A phage nucleus-associated protein from the jumbophage Churi inhibits bacterial growth through protein translation interference.

Antibacterial proteins inhibiting Pseudomonas aeruginosa have been identified in various phages and explored as antibiotic alternatives. Here, we isolated a phiKZ-like phage, Churi, which encodes 364 open reading frames. We examined 15 early-expressed phage proteins for their ability to inhibit bacterial growth, and found that gp335, closely related to phiKZ-gp14, exhibits antibacterial activity. Similar to phiKZ-gp14, recently shown to form a complex with the P. aeruginosa ribosome, we predict experimentally that gp335 interacts with ribosomal proteins, suggesting its involvement in protein translation. GFP-tagged gp335 clusters around the phage nucleus as early as 15 minutes post-infection and remains associated with it throughout the infection, suggesting its role in protein expression in the cell cytoplasm. CRISPR-Cas13-mediated deletion of gp355 reveals that the mutant phage has a prolonged latent period. Altogether, we demonstrate that gp335 is an antibacterial protein of nucleus-forming phages that associates with the ribosomes at the phage nucleus.

Anti-CAMP1 IgG promotes macrophage phagocytosis of Cutibacterium acnes type II.

Among 5 types of the Christie-Atkins-Munch-Petersen factor (CAMP) of Cutibacterium acnes, CAMP1 is highly expressed in phylotype II as well as IB, and thought to be a virulence factor of opportunistic but fatal blood, soft tissue, and implant-related infections. The target of a human single-chain variable antibody fragment (scFv), recently isolated from a phage display library, has been identified as CAMP1 of phylotype II, using immunoprecipitation followed by mass spectrometry, phage display peptide biopanning, 3D-modelling, and ELISA. The IgG1 format of the antibody could enhance phagocytosis of C. acnes DMST 14916 by THP-1 human monocytes. Our results suggest that the antibody-dependent phagocytosis process is mediated by the caveolae membrane system and involves the induction of IL-1ß. This is the first report on the study of a human antibody against CAMP1 of C. acnes phylotype II, of which a potential use as therapeutic antibody against virulence C. acnes infection is postulated.

Nucleus-forming jumbophage PhiKZ therapeutically outcompetes non-nucleus-forming jumbophage Callisto.

With the recent resurgence of phage therapy in modern medicine, jumbophages are currently under the spotlight due to their numerous advantages as anti-infective agents. However, most significant discoveries to date have primarily focused on nucleus-forming jumbophages, not their non-nucleus-forming counterparts. In this study, we compare the biological characteristics exhibited by two genetically diverse jumbophages: 1) the well-studied nucleus-forming jumbophage, PhiKZ; and 2) the newly discovered non-nucleus-forming jumbophage, Callisto. Single-cell infection studies further show that Callisto possesses different replication machinery, resulting in a delay in phage maturation compared to that of PhiKZ. The therapeutic potency of both phages was examined in vitro and in vivo, demonstrating that PhiKZ holds certain superior characteristics over Callisto. This research sheds light on the importance of the subcellular infection machinery and the organized progeny maturation process, which could potentially provide valuable insight in the future development of jumbophage-based therapeutics.

Phage-induced bacterial morphological changes reveal a phage-derived antimicrobial affecting cell wall integrity.

In a looming post-antibiotic era, antibiotic alternatives have become key players in the combat against pathogens. Although recent advances in genomic research allow scientists to fully explore an organism's genome in the search for novel antibacterial molecules, laborious work is still needed in order to dissect each individual gene product for its antibacterial activity. Here, we exploited phage-induced bacterial morphological changes as anchors to explore and discover a potential phage-derived antimicrobial embedded in the phage genome. We found that, upon vibriophage KVP40 infection, Vibrio parahaemolyticus exhibited morphological changes similar to those observed when treated with mecillinam, a cell wall synthesis inhibitor, suggesting the mechanism of pre-killing that KVP40 exerts inside the bacterial cell upon sieging the host. Genome analysis revealed that, of all the annotated gene products in the KVP40 genome that are involved in cell wall degradation, lytic transglycosylase (LT) is of particular interest for subsequent functional studies. A single-cell morphological analysis revealed that heterologous expression of wild-type KVP40-LT induced similar bacterial morphological changes to those treated with the whole phage or mecillinam, prior to cell burst. On the contrary, neither the morphology nor the viability of the bacteria expressing signal-peptide truncated- or catalytic mutant E80A- KVP40-LT was affected, suggesting the necessity of these domains for the antibacterial activities. Altogether, this research paves the way for the future development of the discovery of phage-derived antimicrobials that is guided through phage-induced morphological changes.

