Bacteriophage-Antibiotic Combination Therapy against Pseudomonas aeruginosa.

Phage therapy is an alternative therapy that is being used as the last resource against infections caused by multidrug-resistant bacteria after the failure of standard treatments. Pseudomonas aeruginosa can cause pneumonia, septicemia, urinary tract, and surgery site infections mainly in immunocompromised people, although it can cause infections in many different patient profiles. Cystic fibrosis patients are particularly vulnerable. In vitro and in vivo studies of phage therapy against P. aeruginosa include both bacteriophages alone and combined with antibiotics. However, the former is the most promising strategy utilized in clinical infections. This review summarizes the recent studies of phage-antibiotic combinations, highlighting the synergistic effects of in vitro and in vivo experiments and successful treatments in patients.

Alternatives to Antibiotics against Mycobacterium abscessus.

Mycobacterium abscessus complex is extremely difficult to treat. Intrinsic and acquired bacterial resistance makes this species one of the most challenging pathogens and treatments last from months to years, associated with potential risky antibiotic toxicity and a high number of failures. Nonantibiotic antimicrobial agents against this microorganism have recently been studied so as to offer an alternative to current drugs. This review summarizes recent research on different strategies such as host modulation using stem cells, photodynamic therapy, antibiofilm therapy, phage therapy, nanoparticles, vaccines and antimicrobial peptides against M. abscessus both in vitro and in vivo.

Corrigendum: Considerations for Phage Therapy Against Mycobacterium abscessus.

[This corrects the article DOI: 10.3389/fmicb.2020.609017.].

Advances in Bacteriophage Therapy against Relevant MultiDrug-Resistant Pathogens.

The increase of multiresistance in bacteria and the shortage of new antibiotics in the market is becoming a major public health concern. The World Health Organization (WHO) has declared critical priority to develop new antimicrobials against three types of bacteria: carbapenem-resistant A. baumannii, carbapenem-resistant P. aeruginosa and carbapenem-resistant and ESBL-producing Enterobacteriaceae. Phage therapy is a promising alternative therapy with renewed research in Western countries. This field includes studies in vitro, in vivo, clinical trials and clinical cases of patients receiving phages as the last resource after failure of standard treatments due to multidrug resistance. Importantly, this alternative treatment has been shown to be more effective when administered in combination with antibiotics, including infections with biofilm formation. This review summarizes the most recent studies of this strategy in animal models, case reports and clinical trials to deal with infections caused by resistant A. baumannii, K. pneumoniae, E. coli, and P. aeruginosa strains, as well as discusses the main limitations of phage therapy.

Phages in Food Industry Biocontrol and Bioremediation.

Bacteriophages are ubiquitous in nature and their use is a current promising alternative in biological control. Multidrug resistant (MDR) bacterial strains are present in the livestock industry and phages are attractive candidates to eliminate them and their biofilms. This alternative therapy also reduces the non-desirable effects produced by chemicals on food. The World Health Organization (WHO) estimates that around 420,000 people die due to a foodborne illness annually, suggesting that an improvement in food biocontrol is desirable. This review summarizes relevant studies of phage use in biocontrol focusing on treatments in live animals, plants, surfaces, foods, wastewaters and bioremediation.

Considerations for Phage Therapy Against Mycobacterium abscessus.

There is a global increasing number of Mycobacterium abscessus infections, especially pulmonary infections. Reduced therapeutic options exist against this opportunistic pathogen due to its high intrinsic and acquired levels of antibiotic resistance. Phage therapy is a promising afresh therapy, which uses viruses to lyse bacteria responsible for the infection. Bacteriophages have been recently administered under compassionate use to a 15-year-old patient infected with M. abscessus in combination with antibiotics with excellent results. This mini review highlights different recommendations for future phage administrations such as where to look for new phages, the use of cocktail of mycobacteriophages to broaden phage specificity and to tackle resistance and phage insensitivity due to temperate phages present in bacterial genomes, the combined use of phages and antibiotics to obtain a synergistic effect, the liposomal administration to reach a prolonged effect, intracellular delivery and protection against neutralizing antibodies, and the convenience of using this strategy in patients suffering from cystic fibrosis (CF) since phages are believed to promote immunomodulatory actions and eliminate biofilms.

