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communications biology
Curbing gastrointestinal infections by defensin fragment modifications without harming commensal microbiota
Louis Koeninger ORCID: orcid.org/0000-0001-9868-06701,
Lisa Osbelt2,3,
Anne Berscheid ORCID: orcid.org/0000-0003-1585-87154,5,
Judith Wendler1,
Jürgen Berger6,
Katharina Hipp ORCID: orcid.org/0000-0002-1493-96986,
Till R. Lesker2,
Marina C. Pils7,
Nisar P. Malek1,
Benjamin A. H. Jensen8,
Heike Brötz-Oesterhelt ORCID: orcid.org/0000-0001-9364-18324,5,9,
Till Strowig ORCID: orcid.org/0000-0003-0185-14592,10 na1 &
Jan Wehkamp1,9 na1
Communications Biology volume 4, Article number: 47 (2021) Cite this article
The occurrence and spread of multidrug-resistant pathogens, especially bacteria from the ESKAPE panel, increases the risk to succumb to untreatable infections. We developed a novel antimicrobial peptide, Pam-3, with antibacterial and antibiofilm properties to counter this threat. The peptide is based on an eight-amino acid carboxyl-terminal fragment of human β-defensin 1. Pam-3 exhibited prominent antimicrobial activity against multidrug-resistant ESKAPE pathogens and additionally eradicated already established biofilms in vitro, primarily by disrupting membrane integrity of its target cell. Importantly, prolonged exposure did not result in drug-resistance to Pam-3. In mouse models, Pam-3 selectively reduced acute intestinal Salmonella and established Citrobacter infections, without compromising the core microbiota, hence displaying an added benefit to traditional broad-spectrum antibiotics. In conclusion, our data support the development of defensin-derived antimicrobial agents as a novel approach to fight multidrug-resistant bacteria, where Pam-3 appears as a particularly promising microbiota-preserving candidate.
The spread of antibiotic-resistant bacteria is an urgent public health threat. Specifically, the so-called ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens account for the majority of nosocomial infections worldwide being attributed to ≥700,000 deaths anually1. The World Health Organization (WHO) has recently published a list of 12 bacteria against which new antibiotics are urgently needed, including the ESKAPE pathogens2. While traditional antibiotics fight pathogens, they also have wide-ranging consequences for the commensal gut microbiota3. Administration disrupts the microbial composition and can result in a long-lasting dysbiosis, which is associated with mounting diseases4. Decreased diversity and -taxonomic richness, the spread of antimicrobial resistance as well as increased colonization of opportunistic pathogens, including secondary infections with Clostridioides difficile, are just a few of the many side-effects traditional antibiotics impose5,6. The current antimicrobial crisis is a product of the long-term neglected development of new antibiotics by pharmaceutical companies and governments7. Thus, new strategies more resilient to multidrug resistance are urgently warranted8.
Antimicrobial peptides (AMPs) are small, cationic peptides existing in all multicellular organisms and exhibit a broad range of antimicrobial and immunological properties9. They are considered a promising treatment option and have the potential to be a new generation of antimicrobials against multidrug-resistant bacteria. Recently, de Breij et al. demonstrated the potential of novel antimicrobial peptides by developing SAAP-148, which showed promising effects against biofilm-associated skin infections in ex vivo human skin and murine skin in vivo10. First preclinical trials with SAAP-148 against methicillin-resistant S. aureus infections have already been conducted according to AdisInsight—a database for drug development11.
Defensins, the most prominent class of AMPs in humans, are key effector molecules of innate immunity. These peptides protect the host from infectious microbes and shape the composition of microbiota at mucosal surfaces12,13,14,15. To this end, the first identified human β-defensin, human β-defensin 1 (hBD-1), is constitutively expressed in surface epithelia by monocytes, plasmacyoid dendritic cells, and platelets16,17,18. Previously, the antimicrobial activity of hBD-1 was underestimated until it was analyzed under reduced conditions as found in the human intestine. Reduced hBD-1 has an increased antimicrobial activity, but can be degraded by intestinal proteases19,20. We have recently shown that this creates an eight-amino acid carboxyl-terminal fragment (called octapeptide) with retained antimicrobial activity, albeit low in vivo stabilty21.
Here, we leveraged those findings by developing novel synthetic peptides with improved antimicrobial activity and enhanced in vivo stability. We modified the hBD-1-derived octapeptide with palmitic acid and various spacers, such as sugars or amino acids, to create lipopeptides (Pams) with increased stability and bactericidal activity22,23. The most promising peptide was tested against multidrug-resistant pathogens and biofilms followed by exploratory safety assessment. Lastly, we determined its influence on the murine microbiota after oral application as well as its efficacy in murine gastrointestinal infection models.
Design and screening of octapeptide based lipopeptides identifies candidate with improved antimicrobial activity
Lipopeptides are used as antibiotics which are highly active against multidrug-resistant bacteria and fungi24,25. Previous studies have demonstrated an enhanced activity of HDPs after fatty acid modification at the N-terminal end26. Thereby, C14–C18 long chain fatty acids have proven to be ideal for this purpose27,28. Within this work, we used a C16 long fatty acid, namely palmitic acid together with different spacers such as sugars or amino acids to improve stability and bactericidal activity of the carboxyl-terminal fragment of hBD-1. We designed 5 unique lipopeptides, Pam-1 to Pam-5, based on the recently discovered octapeptide (Fig. 1a). This set of peptides was screened for their antimicrobial activity against several pathogenic bacteria using a radial diffusion assay (Fig. 1b). Pam-1, Pam-4, and Pam-5 were generally inactive against tested strains, thus contrasting the potent inhibition of bacterial growth mediated by Pam-2 and Pam-3. Bacterial growth was most strongly inhibited by Pam-3, either on par (S. aureus) or superior (C. rodentium, P. aeruginosa, and S. Typhimurium) to the octapeptide, pointing toward modification-specific activities. Notably, both Pam-2 and Pam-3 consistently inhibited S. Typhimurium growth, a species the non-modified octapeptide failed to inhibit.
Fig. 1: Screening of differently modified octapeptides reveals peptides with improved antimicrobial activity.
a Octapeptide, the C-terminal eight amino acids of human β-defensin 1, was chemically modified with palmitic acid and different spacers such as sugars or amino acids or 8-amino-3.6-dioxaoctanoic acid (Ado) to generate lipopeptides. b Antimicrobial activity was measured by radial diffusion assay. The diameter of inhibition zones indicates antimicrobial activity; a diameter of 2.5 mm (dotted line) is the diameter of an empty well. Results are means ± SEM of three independent experiments.
Pam-3 displays bactericidal activity against multidrug-resistant bacteria and slow resistance selection
We used a broth microdilution assay to further analyze the potential of our lipopeptides to kill multidrug resistant bacteria belonging to the ESKAPE pathogen panel (Supplementary Fig. 1 and Supplementary Table 1). Pam-1 and Pam-4 displayed no or low bactericidal activity, whereas Pam-5 showed moderate effects against these pathogens. Similar to the results of the radial diffusion assay, both Pam-2 and Pam-3 were highly effective. Remarkably, Pam-2 and Pam-3 inhibited the growth of an A. baumannii isolate (DSM30007), which is otherwise resistant to the last-resort antibiotics, colistin and tigecycline. Despite some bactericidal similarities between Pam-2 and Pam-3, the latter proved superior to all other Pam's and was highly effective against these bacteria at concentrations of 4.69–18.75 µM (Fig. 2a and Supplementary Fig. 1). We accordingly selected Pam-3 for further characterization as a potential therapeutic against multidrug resistant bacteria.
Fig. 2: Pam-3 kills multidrug-resistant ESKAPE pathogens and resistance was not selected.
a Susceptibility of multidrug-resistant ESKAPE (E. faecium, S. aureus, K. pneumoniae, P. aeruginosa and E. coli) pathogens to antibiotics and Pam-3. Bacteria susceptible to all (blue boxes) or intermediate/resistant to at least one (red boxes) of the antibiotics per class. Gray boxes are shown if the susceptibility to agents in that class was not assessed. Results are expressed as the LC99.9, the lowest peptide concentration in micromolar that resulted in ≥99.9% killing. Results are medians (and ranges) of three independent experiments. If no range is indicated, then the LC99.9 was identical in all experiments. b, c Resistance development of S. aureus ATCC 25923 (b) and S. Typhimurium DSM554 (c) to Pam-3 (red line) and the antibiotics rifampicin and ciprofloxacin (black line), respectively. Values are fold changes (in log2) in minimal inhibitory concentration (MIC) relative to the MIC of the first passage.
As the development and selection of antibiotic-resistant bacteria in response to new antibiotic candidates is a significant problem, we assessed the ability of S. aureus and S. Typhimurium to develop resistance against Pam-3. When cultured in the presence of sub-inhibitory concentrations of Pam-3 for 25 passages, no significant increase in the minimal inhibition concentration (MIC) was observed for S. aureus. In contrast, the MIC for the standard antibiotic, rifampicin started to rapidly increase after five passages and had increased ≥4096-fold after 15 passages (Fig. 2b). Similarly, although exposed to Pam-3 for continuous serial passages, no resistant S. Typhimurium isolates emerged, whereas the presence of ciprofloxacin resulted in an increased MIC already after 3 passages, and a ≥256-fold MIC increase after 19 passages (Fig. 2c).