Nucleus-forming vibriophage cocktail reduces shrimp mortality in the presence of pathogenic bacteria.

The global aquaculture industry has suffered significant losses due to the outbreak of Acute Hepatopancreatic Necrosis Disease (AHPND) caused by Vibrio parahaemolyticus. Since the use of antibiotics as control agents has not been shown to be effective, an alternative anti-infective regimen, such as phage therapy, has been proposed. Here, we employed high-throughput screening for potential phages from 98 seawater samples and obtained 14 phages exhibiting diverse host specificity patterns against pathogenic VPAHPND strains. Among others, two Chimallinviridae phages, designated Eric and Ariel, exhibited the widest host spectrum against vibrios. In vitro and in vivo studies revealed that a cocktail derived from these two nucleus-forming vibriophages prolonged the bacterial regrowth of various pathogenic VPAHPND strains and reduced shrimp mortality from VPAHPND infection. This research highlights the use of high-throughput phage screening that leads to the formulation of a nucleus-forming phage cocktail applicable for bacterial infection treatment in aquaculture.

A novel coli myophage and antibiotics synergistically inhibit the growth of the uropathogenic E. coli strain CFT073 in stoichiometric niches.

Urinary tract infections are widespread bacterial infections affecting millions of people annually, with Escherichia coli being the most prevalent. Although phage therapy has recently gained interest as a promising alternative therapy for antibiotic-resistant bacteria, several studies have raised concerns regarding the evolution of phage resistance, making the therapy ineffective. In this study, we discover a novel coli myophage designated as Killian that targets E. coli strains, including the uropathogenic E. coli (UPEC) strain CFT073. It requires at least 20 minutes for 90% of its particles to adsorb to the host cells, undergoes subcellular activities for replication for 30 minutes, and eventually lyses the cells with a burst size of about 139 particles per cell. Additionally, Killian can withstand a wide variety of temperatures (4-50°C) and pHs (4-10). Genome analysis reveals that Killian's genome consists of 169,905 base pairs with 35.5% GC content, encoding 276 open reading frames; of these, 209 are functionally annotated with no undesirable genes detected, highlighting its potential as an antibiotic alternative against UPEC. However, after an 8-hour phage treatment at high multiplicities of infection, bacterial density continuously increases, indicating an onset of bacterial growth revival. Thus, the combination study between the phage and three different antibiotics, including amikacin, ciprofloxacin, and piperacillin, was performed and showed that certain pairs of phage and antibiotics exhibited synergistic interactions in suppressing the bacterial growth revival. These findings suggest that Killian-antibiotic combinations are effective in inhibiting the growth of UPEC. IMPORTANCE Phage therapy has recently been in the spotlight as a viable alternative therapy for bacterial infections. However, several studies have raised concerns about the emergence of phage resistance that occurs during treatment, making the therapy not much effective. Here, we present the discovery of a novel E. coli myophage that, by itself, can effectively kill the uropathogenic E. coli, but the emergence of bacterial growth revival was detected during the treatment. Phage and antibiotics are then combined to improve the efficiency of the phage in suppressing the bacterial re-growth. This research would pave the way for the future development of phage-antibiotic cocktails for the sustainable use of phages for therapeutic purposes.

Combination of genetically diverse Pseudomonas phages enhances the cocktail efficiency against bacteria.