A lipopolysaccharide-free outer membrane vesicle vaccine protects against Acinetobacter baumannii infection.

Outer membrane vesicles (OMVs) were isolated from an Acinetobacter strain deficient in lipopolysaccharide (LPS) due to a mutation in lpxD (IB010). Two immunizations with 10 µg of IB010 OMVs elicited total IgG, IgM, IgG1 and IgG2c titers similar to those observed after immunization with OMVs derived from the parental strain (ATCC 19606), and IB010 OMVs plus purified LPS. Immunization with IB010 OMVs resulted in significantly reduced post-infection spleen bacterial loads and serum IL-1ß and IL-6 levels compared to control mice in a disseminated sepsis model. Mice immunized with 10 µg IB010 OMVs demonstrated significant, but partial, protection (75%) against infection, whereas mice immunized with ATCC 19606 OMVs or IB010 OMVs plus purified LPS were completely protected. Immunization of mice with 100 µg of IB010 OMVs completely protected mice from infection. This study demonstrates that LPS deficient A. baumannii produces OMVs, and that immunization with these OMVs elicits protective immunity against infection.

Deciphering GRINA/Lifeguard1: Nuclear Location, Ca2+ Homeostasis and Vesicle Transport.

The Glutamate Receptor Ionotropic NMDA-Associated Protein 1 (GRINA) belongs to the Lifeguard family and is involved in calcium homeostasis, which governs key processes, such as cell survival or the release of neurotransmitters. GRINA is mainly associated with membranes of the endoplasmic reticulum, Golgi, endosome, and the cell surface, but its presence in the nucleus has not been explained yet. Here we dissect, with the help of different software tools, the potential roles of GRINA in the cell and how they may be altered in diseases, such as schizophrenia or celiac disease. We describe for the first time that the cytoplasmic N-terminal half of GRINA (which spans a Proline-rich domain) contains a potential DNA-binding sequence, in addition to cleavage target sites and probable PY-nuclear localization sequences, that may enable it to be released from the rest of the protein and enter the nucleus under suitable conditions, where it could participate in the transcription, alternative splicing, and mRNA export of a subset of genes likely involved in lipid and sterol synthesis, ribosome biogenesis, or cell cycle progression. To support these findings, we include additional evidence based on an exhaustive review of the literature and our preliminary data of the protein-protein interaction network of GRINA.

A Live Salmonella Vaccine Delivering PcrV through the Type III Secretion System Protects against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a common Gram-negative opportunistic pathogen that is intrinsically resistant to a wide range of antibiotics. The development of a broadly protective vaccine against P. aeruginosa remains a major challenge. Here, we used an attenuated strain of Salmonella enterica serovar Typhimurium as a vehicle to express P. aeruginosa antigens. A fusion between the S. enterica type III secretion effector protein SseJ and the P. aeruginosa antigen PcrV expressed under the control of the sseA promoter was translocated by Salmonella into host cells in vitro and elicited the generation of specific antibodies in mice. Mice immunized with attenuated Salmonella expressing this fusion had reduced bacterial loads in the spleens and lungs and lower serum levels of proinflammatory cytokines than control mice after P. aeruginosa infection. Importantly, immunized mice also showed significantly enhanced survival in this model. These results suggest that type III secretion effectors of S. enterica are appropriate carriers in the design of a live vaccine to prevent infections caused by P. aeruginosaIMPORTANCE The Gram-negative bacterium Pseudomonas aeruginosa is an important opportunistic pathogen that causes infections in cystic fibrosis and hospitalized patients. Therapeutic treatments are limited due to the emergence and spread of new antibiotic-resistant strains. In this context, the development of a vaccine is a priority. Here, we used an attenuated strain of Salmonella enterica serovar Typhimurium as a vehicle to express and deliver the Pseudomonas antigen PcrV. This vaccine induced the generation of specific antibodies in mice and protected them from lethal infections with P. aeruginosa This is an important step toward the development of an effective vaccine for the prevention of infections caused by P. aeruginosa in humans.