Pam-3 eliminates established biofilms and causes rapid killing by permeabilizing the bacterial membrane
Bacterial biofilms are highly resistant to growth inhibitors and bactericidal treatment regimens. Apart from the hindered penetration of antibacterial agents, treatment is further complicated by 10–1000 times increased tolerance exhibited by biofilm protected bacteria compared to planktonic bacteria29. Because of that, we assessed the ability of Pam-3 to eradicate established biofilms in a dose-depended manner. Within 1 h, 300 µM of Pam-3 eliminated P. aeruginosa in biofilms (Kruskal–Wallis test, p = 0.0074, Fig. 3a) and similarly eradicated ~99.99% of S. aureus in biofilms (Kruskal–Wallis test, p = 0.0026, Fig. 3a). A primary target of antimicrobial peptides is the bacterial cell envelope. Disturbing the integrity and function of the outer and/or inner membranes results in loss of the barrier function and dissipation of the membrane potential30. To clarify the mode of action of Pam-3, we used a ypuA promotor-based luciferase reporter strain of B. subtilis to identify cytoplasmic membrane-associated and cell envelope-related stress31. The ypuA promoter was activated (2 fold) by Pam-3, indicating cell envelope impairment (Fig. 3b). Hence, to strengthen this result, we analyzed the influence of Pam-3 on the transmembrane potential of S. aureus NCTC8325. The protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was used as a positive control to depolarize bacteria, i.e., leading to a reduction of their membrane potential. Upon depolarization, DiOC2(3) shifts from green fluorescence toward red emission because of self-association of the dye molecules. Pam-3 treatment caused a breakdown of the membrane potential in a concentration-dependent manner (Fig. 3c). Both results emphasize that Pam-3 acts on bacterial membranes. Since a large variety of AMPs target the membrane as pore formers32, we next analyzed the ability of Pam-3 to induce membrane lesions. To this end, we treated S. aureus NCTC8325 with Pam-3 at 4x MIC and added a mixture of Syto9 and propidium iodide (PI). The membrane-permeant Syto9 stains all living cells green, whereas the red-fluorescent PI can only enter cells through large membrane pores or lesions. Pam-3 led to a strong influx of PI (Fig. 3d). To further assess the mechanism of killing, transmission electron microscopy (TEM) was performed to analyze changes in bacterial morphology to compare bacterial morphology before and after treatment with Pam-3. In agreement with the pore formation and the induction of cell envelope stress and depolarization, Pam-3 treatment resulted in strong cell envelope damage with disrupted membranes and pores in most of the cells. Furthermore, additional membranous structures could be observed in many cells similar to rhesus macaque θ-defensins33. Additionally, scanning electron microscopy (SEM) was employed to observe cell morphological changes after Pam-3 treatment directly. Exposure to Pam-3 resulted in membrane surface disruption and lysed cells similar to hBD120, while control cells exhibited a bright and smooth surface (Fig. 3e). Pore formation was associated with fast killing of these bacteria. We assayed bactericidal kinetics to assess the rapidness of Pam-3 mediated killing. Pam-3 killed more than 90% of P. aeruginosa within 1 min and S. aureus within 15 min (Fig. 3f). Eradication to the level of detection was observed 2 and 30 min after Pam-3 treatment, respectively.
Fig. 3: Pam-3 eliminates established biofilms, causing rapid killing by inducing large pores or lesions.
a Bactericidal activity of Pam-3 against established biofilms of S. aureus ATCC25923 (black line) and P. aeruginosa PAO1 (gray line). Results are expressed as the number of viable bacteria (in log10 CFU) after 1 h treatment of 24 h old biofilms with Pam-3. Values are means ± SEM of three replicates from three independent experiments. Statistics were evaluated by using the Kruskal–Wallis test. b Activation of the B. subtilis ypuA promoter by Pam-3 indicates cell envelope stress. Data are presented as mean ± SEM. Experiments were carried out three independent times. c Effect of Pam-3 on S. aureus NCTC8325 membrane potential after 30 min of treatment. The protonophore CCCP was used as a positive control and 0.01% acetic acid as a negative control. Data are presented as mean ± SEM of three independent experiments. d Fluorescence microscopy of S. aureus NCTC8325 treated with Pam-3 (4x MIC) reveals pore formation by causing a strong influx of red-fluorescent propidium iodide. Scale bars: 1 µm. e Transmission electron microscopy of high-pressure frozen, freeze-substituted, and embedded S. aureus NCTC8325 treated with Pam-3 or 0.01% acetic acid for 30 min to observe membrane disruption. Scale bars, 0.2 µm. S. aureus NCTC8325 was exposed to Pam-3 or 0.01% acetic acid for 60 min. The samples were fixed in Karnovsky's reagent, and morphology was analyzed by scanning electron microscopy. Scale bars, 2 µm. f Killing of S. aureus ATCC25923 (black line) and P. aeruginosa PAO1 (gray line) after 1–120 min exposure to 9.38 µM (1x MIC) Pam-3. Results are expressed as the number of viable bacteria (in log10 CFU) per milliliter. Values are means ± SEM of three independent experiments.
Orally administered Pam-3 showed good acute tolerability in an animal model
Potential side effects of orally administered Pam-3 were assessed in mice. Histological analysis and determination of serum markers 24 h after application of two doses of 250 µg Pam-3 did not reveal acute toxicity. Specifically, there were no alterations in bodyweight and no signs of systemic toxicity or distress (Fig. 4a). Moreover, measurement of serum levels of glutamic oxaloacetic transaminase and creatinine showed no significant differences between the groups suggesting no effect on kidney and liver metabolism (Fig. 4b, c). Finally, histological examination of gastrointestinal tissues, liver, and kidney revealed no alterations, except a minor shortening of intestinal villi the jejunum-ileum of one control and two treated animals (Table 1). As no difference in these histopathological findings was observed between PBS and Pam-3 treated animals, they were regarded as background observations (Table 1 and Supplementary Fig. 2). Thus, we conclude that Pam-3 treatment was not associated with acute toxicity.
Fig. 4: Safety of orally applied Pam-3 in mice.
Dose-depended oral tolerance test in mice. Animals were treated twice with 125 µg (orange circles) or 250 µg Pam-3 (red circles) or PBS (black circles). a Weight change of mice (125 µg Pam-3, N = 5; 250 µg Pam-3, N = 6 and PBS, N = 5), b glutamic oxaloacetic transaminase (GOT) levels of treated animals with 125 µg Pam-3 (N = 4) or 250 µg Pam-3 (N = 6) or PBS (N = 5) and c creatinine levels (125 µg Pam-3, N = 4; 250 µg Pam-3, N = 6 and PBS, N = 5) one day after Pam-3 application. Results are presented as mean ± SEM of biologically independent animals.
Table 1 Safety of orally application of Pam-3 in mice.
Pam-3 preserves the core gut microbiota
To investigate the effect of Pam-3 on the intestinal microbiota, we treated mice twice at an 8-h interval with Pam-3 (125 or 250 µg/each dose) or PBS orally and collected fresh fecal samples before and 24 h after application. Microbiota composition was analyzed using 16S rRNA sequencing. Analysis of beta diversity and calculation of weighted Unifrac Distances demonstrated that changes in the microbiota between the before and after samples were similar between Pam-3 treated mice and PBS gavaged control mice (Fig. 5a, b). Similarly, while minor changes in the community structure were observed in both groups (i.e., treated and untreated), the number of detected species as well as the complexity (Wilcoxon-Test, Fig. 5c, d) remained comparable, thus contrasting treatment with traditional antibiotics, such as ampicillin (Supplementary Fig. 3). In line with the analysis of alpha and beta diversity, Pam-3 treatment did not affect the abundance of bacterial genera (Fig. 5e, f). Combined, these results demonstrate that, with the application regime conducted, Pam-3 treatment of healthy chow-fed mice does not affect the overall community structure or diversity of the microbiota.
Fig. 5: Pam-3 treatment does not affect microbiota diversity but has influence on certain bacteria phyla.
Chow-fed mice were treated orally twice at an 8-h interval with 125 µg Pam-3 (N = 5) or 250 µg Pam-3 (N = 6) or PBS (N = 5) as a control. Feces samples were collected before and after treatment to observe short term changes in the microbiome. a Principal coordinate analysis (PCoA including group mean) of fecal microbiota composition using Weighted UniFrac Distances before and after treatment, respectively. b PCoA including group mean of fecal microbiota composition using Unweighted UniFrac Distances, respectively before and after treatment. c Richness (observed species) before and after treatment. d Fecal microbiota was calculated by Shannon's Diversity index. The statistical significance was calculated by using Wilcoxon test. e Pam-3 treatment affects the abundance of bacterial genera. f Aggregated by genus. Statistical analysis performed by the LEfSe platform (https://galaxyproject.org/learn/visualization/custom/lefse/) using default settings.
Pam-3 treatment combats acute and established gastrointestinal infections of S. Typhimurium and Citrobacter rodentium
To assess the efficacy of Pam-3 on acute intestinal bacterial infections, mice were infected with S. Typhimurium and treated orally 6 and 22 h post infection with 250 µg peptide or PBS (Fig. 6a). Pam-3 treated animals showed significantly reduced colony-forming units (CFU) of S. Typhimurium in cecum content and tissue (Mann–Whitney test, p < 0.0001 and p = 0.0409, Fig. 6b). Furthermore, Pam-3 also lowered the bacterial load in the small intestine content without affecting the small intestine tissue (Mann–Whitney test, p = 0.0024 and p = 0.8621, Fig. 6c) and tends to reduce weight loss (Fig. 6d).
