Bactericidal effect of bacteria isolated from the marine sponges Hymeniacidon perlevis and Halichondria panicea against carbapenem-resistant Acinetobacter baumannii.

In this study, we evaluated the antimicrobial activity of bacteria isolated from the marine sponges Hymeniacidon perlevis and Halichondria panicea against seven Acinetobacter baumannii strains, the majority of which were clinically relevant carbapenem-resistant A. baumannii strains. We observed the inhibitory activity of 18 (out of 114) sponge-isolated bacterial strains against all A. baumanii strains using medium-throughput solid agar overlay assays. These inhibitory strains belonged to the genera Lactococcus, Pseudomonas, and Vagococcus. In addition, this antimicrobial activity was validated through a liquid co-cultivation challenge using an inhibitory strain of each genus and a green fluorescent protein-tagged A. baumanii strain. Fluorescence measurements indicated that the growth of A. baumanii was inhibited by the sponge isolates. In addition, the inability of A. baumanii to grow after spreading the co-cultures on solid medium allowed us to characterize the activity of the sponge isolates as bactericidal. In conclusion, this study demonstrates that marine sponges are a reservoir of bacteria that deserves to be tapped for antibiotic discovery against A. baumanii.

Synergistic Inhibitory Effect of Polymyxin B in Combination with Ceftazidime against Robust Biofilm Formed by Acinetobacter baumannii with Genetic Deficiency in AbaI/AbaR Quorum Sensing.

Carbapenem resistance of Acinetobacter baumannii poses challenges to public health. Biofilm contributes to the persistence of A. baumannii cells. This study was designed to investigate the genetic relationships among carbapenem resistance, polymyxin resistance, multidrug resistance, biofilm formation, and surface-associated motility and evaluate the antibiofilm effect of polymyxin in combination with other antibiotics. A total of 103 clinical A. baumannii strains were used to determine antibiotic susceptibility, biofilm formation capacity, and motility. Enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting was used to determine the genetic variation among strains. The distribution of 17 genes related to the resistance-nodulation-cell division (RND)-type efflux, autoinducer-receptor (AbaI/AbaR) quorum sensing, oxacillinases (OXA)-23, and insertion sequence of ISAba1 element was investigated. The representative strains were chosen to evaluate the gene transcription and the antibiofilm activity by polymyxin B (PB) in combination with merapenem, levofloxacin, and ceftazidime, respectively. ERIC-PCR-dependent fingerprints were found to be associated with carbapenem resistance and multidrug resistance. The presence of blaOXA-23 was found to correlate with genes involved in ISAba1 insertion, AbaI/AbaR quorum sensing, and AdeABC efflux. Carbapenem resistance was observed to be negatively correlated with biofilm formation and positively correlated with motility. PB in combination with ceftazidime displayed a synergistic antibiofilm effect against robust biofilm formed by an A. baumannii strain with deficiency in AbaI/AbaR quorum sensing. Our results not only clarify the genetic correlation among carbapenem resistance, biofilm formation, and pathogenicity in a certain level but also provide a theoretical basis for clinical applications of polymyxin-based combination of antibiotics in antibiofilm therapy. IMPORTANCE Deeper explorations of molecular correlation among antibiotic resistance, biofilm formation, and pathogenicity could provide novel insights that would facilitate the development of therapeutics and prevention against A. baumannii biofilm-related infections. The major finding that polymyxin B in combination with ceftazidime displayed a synergistic antibiofilm effect against robust biofilm formed by an A. baumannii strain with genetic deficiency in AbaI/AbaR quorum sensing further provides a theoretical basis for clinical applications of antibiotics in combination with quorum quenching in antibiofilm therapy.

In Vitro Activity of KBP-7072 against 536 Acinetobacter baumannii Complex Isolates Collected in China.