Carrion's disease: more than a neglected disease.

Infections with Bartonella bacilliformis result in Carrion's disease in humans. In the first phase of infection, the pathogen causes a hemolytic fever ("Oroya fever") with case-fatality rates as high as ~90% in untreated patients, followed by a chronical phase resulting in angiogenic skin lesions ("verruga peruana"). Bartonella bacilliformis is endemic to South American Andean valleys and is transmitted via sand flies (Lutzomyia spp.). Humans are the only known reservoir for this old disease and therefore no animal infection model is available. In the present review, we provide the current knowledge on B. bacilliformis and its pathogenicity factors, vectors, possible unknown reservoirs, established and potential infection models and immunological aspects of the disease.

Immunization with lipopolysaccharide-free outer membrane complexes protects against Acinetobacter baumannii infection.

Outer membrane complex (OMC) vaccines, which contain antigens from the bacterial outer membrane, have been developed for multiple Gram-negative bacteria. However, OMC vaccines demonstrate high endotoxin activity due to the presence of lipopolysaccharide in the bacterial outer membrane, thus precluding their use in humans. We isolated OMCs from an LPS-deficient strain of A. baumannii (IB010) which completely lacks LPS due to a mutation in the lpxD gene. OMCs from IB010 demonstrated a more than 10,000-fold reduction in endotoxin activity compared to OMCs from wild type A. baumannii. Vaccination with IB010 OMCs produced similar levels of antigen-specific IgG and IgM after two administrations compared to wild type OMCs, and resulted in a similar reduction in post-infection spleen bacterial loads and serum pro-inflammatory cytokine levels. Vaccination with IB010 OMCs provided significant protection against infection compared to control mice, indicating the LPS-free OMCs could contribute to vaccine strategies for preventing infection by A. baumannii.

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

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

CHROMagar mSuperCARBA and RAPIDEC® Carba NP test for detection of carbapenemase-producing Enterobacteriaceae.

A novel chromogenic medium CHROMagar mSuperCARBA was evaluated to detect carbapenem-resistant Gram-negatives. This medium is as sensitive and as specific as the SUPERCARBA medium for detecting KPC, MBL and OXA-48-type producers (100% and 100%, respectively) and is compatible with subsequent testing of carbapenemase activity using the RAPIDEC® CARBA NP.

Inhibition of LpxC Increases Antibiotic Susceptibility in Acinetobacter baumannii.

LpxC inhibitors have generally shown poor in vitro activity against Acinetobacter baumannii We show that the LpxC inhibitor PF-5081090 inhibits lipid A biosynthesis, as determined by silver staining and measurements of endotoxin levels, and significantly increases cell permeability. The presence of PF-5081090 at 32 mg/liter increased susceptibility to rifampin, vancomycin, azithromycin, imipenem, and amikacin but had no effect on susceptibility to ciprofloxacin and tigecycline. Potentiating existing antibiotics with LpxC inhibitors may represent an alternative treatment strategy for multidrug-resistant A. baumannii.

Impaired growth under iron-limiting conditions associated with the acquisition of colistin resistance in Acinetobacter baumannii.