Fig. 6: Oral application of Pam-3 combats acute and established infections of S. Typhimurium and C. rodentium.
a Overview of experimental setup. Mice were infected with S. Typhimurium (red circles) and treated orally with either 250 µg Pam-3 (N = 12) or PBS (N = 9) after 6 h and 22 h post infection. Mice were infected with C. rodentium (blue circles) and treated orally with either 250 µg Pam-3 (N = 11) or PBS (N = 11) after 5 days post infection. b CFU/ml of S. Typhimurium in cecum content and tissue. c CFU/ml of S. Typhimurium in small intestine content and tissue. d Body weight change during acute S. Typhimurium infection. e CFU/g of C. rodentium in cecum content and tissue. f CFU/g of C. rodentium in colon content and tissue. Results are expressed as the number of viable bacteria (in log10 CFU) in the lumen and tissue and presented as mean ± SEM of biologically independent animals. Statistics were evaluated by using the Mann–Whitney test.
We subsequently assessed the therapeutic potential of Pam-3 against an already established intestinal infection. To this end, mice were infected with C. rodentium and received two doses of 250 µg Pam-3 or PBS 5 days post infection (Fig. 6a). Pam-3 treatment reduced the number of bacteria in the cecum content and cecum tissue (Mann–Whitney test, p = 0.0104 and p = 0.0473, Fig. 6e). Treatment also significantly reduced the number of viable Citrobacter in colon content and colon tissue (Mann–Whitney test, p = 0.0010 and p = 0.0104, Fig. 6f). Together these data corroborate the in vivo efficacy of Pam-3 against two different enteric pathogens.
The increasing number of multidrug-resistant pathogens is one of the greatest challenges of our time34. The Centers for Disease Control and Prevention (CDC) estimates that the annual infection rate exceeds 2.8 million cases in USA alone, resulting in more than 35,000 deaths caused by multidrug-resistant bacteria and fungi35. Due to this alarming development, alternatives to conventional antibiotics are urgently needed36,37. Herein, we report that Pam-3, a palmitoleic acid-modified octapeptide fragment from hBD-1, is effective against multidrug-resistant ESKAPE pathogens in vitro. Moreover, Pam-3 is also highly effective in vitro against A. baummanii resistant to the last-resort antibiotics, colistin and tigecycline. Biofilm-encased bacteria are much less susceptible to conventional antibiotics than their identical planktonic counterparts complicating treatments38. In contrast to most antibiotics, our experiments showed that Pam-3 was able to eradicate established S. aureus and P. aeruginosa biofilms in vitro.
Development of antibiotic resistance is increasing at an alarming rate39. Here, we demonstrated the lack of resistance development to Pam-3 in Gram-positive (S. aureus) and Gram-negative (S. Typhimurium) bacteria compared to conventional antibiotics, when cultured for 25 passages in the presence of sub-inhibitory concentrations. This result indicates that resistance development against Pam-3 is a rare event40.
One of the reasons for this observation could be the rapid killing of bacteria by Pam-3 and its associated mode of action. AMPs with bactericidal effects often interact with membranes as part of their mode(s) of action. Apart from membrane-disruptive mechanisms, like pore formation, AMPs can kill through electrostatics and localized perturbations or non-membrane-disruptive mechanisms, which targets multiple microbial processes and/or physiological functions41,42,43.
Our analysis revealed that Pam-3 causes cell envelope stress by breaking down the membrane potential and inducing pore formation. Taken together, the capacity to induce rapid killing of Gram-positive and Gram-negative bacteria, a low risk of resistance selection, combined with biofilm eradication underscores the value of Pam-3 for further drug development.
The rise in antibiotic resistance and the urgently search for potential alternatives shifted greater research focus on AMPs37,44. Most AMPs in pre- and clinical development are evaluated for topical rather than oral administration, for different reasons such as in vivo efficacy and stability45. We demonstrated the efficiency of orally administered Pam-3 in two different in vivo models of gastrointestinal infections, namely S. Typhimurium and C. rodentium. In both models, Pam-3 treatment resulted in significantly reduced bacterial burden in the gastrointestinal tract. Further studies, including proper dose regimens, are necessary for full eradication of both infections.
Antibiotic treatment has non negligible consequences. Observational, clinical, and epidemiologic studies have demonstrated that antibiotic treatment affects the gut microbiota composition with immediate effects on health5,46. Changes in the microbiota composition, decreased diversity, reduced taxonomic richness, and a so-called dysbiosis are the main consequences47,48. Further, antibiotics can have long-term effects such as increased susceptibility to infections, obesity, and obesity-associated metabolic diseases5,49. In contrast to conventional antibiotics, Pam-3 treatment had unexpectedly no appreciable effects on commensal microbes. Although further studies are warranted to rigorously address this assumption, our data points toward pathogen specific killing by Pam-3 treatment, which would be a significant advantage over conventional antibiotics where disruptive effect on the resident microbiota as well as a rapid drop in diversity is commonly observed50. Furthermore, after antibiotic treatment, the intestine is often colonized by non-commensal bacteria, which can result in long-term environmental changes51. Instead of a loss of diversity, high-dose Pam-3 treated mice showed an unaffected bacterial diversity. Our data points toward an increased number of observed species and nominally increased diversity. This observation is in line with previous studies on other fragments from our group52 suggesting that targeted attack on certain (rather high-abundant) bacterial strains frees up new niches for low-abundant taxa, which were previously below the limit of detection. Microbiota-modulating capabilities of AMPs could make a difference in the treatment of many gut microbiota associated diseases, including inflammatory bowel disease53.
In conclusion, the results presented here demonstrate that Pam-3 is a promising alternative to fight multidrug-resistant infections in a post-antibiotic world because of its broad antimicrobial activity against Gram-positive and Gram-negative pathogens and its efficacy against gastrointestinal infections without disrupting the resident microbiota. Future studies are therefore warranted to examine the full potential of this and other biostable novel peptides. Pam-3 could open a new chapter of an effective, microbiota-saving treatment strategy of bacterial infections.
Microorganisms and culture
Clinical isolates of A. baumannii DSM30007, E. faecium DSM2918, K. pneumoniae DSM30104, and P. aeruginosa DSM1117 were provided by the Department for Laboratory Medicine at Robert-Bosch-Hospital Stuttgart, Germany. C. rodentium DSM16636 and E. coli DSM8695 were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkultur GmbH Braunschweig, Germany. Clinical isolates of A. baumannii LMG944, A. baumannii ECII, E. coli 6940, E. coli DSM682, E. faecium 11037 CHB, E. faecium 20218 CHB, K. pneumoniae 6727 and K. pneumoniae 6970 as well as S. aureus DSM20231, S. aureus ATCC25923, S. aureus ATCC33592, S. aureus ATCC43300, S. enterica serovar Typhimurium DSM554, P. aeruginosa ATCC27853, P. aeruginosa NRZ01677 and P. aeruginosa PAO1 were provided by the Institute of Medical Microbiology and Hygiene Tübingen, Germany. B. subtilis ypuA and S. aureus NCTC8325 was obtained from the Interfaculty Institute for Microbiology and Infection Medicine, Tübingen, Germany. The wild-type S. Typhimurium strain SL1344 harboring a chromosomally integrated luxCDABE cassette, which is confirmed by kanamycin resistance54 and the nalidixic acid and kanamycin-resistant, bioluminescent C. rodentium strain ICC18055 were obtained from Helmholtz Centre for Infection Research Braunschweig, Germany.
All bacteria were stored in cryo vials (Roth) at −80 °C. Before each experiment, inocula from the frozen stocks were grown overnight at 37 °C on LB or Columbia blood agar plates (BD). For experiments, fresh cultures were prepared in tryptic soy broth (BD).
All lipopeptides were chemically synthesized by EMC Microcollections GmbH (Tübingen, Germany) and purified by precipitation. In detail, peptide synthesis was performed by solid-phase Fmoc/tert-butyl chemistry on Chloro-(2′-chloro)trityl polystyrene resin (Rapp Polymere, Tübingen, Germany) using an automated peptide synthesizer for multiple peptide synthesis (Syro, MultiSynTech, Germany). Side chain-protecting groups of Fmoc-amino acids were 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Arg), trityl (Cys) and tert-butyloxycarbonyl (Lys). Fmoc-protected amino acids including Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc-Ado-OH, IRIS Biotech GmbH, Germany) were coupled twice (double couplings, sevenfold molar excess of amino acids) by in situ activation using DIC/HOBt and TCTU with DIEA. The removal of the Fmoc-protecting group was carried out twice by treatment with piperidine/DMF (1:4, v/v). Resins were washed with DMF (6x) after each coupling and deprotection step. N-terminal acylation of the resin bound peptide was performed manually using palmitic acid (1.2 eq), DIC/HOBt (1.5 eq) with DIEA (2 eq) in NMP/DCM (4/1) for 16 h. After washing with DMF (8x) and DCM (5x) completeness of acylation was confirmed by Kaiser-Test. The lipopeptide was cleaved off the resin and side-chain deprotected by treatment with TFA/reagent K/H2O (80/15/5, v/v/v) for 3 h and precipitated by adding diethyl ether. After centrifugation, the peptides were dissolved in tert-butyl alcohol/H2O (4:1, v/v) and lyophilized. The structure of the lipopeptide was confirmed by RP-HPLC-ESI-MS and was >>90% (Supplementary Fig. 4). All peptides were dissolved in 0.01% acetic acid.
Antimicrobial activity
Antimicrobial activity was analyzed by radial diffusion assay56. Log-phase bacteria were cultivated for up to 18 h in TSB (TSB, Becton Dickinson, USA), washed and diluted to 4 × 106 CFU in 10 ml agar. Bacteria were incubated in 10 ml of 10 mM sodium phosphate, pH 7.4, containing 0.3 mg/ml of TSB powder and 1% (w/v) low EEO-agarose (AppliChem). 1 µg of each lipopeptide was pipetted into punched wells in a final volume of 4 µl and diffused into the gel for 3 h at 37 °C. After that, a nutrient-rich gel with 6% TSB (w/v) and 1% agarose in 10 mM sodium phosphate buffer was poured on top of the first gel and incubated for up to 24 h at 37 °C. Then the diameter of inhibition zones was measured.