Acinetobacter baumannii has emerged globally as a difficult-to-treat nosocomial pathogen and become resistant to carbapenems, resulting in limited treatment options. KBP-7072 is a novel semisynthetic aminomethylcycline, expanded spectrum tetracycline antibacterial agent with completed phase 1 clinical development studies. This study aimed to evaluate the in vitro activity of KBP-7072 and several comparators against clinical A. baumannii isolates collected from China. A collection of 536 A. baumannii clinical isolates were isolated from 20 hospitals across 13 provinces and cities in China between 2018 and 2019. Antimicrobial susceptibility testing of 12 antimicrobial agents was performed utilizing the broth microdilution method recommended by CLSI. KBP-7072 has shown active antibacterial activity against 536 A. baumannii isolates. It inhibited the growth of all isolates at 4 mg/liter, including 372 carbapenem-resistant isolates, 37 tigecycline MIC ≥ 4 mg/liter isolates, and 138 omadacycline MIC ≥ 4 mg/liter isolates. Compared with other expanded spectrum tetracyclines, KBP-7072 (MIC90, 1 mg/liter) outperformed 2-fold and 4-fold more active against 536 A. baumannii isolates than tigecycline (MIC90, 2 mg/liter) and omadacycline (MIC90, 4 mg/liter). KBP-7072 was as equally active as colistin (MIC90, 1 mg/liter, 99.4% susceptible). Doxycycline (33.4% susceptible), gentamicin (31.3% susceptible), meropenem (30.6%, susceptible), imipenem (30.2% susceptible), ceftazidime (27.8% susceptible), piperacillin-tazobactam (27.2% susceptible), and levofloxacin (27.2% susceptible) showed marginally poor antibacterial activity against tested isolates according to CLSI breakpoints, except for minocycline (73.7% susceptible). KBP-7072 is a potential alternative agent for the treatment of infection caused by A. baumannii, including carbapenem-resistant species. IMPORTANCE It is reported that A. baumannii has emerged as an intractable nosocomial pathogen in hospitals especially when it develops resistance to carbapenems and other antibiotics, which limits treatment options and leads to high mortality. In February 2017, the WHO published a list of ESKAPE pathogens designated "priority status" for which new antibiotics are urgently needed. Therefore, the epidemiological surveillance and new therapeutic development of A. baumannii must be strengthened to confront an emerging global epidemic. KBP-7072 is a novel, expanded spectrum tetracycline antibacterial and has demonstrated good in vitro activity against recent geographically diverse A. baumannii isolates collected from North America, Europe, Latin America, and Asia-Pacific. This study has shown excellent in vitro activity of KBP-7072 against clinical A. baumannii isolates collected from different regions of China, regarded as supplementary to KBP-7072 pharmacodynamics data, which is of great significance, as it is promising an alternative treatment in CRAB isolates infections in China.

Amikacin potentiator activity of zinc complexed to a pyrithione derivative with enhanced solubility.

Resistance to amikacin in Gram-negatives is usually mediated by the 6'-N-acetyltransferase type Ib [AAC(6')-Ib], which catalyzes the transfer of an acetyl group from acetyl CoA to the 6' position of the antibiotic molecule. A path to continue the effective use of amikacin against resistant infections is to combine it with inhibitors of the inactivating reaction. We have recently observed that addition of Zn2+ to in-vitro enzymatic reactions, obliterates acetylation of the acceptor antibiotic. Furthermore, when added to amikacin-containing culture medium in complex to ionophores such as pyrithione (ZnPT), it prevents the growth of resistant strains. An undesired property of ZnPT is its poor water-solubility, a problem that currently affects a large percentage of newly designed drugs. Water-solubility helps drugs to dissolve in body fluids and be transported to the target location. We tested a pyrithione derivative described previously (Magda et al. Cancer Res 68:5318-5325, 2008) that contains the amphoteric group di(ethyleneglycol)-methyl ether at position 5 (compound 5002), a modification that enhances the solubility. Compound 5002 in complex with zinc (Zn5002) was tested to assess growth inhibition of amikacin-resistant Acinetobacter baumannii and Klebsiella pneumoniae strains in the presence of the antibiotic. Zn5002 complexes in combination with amikacin at different concentrations completely inhibited growth of the tested strains. However, the concentrations needed to achieve growth inhibition were higher than those required to achieve the same results using ZnPT. Time-kill assays showed that the effect of the combination amikacin/Zn5002 was bactericidal. These results indicate that derivatives of pyrithione with enhanced water-solubility, a property that would make them drugs with better bioavailability and absorption, are a viable option for designing inhibitors of the resistance to amikacin mediated by AAC(6')-Ib, an enzyme commonly found in the clinics.

A New Class of Cell Wall-Recycling l,d-Carboxypeptidase Determines ß-Lactam Susceptibility and Morphogenesis in Acinetobacter baumannii.

