcal disease in developed countries has raised major public health concern, since the difficulties to treat these infections due to increasing antibiotic resistance. Vaccination is still the best way to combat pneumococcal infections. One of the mechanisms that bacterial pathogens use to combat the defense responses of invaded hosts is the production and release of extracellular vesicles derived from the outer surface. Little is known about this phenomenon in Gram-positives. We show that pneumococcus produces membrane-derived vesicles particularly enriched in lipoproteins. We also show the utility of pneumococcal vesicles as a new type of vaccine, as they induce protection in immunized mice against infection with a virulent strain. This work will contribute to understand the role of these structures in important biological processes such as host-pathogen interactions and prevention of human disease.

Activity of host antimicrobials against multidrug-resistant Acinetobacter baumannii acquiring colistin resistance through loss of lipopolysaccharide.

Acinetobacter baumannii can acquire resistance to the cationic peptide antibiotic colistin through complete loss of lipopolysaccharide (LPS) expression. The activities of the host cationic antimicrobials LL-37 and human lysozyme against multidrug-resistant clinical isolates of A. baumannii that acquired colistin resistance through lipopolysaccharide loss were characterized. We demonstrate that LL-37 has activity against strains lacking lipopolysaccharide that is similar to that of their colistin-sensitive parent strains, whereas human lysozyme has increased activity against colistin-resistant strains lacking LPS.

Cellular HIV reservoir replenishment is not affected by blip or intermittent viremia episodes during darunavir/ritonavir monotherapy.

OBJECTIVE: To assess the impact of blips and persistent viremia episodes on cell-associated HIV-DNA reservoir in extensively pretreated patients receiving ritonavir-boosted darunavir monotherapy (MtDRV/rtv) for 24 months. DESIGN AND METHODS: Patients from the MonDAR prospective study (NCT01606722) who received at least 6 months of MtDRV/rtv and had at least two available peripheral blood mononuclear cells (PBMCs) samples were selected and classified according to the viral outcome as continuous undetectable viremia (cUV; n = 40), blips (n = 31), intermittent viremia (IV; n = 23), and virological failure (VF, two consecutive viral loads >200 copies/ml; n = 20). Proviral HIV-DNA was quantified by real-time PCR in PBMCs samples at baseline, and months 6, 12, 18 and 24. Additionally, HIV-DNA levels were exhaustively analyzed at virological failure and blip episodes. RESULTS: The HIV-DNA levels remained constant during the 24 months in every group. Neither blips nor intermittent viremia influenced the HIV-DNA levels at short-term or at middle term. By contrast, virological failure episodes gave rise to a significant increase in proviral DNA (2.15 vs. 2.32 log10 HIV-DNA copies/10 PBMCs; P = 0.042). Basal proviral DNA levels more than 2 log10 copies/10 PBMCs predicted the time to viral rebound at any given cut-off point (>20, >50, and >200 copies/ml. HR: 3.02, 2.61, and 3.02, respectively; P ≤ 0.03. Besides, an adherence less than 95% was also strongly associated with virological failure (HR, 3.17; P = 0.021). CONCLUSION: Blip episodes and intermittent viremia did not affect the cellular HIV reservoir dynamic during MtDRV/rtv. Higher adherence and an HIV-DNA levels less than 2 log10 copies/10 PBMCs at baseline were associated with a lower risk of virological failure.

Progress on the development of rapid methods for antimicrobial susceptibility testing.