Bactericidal activity
Bactericidal activity was assessed by broth microdilution assay57. Log-phase bacteria were collected by centrifugation (2500 rpm, 10 min, 4 °C), washed twice with 10 mM sodium phosphate buffer containing 1% (w/v) TSB and the optical density at 600 nm was adjusted to 0.1. Approximately 5 × 105 CFU/ml bacteria were incubated with serial peptide concentrations (1.17–150 µM) in a final volume of 100 µl in 10 mM sodium phosphate buffer containing 1% (w/v) TSB for 2 h at 37 °C. After incubation, 100 µl of 6% TSB (w/v) were added and absorbance was measured at 600 nm (Tecan, Switzerland) and monitored for 18 h. Afterward, 100 µl per well were plated on LB agar to determine the numbers of viable bacteria microbiologically. Bactericidal activity is expressed as the LC99.9, the lowest concentration that killed ≥99.9 % of bacteria.
For time-kill experiments, bacteria (5 × 105 CFU/ml) were incubated with 9.38 µM Pam-3 in 10 mM sodium phosphate buffer containing 1% (w/v) TSB in LoBind tubes (Eppendorf) in a total volume of 550 µl. As an untreated control, bacteria were incubated in 10 mM sodium phosphate buffer containing 1% (w/v) TSB. After incubation at 37 °C and 150 rpm for 1–120 min, a sample of 50 µl was taken from the suspension and added to 50 µl of a 0.05% (v/v) sodium polyanethol sulfonate (Sigma-Aldrich) solution, which neutralizes remaining peptide activity, and plated on LB agar to determine the number of viable bacteria.
Resistance development
Development of resistance to the peptides was assessed with S. aureus and S. Typhimurium. For comparison, the development of resistance to the clinically relevant antibiotics rifampicin and ciprofloxacin (Sigma-Aldrich) was determined. Bacteria were cultured overnight at 37 °C at 150 rpm in TSB. Bacteria were washed twice with 10 mM sodium phosphate buffer containing 1% (w/v) TSB. Washed bacteria were incubated with serial Pam-3 or antibiotic concentrations (with final concentrations of 1.17–150 µM peptide or 0.0156–0.5 µg/ml rifampicin or ciprofloxacin) in a final volume of 100 µl in 10 mM sodium phosphate buffer containing 1% (w/v) TSB for 2 h at 37 °C. After incubation, 100 µl of 6% TSB (w/v) was added and plates incubated in a humidified atmosphere for 21 h at 37 °C and 150 rpm.
The MIC, the lowest concentration of peptide/antibiotic that caused a lack of visible bacterial growth, was determined for each bacterial species. Thereafter, 5 × 105 CFU/ml of the 0.5-fold MIC suspension was added to a fresh medium containing peptides/antibiotics and these mixtures were incubated as described above. This was repeated for 25 passages.
Treatment of established biofilms
A log-phase culture of P. aeruginosa was diluted in BM2 medium and of S. aureus in TSB to 5 × 105 CFU/ml. 100 µl of each bacterial suspension was added to a round-bottom polystyrene microtiter plate and incubated for 24 h at 37 °C in a humidified atmosphere. Then, planktonic bacteria were removed by two wash steps with PBS. Next, biofilms were exposed to serial peptide dilutions (9.38–300 µM) in a final volume of 100 µl in 10 mM sodium phosphate buffer containing 1% (w/v) TSB for 1 h at 37 °C in a humidified atmosphere. As a control, bacteria were exposed to 10 mM sodium phosphate buffer containing 1% (w/v) TSB without peptide. Afterward, adherent bacteria in each well were resuspended, and the number of viable bacteria was determined microbiologically. To visualize the data on a logarithmic scale, a value of 1 CFU was assigned when no growth occurred. The biofilm degradation assay was performed in agreement with the original report describing this method first58.
Interaction with the bacterial membrane
A specific bacterial reporter strain with the genetic background of Bacillus subitilis 1S34, carrying the promoter of the ypuA gene, fused to the firefly luciferase reporter gene, was used to identify cell envelope-related damage caused by treatment with antimicrobial compounds31. The assay was carried out in agreement with former reports describing this method20,59. Bacteria were cultured to an OD600 of 0.9 in LB broth with 5 µg/ml erythromycin at 37 °C and diluted to an OD600 of 0.02. Serial peptide dilutions (0.15–150 µM) were prepared in a microtiter plate and incubated with the adjusted bacterial suspension at 37 °C for 1 h. Subsequently, citrate buffer (0.1 M, pH 5) containing 2 mM luciferin (Iris Biotech, Germany) was added and luminescence was measured using a microplate reader (Tecan, Switzerland).
Bacterial membrane potential
For determination of membrane potential changes, S. aureus NCTC8325 was grown to log-phase in LB + 0.1% glucose, harvested and the optical density at 600 nm (OD600) was adjusted to 0.5. Bacteria were incubated with 30 µM 3,3′-diethyloxacarbocyanine iodide (DiOC2(3), Invitrogen™) for 15 min in the dark and treated with serial peptide concentrations for 30 min. The protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP, Sigma Aldrich) was used as a positive control and DMSO or 0.01% acetic acid as negative controls. Fluorescence was measured at an excitation wavelength of 485 nm and two emission wavelengths, 530 nm (green) and 630 (nm) red, using a microplate reader (Tecan, Switzerland).
Bacterial pore formation
Pore formation was monitored using the Live/Dead BacLight bacterial viability kit (Molecular Probes)60. S. aureus was grown in LB at 37 °C to log-phase and, 100 µl aliquots were treated with 37.5 µM Pam-3 (4x MIC) or left untreated as a control. Samples were taken after 10 min of peptide treatment, then 0.2 µl of a 1:1 mixture of SYTO9 and propidium iodide (PI) was added and further incubated for 15 min at RT in the dark. Fluorescence microscopy was carried out using a Zeiss Axio Observer Z1 automated microscope. Images were acquired with an Orca Flash 4.0 V2 camera (Hamamatsu), C Plan-Apo 63x/1.4 Oil DIC and alpha Plan-Apochromat 100x/1.46 Oil Ph3 objectives (Zeiss) and processed using the Zen software package (Zeiss).
Transmission and scanning electron microscopy
Morphologic analysis of bacteria was characterized by electron microscopy21. Approximately 1.2 × 109 CFU/ml bacteria were incubated with 37.5 µM Pam-3 (4x MIC) in 10 mM sodium phosphate buffer containing 1% (w/v) TSB broth for 30 or 120 min at 37 °C. As a control, bacteria were exposed to 0.01% acetic acid. Afterward bacteria were fixed in Karnovsky's reagent.
For transmission electron microscopy (TEM), bacteria were high-pressure frozen (HPF Compact 03, Engineering Office M. Wohlwend GmbH) in capillaries, freeze-substituted (AFS2, Leica Microsystems) with 2% OsO4 and 0.4% uranyl acetate in acetone as substitution medium and embedded in EPON. Ultrathin sections were stained with uranyl acetate and lead citrate and analyzed with a Tecnai Spirit (Thermo Fisher Scientific) operated at 120 kV.
For scanning electron microscopy (SEM), bacteria were washed in PBS and finally fixed with 1% OsO4 on ice for 1 h. Next, samples were prepared on polylysin-coated coverslips, dehydrated in a graduated series to 100% ethanol and critical point dried (Polaron) with CO2. Finally, samples were sputter-coated with a 3 nm thick layer of platinum (Safematic CCu-010) and examined with a Hitachi Regulus 8230 field emission scanning electron microscope (Hitachi) at an accelerating voltage of 5 kV.
C57BL/6N mice were generated and maintained (including breeding and housing) at the animal facilities of the Helmholtz Centre for Infection Research (HZI) under enhanced specific pathogen-free (SPF) conditions61. Animals used in the experiments were gender and age matched. Female and male mice with an age of 8–12 weeks were used. Sterilized food and water ad libitum was provided. Mice were kept under a strict 12-h light cycle (lights on at 7:00 am and off at 7:00 pm) and housed in groups of two to six mice per cage. All mice were euthanized by asphyxiation with CO2 and cervical dislocation. All animal experiments were performed in agreement with the local government of Lower Saxony, Germany (approved permission No. 33.19-42502-04-18/2499).
Age- and gender-matched mice received two doses of peptides (0, 125 µg or 250 µg) solved in 100 µl PBS orally per day. Bodyweight and appearance were recorded. The following day, mice were sacrificed and stomach, kidney, spleen, liver, small intestine, cecum, and colon were removed for histological scoring. Around 1 ml of blood was taken from the heart to measure inflammatory markers, including creatinine in the kidney and the enzyme levels of glutamate-oxalacetate-transaminase (GOT) in the liver.
Infection and treatment of mice
Salmonella Typhimurium infection
For S. Typhimurium infections experiments, age- and sex-matched mice between 10 and 14 weeks of age were used. Both- female and male mice were used in experiments. Water and food were withdrawn for 4 h before mice were treated with 20 mg/mouse of streptomycin by oral gavage. Afterward, mice were supplied with water and food ad libitum. 20 h after streptomycin treatment, water and food were withdrawn again, 4 h before the mice were orally infected with 105 CFU of S. Typhimurium in 200 μl PBS. Drinking water ad libitum was supplied immediately and food 2 h post infection (p.i.). After 6 and 22 h p.i. mice received 250 µg peptide solved in 100 µl PBS or only PBS orally. 48 h after infection, mice were sacrificed, and intestinal organs were removed to assess the bacterial burden in the lumen and tissues. Mice were weighed every day to record potential body-weight loss.