The hospital-acquired pathogen Acinetobacter baumannii possesses a complex cell envelope that is key to its multidrug resistance and virulence. The bacterium, however, lacks many canonical enzymes that build the envelope in model organisms. Instead, A. baumannii contains a number of poorly annotated proteins that may allow alternative mechanisms of envelope biogenesis. We demonstrated previously that one of these unusual proteins, ElsL, is required for maintaining a characteristic short rod shape and for withstanding antibiotics that attack the septal cell wall. Curiously, ElsL is composed of a leaderless YkuD-family domain usually found in secreted, cell wall-modifying l,d-transpeptidases (LDTs). Here, we show that, rather than being an LDT, ElsL is actually a new class of cytoplasmic l,d-carboxypeptidase (LDC) that provides a critical step in cell wall recycling previously thought to be missing from A. baumannii. Absence of ElsL impairs cell wall integrity, morphology, and intrinsic resistance due to buildup of murein tetrapeptide precursors, toxicity of which is bypassed by preventing muropeptide recycling. Multiple pathways in the cell become sites of vulnerability when ElsL is inactivated, including l,d-cross-link formation, cell division, and outer membrane lipid homoeostasis, reflecting its pleiotropic influence on envelope physiology. We thus reveal a novel class of cell wall-recycling LDC critical to growth and homeostasis of A. baumannii and likely many other bacteria. IMPORTANCE To grow efficiently, resist antibiotics, and control the immune response, bacteria recycle parts of their cell wall. A key step in the typical recycling pathway is the reuse of cell wall peptides by an enzyme known as an l,d-carboxypeptidase (LDC). Acinetobacter baumannii, an "urgent-threat" pathogen causing drug-resistant sepsis in hospitals, was previously thought to lack this enzymatic activity due to absence of a known LDC homolog. Here, we show that A. baumannii possesses this activity in the form of an enzyme class not previously associated with cell wall recycling. Absence of this protein intoxicates and weakens the A. baumannii cell envelope in multiple ways due to the accumulation of dead-end intermediates. Several other organisms of importance to health and disease encode homologs of the A. baumannii enzyme. This work thus reveals an unappreciated mechanism of cell wall recycling, manipulation of which may contribute to enhanced treatments targeting the bacterial envelope.

Acinetobacter baumannii Can Survive with an Outer Membrane Lacking Lipooligosaccharide Due to Structural Support from Elongasome Peptidoglycan Synthesis.

Gram-negative bacteria resist external stresses due to cell envelope rigidity, which is provided by two membranes and a peptidoglycan layer. The outer membrane (OM) surface contains lipopolysaccharide (LPS; contains O-antigen) or lipooligosaccharide (LOS). LPS/LOS are essential in most Gram-negative bacteria and may contribute to cellular rigidity. Acinetobacter baumannii is a useful tool for testing these hypotheses as it can survive without LOS. Previously, our group found that strains with naturally high levels of penicillin binding protein 1A (PBP1A) could not become LOS deficient unless the gene encoding it was deleted, highlighting the relevance of peptidoglycan biosynthesis and suggesting that high PBP1A levels were toxic during LOS deficiency. Transposon sequencing and follow-up analysis found that axial peptidoglycan synthesis by the elongasome and a peptidoglycan recycling enzyme, ElsL, were vital in LOS-deficient cells. The toxicity of high PBP1A levels during LOS deficiency was clarified to be due to a negative impact on elongasome function. Our data suggest that during LOS deficiency, the strength of the peptidoglycan specifically imparted by elongasome synthesis becomes essential, supporting that the OM and peptidoglycan contribute to cell rigidity. IMPORTANCE Gram-negative bacteria have a multilayered cell envelope with a layer of cross-linked polymers (peptidoglycan) sandwiched between two membranes. Peptidoglycan was long thought to exclusively provide rigidity to the cell providing mechanical strength. Recently, the most outer membrane of the cell was also proposed to contribute to rigidity due to properties of a unique molecule called lipopolysaccharide (LPS). LPS is located on the cell surface in the outer membrane and is typically required for growth. By using Acinetobacter baumannii, a Gram-negative bacterium that can grow without LPS, we found that key features of the peptidoglycan structure also become essential. This finding supports that both the outer membrane and peptidoglycan contribute to cell rigidity.

Enzymatically-crosslinked gelatin hydrogels containing paenipeptin and clarithromycin against carbapenem-resistant pathogen in murine skin wound infection.

BACKGROUND: The recent rise and spread of carbapenem-resistant pathogens pose an urgent threat to public health and has fueled the search for new therapies. Localized delivery of topical antibiotics is an alternative for the treatment of infected wounds caused by drug-resistant pathogens. In this study, we aimed to develop antimicrobial-loaded hydrogels for topical treatment of wound infections in a murine skin wound infection. RESULTS: Paenipeptin analogue 1, a linear lipopeptide, potentiated clarithromycin against multidrug-resistant Acinetobacter baumannii, Enterobacter cloacae, Escherichia coli, and Klebsiella pneumoniae. Enzymatically-crosslinked gelatin hydrogels were developed to encapsulate paenipeptin analogue 1 and clarithromycin. The encapsulated antimicrobials were gradually released from hydrogels during incubation, reaching 75.43 and 53.66% for paenipeptin and clarithromycin, respectively, at 24 h. The antimicrobial-loaded hydrogels containing paenipeptin and clarithromycin synergistically resulted in 5-log reduction in carbapenem-resistant A. baumannii within 6 h in vitro. Moreover, the antimicrobial-loaded hydrogels reduced 3.6- and 2.5-log of carbapenem-resistant A. baumannii when treated at 4 or 20 h post infection, respectively, in a murine skin wound infection. CONCLUSIONS: Enzymatically-crosslinked gelatin hydrogels loaded with paenipeptin analogue 1 and clarithromycin exhibited potent therapeutic efficacy against carbapenem-resistant A. baumannii in murine skin wound infection.