Phage treatment has been used as an alternative to antibiotics since the early 1900s. However, bacteria may acquire phage resistance quickly, limiting the use of phage treatment. The combination of genetically diverse phages displaying distinct replication machinery in phage cocktails has therefore become a novel strategy to improve therapeutic outcomes. Here, we isolated and studied lytic phages (SPA01 and SPA05) that infect a wide range of clinical Pseudomonas aeruginosa isolates. These relatively small myophages have around 93 kbp genomes with no undesirable genes, have a 30-min latent period, and reproduce a relatively high number of progenies, ranging from 218 to 240 PFU per infected cell. Even though both phages lyse their hosts within 4 h, phage-resistant bacteria emerge during the treatment. Considering SPA01-resistant bacteria cross-resist phage SPA05 and vice versa, combining SPA01 and SPA05 for a cocktail would be ineffective. According to the decreased adsorption rate of the phages in the resistant isolates, one of the anti-phage mechanisms may occur through modification of phage receptors on the target cells. All resistant isolates, however, are susceptible to nucleus-forming jumbophages (PhiKZ and PhiPA3), which are genetically distinct from phages SPA01 and SPA05, suggesting that the jumbophages recognize a different receptor during phage entry. The combination of these phages with the jumbophage PhiKZ outperforms other tested combinations in terms of bactericidal activity and effectively suppresses the emergence of phage resistance. This finding reveals the effectiveness of the diverse phage-composed cocktail for reducing bacterial growth and prolonging the evolution of phage resistance.

Roles of qseC mutation in bacterial resistance against anti-lipopolysaccharide factor isoform 3 (ALFPm3).

Propelled by global climate changes, the shrimp industry has been facing tremendous losses in production due to various disease outbreaks, particularly early mortality syndrome (EMS), a disease caused by Vibrio parahaemolyticus AHPND. Not only is the use of antibiotics as EMS control agents not yet been proven successful, but the overuse and misuse of antibiotics could also worsen one of the most challenging global health issues-antimicrobial resistance. To circumvent antibiotic usage, anti-lipopolysaccharide factor isoform 3 (ALFPm3), an antimicrobial peptide (AMP) derived from the shrimp innate immune system, was proposed as an antibiotic alternative for EMS control. However, prolonged use of AMPs could also lead to bacterial cross resistance with life-saving antibiotics used in human diseases. Here, we showed that ALFPm3-resistant strains of E. coli could be induced in vitro. Genome analysis of the resistant mutants revealed multiple mutations, with the most interesting being a qseC(L299R). A study of antibiotic susceptibility profile showed that the resistant strains harboring the qseC(L299R) not only exhibited higher degree of resistance towards polymyxin antibiotics, but also produced higher biofilm under ALFPm3 stress. Lastly, a single cell death analysis revealed that, at early-log phase when biofilm is scarce, the resistant strains were less affected by ALFPm3 treatment, suggesting additional mechanisms by which qseC orchestrates to protect the bacteria from ALFPm3. Altogether, this study uncovers involvement of qseC mutation in mechanism of resistance of the bacteria against ALFPm3 paving a way for future studies on sustainable use of ALFPm3 as an EMS control agent.

High-Resolution Bacterial Cytological Profiling Reveals Intrapopulation Morphological Variations upon Antibiotic Exposure.

Phenotypic heterogeneity is crucial to bacterial survival and could provide insights into the mechanism of action (MOA) of antibiotics, especially those with polypharmacological actions. Although phenotypic changes among individual cells could be detected by existing profiling methods, due to the data complexity, only population average data were commonly used, thereby overlooking the heterogeneity. In this study, we developed a high-resolution bacterial cytological profiling method that can capture morphological variations of bacteria upon antibiotic treatment. With an unprecedented single-cell resolution, this method classifies morphological changes of individual cells into known MOAs with an overall accuracy above 90%. We next showed that combinations of two antibiotics induce altered cell morphologies that are either unique or similar to that of an antibiotic in the combinations. With these combinatorial profiles, this method successfully revealed multiple cytological changes caused by a natural product-derived compound that, by itself, is inactive against Acinetobacter baumannii but synergistically exerts its multiple antibacterial activities in the presence of colistin. The findings have paved the way for future single-cell profiling in bacteria and have highlighted previously underappreciated intrapopulation variations caused by antibiotic perturbation.