Citrobacter rodentium infection
Bioluminescence expressing C. rodentium strain ICC180 was used for all infection experiments55. C. rodentium inocula were prepared by culturing bacteria overnight at 37 °C in LB broth with 50 µg/ml kanamycin. Subsequently, the culture was diluted 1:100 in fresh medium, and sub-cultured for 4 h at 37 °C in LB broth62. Bacteria were washed twice in phosphate-buffered saline (PBS). Mice were orally inoculated with 108 CFU of C. rodentium diluted in 200 µl PBS. Weight of the mice was monitored and feces were collected for measurements of the pathogen burden. After 5 days post infection mice received twice 250 µg peptide solved in 100 µl PBS or only PBS orally. The following day, mice were sacrificed to assess bacterial burden in the lumen and tissues of the cecum and the colon.
Analysis of bacterial loads in feces
Fresh fecal samples were collected and weighted. Samples were homogenized in 1 ml LB media by bead-beating with 1 mm zirconia/silica beads twice for 25 s using a Mini-Beadbeater-96 (BioSpec). To determine CFUs, dilutions of homogenized samples were plated on LB plates with 50 μg/ml kanamycin.
Analysis of bacterial loads in intestinal content and systemic organs
All mice were euthanized by asphyxiation with CO2 at indicated time points. Intestinal tissues (small intestine, cecum, colon) were removed aseptically. To collect intestinal content, organs were flushed with PBS. Organs were opened longitudinally, cleaned thoroughly with PBS and weighted. Organs and content were homogenized in PBS using a Polytron homogenizer (Kinemtatica). Dilutions of homogenized samples were plated on LB plates containing 50 μg/ml kanamycin to determine CFUs.
Microbiota analysis
Feces samples were collected at different time points (before and after infection), and bacterial DNA was extracted from snap-frozen feces using a phenol-chloroform-based method previously described63. In brief, 500 µl of extraction buffer (200 mM Tris (Roth), 20 mM EDTA (Roth), 200 mM NaCl (Roth), pH 8.0), 200 µl of 20% SDS (AppliChem), 500 µl of phenol:chloroform:isoamyl alcohol (PCI) (24:24:1) (Roth) and 100 µl of zirconia/silica beads (0.1 mm diameter) (Roth) were added per feces sample. Lysis of bacteria was performed by mechanical disruption using a Mini-BeadBeater-96 (BioSpec) for two times 2 min. After centrifugation, the aqueous phase was processed by another phenol:chloroform:isoamyl alcohol extraction before precipitation of DNA using 500 µl isopropanol (J.T. Baker) and 0.1 volume of 3 M sodium acetate (Applichem). Samples were incubated at −20 °C for at least several hours or overnight and centrifuged at 4 °C at maximum speed for 20 min. The resulting DNA pellet was washed, dried using a speed vacuum and resuspended in TE Buffer (Applichem) with 100 µg/ml RNase I (Applichem). Crude DNA was column purified (BioBasic Inc.) to remove PCR inhibitors.
16S rRNA gene amplification of the V4 region (F515/R806) was performed according to an established protocol previously described64. Briefly, DNA was normalized to 25 ng/µl and used for sequencing PCR with unique 12-base Golary barcodes incorporated via specific primers (obtained from Sigma). PCR was performed using Q5 polymerase (NewEnglandBiolabs) in triplicates for each sample, using PCR conditions of initial denaturation for 30 s at 98 °C, followed by 25 cycles (10 s at 98 °C, 20 s at 55 °C, and 20 s at 72 °C). After pooling and normalization to 10 nM, PCR amplicons were sequenced on an Illumina MiSeq platform via 250 bp paired-end sequencing (PE250). Using Usearch8.1 software package (http://www.drive5.com/usearch/) the resulting reads were assembled, filtered and clustered. Sequences were filtered for low-quality reads and binned based on sample-specific barcodes using QIIME v1.8.065. Merging was performed using -fastq_mergepairs—with fastq_maxdiffs 30. Quality filtering was conducted with fastq_filter (-fastq_maxee 1), using a minimum read length of 250 bp and a minimum number of reads per sample = 1000. Reads were clustered into 97% ID OTUs by open-reference OTU picking and representative sequences were determined by use of UPARSE algorithm66. Abundance filtering (OTUs cluster >0.5%) and taxonomic classification were performed using the RDP Classifier executed at 80% bootstrap confidence cut off67. Sequences without matching reference dataset were assembled as de novo using UCLUST. Phylogenetic relationships between OTUs were determined using FastTree to the PyNAST alignment68. Resulting OTU absolute abundance table and mapping file were used for statistical analyses and data visualization in the R statistical programming environment package phyloseq69.
Statistics and reproducibility
Experimental results were analyzed for statistical significance using GraphPad Prism v8.2 (GraphPad Software Inc.). Apart from microbiota analysis, all data were analyzed by Mann–Whitney test or Kruskal–Wallis test. Here, differences were analyzed by Wilcoxon signed-rank test or Kruskal–Wallis test comparison of totals between groups70. OTUs with Kruskal–Wallis test <0.05 were considered for analysis.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
16S rRNA gene sequencing data have been deposited in the NCBI (Bioproject Database) under the accession number: PRJEB37278. All source data underlying the graphs presented in the main figures are available in Supplementary Data 1. Please contact the corresponding author (L.K.) for access of other data or information, which is stored electronically, and will be made available upon reasonable requests.
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We thank Marion Strauss and Jutta Bader for excellent technical assistance; Stefanie Fehrenbacher for providing the Antibiograms of ESKAPE pathogens. This study was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) to Jan Wehkamp—Project ID WE4336/7-1 and also under Germany´s Excellence Strategy—EXC 2124 "CMFI"—Project ID 390838134. Till Strowig was supported by the Helmholtz Association and the DFG under Germany's Excellence Strategy—EXC 2155 "RESIST"—Project ID 39087428. Heike Brötz-Oesterhelt and Anne Berscheid acknowledge funding by the German Center of Infection Research (DZIF), project TTU 09.818, and infrastructural support by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany´s Excellence Strategy—EXC 2124 "CMFI"—Project ID 390838134. Lisa Osbelt was supported by the federal state of Saxony-Anhalt and the European Structural and Investment Funds (ESF, 2014–2020), project number 4410032030ZS/2016/08/80645. We acknowledge support by Open Access Publishing Fund of University of Tübingen.
These authors contributed equally: Till Strowig, Jan Wehkamp.
Department of Internal Medicine I, University Hospital Tübingen, Tübingen, Germany
Louis Koeninger, Judith Wendler, Nisar P. Malek & Jan Wehkamp
Department of Microbial Immune Regulation, Helmholtz Centre for Infection Research, Braunschweig, Germany
Lisa Osbelt, Till R. Lesker & Till Strowig
ESF International Graduate School on Analysis, Imaging and Modelling of Neuronal and Inflammatory Processes, Otto-von-Guericke University, Magdeburg, Germany
Lisa Osbelt
Department for Microbial Bioactive Compounds, Interfaculty Institute of Microbiology and Infection Medicine, University of Tübingen, Tübingen, Germany
Anne Berscheid & Heike Brötz-Oesterhelt
German Center for Infection Research (DZIF), Partner Site Tübingen, Tübingen, Germany
Max-Planck Institute for Developmental Biology, Electron Microscopy, Tübingen, Germany
Jürgen Berger & Katharina Hipp
Mouse Pathology and Histology, Helmholtz Centre for Infection Research, Braunschweig, Germany
Marina C. Pils
Novo Nordisk Foundation Center for Basic Metabolic Research, Human Genomics and Metagenomics in Metabolism, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
Benjamin A. H. Jensen
Cluster of Excellence - Controlling Microbes to Fight Infections, Tübingen, Germany
Heike Brötz-Oesterhelt & Jan Wehkamp
Cluster of Excellence - Resolving Infection Susceptibility, Hannover, Germany
Till Strowig
Louis Koeninger
Anne Berscheid
Judith Wendler
Jürgen Berger
Katharina Hipp
Till R. Lesker
Nisar P. Malek
Heike Brötz-Oesterhelt
Jan Wehkamp
L.K conceived the research, designed and carried out experiments and data analysis, and wrote the manuscript. L.O. designed, conducted the in vivo infection model experiments and analyzed the data. M.P. conducted histology and analyzed the data. A.B. performed in vitro MoA experiments and data analysis. J.B. and K.H. were in charge for the electron microscopy experiments. T.L. performed the bioinformatics. A.B., J.u.W., N.P.M., B.A.H.J., and H.B.-O. assisted with data interpretation and manuscript editing. T.S. and J.W. conceived the research, assisted with data interpretation and wrote the manuscript. All authors were involved in data discussion and the final version of the manuscript.
Correspondence to Louis Koeninger.
J. Wehkamp and L. Koeninger are planning to apply for a patent on lipopeptides of the "Pam" series. The other authors declare no competing interests.
Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Koeninger, L., Osbelt, L., Berscheid, A. et al. Curbing gastrointestinal infections by defensin fragment modifications without harming commensal microbiota. Commun Biol 4, 47 (2021). https://doi.org/10.1038/s42003-020-01582-0
Communications Biology ISSN 2399-3642 (online)


text:
Obama To Address Health Care Website Problems
By Mara Liasson
Steve Inskeep
STEVE INSKEEP, HOST:
Now let's look at the politics of the Obamacare rollout with NPR's national political correspondent, Mara Liasson. She's on the line.
Hi, Mara.