The Impact of Omega-3 Fatty Acids on the Evolution of Acinetobacter baumannii Drug Resistance.

The bacterial pathogen Acinetobacter baumannii has emerged as an urgent threat to health care systems. The prevalence of multidrug resistance in this critical human pathogen is closely associated with difficulties in its eradication from the hospital environment and its recalcitrance to treatment during infection. The development of resistance in A. baumannii is in part due to substantial plasticity of its genome, facilitating spontaneous genomic evolution. Many studies have investigated selective pressures imposed by antibiotics on genomic evolution, but the influence of high-abundance bioactive molecules at the host-pathogen interface on mutation and rates of evolution is poorly understood. Here, we studied the roles of host fatty acids in the gain in resistance to common antibiotics. We defined the impact of the polyunsaturated fatty acids arachidonic acid and docosahexaenoic acid on the development of resistance to erythromycin in A. baumannii strain AB5075_UW using a microevolutionary approach. We employed whole-genome sequencing and various phenotypic analyses to characterize microbe-lipid-antibiotic interactions. Cells exposed to erythromycin in the presence of the fatty acids displayed significantly lower rates of development of resistance to erythromycin and, importantly, tetracycline. Subsequent analyses defined diverse means by which host fatty acids influence the mutation rates. This work has highlighted the critical need to consider the roles of host fatty acids in A. baumannii physiology and antimicrobial resistance. Collectively, we have identified a novel means to curb the development of resistance in this critical human pathogen. IMPORTANCE The global distribution of multidrug resistance in A. baumannii has necessitated seeking not only alternative therapeutic approaches but also the means to limit the development of resistance in clinical settings. Highly abundant host bioactive compounds, such as polyunsaturated fatty acids, are readily acquired by A. baumannii during infection and have been illustrated to impact the bacterium's membrane composition and antibiotic resistance. In this work, we show that in vitro supplementation with host polyunsaturated fatty acids reduces the rate at which A. baumannii gains resistance to erythromycin and tetracycline. Furthermore, we discover that the impact on resistance development is closely associated with the primary antimicrobial efflux systems of A. baumannii, which represent one of the major drivers of clinical resistance. Overall, this study emphasizes the potential of host macromolecules in novel approaches to circumvent the difficulties of multidrug resistance during A. baumannii treatment, with fatty acid supplements such as fish oil providing safe and cost-effective ways to enhance host tolerance to bacterial infections.

CsrA Coordinates Compatible Solute Synthesis in Acinetobacter baumannii and Facilitates Growth in Human Urine.

CsrA is a global regulator widespread in bacteria and known to be involved in different physiological processes, including pathogenicity. Deletion of csrA of Acinetobacter baumannii strain ATCC 19606 resulted in a mutant that was unable to utilize a broad range of carbon and energy sources, including amino acids. This defect in amino acid metabolism was most likely responsible for the growth inhibition of the ΔcsrA mutant in human urine, where amino acids are the most abundant carbon source for A. baumannii. Recent studies revealed that deletion of csrA in the A. baumannii strains AB09-003 and ATCC 17961 resulted in an increase in hyperosmotic stress resistance. However, the molecular basis for this observation remained unknown. This study aimed to investigate the role of CsrA in compatible solute synthesis. We observed striking differences in the ability of different A. baumannii strains to cope with hyperosmotic stress. Strains AB09-003 and ATCC 17961 were strongly impaired in hyperosmotic stress resistance in comparison to strain ATCC 19606. These differences were abolished by deletion of csrA and are in line with the ability to synthesize compatible solutes. In the salt-sensitive strains AB09-003 and ATCC 17961, compatible solute synthesis was repressed by CsrA. This impairment is mediated via CsrA and could be overcome by deletion of csrA from the genome. IMPORTANCE The opportunistic human pathogen Acinetobacter baumannii has become one of the leading causes of nosocomial infections around the world due to the increasing prevalence of multidrug-resistant strains and their optimal adaptation to clinical environments and the human host. Recently, it was found that CsrA, a global mRNA binding posttranscriptional regulator, plays a role in osmotic stress adaptation, virulence, and growth on amino acids of A. baumannii AB09-003 and ATCC 17961. Here, we report that this is also the case for A. baumannii ATCC 19606. However, we observed significant differences in the ΔcsrA mutants with respect to osmostress resistance, such as the AB09-003 and 17961 mutants being enhanced in osmostress resistance whereas the ATCC 19606 mutant was not. This suggests that the role of CsrA in osmotic stress adaptation is strain specific. Furthermore, we provide clear evidence that CsrA is essential for growth in human urine and at high temperatures.