Antimicrobial susceptibility testing is essential for guiding the treatment of many types of bacterial infections, especially in the current context of rising rates of antibiotic resistance. The most commonly employed methods rely on the detection of phenotypic resistance by measuring bacterial growth in the presence of the antibiotic being tested. Although these methods are highly sensitive for the detection of resistance, they require that the bacterial pathogen is isolated from the clinical sample before testing and must employ incubation times that are sufficient for differentiating resistant from susceptible isolates. Knowledge regarding the molecular determinants of antibiotic resistance has facilitated the development of novel approaches for the rapid detection of resistance in bacterial pathogens. PCR-based techniques, mass spectrometry, microarrays, microfluidics, cell lysis-based approaches and whole-genome sequencing have all demonstrated the ability to detect resistance in various bacterial species. However, it remains to be determined whether these methods can achieve sufficient sensitivity and specificity compared with standard phenotypic resistance testing to justify their use in routine clinical practice. In the present review, we discuss recent progress in the development of methods for rapid antimicrobial susceptibility testing and highlight the limitations of each approach that still remain be addressed.

Rapid detection of antibiotic resistance in Acinetobacter baumannii using quantitative real-time PCR.

OBJECTIVES: The rapid detection of antibiotic resistance in clinical isolates of Acinetobacter baumannii would shorten the period during which patients receive empirical therapy and facilitate the early initiation of directed antibiotic therapy. The objective of this study was to evaluate the ability of a real-time PCR assay to detect antibiotic resistance to four clinically relevant antibiotics from different antibiotic classes in clinical isolates of A. baumannii. METHODS: The growth of 48 clinical isolates of A. baumannii with a broad range of MICs of imipenem, ciprofloxacin, colistin and amikacin was evaluated using a real-time PCR assay targeting a highly conserved region of the ompA gene. Fold changes in the number of copies of genomic DNA after 6 h of growth were used to determine resistance and the results were compared with those obtained using broth microdilution. RESULTS: The results obtained using the real-time PCR assay were concordant with broth microdilution for 184 of 192 determinations (95.8%). The global values for specificity (97.5%), sensitivity (92.9%), positive predictive value (95.6%) and negative predictive value (96.0%) indicated that the real-time PCR assay was able to reliably differentiate between resistant and non-resistant strains. CONCLUSIONS: The use of real-time PCR to monitor bacterial growth in the presence of antibiotics is effective for rapidly identifying antibiotic resistance in A. baumannii.

Intracellular and plasma pharmacokinetics of 400 mg of etravirine once daily versus 200 mg of etravirine twice daily in HIV-infected patients.

OBJECTIVES: To compare intracellular and plasma etravirine concentrations when etravirine was given at 200 mg/12 h versus 400 mg/24 h and to evaluate whether the results would support once-daily dosing. METHODS: This was an open-label sequential study in which eight patients on protease inhibitor (PI)-sparing regimens containing etravirine were included. Full pharmacokinetic profiles were performed while on 200 mg of etravirine/12 h and after switching to 400 mg of etravirine/24 h. Intracellular and plasma levels were determined by liquid chromatography coupled with mass spectrometry. Pharmacokinetic parameters were calculated by non-compartmental analysis and compared by geometric mean ratios (GMRs) using 200 mg of etravirine/12 h as the reference group. TRIAL REGISTRATION: ClinicalTrials.gov NCT01121809. RESULTS: The geometric mean (GM) for etravirine AUC(0-τ) (5602 versus 5076 ng · h/mL, GMR 0.91), C(max) (403 versus 495 ng/mL, GMR 1.23) and C(min) (139 versus 102 ng/mL, GMR 0.74) were similar with both dosing schedules at the intracellular level. In plasma, the GMRs for AUC(0-τ), C(max) and C(min) were 1.31, 1.76 and 0.99, respectively. The mean intracellular penetration, evaluated as intracellular and plasma AUC(0-τ) ratios, was 81% when etravirine was dosed twice daily and 56% with once-daily dosing. CONCLUSIONS: Our results show that intracellular etravirine levels were similar with both dosing regimens in patients with PI-sparing regimens, while etravirine plasma AUC(0-τ) and C(max) were 30% and 76% higher with the once-daily regimen, respectively. Thus, a once-daily dosing regimen is supported not only by plasma etravirine pharmacokinetic profiles but also by intracellular levels.