MARA LIASSON, BYLINE: Hi, Steve.
INSKEEP: OK. So the standoff is over for the moment. The government is reopened. The battle over the debt ceiling is behind us. And now the focus is on this actual law.
LIASSON: That's right. We're now not fighting about whether the government should be shut down until Obamacare is defunded. We're going to be fighting about the success of the actual law. This is where many Republicans thought the fight should be waged in the first place. So today in the Rose Garden, the president is going to directly address the technical problems. The Department of Health and Human Services has announced a new tech surge, a new team of computer experts that's going to be repairing the website.
On the Hill, Republicans are going to hold a hearing on Thursday in the House Energy and Commerce Committee, which so far HHS director Kathleen Sebelius is refusing to attend and Republicans have been calling for her resignation because of the rollout problems. So that's where we are in the fight now.
INSKEEP: Isn't this giving Republicans just endless opportunities to say, see, I told you so, it's not going to work.
LIASSON: Absolutely. This is an I-told-you-so moment. And the problem is that these technical problems are big problems. This is the president's most significant legislative accomplishment, and whether the healthcare law is implemented successfully or not will in large part determine his legacy.
And these are more than glitches, or as the administration initially suggested problems of too many people trying to visit the site at once. And as you heard Yuki explain, part of this problem is practical. If the website problems discourage enough young healthy people from signing up, and only sick people are willing to wade through these delays, the system could collapse of its own weight, which is what opponents have been predicting, and some supporters fear that the penalty for not signing up, one percent of your income or $95 in the first year, is not only too low but it's almost impossible to enforce.
So as Yuki said, we're not going to get actual enrollment numbers for another couple of weeks, but all of these problems are giving fuel to opponents, and ironically all of them would've received a heck of a lot more attention if the shutdown standoff had not been the overwhelming story in the last two weeks.
INSKEEP: Oh, well, let's talk about that, Mara, because Republicans, some of them, were urging another conversation, a bigger conversation about Obamacare. They were hoping that the law would become even less popular. What seems to have happened is the Republican Party became less popular, but what about the law itself? How has public opinion changed?
LIASSON: Well, during the shutdown polls showed an actual uptick in the popularity of the law. The law was still overall unpopular, but not by as big a margin. Now, that could just be a reflection of the negative reaction to the Republican shutdown strategy. But what we're waiting for now is to find out not only what uninsured people think of the law after they get on the website and they are able to or not able to sign up, but also what do insured people think of the law.
Do people's premiums go up, do their employer-provided coverage get worse, and will they blame Obamacare for that? Right now everything that happens in the healthcare delivery system, and there are big changes happening in healthcare, some of them because of Obamacare and some of them have nothing at all to do with the law, but I do think that all of them are going to get wrapped up in the public debate over Obamacare.
INSKEEP: Big debate within the Republican Party now, isn't there, Mara Liasson? You can think of someone like John McCain, who said this is a terrible law, but it went through the process, Republicans had their say, and people like Ted Cruz, who are arguing that it is still worth doing anything possible to block it.
LIASSON: Well, there's no debate in the Republican Party about whether Obamacare is bad and needs to be repealed. The question is about tactics. That's the big debate in the Republican Party. Do you need to have a Republican majority in the Senate and a Republican in the White House in order to do this or is there something that Republicans can do right now.
So that debate will continue.
INSKEEP: And Republicans themselves have not agreed on the tactics for the next couple of months?
LIASSON: No. Although it sounds like they're not going to use the shutdown strategy again when we get to the next deadlines in January and February.
INSKEEP: Oh, Ted Cruz has been talking about not ruling out a shutdown, but powerful Republicans like Mitch McConnell say forget it.
LIASSON: Right, right.
INSKEEP: Okay, Mara, thanks very much.
LIASSON: Thank you.
INSKEEP: That's NPR national political correspondent Mara Liasson on this Monday morning. Transcript provided by NPR, Copyright NPR.
Mara Liasson
Mara Liasson is a national political correspondent for NPR. Her reports can be heard regularly on NPR's award-winning newsmagazine programs Morning Edition and All Things Considered. Liasson provides extensive coverage of politics and policy from Washington, DC — focusing on the White House and Congress — and also reports on political trends beyond the Beltway.
See stories by Mara Liasson
Steve Inskeep is a host of NPR's Morning Edition, as well as NPR's morning news podcast Up First.
See stories by Steve Inskeep

redpajama_set_name: RedPajamaCommonCrawl


text:
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Daniel J. Lee
NCRP Vice-Chair
Daniel Lee is executive director of the Levi Strauss Foundation. He joined Levi Strauss & Co. (LS&CO.), and the Levi Strauss Foundation in 2003 as a Community Affairs Manager for the Asia Pacific Division in Singapore, where he managed and implemented corporate social responsibility programs, employee volunteerism and grantmaking in three global giving areas — HIV/AIDS, worker rights, and asset building.
Subsequently, he relocated to San Francisco and assumed the role of Director of Global Grantmaking Programs. Lee has extensive experience with international non-governmental organizations in the fields of human rights, HIV/AIDS and social justice.
Before joining LS&Co., he served as Senior Program Officer for Asia and Pacific at the International Gay and Lesbian Human Rights Commission (IGLHRC) and on the founding board of the Massachusetts Asian AIDS Prevention Project. In addition to FCAA, Mr. Lee currently serves on the International Advisory Board of the Astraea Lesbian Foundation for Justice and the Asia-Oceania advisory panel of the Global Fund for Women. He received an B.A. in Religion and History from Princeton University and a Master of Divinity from Harvard University.


Understand something important right away, bone conduction headphones are different. Some of you may have never even heard of them and due to the nature of their design, you can't really compare them to traditional models. It all comes down to the technology behind bone conduction as this is the conduction of sound through the bones of the skull. When implemented into headphones, what you get are devices that are lightweight, convenient, and ideal for people with normal or impaired hearing to use. But, if you are looking for sound quality that rivals the best Bose headphones out there, you will simply be disappointed. Of course, with that being said, some models will naturally sound better than others (though that is just one of the factors in the buying process). And, this "buying process" is exactly why you are here today.
If you had a theoretical draft and all the bone conduction headphones on the market were eligible, the Trekz Air would be the consensus number one selection. They simply are the standard bearer for this technology.
Look, for this technology, these are about as good as it is going to get for sound quality. This is thanks to the Premium Pitch technology that provides a premium audio experience.
Okay, the good news is that these are 20 percent lighter than its counterpart and you will barely feel them when you have them on. The bad news is the band is not adjustable, for some odd reason.
Without a doubt, the Trekz Air was designed with athletes in mind. After all, they are fully sweatproof so even during long and grueling runs, they will stay securely on your head.
Though these are equipped with buttons on the side that can change various audio settings, there was one user who felt that they were awkwardly placed.
Thanks to the wireless and Bluetooth design (these sport version 4.2, for the record), you can enjoy these without the hassle of a cord. Also, the integrated battery will charge in two hours and last for six (if you are listening to music non-stop).
For sure, this technology has a long way to go before it ever achieves the same level of sound quality as over-ear headphones but without question, these right here are the current measuring stick.
Even though these do not quite reach the sound quality of the models designed by AfterShokz, you can argue that they are superiorly built. For certain, that is the main selling point here.
It is noted by the seller that they believe their model is one of the best at preventing sound leakage. As for previous users, most feel the overall sound quality is merely okay.
These have been both ergonomically designed and also streamlined for comfort and balance. The result is a more pleasurable experience that is for sure.
Even though some previous buyers have had longevity issues, these are brilliantly designed with nano-material and superior sealing. So, you will never need to worry about sweat damaging the interior circuitry.
Right on the outside of the earbuds, there is a multi-function button that allows you to conveniently adjust specific settings.
A very impressive battery is integrated here (a 220mAh lithium battery) and it allows for 1000 hours of standby time and up to 11 hours of continuous playing time.
What you need to appreciate about this design are the physical characteristics. Sure, the sound quality could use improved but that is not necessarily something new.
If it was not evident before then it should be now that AfterShokz is the leader of this technology. Their Bluez 2S model sports their patented technology that minimizes sound leakage and enhances the audio overall.
Once again, AfterShokz sets the standard for sound quality. While still not amazing, the Bluez 2s sports PremiumPitch technology and also LeakSlayer technology. The latter actually reduces the amount of natural sound leakage.
It seems to be the general consensus that the Bluez 2S headphones are small. Previous buyers have claimed they have been unable to achieve a comfortable fit because of it, unfortunately.
Due to the nanotechnology treatment that has been integrated, these are going to repel sweat when you exercise. Of course, in regard to the longevity of this model, some previous buyers have had issues.
As with most of the top-end models on the market, there are specific buttons integrated here that allow you to make changes on the go quickly and easily.
These sport the somewhat standard Bluetooth range of 33 feet. Additionally, the battery can charge in two short hours and last for 10 days on standby and six hours during continuous play.
In a way, you could consider this model a nice alternative option to the gold standard of this technology. After all, it is nearly just as good at a significantly lower price (and comes from the same designers).
Even though this type of sound technology is not as expensive as others, the BHS-530 is an astounding revelation of affordability and quality. Literally, thousands of users have raved about them and will back up that claim.
When you look at the track record of this technology, it is actually surprising when you look at the sound quality of this model. Loud audio and decent bass can be achieved, which can be a rarity.
Not only does the flexible Memory-Wire adapt to the size of your head but the memory foam ear tips also enhance the comfort overall. Not a bad combination at all.
To better withstand sweat and rain, a nanocoating finish has been applied to these. In spite of this integration, longevity issues have been documented by previous buyers.