Phage-resistant Pseudomonas aeruginosa against a novel lytic phage JJ01 exhibits hypersensitivity to colistin and reduces biofilm production.

Pseudomonas aeruginosa, a major cause of nosocomial infections, has been categorized by World Health Organization as a critical pathogen urgently in need of effective therapies. Bacteriophages or phages, which are viruses that specifically kill bacteria, have been considered as alternative agents for the treatment of bacterial infections. Here, we discovered a lytic phage targeting P. aeruginosa, designated as JJ01, which was classified as a member of the Myoviridae family due to the presence of an icosahedral capsid and a contractile tail under TEM. Phage JJ01 requires at least 10 min for 90% of its particles to be adsorbed to the host cells and has a latent period of 30 min inside the host cell for its replication. JJ01 has a relatively large burst size, which releases approximately 109 particles/cell at the end of its lytic life cycle. The phage can withstand a wide range of pH values (3-10) and temperatures (4-60°C). Genome analysis showed that JJ01 possesses a complete genome of 66,346 base pairs with 55.7% of GC content, phylogenetically belonging to the genus Pbunavirus. Genome annotation further revealed that the genome encodes 92 open reading frames (ORFs) with 38 functionally predictable genes, and it contains neither tRNA nor toxin genes, such as drug-resistant or lysogenic-associated genes. Phage JJ01 is highly effective in suppressing bacterial cell growth for 12 h and eradicating biofilms established by the bacteria. Even though JJ01-resistant bacteria have emerged, the ability of phage resistance comes with the expense of the bacterial fitness cost. Some resistant strains were found to produce less biofilm and grow slower than the wild-type strain. Among the resistant isolates, the resistant strain W10 which notably loses its physiological fitness becomes eight times more susceptible to colistin and has its cell membrane compromised, compared to the wild type. Altogether, our data revealed the potential of phage JJ01 as a candidate for phage therapy against P. aeruginosa and further supports that even though the use of phages would subsequently lead to the emergence of phage-resistant bacteria, an evolutionary trade-off would make them more sensitive to antibiotics.

Mansonone G and its derivatives exhibit membrane permeabilizing activities against bacteria.

In an era where the rate of bacteria evolving to be resistant to clinically-used antibiotics far exceeds that of antibiotic discovery, the search for new sources of antibacterial agents has expanded tremendously. In recent years, interest in plant-based natural products as promising sources of antibacterial agents has taken an upward trend. Mansonones, botanically-derived naphthoqionones, having many uses in Asian traditional medicine-including anti-infective roles-have sparked interest as a possible source of antibacterial agents. Here, we show that mansonone G, extracted from Mansonia gagei Drumm. heartwoods, possessed antibacterial activities towards Bacillus subtilis, Staphylococcus aureus and Escherichia coli lptD4213, inhibiting the growth of the bacteria at 15.6 µM, 62.5 µM and 125 µM, respectively. Fourteen derivatives of mansonone G were synthesized successfully and were found to have a similar antibacterial spectrum to that of the parent compound, with some derivatives possessing improved antibacterial activities. Bacterial cytological profiling analysis showed that mansonone G harbors membrane permeabilizing activities against B. subtilis and E. coli lptD4213. Temporal analysis of SYTOX Green staining among individual cells showed that mansonone G rapidly permeabilized bacterial membrane within 10 min, with SYTOX Green intensity reaching 13-fold above that of the control. Collectively, these findings highlight the importance of mansonone G and its derivatives as potential antibacterial agents, paving the way for further modifications in order to improve their antibacterial spectrum.

Viral speciation through subcellular genetic isolation and virogenesis incompatibility.