A Slam-dependent hemophore contributes to heme acquisition in the bacterial pathogen Acinetobacter baumannii.

Nutrient acquisition systems are often crucial for pathogen growth and survival during infection, and represent attractive therapeutic targets. Here, we study the protein machinery required for heme uptake in the opportunistic pathogen Acinetobacter baumannii. We show that the hemO locus, which includes a gene encoding the heme-degrading enzyme, is required for high-affinity heme acquisition from hemoglobin and serum albumin. The hemO locus includes a gene coding for a heme scavenger (HphA), which is secreted by a Slam protein. Furthermore, heme uptake is dependent on a TonB-dependent receptor (HphR), which is important for survival and/or dissemination into the vasculature in a mouse model of pulmonary infection. Our results indicate that A. baumannii uses a two-component receptor system for the acquisition of heme from host heme reservoirs.

Antimicrobial, antioxidant and cytotoxic properties of Chenopodium glaucum L.

We evaluated phytochemical composition, antibacterial, antifungal, anti-oxidant and cytotoxic properties of aqueous (water) and organic extracts (methanol, ethyl acetate and n-hexane) of Chenopodium glaucum. Highest phenolic content 45 mg gallic acid equivalents (GAE)/g d.w was found in aqueous extract followed by ethyl acetate (41mg GAE/g d.w) and methanol extract (34.46 mg GAE/g d.w). Antibacterial potential of aqueous and organic extracts of C. glaucum was examined against Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli and Staphylococcus epidermidis. The aqueous, methanolic, ethyl acetate, and n-hexane extract showed antibacterial activity against A. baumannii, K. pneumoniae, E. coli and S. epidermidis. However, against A. baumannii significantly higher inhibition zone (19 mm and 18.96 mm respectively) was shown by ethyl acetate and methanol extracts. Aqueous extract possessed highest growth inhibition (11 mm) against E. coli. Aqueous, ethyl acetate and methanol extracts showed 9 mm, 10 mm, and 10.33 mm zone of inhibition against the K. pneumoniae. For antifungal activity, the extracts were less effective against Aspergillus niger but showed strong antifungal activity against Aspergillus flavus (A. flavus). The antioxidant activity was measured as DPPH (2, 2-diphenyl-1-picrylhydrazyl), H2O2 and ABTS (2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) scavenging activity of free radicals. All the organic extracts of C. glaucum possessed ABTS, DPPH and H2O2 scavenging properties. The highest cytotoxic activity measured as half maximal inhibitory concentration (IC50) against human lungs carcinoma cells was recorded for methanolic (IC50 = 16 µg/mL) and n-hexane (IC50 = 25 µg/mL) extracts, respectively. The Gas chromatography-mass spectrometry (GC-MS) analysis showed 4 major and 26 minor compounds in n-hexane extract and 4 major and 7 minor compounds in methanol extract of the C. glaucum. It is concluded that aqueous and organic extracts of C. glaucum would be potential therapeutic agents and could be exploited on a pilot scale to treat human pathogenic diseases.

In Vitro Activities of Tigecycline in Combination with Amikacin or Colistin Against Carbapenem-Resistant Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii (CRAB) has been a common pathogen of nosocomial infections and severely threatened the public health for decades. Tigecycline is a new type of antibacterial glycylcycline and minocycline derivative and has been used to treat CRAB in clinical practice. However, the synergistic effects of tigecycline in combination with other antibiotics including colistin or amikacin remain unclear. A total of 216 CRAB isolates were collected from multiple body parts of different patients. The gene types of these isolates were analyzed and their resistance to carbapenems was determined by Etest. Broth microdilution method was utilized to evaluate the minimum inhibitory concentration (MIC) of each sample. Checkerboard screening technique was performed to demonstrate the synergistic effects of antibiotics and fractional inhibitory concentration index (FICI) was established. Therefore, the joint treatment of tigecycline and colistin (1:1) could effectively improve the sensitivity of AB to antibiotics. OXA-24-like isolates were more sensitive to the combination of tigecycline and amikacin. On the other hand, OXA-23-like isolates were more sensitive to the combination of tigecycline and colistin. Tigecycline exhibited synergistic effects with amikacin and colistin to inhibit CRAB.