Though these are not overflowing with features, you can quickly and conveniently switch between music and phone calls with the simple push of a button.
Bluetooth 4.1 is the wireless protocol that is utilized here and as for the integrated battery, it can last for up to 11 hours of play time.
You will simply be hard-pressed to find a better affordable option on the market. In fact, you might as well stop looking because or the price, these are unbeatable.
Back once again, this here is yet another option from AfterShokz. And, even though this model does not top the list, it can be argued that this right here is the most popular option from the juggernaut.
All the classic technologies that AfterShokz implements in their bone conduction headphones are present here so what more is there to say?
Even though these are lightweight and flexible, there was a user who had strong negative feelings about the design of the battery and control compartments (claiming they dug into their ears).
In addition to the fact that these sports titanium, they are also water-resistant to IP55 standards. This means that they can handle sweat, dust, and moisture very well.
Now, there is an included manual that will walk you through the features of this model. Yet, this manual has been criticized for being too short and confusing.
The implemented Bluetooth 4.1 technology allows these to be connected to virtually all your smart devices and the battery can last for up to six hours of continuous use (with music playing).
These right here end the so-called "party" for AfterShokz models. Again, you can see why they are so popular as the quality truly speaks for itself.
Admittedly, in terms of popularity, the drop off here is noticeable as many people have probably never even heard of these. What a shame that is because as you will see, they can compete with the very best on the market.
Though the microphone quality on these is admirable, the bass and maximum volume simply are not. Do not get your hopes up very high for those, let's just say that.
This is where these truly shine. In addition to the rubber coating and lightweight nature of these, an adjustable strap is also included (though you do not need to use it).
Being water-resistant to IPX6 standards, these can handle most of the sweat that drips down your forehead and also light rainfall.
Thanks to the buttons on the side panel of the band, you can adjust both the volume of these and also the current audio track that you are listening to.
Admittedly, the battery is pretty weak here. While it is not all that much of a downgrade from other models, you can expect around two to three hours of continuous use (with the volume maxed).
Overall, it is safe to say that you should give these a chance. They really are a low-risk high reward option and as long as you do not expect too much from the sound quality, you should enjoy them.
As with most of the other models on the market, the F1 Headphones are made of titanium and are lightweight and flexible as a result. Oh yeah, and they are also water-resistant to IP55 standards.
Though the audio is not the loudest here, what is really nice is the built-in microphone will pick up your voice very well so the person on the other end can hear you clearly.
The ergonomic design and lightweight nature of these help the comfort. The real problem, though, lies in the fact that they are very small.
In addition to the titanium build of these headphones, they are also rated with an IP55 waterproof grade which makes them resistant to rain and sweat.
Vidonn designed their model in the same nature as so many others as they included buttons on the side panel. With these, again, you can quickly make modifications on the fly.
Thanks to the power of the 180mAh lithium battery, you can enjoy up to six hours of continuous music or up to 240 hours on standby mode.
Granted you do not sport a large cranium, these should be an optimal choice for you. The built-in microphone and fancy design features make this a compelling choice for sure.
Yep, this right here is another option that utilizes Bluetooth technology for its wireless protocol and this will not be problematic for most of you. The titanium alloy band, among other features, is also worth mentioning, though.
Though not too many users have raved about the sound quality of these, a few have complimented the quality of the built-in microphone.
On the one hand, it will not even feel like these are on they are so light in weight. On the flipside, they do tend to move around if you are partaking in rigorous exercises.
Do not think that just because these are light in weight that they are cheaply built. The titanium band can withstand at least 15000 times bending which is very impressive, to say the least.
With the use of just one simple button and two volume keys, you are able to quickly hang up on incoming calls, adjust the volume and switch between songs (among other things).
When you compare the battery of this model to others, it does well. In fact, with a two-hour charge time and six-hour runtime, it is about what you expect for this technology.
What more could really be said here? If you summarize these headphones, you would have to say that they perfectly epitomize the greatness and also the limitations of this technology.
Probably one of the hottest topics of this next model is that it supports Bluetooth 5.0 technology. While this may sound trivial to some of you, this is actually one of the latest versions of the wireless protocol. Of course, this excels in other areas, as well.
Let's just say these do not sound like AfterShokz models. The sound quality is limited and not that great and that is simply the reality of the situation.
If you have a smaller head, you may struggle to keep these stable. They seem better optimized for users with larger craniums.
No, these are not going to break very easily and this is thanks to the titanium-nickel memory alloy material that was utilized. Also, these are sweatproof.
Now, there is only one button integrated but it does serve multiple purposes. While it is not exactly complex to learn and operate, you may experience user error from time to time.
As mentioned, these are outfitted with Bluetooth 5.0 technology. In the world of Bluetooth, this is about as good as it gets.
For some of you, the allure of Bluetooth 5.0 is not going to be enough and that is fair. But, these really are a solid pair of bone conduction headphones overall.
Without question, this is one of the most popular options on the entire market. Some of you may say it is simply due to the price and while that does play a role, the quality is also solid.
Implemented into these is aptX technology and this enhances the audio. In spite of this, however, the sound quality is what you would expect from headphones at this price range.
All in all, the comfort has not exactly been raved by previous users. Some of which have even gone out of their way to criticize the provided comfort.
The strong adhesion and shape memory of these bodes well when you need to store them away as they can roll up in a fixed shape, which is really nice.
Much like the last model, these do sport buttons for easier adjusting. However, they also take some time to get accustomed to as they perform multiple actions.
In addition to the eight hours of talk and play time, the Bluetooth reception of these has been celebrated by various reviewers.
Look, when you are in the market for any type of headphones, the price will play a role. While there are clearly superior options out there, for this price, few will complain.
At the end of the day, these are headphones which means their sound quality is a top priority.
It was mentioned earlier and it really does need to be reiterated, bone conduction headphones simply can't compete with standard models when it comes to sound quality. For the record, standard models will use air conduction to transfer audio and while this is not necessarily superior to bone conduction, it comes down to the manner in which they are designed. You see, standard designs will typically either be over-ear or on-ear. However, that is not the case with this type of technology as you will see open-ear headphones and this is the main reason why the sound quality is not as great. These simply are not designed to go in your ear and due to this, you are not receiving a truly direct transmission of sound.
However, that does not mean that certain models out there will not sport superior sound quality. And, when you look at AfterShokz, that becomes apparent very quickly. You see, what separates a brand like this is the fact that they implement specific technologies that allow their models to stand out. Two that immediately come to mind are Premium Pitch and LeakSlayer. The latter is really the more noteworthy of the two as a common problem with this type of technology is sound leakage. Well, LeakSlayer technology solves this issue, to an extent.
Quick question, do you really want to wear a pair of headhpones that are uncomfortable?
Now, as opposed to the last section that showed how inferior this type of technology is to other headphones, this section shows why it holds an advantage. Generally speaking, bone conduction headphones will offer superior comfort and it comes down to the manner in which they are designed. As they are open-ear designs, they do not technically fit in or on your ear and are more comfortable, as a result. Of course, their lightweight nature also helps with this. It is not uncommon for you to forget that you even have them on and even though it sounds outlandish, many previous reviewers have shared their experiences of such.
However, much like with the sound quality, this does not mean that each and every model on the market will offer the same amount of comfort. Instead, while all models will have the same properties as listed above, certain options may have unique features such as adjustable bands and additional cushioning materials. Either way, you slice it, you need to ensure the model you get fits you properly and is comfortable to wear.
Outside of the main materials, you also need to pay attention to any coatings that are present.
So, what are these headphones made out of anyway? Speaking for the band (which is the section that connects the two earbuds together), you will oftentimes see them made out of titanium. You are probably aware that titanium is a metal but do you know why it is such a popular material? For starters, as opposed to its steel counterpart, it is much, much lighter (40 percent lighter, as a matter of fact). At the same time, though, this metal does not sacrifice in other areas such as its strength and corrosion resistance. Speaking for the latter, this is one of the most highly touted properties of the entire metal.
Of course, outside of the main material, you will also want to look out for coatings. In specific, what is known as a nanocoating as this is basically going to provide a surface that is going to repel various particles (such as dirt and water). Oh, and speaking of which, this is a good time to discuss the IP rating. Better known as the International Protection Rating, this is used to classify how well a certain object resists both solids and liquids. The first number represents solids and the second liquids. And, the higher the number the more resistant the object is. For the record, the highest number the system goes to (for liquids) is eight.
The main thing here is that most models on the market will sport button controls for easier adjustments.
To be honest, when compared to other headphones that are oftentimes loaded with design features, it is safe to say that bone conduction models are pretty simple designs. In spite of this, however, one of the most important features that they often integrate is the use of controls. As you know, you can control almost everything with your connected device. However, sometimes, it is more convenient to simply press a corresponding button on the headphones themselves. Indeed, this is what you want to look for here.
Yet, it is not as simple as it sounds. First off, the buttons need to be placed practically. And, secondly, some models will only include one button and it will then be up to you to memorize the different functions that the button can perform. After all, most of the time, with the use of these buttons, you will be able to adjust the volume, switch between music tracks, and answer or hang up on phone calls. Oh, and speaking of which, if you do want to make calls, the model you pick needs to have a high-quality microphone built-in.
Since most options on the market will be wireless, you need to pay close attention to the battery life and wireless protocol.
As you noticed from this list, many top-end models out there are wireless. What this means is two huge factors come to mind. The first of which is the battery life and while this is pretty self-explanatory, there are a few things to point out. For the most part, the battery lives of these types of audio devices will range anywhere from 4 to 11 hours. However, this is when used continuously. Sellers will also typically inform you of how long their model can run on standby (and this merely kicks in after the device has been idle for too long, much like a computer). Do not get the two mixed up.