Understanding how biological species arise is critical for understanding the evolution of life on Earth. Bioinformatic analyses have recently revealed that viruses, like multicellular life, form reproductively isolated biological species. Viruses are known to share high rates of genetic exchange, so how do they evolve genetic isolation? Here, we evaluate two related bacteriophages and describe three factors that limit genetic exchange between them: 1) A nucleus-like compartment that physically separates replicating phage genomes, thereby limiting inter-phage recombination during co-infection; 2) A tubulin-based spindle that orchestrates phage replication and forms nonfunctional hybrid polymers; and 3) A nuclear incompatibility factor that reduces phage fitness. Together, these traits maintain species differences through Subcellular Genetic Isolation where viral genomes are physically separated during co-infection, and Virogenesis Incompatibility in which the interaction of cross-species components interferes with viral production.

A novel vibriophage exhibits inhibitory activity against host protein synthesis machinery.

Since the emergence of deadly pathogens and multidrug-resistant bacteria at an alarmingly increased rate, bacteriophages have been developed as a controlling bioagent to prevent the spread of pathogenic bacteria. One of these pathogens, disease-causing Vibrio parahaemolyticus (VPAHPND) which induces acute hepatopancreatic necrosis, is considered one of the deadliest shrimp pathogens, and has recently become resistant to various classes of antibiotics. Here, we discovered a novel vibriophage that specifically targets the vibrio host, VPAHPND. The vibriophage, designated Seahorse, was classified in the family Siphoviridae because of its icosahedral capsid surrounded by head fibers and a non-contractile long tail. Phage Seahorse was able to infect the host in a broad range of pH and temperatures, and it had a relatively short latent period (nearly 30 minutes) in which it produced progeny at 72 particles per cell at the end of its lytic cycle. Upon phage infection, the host nucleoid condensed and became toroidal, similar to the bacterial DNA morphology seen during tetracycline treatment, suggesting that phage Seahorse hijacked host biosynthesis pathways through protein translation. As phage Seahorse genome encodes 48 open reading frames with many hypothetical proteins, this genome could be a potential untapped resource for the discovery of phage-derived therapeutic proteins.

Viral Capsid Trafficking along Treadmilling Tubulin Filaments in Bacteria.

Cargo trafficking along microtubules is exploited by eukaryotic viruses, but no such examples have been reported in bacteria. Several large Pseudomonas phages assemble a dynamic, tubulin-based (PhuZ) spindle that centers replicating phage DNA sequestered within a nucleus-like structure. Here, we show that capsids assemble on the membrane and then move rapidly along PhuZ filaments toward the phage nucleus for DNA packaging. The spindle rotates the phage nucleus, distributing capsids around its surface. PhuZ filaments treadmill toward the nucleus at a constant rate similar to the rate of capsid movement and the linear velocity of nucleus rotation. Capsids become trapped along mutant static PhuZ filaments that are defective in GTP hydrolysis. Our results suggest a transport and distribution mechanism in which capsids attached to the sides of filaments are trafficked to the nucleus by PhuZ polymerization at the poles, demonstrating that the phage cytoskeleton evolved cargo-trafficking capabilities in bacteria.

Bacterial Cytological Profiling as a Tool To Study Mechanisms of Action of Antibiotics That Are Active against Acinetobacter baumannii.

An increasing number of multidrug-resistant Acinetobacter baumannii (MDR-AB) infections have been reported worldwide, posing a threat to public health. The establishment of methods to elucidate the mechanism of action (MOA) of A. baumannii-specific antibiotics is needed to develop novel antimicrobial therapeutics with activity against MDR-AB We previously developed bacterial cytological profiling (BCP) to understand the MOA of compounds in Escherichia coli and Bacillus subtilis Given how distantly related A. baumannii is to these species, it was unclear to what extent it could be applied. Here, we implemented BCP as an antibiotic MOA discovery platform for A. baumannii We found that the BCP platform can distinguish among six major antibiotic classes and can also subclassify antibiotics that inhibit the same cellular pathway but have different molecular targets. We used BCP to show that the compound NSC145612 inhibits the growth of A. baumannii via targeting RNA transcription. We confirmed this result by isolating and characterizing resistant mutants with mutations in the rpoB gene. Altogether, we conclude that BCP provides a useful tool for MOA studies of antibacterial compounds that are active against A. baumannii.