Synergism versus Additivity: Defining the Interactions between Common Disinfectants.

Many of the most common disinfectant and sanitizer products are formulations of multiple antimicrobial compounds. Products claiming to contain synergistic formulations are common, although there is often little supporting evidence. The antimicrobial interactions of all pairwise combinations of common disinfectants (benzalkonium chloride, didecyldimethylammonium chloride, polyhexamethylene biguanide, chlorocresol, and bronopol) were classified via checkerboard assay and validated by time-kill analyses. Combinations were tested against Acinetobacter baumannii NCTC 12156, Enterococcus faecalis NCTC 13379, Klebsiella pneumoniae NCTC 13443, and Staphylococcus aureus NCTC 13143. Synergistic interactions were identified only for the combinations of chlorocresol with benzalkonium chloride and chlorocresol with polyhexamethylene biguanide. Synergism was not ubiquitously demonstrated against all species tested and was on the borderline of the synergism threshold. These data demonstrate that synergism between disinfectants is uncommon and circumstantial. Most of the antimicrobial interactions tested were characterized as additive. We suggest that this is due to the broad, nonspecific mechanisms associated with disinfectants not providing an opportunity for the combined activities of these compounds to exceed the sum of their parts. IMPORTANCE The scarcity of observed synergistic interactions suggests that in the case of many disinfectant-based products, combined mechanisms of interaction may be being misinterpreted. We emphasize the need to correctly differentiate between additivity and synergism in antimicrobial formulations, as inappropriate classification may lead to unnecessary issues in the event of regulatory changes. Furthermore, we question the need to focus on synergism and disregard additivity when considering combinations of disinfectants, as the benefits that synergistic interactions provide are not necessarily relevant to the application of the final product.

The roles of history, chance, and natural selection in the evolution of antibiotic resistance.

History, chance, and selection are the fundamental factors that drive and constrain evolution. We designed evolution experiments to disentangle and quantify effects of these forces on the evolution of antibiotic resistance. Previously, we showed that selection of the pathogen Acinetobacter baumannii in both structured and unstructured environments containing the antibiotic ciprofloxacin produced distinct genotypes and phenotypes, with lower resistance in biofilms as well as collateral sensitivity to ß-lactam drugs (Santos-Lopez et al., 2019). Here we study how this prior history influences subsequent evolution in new ß-lactam antibiotics. Selection was imposed by increasing concentrations of ceftazidime and imipenem and chance differences arose as random mutations among replicate populations. The effects of history were reduced by increasingly strong selection in new drugs, but not erased, at times revealing important contingencies. A history of selection in structured environments constrained resistance to new drugs and led to frequent loss of resistance to the initial drug by genetic reversions and not compensatory mutations. This research demonstrates that despite strong selective pressures of antibiotics leading to genetic parallelism, history can etch potential vulnerabilities to orthogonal drugs.

In vitro and in vivo toxicity and antibacterial efficacy of melittin against clinical extensively drug-resistant bacteria.

BACKGROUND: Melittin is one of the most studied antimicrobial peptides, and several in vitro experiments have demonstrated its antibacterial efficacy. However, there is evidence showing melittin has non-promising effects such as cytotoxicity and hemolysis. Therefore, concerns about unwanted collateral toxicity of melittin lie ahead in the path toward its clinical development. With these considerations, the present study aimed to fill the gap between in vitro and in vivo studies. METHODS: In the first step, in vitro toxicity profile of melittin was assessed using cytotoxicity and hemolysis tests. Next, a maximum intraperitoneal (i.p.) sub-lethal dose was determined using BALB/c mice. Besides toxicity, antimicrobial efficacy of melittin against extensively drug-resistant (XDR) Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus (MRSA), and KPC-producing Klebsiella pneumonia (KPC-KP) pathogens were tested using both in vitro and in vivo methods. RESULTS: Melittin showed extensive hemolysis (HD50 = 0.44 µg/mL), and cytotoxicity (IC50 = 6.45 µg/mL) activities with i.p. LD50 value of 4.98 mg/kg in BALB/c mice. In vitro antimicrobial evaluation showed melittin MIC range from 8 to 32 µg/mL for the studied pathogens. Treatment of infected mice with repeated sub-lethal doses of melittin (2.4 mg/kg) displayed no beneficial effect on their survival and peritoneal bacterial loads. CONCLUSIONS: These results indicate that melittin at its safe dose could not exhibit antimicrobial activity, which hinders its application in clinical practice.