The second factor is the wireless protocol. Nine times out of 10 (and probably 10 times out of 10), this will be Bluetooth. And, even though Bluetooth is backwards compatible and included on nearly all smart devices, you may want to look for the latest version (it does get updated quite frequently) as this can deliver performance enhancements.
Are you starting to get the point that these types of sound devices can't hang with others when it comes to their sound quality? Instead of beating the dead horse some more, let's point out that even though this is true, there are still options out there that will offer superior sound clarity and crispness. Even still, though, you really should just set your expectations low here and work from there.
First and foremost, the most notable aspect of any pair of bone conduction headphones is how light they are. Just about every single model on the market will be this way but that does not mean that they all will be comfortable. Although this is a major factor in the overall comfort, it is far from the only one. After all, proper fitting and stability will also play vital roles.
As these types of devices are not your prototypical designs, it also means that they will not be made from the same cliché materials. In fact, one you see quite common is titanium (instead of steel or aluminum). Past this, as these are also generally optimized for athletes, you will also commonly see engineers apply special coatings to their headphones to make them sweatproof and also highly resistant to water.
q: How exactly does this technology work?
When you really break down how these devices work, it is incredibly fascinating. You see, bone conduction itself is a natural part of the hearing process as sound travels through the bones and eardrums at the same time. When this technology is implemented into headphones, what happens is sound will be produced with mini vibrations and it will travel through your cheekbones. All in the meanwhile, these vibrations completely bypass your eardrums.
q: Can these be worn with hearing aids?
This was something that was briefly alluded to at the beginning of this guide but it is a question that gets brought up quite a lot on the internet so buyers must be pondering it. Here is what is fantastic because not only can people with hearing aids use this type of audio device but this is actually one of the major reasons why one would. Of course, it also depends on what type of hearing aid you currently use because it may conflict with the band when you go to put it on.
q: What about with helmets on?
Again, as these headphones are great for physical activity, this may be a question that you have. As with hearing aids, it is going to be a piece of cake to wear any pair of these with a helmet on. It comes back to the manner in which they are designed as they will merely be resting around your neck and not on the top of your head. Due to this, there should be no conflictions or restrictions if you want to use them with hats, helmets, or any other type of headwear.
q: In general, what is the correct way to wear them?
For over-ear or on-ear models, it will basically be common sense when it comes to putting them on. On the flipside, these are a little different. As they will be designed to go in front of your ears, you actually will not place the earbuds in your ears. Instead, the looped sections will go above your ears comfortably. From there, the band needs to be behind your head. Now, if the band is adjustable then you can mess around with where you need it to ensure you achieve the most optimal fit possible.
q: Is it legal to drive with them on?
Wow, is this ever a loaded question? First off, understand that all states and countries have different laws and regulations so you should always check with your jurisdiction beforehand. In the United States, the laws vary when it comes to driving with headphones. Now, in theory, these types of sound devices do not inhibit your awareness very much as you will still be able to hear background noise. With that said, it is not a guarantee that a police officer will not pull you over with a pair of these on.
q: Can people with hearing loss enjoy them?
If you recall back to how bone conduction works, in the first place, then it should not surprise you to know that the answer to this question is yes. Remember, the sound is going to bypass the eardrums and, thus, users with hearing loss will still be able to enjoy the emitted sound. And, truth be told, hearing aids will not even be required for some users.


So make sure to complete each one before challenging the quiz!
Rescue Tyler before it's too late!
Help Tyler complete the maintenance checks.
Get the plant up and running!
Gary has had enough of those pumps! Help him out.
See if you are really a qualified electrical worker!


a Polish economist (Ph.D.), computer scientist, senior IT security specialist at IBM, and assistent professor at University of Łódź, and before, professor at Technical University of Łódź and lecturer at Poznań University of Economics and Business. His research interests include analysis of time series along with exponential smoothing as well as pseudorandom number generators.
Maciej Szmit organized the Polish Computer Chess Championships, and is author of the Akiba chess program and WBUnit, a Delphi unit for Chess Engine Communication Protocol support .


I HAVE A LAPTOP! Which means I can get back to blogging and videos, finally!
I have also just got back yesterday from a week in the sun with my mum doing nothing but reading, sunbathing and drinking cocktails, pure bliss! But I also feel that I am massively behind because I want to do blog posts all about my trip to California in September that I wasn't able to do when I got home due to the lack of a laptop.
So I thought I would start with the one and only 'kind of' outfit photos that I got while I was in Cali. I had planned to do loads of outfit photos but with all the sightseeing and running around we were doing I didn't really have time to take any outfit photos so instead I am going to put together posts about what we did and where we stayed etc. If you're interested in any of that kind of stuff then those posts should be going up soon!
These photos were taken on our last day of the trip in LA by the Instagram-famous pink wall of Melrose Avenue. After seeing it on so many of the people I follow's Instagrams it was definitely on my must-see list for the trip. It really is crazy bright in real-life and we did spot quite a few people heading there to take pictures. It's the side of the Paul Smith building and is deffo one to check out if you're on Melrose and fancy a few snaps!
I did take a few outfit photos on the holiday with my mum but I think I'll post things chronologically to catch up, so expect lots of California posts, followed by outfit posts from the sunny shores of Cape Verde coming up on my blog.


text:
Mapping Jewish San Francisco
The House of Love and Prayer
Exhibition | The House of Love and Prayer
Confluence of Time and Place
BY OREN KROLL-ZELDIN
A study session at the House of Love and Prayer, 1973. Photo courtesy of Yehudit and Reuven Goldfarb.
A Brief History of the House of Love and Prayer
Carlebach and Controversy
Everyday Life at the House of Love and Prayer
Shlomo Carlebach's Role
Getting High on Torah and Prayer
Food and Shabbat Meals
Financial Difficulties and Fundraising
The Mechitzah Controversy
Life Magazine Wedding
The Holy Beggars' Gazette
Global Offshoots of the House of Love and Prayer
This diagram provides a visual representation of how the House of Love and Prayer could only have existed at this particular place and time.
Despite the seemingly impossible task of fusing tradition with a Countercultural Hippie movement that bucked convention, the House of Love and Prayer successfully mixed Jewish customs and practices with the Hippie Counterculture in a way that was perhaps only possible in the late 1960s Haight-Ashbury district of San Francisco.
Aryae Coopersmith, co-founder of the House of Love and Prayer, discusses what drew people to the House and why it was a particular moment in history that …
In a sense, the story of the House of Love and Prayer actually begins during the Shoah—the Jewish genocide of WWII—and not in 1968 or even 1949. In immigrating to the United States in 1939 and 1941, respectively, both Shlomo Carlebach and Zalman Schachter escaped the Nazi regime. After arriving in the United States they each wanted to play a role in rebuilding in the United States the decimated European Jewish community. Eventually, the Sixth Lubavitch Rebbe, Yosef Yitzchak Schneersohn, sent the two of them on a mission to meet Jewish students on college campuses across the United States in order to bring non-Orthodox Jews "back" to Judaism. The Rebbe felt a sense of urgency as he believed that American materialistic culture would destroy the souls of Jews in the same way that the Nazis destroyed the bodies of the Jews. The Rebbe wanted Shlomo and Zalman to meet and engage these Jews before it was too late.[1] Over the next decade Shlomo and Zalman traveled across the United States developing their missionary bonafides.
Meanwhile, with the increase in popularity of the Beatniks and Hippie movements, in the 1950s and 1960s respectively, people from all over the world were arriving in San Francisco. Many of those who came to San Francisco were spiritual seekers and/or individuals looking to liberate themselves from the constraints of post-WWII America, the Korean War, or the Vietnam War American society.
By the time Carlebach came to the San Francisco Bay Area in 1966, for the Berkeley Folk Festival, San Francisco and those living there were open and ready for what he had to offer. In particular, young Jews were open to new pathways of Jewish living. Along with other spiritual teachers and healers of the time, such as Sufi Sam, who frequently visited the House, and the renowned rock band the Grateful Dead, who were based in Haight-Ashbury and, on occasion, with whom Shlomo reportedly jammed, Shlomo was successfully able to tap into the collective energy and desire for spirituality already present among youth, particularly the Jewish youth.
America of the 1960s and 1970s also had another popular trend, primarily taking place in urban settings: the rise of new religious movements. Some of these groups, such as ISKCON (more commonly known as the Hare Krishnas), the Church of Scientology, and Transcendental Meditation, offered the American public religious ideas and forms of religious practice previously unknown to American society. Others served as revitalization movements within established American religious traditions, and were new in organization but not in core religious tenets. The Christian American and Jewish American communities saw many of these revitalization groups within their respective milieus. Though quite different in form and practice from groups like Aish Hatorah, another group that emerged at this time, the House of Love and Prayer should be seen within this historical context as well.[2]
Aryae Coopersmith discusses how the House of Love and Prayer was able to capture the magic of San Francisco in the late 1960s.
The House of Love and Prayer could only have taken root in San Francisco in the late 1960s. It emerged at this specific time and place as a direct outgrowth of multiple forces that came together perfectly: the Counterculture movement, Hippies, New Religious Movements, Post-Holocaust American Judaism, a charismatic leader, and the city of San Francisco. This is further reflected by the fact that the House of Love and Prayer fell apart and disbanded by the late 1970s, the same time that the Counterculture movement was waning.
[1] Interview with member of the House of Love and Prayer, April 2017.
[2] Aaron J. Tapper, "The 'Cult' of Aish Hatorah: Ba'alei Teshuva and the New Religious Movement Phenomenon," Jewish Journal of Sociology, 44, Nos. 1-2 (2002) 5-29.
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