Acquisition of colistin resistance in Acinetobacter baumannii has been associated with reduced bacterial fitness and virulence, although the mechanisms underlying this fitness loss have not been well characterised. In this study, the role played by environmental iron levels on the growth and survival of colistin-resistant strains of A. baumannii was assessed. Growth assays with the colistin-susceptible ATCC 19606 strain and its colistin-resistant derivative RC64 [colistin minimum inhibitory concentration (MIC) of 64 mg/L] demonstrated that the strains grew similarly in rich laboratory medium (Mueller-Hinton broth), whereas RC64 demonstrated impaired growth compared with ATCC 19606 in human serum (>100-fold at 24 h). Compared with RC64, ATCC 19606 grew in the presence of higher concentrations of the iron-specific chelator 2,2'-bipyridine and grew more readily under iron-limiting conditions in solid and liquid media. In addition, iron supplementation of human serum increased the growth of RC64 compared with unsupplemented human serum to a greater extent than ATCC 19606. The ability of 11 colistin-resistant clinical isolates with mutations in the pmrB gene to grow in iron-replete and iron-limiting conditions was assessed, demonstrating that eight of the strains showed reduced growth under iron limitation. Individual mutations in the pmrB gene did not directly correlate with a decreased capacity for growth under iron limitation, suggesting that mutations in pmrB may not directly produce this phenotype. Together these results indicate that acquisition of colistin resistance in A. baumannii can be associated with a decreased ability to grow in low-iron environments.

Vaccines for Antibiotic-Resistant Bacteria: Possibility or Pipe Dream?

The increasing incidence of infections caused by antibiotic-resistant bacteria from multiple species, together with the paucity of new antibiotics in the development pipeline, indicates that vaccines could play a role in combating these infections. The development of vaccines for these infections presents unique challenges related to target population selection, vaccine administration, and antigen identification. Advances in genomic, transcriptomic, and proteomic technologies offer great potential for identifying promising antigens that are highly conserved and expressed during human infections. Although important challenges remain, the potential health and economic benefits associated with the clinical implementation of vaccination strategies for the prevention of antibiotic-resistant infections warrant their continued development.

Identifying targets for antibiotic development using omics technologies.

The lack of new compounds in the antibiotic development pipeline together with the increasing incidence of infections caused by antibiotic-resistant bacteria on a global scale represents an alarming public health problem. Advances in genomic, transcriptomic and proteomic technologies permit the characterization of bacterial physiology at an unprecedented scale, and thus can facilitate the identification of bacterial factors that could serve as targets for the development of new antibiotics. Recent studies employing these technologies have permitted the elucidation of key components in multiple bacterial processes such as bacterial survival, persistence in the host and infection. The continued use of these approaches and the incorporation of emerging omics technologies hold great potential in elucidating high value targets for antibiotic development.

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

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

Immunization with lipopolysaccharide-deficient whole cells provides protective immunity in an experimental mouse model of Acinetobacter baumannii infection.

The increasing clinical importance of infections caused by multidrug resistant Acinetobacter baumannii warrants the development of novel approaches for prevention and treatment. In this context, vaccination of certain patient populations may contribute to reducing the morbidity and mortality caused by this pathogen. Vaccines against Gram-negative bacteria based on inactivated bacterial cells are highly immunogenic and have been shown to produce protective immunity against a number of bacterial species. However, the high endotoxin levels present in these vaccines due to the presence of lipopolysaccharide complicates their use in human vaccination. In the present study, we used a laboratory-derived strain of A. baumannii that completely lacks lipopolysaccharide due to a mutation in the lpxD gene (IB010), one of the genes involved in the first steps of lipopolysaccharide biosynthesis, for vaccination. We demonstrate that IB010 has greatly reduced endotoxin content (<1.0 endotoxin unit/106 cells) compared to wild type cells. Immunization with formalin inactivated IB010 produced a robust antibody response consisting of both IgG1 and IgG2c subtypes. Mice immunized with IB010 had significantly lower post-infection tissue bacterial loads and significantly lower serum levels of the pro-inflammatory cytokines IL-1ß, TNF-α and IL-6 compared to control mice in a mouse model of disseminated A. baumannii infection. Importantly, immunized mice were protected from infection with the ATCC 19606 strain and an A. baumannii clinical isolate. These data suggest that immunization with inactivated A. baumannii whole cells deficient in lipopolysaccharide could serve as the basis for a vaccine for the prevention of infection caused by A. baumannii.

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

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