Caerin 1.1 and 1.9 Peptides from Australian Tree Frog Inhibit Antibiotic-Resistant Bacteria Growth in a Murine Skin Infection Model.

The host defense peptide caerin 1.9 was originally isolated from skin secretions of an Australian tree frog and inhibits the growth of a wide range of bacteria in vitro. In this study, we demonstrated that caerin 1.9 shows high bioactivity against several bacteria strains, such as Staphylococcus aureus, Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus (MRSA), and Streptococcus haemolyticus in vitro. Importantly, unlike the antibiotic Tazocin, caerin 1.9 does not induce bacterial resistance after 30 rounds of in vitro culture. Moreover, caerin 1.1, another peptide of the caerin family, has an additive antibacterial effect when used together with caerin 1.9. Furthermore, caerin 1.1 and 1.9 prepared in the form of a temperature-sensitive gel inhibit MRSA growth in a skin bacterial infection model of two murine strains. These results indicate that caerin 1.1 and 1.9 peptides could be considered an alternative for conventional antibiotics. IMPORTANCE Antibiotic-resistant bacteria cause severe problems in the clinic. We show in our paper that two short peptides isolated from an Australian frog and prepared in the form of a gel are able to inhibit the growth of antibiotic-resistant bacteria in mice, and, unlike antibiotics, these peptides do not lead to the development of peptide-resistant bacteria strains.

Octopromycin: Antibacterial and antibiofilm functions of a novel peptide derived from Octopus minor against multidrug-resistant Acinetobacter baumannii.

The emergence of carbapenem-resistant Acinetobacter baumannii has increased the risk of nosocomial infections, which pose a huge health threat. There is an urgent need to develop alternative therapies, including broad-spectrum antimicrobial peptides. In this study, we designed, characterized, and studied the antibacterial, antibiofilm effects and possible mode of actions of a novel synthetic peptide Octopromycin, derived from the proline-rich protein 5 of Octopus minor. Octopromycin consists of 38 amino acids, (+5) net positive charge, high hydrophobic residue ratio (36%), and two α-helix secondary structures. The minimum inhibitory concentration and minimum bactericidal concentration against A. baumannii were 50 and 200 µg/mL, respectively. Time-kill kinetics and bacterial viability assays confirmed the concentration-dependent antibacterial activity of Octopromycin. Field emission scanning electron microscopy images clearly showed ultrastructural alterations in Octopromycin-treated A. baumannii cells. Propidium iodide penetrated into Octopromycin-treated A. baumannii cells, demonstrating the loss of cell membrane integrity. Octopromycin treatment increased the production of reactive oxygen species in a concentration-dependent manner, and it inhibited the biofilm formation and showed biofilm eradication activity against A. baumannii. In vitro and in vivo safety evaluation revealed that Octopromycin was nontoxic to HEK293T and Raw 264.7 cells (<400 µg/mL), as well as mice red blood cells (<300 µg/mL), and zebrafish embryos (<4 µg/mL). An in vivo study results revealed that the A. baumannii-infected fish treated with Octopromycin exhibited a significantly higher relative percent survival (37.5%) than the infected mock-treated fish with PBS (16.6%). Furthermore, a decreased bacterial load and fewer alterations in histological analysis confirmed the successful control of A. baumannii by Octopromycin in vivo. Collectively, the results indicate that the antibacterial peptide Octopromycin may achieve rapid control of A. baumannii through multi-target interactions; it presents a desirable therapeutic option for the prevention and control of the infections.

Therapeutic Potential of Novel Mastoparan-Chitosan Nanoconstructs Against Clinical MDR Acinetobacter baumannii: In silico, in vitro and in vivo Studies.