Listeria monocytogenes endocarditis: case report, review of the literature, and laboratory evaluation of potential novel antibiotic synergies.

Endocarditis is a rare but serious manifestation of Listeria monocytogenes (LM). However, the optimal treatment strategy for LM endocarditis has yet to be established. Current antibiotic strategies for listeriosis include penicillin G or ampicillin (AMP) monotherapy, or AMP + gentamicin combination therapy which is often favored for endocarditis. The primary objective of our investigation was to assess the utility of AMP + ceftriaxone (CRO) and AMP + daptomycin (DAP) against LM, modeling less nephrotoxic antibiotic combinations traditionally used to manage resistant enterococcal endocarditis. Here we report a case of LM endocarditis, review the world literature, and evaluate alternative treatment strategies for listeriosis utilizing in vitro and ex vivo studies. The combination of AMP + CRO and AMP + DAP were each noted to have synergistic activity against a LM endocarditis isolate. Additionally, co-incubation of the isolate with sub-lethal concentrations of antibiotics (AMP, CRO, DAP, AMP + CRO or AMP + DAP) sensitized the bacterium to whole blood killing while pretreatment with CRO and DAP (at 1/4 MIC) sensitized the bacterium to neutrophil killing. However, these effects did not reflect potentiation of antibiotic activity to human cathelicidin peptide LL-37, which is abundant in neutrophils and highly active against LM. Interestingly, AMP pretreatment of the LM endocarditis isolate resulted in increased DAP binding to the bacterium when assessed by fluorescence microscopy. These in vitro and ex vivo studies suggest further investigation of combination therapy using AMP + CRO or AMP + DAP as an alternative treatment for LM infection is warranted.

Group A Streptococcal M1 Protein Provides Resistance against the Antimicrobial Activity of Histones.

Histones are essential elements of chromatin structure and gene regulation in eukaryotes. An unexpected attribute of these nuclear proteins is their antimicrobial activity. A framework for histone release and function in host defense in vivo was revealed with the discovery of neutrophil extracellular traps, a specialized cell death process in which DNA-based structures containing histones are extruded to ensnare and kill bacteria. Investigating the susceptibility of various Gram-positive pathogens to histones, we found high-level resistance by one leading human pathogen, group A Streptococcus (GAS). A screen of isogenic mutants revealed that the highly surface-expressed M1 protein, a classical GAS virulence factor, was required for high-level histone resistance. Biochemical and microscopic analyses revealed that the N-terminal domain of M1 protein binds and inactivates histones before they reach their cell wall target of action. This finding illustrates a new pathogenic function for this classic GAS virulence factor, and highlights a potential innate immune evasion strategy that may be employed by other bacterial pathogens.

Classical ß-Lactamase Inhibitors Potentiate the Activity of Daptomycin against Methicillin-Resistant Staphylococcus aureus and Colistin against Acinetobacter baumannii.

We asked whether beta-lactamase inhibitors (BLIs) increased the activity of daptomycin (DAP) against methicillin-resistant Staphylococcus aureus (MRSA), the peptide antibiotic colistin (COL) against the emerging Gram-negative nosocomial pathogen Acinetobacter baumannii, and the human host defense peptide cathelicidin LL37 against either pathogen. DAP and LL37 kill curves were performed with or without BLIs against MRSA, vancomycin-intermediate S. aureus (VISA), and heterogeneous VISA (hVISA). COL and LL37 kill curves were performed against A. baumannii Boron-dipyrromethene (BODIPY)-labeled DAP binding to MRSA grown with the BLI tazobactam (TAZ) was assessed microscopically. The combination of COL plus TAZ was studied in a murine model of A. baumannii pneumonia. TAZ alone lacked in vitro activity against MRSA or A. baumannii The addition of TAZ to DAP resulted in a 2- to 5-log10 reduction in recoverable MRSA CFU at 24 h compared to the recoverable CFU with DAP alone. TAZ plus COL showed synergy by kill curves for 4 of 5 strains of A. baumannii tested. Growth with 20 mg/liter TAZ resulted in 2- to 2.5-fold increases in the intensity of BODIPY-DAP binding to MRSA and hVISA strains. TAZ significantly increased the killing of MRSA and A. baumannii by LL37 in vitro TAZ increased the activity of COL in a murine model of A. baumannii pneumonia. Classical BLIs demonstrate synergy with peptide antibiotics. Since BLIs have scant antimicrobial activity on their own and are thus not expected to increase selective pressure toward antibiotic resistance, their use in combination with peptide antibiotics warrants further study.