PURPOSE: Acinetobacter baumannii antibiotic resistant infections in high-risk patients are a great challenge for researchers and clinicians worldwide. In an effort to achieve potent bactericidal outcomes, a novel chitosan-mastoparan nanoconstruct (Mast-Cs NC) was designed and assessed for its therapeutic potential through in silico, in vitro and in vivo experimentation against clinical multidrug-resistant (MDR) A. baumannii. METHODS: Optimized 3D structures of mastoparan and chitosan were coupled computationally through an ionic cross-linker to generate a circular ring of chitosan encasing mastoparan. The complex was assessed for interactions and stability through molecular dynamic simulation (MDS). Binding pocket analysis was used to assess the protease-peptide interface. Mast-Cs NC were prepared by the ionic gelation method. Mast-Cs NC were evaluated in vitro and in vivo for their therapeutic efficacy against drug-resistant clinical A. baumannii. RESULTS: MDS for 100 ns showed stable bonds between chitosan and mastoparan; the first at chitosan oxygen atom-46 and mastoparan isoleucine carbon atom with a distance of 2.77 Å, and the second between oxygen atom-23 and mastoparan lysine nitrogen atom with a distance of 2.80 Å, and binding energies of -3.6 and -7.4 kcal/mol, respectively. Mast-Cs complexes approximately 156 nm in size, with +54.9 mV zeta potential and 22.63% loading capacity, offered >90% encapsulation efficiency and were found to be geometrically incompatible with binding pockets of various proteases. The MIC90 of Mast-Cs NC was significantly lower than that of chitosan (4 vs 512 µg/mL, respectively, p<0.05), with noticeable bacterial damage upon morphological analysis. In a BALB/c mouse sepsis model, a significant reduction in bacterial colony count in the Mast-Cs treated group was observed compared with chitosan and mastoparan alone (p<0.005). Mast-Cs maintained good biocompatibility and cytocompatibility. CONCLUSION: Novel mastoparan-loaded chitosan nanoconstructs signify a successful strategy for achieving a synergistic bactericidal effect and higher therapeutic efficacy against MDR clinical A. baumannii isolates. The Mast-Cs nano-drug delivery system could work as an alternative promising treatment option against MDR A. baumannii.

In vitroassessment of green polyhydroxybutyrate/chitosan blend loaded with kaempferol nanocrystals as a potential dressing for infected wounds.

Despite the major medical advancements in recent decades, treating infected wounds successfully remains a challenge. In this research, a functional blend of Polyhydroxybutyrate (PHB) and Chitosan (Cs) was developed for wound infection mitigation with tailored biological and physicochemical properties. Water insoluble kaempferol (KPF) was pre-formulated to water soluble KPF nanocrystals (KPF-NCs) with fine particle size of 145 ± 11 nm, and high colloidal stability (-31 ± 0.4 mV) to improve its drug transdermal delivery. PHB-Cs-KPF-NCs (1:2 ratio) film owned the best physical properties in terms of high breathability, thermal stability and mechanical strength (33 ± 1 MPa). Besides, XRD and FTIR findings indicated the interaction between Cs, PHB and KPF, reducing the film crystallinity. The scanning electron microscopy of the film displayed a highly interconnected porous morphology. KPF-NCs were integrated in PHB-Cs matrix with a marked encapsulation efficiency of 96.6%. The enhanced drug-loading film showed a sustain release pattern of KPF-NCs over 48 h. Interestingly, the developed blend possessed an impressive blood clotting capacity within 20 min. Furthermore, we presented a new naturally-sourced mixture of Cs+KPF-NCs with powerful antibacterial effects against MDRStaphylococcus aureusandAcentibacter baumanniiat very low concentrations. The membrane evidenced a remarkable antibacterial naturein vitrowith almost 100% cell viability reduction against the study strains after 48 h. By virtue of these advantages, this green blend is highly proposed for optimal wound care.

Hydrogen peroxide-producing electrochemical bandage controlled by a wearable potentiostat for treatment of wound infections.

Chronic wound infections caused by biofilm-forming microorganisms represent a major burden to healthcare systems. Treatment of chronic wound infections using conventional antibiotics is often ineffective due to the presence of bacteria with acquired antibiotic resistance and biofilm-associated antibiotic tolerance. We previously developed an electrochemical scaffold that generates hydrogen peroxide (H2 O2 ) at low concentrations in the vicinity of biofilms. The goal of this study was to transition our electrochemical scaffold into an H2 O2 -generating electrochemical bandage (e-bandage) that can be used in vivo. The developed e-bandage uses a xanthan gum-based hydrogel to maintain electrolytic conductivity between e-bandage electrodes and biofilms. The e-bandage is controlled using a lightweight, battery-powered wearable potentiostat suitable for use in animal experiments. We show that e-bandage treatment reduced colony-forming units of Acinetobacter buamannii biofilms (treatment vs. control) in 12 h (7.32 ± 1.70 vs. 9.73 ± 0.09 log10 [CFU/cm2 ]) and 24 h (4.10 ± 12.64 vs. 9.78 ± 0.08 log10 [CFU/cm2 ]) treatments, with 48 h treatment reducing viable cells below the limit of detection of quantitative and broth cultures. The developed H2 O2 -generating e-bandage was effective against in vitro A. baumannii biofilms and should be further evaluated and developed as a potential alternative to topical antibiotic treatment of wound infections.