Fosfomycin Enhances the Activity of Daptomycin against Vancomycin-Resistant Enterococci in an In Vitro Pharmacokinetic-Pharmacodynamic Model.

Daptomycin (DAP) is being used more frequently to treat infections caused by vancomycin-resistant enterococcus (VRE). DAP tends to be less active against enterococci than staphylococci and may require high doses or combination therapy to be bactericidal. Fosfomycin (FOF) has activity against VRE and has demonstrated synergistic bactericidal activity with DAP in vitro The objective of this study was to evaluate the activity of DAP alone and in combination with FOF against VRE in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model. The activity of DAP at 8 and 12 mg/kg of body weight/day (DAP 8 and DAP 12, respectively) and FOF of 40 mg/kg intravenously every 8 h, alone and in combination, were evaluated against 2 vancomycin-resistant Enterococcus faecium strains (8019 and 5938) and 2 vancomycin-resistant E. faecalis strains (V583 and R7302) in an in vitro PK/PD model over 72 h. Cell surface charge in the presence and absence of FOF was evaluated by zeta potential analysis. Daptomycin-boron-dipyrromethene (bodipy) binding was assessed by fluorescence microscopy. The addition of FOF to DAP 8 and DAP 12 resulted in significantly increased killing over DAP alone at 72 h for 8019, V583, and R7302 (P < 0.05). Therapeutic enhancement was observed with DAP 12 plus FOF against 8019, V583, and R7302. Cell surface charge became more negative after exposure to FOF by ∼2 to 8mV in all 4 strains. Daptomycin-bodipy binding increased by 2.6 times in the presence of fosfomycin (P < 0.0001). The combination of DAP plus FOF may provide improved killing against VRE (including DAP-resistant strains) through modulation of cell surface charge. Further studies to clarify the role of intravenous FOF are warranted.

Standard susceptibility testing overlooks potent azithromycin activity and cationic peptide synergy against MDR Stenotrophomonas maltophilia.

OBJECTIVES: The Gram-negative bacillus Stenotrophomonas maltophilia (SM) is an emerging MDR opportunistic pathogen. Recent studies identify a potentially relevant activity of azithromycin against Gram-negative bacteria overlooked in standard bacteriological testing. We investigated azithromycin activity against SM in testing conditions incorporating mammalian tissue culture medium and host defence factors. METHODS: MIC testing, chequerboard assays, time-kill assays and fluorescence microscopy were performed for azithromycin, the cationic peptide antibiotic colistin and the human defence peptide cathelicidin LL-37 alone or in combination in cation-adjusted Mueller-Hinton broth or mammalian tissue culture media. Azithromycin sensitization of SM to host immune clearance was tested in a human neutrophil killing assay and a murine pneumonia model. RESULTS: We observed potent bactericidal activity of azithromycin against SM in mammalian tissue culture medium absent in bacteriological medium. Colistin and LL-37 strongly potentiated azithromycin killing of SM by increasing drug entry. Additionally, azithromycin sensitized SM to neutrophil killing and increased SM clearance in the murine pneumonia model. CONCLUSIONS: Despite lack of activity in standard MIC testing, azithromycin synergizes with cationic peptide antibiotics to kill SM in medium mimicking tissue fluid conditions. Azithromycin, alone or in combination with colistin, merits further exploration in therapy of drug-resistant SM infections.